Peptides and their medical uses

Abstract

The present invention relates to peptides, compositions comprising said peptides and their use as inhibitors of angiogenesis and / or neovascularization. In addition, the present invention relates to the use of said peptides and said compositions for treating diseases associated with improper angiogenesis and / or neovascularization. In particular, angiogenesis and / or neovascularization associated with VEGFR1 is described herein.

Description

Technical Field

[0001] The present invention relates to peptides, compositions comprising said peptides and their use as inhibitors of angiogenesis and / or neoangiogenesis. In addition, the present invention relates to the use of said peptides and said compositions for treating diseases associated with incorrect angiogenesis and / or neoangiogenesis. In particular, angiogenesis and / or neoangiogenesis associated with VEGFR1 are described herein. Prior art

[0002] In view of the fact that inhibition of VEGFR-1 activation can be applicable to a rather severe and wide range of diseases, it is conceivable that there will be a strong need for synthetic compounds that can bind to VEGFR-1 and can interfere with the interaction between the ligands VEGF-A, PlGF, VEGF-B and VEGF-A / PlGF heterodimers and VEGFR-1. In fact, advantageously, synthetic compounds are essentially free of pollutants of biological origin, and they can also be produced at a much lower cost than biotherapeutics of recombinant origin.

[0003] To neutralize the ligand, many therapeutic approaches use monoclonal antibodies, as they are molecules characterized by high specificity and affinity. However, synthetic molecules also have their advantages, as they are 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 peptide chains with the formula (R-Glu)-(S-Cys(Bzl))-(S-Cha) were tetramerized on a "core" of three lysines (Tam, JP 1988. Proc. Natl. Acad. Sci. USA 85:5409-5413).

[0005] The tetrameric peptide has the following structure:

[0006] Formula (I)

[0007]

[0008] Ponticelli et al. reported scientific evidence showing that the tetrameric peptide can bind to VEGFR1 in vitro and inhibit the interaction between PlGF, VEGF-A and VEGF-B. 50 The peptide was not able to bind to VEGFR-2 and did not interfere with the activation of VEGFR-2 by VEGF-A.

[0009] Finally, the peptide:

[0010] 1) It exhibits anti-angiogenic activity in vitro and interferes with the pro-angiogenic activity of PlGF and VEGF-A;

[0011] 2) It can replace VEGF-A-sFlt1 binding in the cornea (no blood vessels are formed under physiological conditions), thus making the cornea VEGF-A free (VEGF-A free) and inhibiting new blood vessel formation;

[0012] 3) when administered intraperitoneally, reduce tumor growth, angiogenesis and arteriogenesis, and metastasis; and

[0013] 4) Reduces choroidal neovascularization when administered intravitreally (Cicatiello et al. 2015).

[0014] The anti-angiogenic activity of the peptide is due to both the ability to inhibit the formation of new blood vessels and the ability to inhibit the recruitment of inflammatory cells, preferably mononuclear macrophages, to sites of neoangiogenesis.

[0015] The anti-arteriogenic activity is based on the ability to inhibit the recruitment of smooth muscle cells to sites of neovascularization.

[0016] Purpose of the Invention

[0017] In this context, the authors of the present invention surprisingly found that by inserting chemical groups at the C-terminus of the peptide, in particular amino acids characterized by side chains with steric hindrance comparable to thiol or thioether groups, one significantly increases the activity of the molecule.

[0018] In fact, the above modifications do not impair the selective binding to VEGFR1 and the ability to compete with VEGF-A and / or PlGF for binding to VEGFR1 in a dose-dependent manner. On the contrary, these modifications can produce a 50% inhibition (IC 2000) of the interaction between PlGF or VEGF and VEGFR1 at a concentration below 1000 nM. 50 ); Considering that the affinity of the peptides reported by Ponticelli et al. for VEGFR1 is equal to 10000 nM or higher (expressed as IC 50 ), which is a completely unexpected result. In other words, the peptides of the present invention have an inhibitory ability about one order of magnitude higher than the peptides reported by Ponticelli et al.

[0019] Furthermore, the authors of the present invention surprisingly found that both the peptides described in Ponticelli et al. and the peptides of the present invention exhibited a significant ability to inhibit choroidal neovascularization when administered orally or by gavage. Therefore, these molecules are therapeutically effective for treating (preferably by oral administration) diseases associated with or in any case caused by altered angiogenesis (preferably VEGFR1-dependent angiogenesis) changes.

[0020] The following is a detailed description of the invention, together with non-limiting illustrative examples with reference to the following figures and definitions. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] - Figure 1 The inhibitory activity of iVR1 and iVR1-Cys and anti-PlGF monoclonal antibody on PlGF-induced VEGFR-1 phosphorylation is shown. 293-VEGFR-1 cells were analyzed for VEGFR-1 phosphorylation induced by 20ng / ml PlGF by Western blotting. iVR1-Cys and iVR1 were added to PlGF simultaneously at a concentration of 5μM. A human anti-PlGF neutralizing monoclonal antibody at a concentration of 3.3nM was used as an inhibition control. PBS was used as a negative control.

[0023] - Figure 2 It is shown that iVR1-Cys administered intravitreally inhibits laser-induced choroidal neovascularization in a dose-dependent manner. A single intravitreal injection of 10 μg or 50 μg of iVR1-Cys caused a dose-dependent reduction in choroidal neovascularization equal to 48.9% and 75.9% compared to the injection of vehicle (DMSO). The same amount of iVR1 caused an inhibition of CNV equal to 37.8% and 73.9%. The control peptide (PC) did not show inhibitory ability. Quantification of the volume of neovascularization was performed on n=12 and 15 spots for iVR1 10 μg and 50 μg, on n=10 and 8 spots for iVR1-Cys 10 μg and 50 μg; quantification of the volume of neovascularization was performed on n=15 spots for PC, and on n=14 spots for DMSO. Data are expressed as mean ± SEM relative to control. #p<0.05; *p>0.0002; §p>0.002; vs. PC and DMSO. Below are images representing CNV. Scale bar represents 100 μm.

[0024] - Figure 3It is shown that orally administered iVR1-Cys inhibits laser-induced choroidal neovascularization. Oral administration of 50 mg / Kg of iVR1-Cys twice daily for 7 days caused a 45.9% reduction in choroidal neovascularization compared to vehicle. The same amount of iVR1 caused similar inhibition of CNV (49.7%). Quantification of neovascularization volume was performed on n=18 spots for iVR1-Cys, on n=20 spots for iVR1, and on n=10 spots for vehicle. Data are expressed as mean ± SEM relative to control. *p=0.001 and §p=0.007, compared to DMSO. Below are images representing CNV. Scale bars represent 100 μm.

[0025] definition

[0026] In this article, the term "VEGF" means vascular endothelial growth factor. There are 5 different vascular endothelial growth factors in humans, VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF, which are encoded by 5 different genes. All are glycosylated dimeric proteins.

[0027] In this article, the term "VEGF-A" means vascular endothelial growth factor-A, formerly also known as VPF (vascular permeability factor). It is the most powerful factor in the VEGF family and plays a decisive role in physiological and pathological angiogenesis. At least six different subtypes obtained by alternative splicing have been described in humans. They are all able to interact with two receptors, which are called VEGFR-1 and VEGFR-2.

[0028] In this article, the term "PlGF" means placental growth factor, and its effect is limited to the angiogenesis conditions associated with pathological conditions. Four different subtypes have been described in humans. All can specifically bind to VEGFR-1. VEGF-A and PlGF work with strong synergy in pathological conditions because both interact with VEGFR-1, and because when two corresponding genes are expressed in the same cell, they can produce VEGF-A / PlGF heterodimers that can interact with VEGFR-1 or induce VEGFR-1 / VEGFR-2 heterodimerization.

[0029] In this article, the term "VEGFR-1" means VEGF receptor 1, also known as Flt-1. VEGFR-1 has an intracellular tyrosine-kinase domain, and the extracellular part is composed of seven IgG-like domains. VEGF-A, VEGF-B or PlGF cause receptor dimerization, which is subsequently activated by the autophosphorylation of the tyrosine-kinase domain. In addition to being expressed in endothelial cells, VEGFR-1 is also expressed in many other types of cells, including smooth muscle cells, mononuclear macrophages, fibroblasts and endothelial progenitor cells. It plays an important role in the cells that raise different types of angiogenesis. In this article, the term "soluble VEGFR-1" (sVEGFR-1) means the soluble form of VEGF receptor 1, also known as sFlt-1. It is composed of the first six IgG-like extracellular domains of VEGFR-1 plus a tail, and is produced by alternative splicing of the VEGFR-1 gene. It is usually expressed by the same cells that express the full-length form of VEGFR-1, except in the cornea, where the soluble form is preferentially expressed, which is essential for maintaining the cornea in an avascular state. The messenger sequences of full-length and soluble human VEGFR1 are preferably SEQ ID NO: 1 and SEQ ID NO: 2, respectively, while the protein sequences of full-length human VEGFR1 are SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Sequences characterized by having 80%-99.9% identity with the sequences described herein must be considered as part of this description.

[0030] As used herein, the term "VEGFR-2" means VEGF receptor 2, also known as KDR in humans and Flk-1 in mice. VEGFR-2 is specifically bound by VEGF-A and has a domain structure and activation mechanism similar to that described for VEGFR-1. Unlike receptor 1, it is primarily expressed in endothelial cells. It plays an important role in stimulating the proliferation, migration and differentiation of endothelial cells.

[0031] In the present context, the term "angiogenesis" means the process of formation of new blood vessels from existing blood vessels; in the present context, angiogenesis is preferably referred to as a neovascularization process associated with various types of pathological conditions, preferably selected from:

[0032] - neovascular eye disease, preferably selected from the group consisting of macular edema, wet age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinopathy due to central retinal vein occlusion, vitreous hemorrhage and retinal detachment and combinations thereof; and / or

[0033] - solid tumors and / or tumor metastases, the tumors being preferably selected from the group consisting of: leukemias and lymphomas, preferably acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infantile 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, cancers of the urinary system, bladder cancer, oral tumors, pancreatic tumors, melanoma and skin tumors, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors; and / or

[0034] - bone or joint diseases, preferably selected from: rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, synovial tissue hypertrophy and / or hyperplasia, osteophyte formation, neoplasms and / or metastases and combinations thereof; and / or

[0035] - vascular disease, preferably selected from: atherosclerosis, hemangioma, hemangioendothelioma and combinations thereof; and / or

[0036] - a skin disease, preferably selected from the group consisting of: psoriasis, warts, pyogenic granuloma, hair growth, Kaposi's sarcoma, wound keloid, allergic edema, neoplasms and combinations thereof; and / or

[0037] - angiogenesis observed in adipose tissue diseases, preferably obesity; and / or

[0038] - Diabetes and / or its consequences, preferably retinopathy and / or diabetic foot; and / or

[0039] - Hematopoietic diseases, preferably AIDS and / or Kaposi's sarcoma.

[0040] In this context, the term "neoangiogenesis" means new blood vessel formation, preferably the formation of new blood vessels in tissues that previously had no new blood vessels and / or the increase in the number of blood vessels in already vascularized tissues; in this context, neoangiogenesis is preferably dependent on the activity of VEGFR-1.

[0041] In this context, the term "vascularization" means angiogenesis, ie they are used as synonyms.

[0042] As used herein, the term "neovascularization" means neoangiogenesis, preferably dependent on the activity of VEGFR-1.

[0043] As used herein, the term "arteriogenesis" refers to the process of stabilizing new blood vessels by covering them with smooth muscle cells.

[0044] As used herein, "inhibitor" means a chemical and / or biological entity that is capable of antagonizing the activity of a receptor by binding to the receptor itself and / or its soluble ligand, thereby preventing their interaction.

[0045] In this article, the term "effective dose" means the dose interval in which the administration of the active substance described in the present invention can determine the desired biological effect. As is well known to those skilled in the art, the effective dose can vary according to the following factors: health status, physical condition of the individual in need of treatment, age, formulation of the active substance, assessment of the doctor who takes care of the patient, the ability of the system of a single individual to respond effectively, the degree of response desired, taxonomic groups (e.g., humans, non-human primates, primates, etc.) and other relevant factors. It is expected that the effective dose of the active substance described in the present invention will fall within a sufficiently wide interval that can be determined by routine experiments. In general, as reported by Ragan-Shaw et al. (FASEBJ. 2008 Mar; 22 (3): 659-61), and therefore also herein, when preferably systemically administered, preferably systemically administered by enteral route, more preferably orally, sublingually or rectally administered, the effective dose administered is preferably in the range between 10 mg / dose and 2000 mg / dose. Alternatively, when preferably intravitreal administration, the effective dose administered is in the range between 1 mg / dose and 100 mg / dose. Alternatively, the effective dose administered is preferably in the range of 0.16 mg / Kg body weight to 33.3 mg / Kg body weight. The treatment regimen provides a single dose or multiple doses.

[0046] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0047] A first aspect of the present invention relates to a peptide, preferably a multimeric peptide, which is an isolated peptide and is characterized by the following general formula (II):

[0048] {{{[Y1–Glu–Cys(Bzl)–Cha]2–Z1}i–Z2}j–Z3}z–Y2–Y3

[0049] (Formula II)

[0050] in

[0051] -Y1 is the amino terminal functional group (NH2) of the peptide or at least one chemical group preferably selected in Table I. This list is also to be understood as including chemical groups (preferably amino acids) having steric hindrance and / or chemical properties (particularly side chains in the case of amino acids) that mimic the steric hindrance and / or chemical properties (particularly side chains in the case of amino acids) of the chemical groups (preferably amino acids) listed in Table I and / or are characterized by a similarity, preferably a similarity of at least 70%, determined by methods known to those skilled in the art, for example, but not exclusively, by the methods described in Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862.

[0052] In this context it should be clarified that the D / L notation applicable to defining the absolute configuration of chiral centers present in the groups of the present specification is interchangeable with the R / S notation following the rules reported in the literature, as known to those skilled in the art.

[0053] Table I

[0054]

[0055] -Glu represents glutamic acid, preferably in the absolute configuration R (R-Glu) on the Cα of the amino acid.

[0056] -Cys(Bzl) denotes benzylcysteine, preferably in the absolute configuration S on the Cα of an amino acid containing a benzyl group attached to the sulfur of the amino acid side chain (S-benzyl-cysteine / S-Cys(Bzl)).

[0057] -Cha represents cyclohexylalanine, preferably in the absolute configuration S at the Cα of the amino acid (S-cyclohexylalanine / S-Cha).

[0058] -Y2 is preferably selected from:

[0059] 1. The tripeptide R-Glu–S-Cys(Bzl)–S-Cha, and

[0060] • an α-amino acid, preferably selected from glycine or an α-amino acid characterized by at least one thiol or thioether group, preferably selected from those shown in Table II and combinations thereof.

[0061] This list is also to be understood as including chemical groups (preferably amino acids) having steric hindrance and / or chemical properties (particularly side chains in the case of amino acids) that mimic the steric hindrance and / or chemical properties (particularly side chains in the case of amino acids) of the chemical groups (preferably amino acids) listed in Table II and are characterized by a similarity, preferably a similarity of at least 70%, determined by methods known to those skilled in the art, for example, but not exclusively, by the methods described in Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862.

[0062] Table II

[0063]

[0064]

[0065] -Y3 is preferably selected from the group consisting of: a carboxyl group, a carboxamide group, an N-methyl substituted carboxamide or a disubstituted N,N-dimethyl group, a hydroxyl group and hydrogen.

[0066] - Z1, Z2 and Z3 preferably represent a trifunctional group, preferably characterized by the following formula (III):

[0067]

[0068] wherein k is an integer, preferably comprised between 1 and 4, and B is preferably an amino group or a hydroxyl group. The trifunctional molecule is preferably in the R or S absolute configuration.

[0069] Preferably, Z1, Z2 and Z3 are used to obtain the purpose of branched structure. In fact, this type of structure is usually used for peptide multimerization 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.

[0070] Z1, Z2 and Z3 may be assembled in such a way as to obtain a structure of formula (II) having more than one group Z1, Z2 and Z3, preferably comprising 1, 3 or 7 trifunctional molecules.

[0071] According to a preferred embodiment of the present invention, Z1 and / or Z2 and / or Z3 are preferably connected to each other via an amide bond to form a branched structure. Alternatively, they may be connected to each other via an ester bond, for example when B is preferably a hydroxyl group.

[0072] -i is preferably 4, 2 or 1.

[0073] -j is preferably 2, 1 or 0.

[0074] -z is preferably 1 or 0.

[0075] According to a preferred embodiment, when i=4, j=2 and z=1. According to another preferred embodiment, when i=2, j=1 and z=0.

[0076] According to another preferred embodiment, when i=1, j=z=0.

[0077] If j=0, the Z2 group is omitted, and if z=0, the Z3 group is omitted.

[0078] For the purposes of the present invention, particularly preferred embodiments envisage that i is equal to 2, j is equal to 1 and Z2 is 0 or omitted (in other words, Z3 does not occur, ie, Z3 is absent).

[0079] In a particularly preferred embodiment of the present invention, Z1, Z2 and Z3 are R-lysine or S-lysine (k=4), and i is preferably equal to 2.

[0080] The preferred formula of the multimeric peptide of the present invention is represented by the following formula (Formula IIa):

[0081]

[0082] (Formula IIa)

[0083] According to a particularly preferred embodiment of the present invention, the peptide is a tetrameric peptide characterized by formula (IIb):

[0084]

[0085] (Formula IIb)

[0086] in:

[0087] -Y1 is NH2;

[0088] -Y2 is D-cysteine;

[0089] -Y3 is a carboxyl group;

[0090] - Z1, Z2 and Z3 are as defined above;

[0091] -i is equal to 2;

[0092] -j is equal to 1; and

[0093] -z is equal to zero, which means it does not exist.

[0094] For the sake of convenience, from now on, a particularly preferred embodiment of the peptide characterized by formula lib will be referred to as iVR1-Cys.

[0095] The above peptides show biological activity, preferably modulating activity, more preferably inhibiting angiogenesis and / or neovascularization activity, which is improved compared to the activity of the peptides described by Ponticelli et al., as reported and discussed in the following experimental results, which have non-limiting illustrative purposes herein. The angiogenesis and / or neovascularization described herein is preferably VEGFR1-dependent as defined above.

[0096] The peptide described in Ponticelli et al. is also a tetrameric peptide characterized by formula (IIc):

[0097]

[0098] (Formula IIc)

[0099] in:

[0100] -Y1 is NH2;

[0101] -Y2 is glycine;

[0102] -Y3 is a carboxyl group;

[0103] - Z1, Z2 and Z3 are as defined above;

[0104] -i is equal to 2;

[0105] -j is equal to 1; and

[0106] -z is equal to zero.

[0107] For convenience, from now on, a particularly preferred embodiment of the peptide characterized by formula IIc will be referred to as iVR1.

[0108] The authors of the present invention surprisingly found that by modifying IVR1, in particular at the carboxyl terminus, preferably by inserting a R-Glu–S-Cys(Bzl)–S-Cha group or an α-amino acid, preferably chosen from α-amino acids characterized by at least one thiol or thioether group, preferably chosen from those presented in Table II and combinations thereof, one obtains peptides characterized by an improved biological activity, preferably an improved regulatory capacity, preferably by inhibiting angiogenesis and / or neoangiogenesis as defined above.

[0109] Indeed, as shown and discussed in more detail in the Examples, iVR1-Cys exhibited an improved ability to inhibit the interaction of both PlGF and VEGF-A with VEGFR-1 in a dose-dependent manner compared to iVR1. In particular, iVR1-Cys was able to inhibit the interaction of PlGF with VEGFR-1 by 50% (IC 50 ) was lower than 1000nM, while the IC of VEGF-A / VEGFR-1 was inhibited 50 On the other hand, at a concentration close to 10000 nM, iVR1 was able to inhibit 50% (IC 50 ). Similarly, the IC of iVR1 for VEGF-A / VEGFR-1 inhibition 50 Close to or slightly above 10000nM.

[0110] Thus, iVR1-Cys showed an inhibitory capacity 10-fold greater than that reported for iVR1.

[0111] Furthermore, the present authors demonstrated by in vivo assays that iVR1 caused an inhibition of choroidal neovascularization of 37.8% and 39.3% compared to vehicle and PC (p<0.05), whereas iVR1-Cys caused an inhibition of 48.9% and 51.0% compared to vehicle and PC (p<0.02). Thus, iVR1-Cys showed a greater inhibitory potency than peptide iVR1, as it further reduced neovascularization by 19.3%.

[0112] Finally, both peptides tested by the Examples were able to induce significant inhibition of neovascularization compared to vehicle when administered orally or by gavage.

[0113] The latter fact is particularly important, since, although Ponticelli et al. and Cicatiello et al. 2015 have demonstrated the ability of iVR1 to inhibit choroidal angiogenesis and neovascularization by intravitreal injection, it was absolutely unexpected that administration of the peptide by a different route, in particular by oral gavage, would be able to maintain or even improve the therapeutic efficacy, especially in the case of a highly complex organ such as the eye, and in the case of diseases affecting the eye caused by or in any case associated with unregulated (preferably increased) angiogenesis / neovascularization. In particular, the neovascular diseases of the eye in question are preferably selected from the group consisting of: macular edema, wet age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and combinations thereof.

[0114] Based on this evidence, it is clear that the peptides of the invention, administered by the oral route or by gavage, are also therapeutically effective for the treatment of diseases typically associated with angiogenesis / neoangiogenesis, such as, for example, cancer. Said angiogenesis or neoangiogenesis is preferably VEGFR1 dependent.

[0115] According to one embodiment of the invention, the peptides may be modified to facilitate or improve delivery, preferably by PEGylation, or using a container / shuttle / carrier system, preferably a liposome, micelle, capsule, emulsion, matrix, gel, etc.

[0116] Another aspect of the invention relates to a composition comprising a peptide as described in detail and at least one further pharmaceutically acceptable ingredient.

[0117] The composition preferably comprises at least one peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, ie iVR1-Cys.

[0118] As used herein, pharmaceutically acceptable ingredients refer to compounds selected from the group consisting of excipients, diluents, carriers, adjuvants, preservatives, antibiotics, anti-inflammatory agents, oils, vitamins, antioxidants, chelating agents, solubilizers, viscosity agents, inert gases, surfactants, emulsifiers, buffer substances, immunosuppressants, anti-tumor agents, and combinations thereof.

[0119] For example, according to one embodiment, the composition comprises a peptide of the present invention in combination with at least one anti-angiogenic / anti-neoangiogenic molecule, an antibody that neutralizes the effects 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.

[0120] Another aspect of the present invention relates to a peptide as described above, preferably a peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, ie iVR1-Cys, for use as a medicament.

[0121] Another aspect of the invention relates to a peptide as described above, preferably a peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, i.e. iVR1-Cys, or a composition comprising said peptide as described above, for use in the treatment of pathological conditions associated with or caused by improper angiogenesis / neoangiogenesis, i.e. diseases in which angiogenesis / neoangiogenesis is unregulated; angiogenesis / neoangiogenesis is preferably already increased and therefore needs to be inhibited.

[0122] In addition to being useful for treating the disease, the peptide as described above, preferably a peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, i.e. iVR1-Cys, or a composition comprising the peptide as described above can also be used for subsequent further alternative therapeutic treatment of the disease.

[0123] As already described previously, angiogenesis / neoangiogenesis as defined previously is preferably dependent on or induced / regulated by VEGFR1 or the VEGFR1 pathway.

[0124] The disease / condition is preferably selected from:

[0125] - neovascular eye disease, preferably selected from the group consisting of macular edema, wet age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinopathy due to central retinal vein occlusion, vitreous hemorrhage and retinal detachment and combinations thereof; and / or

[0126] - solid or liquid tumors and / or tumor metastases, the tumors being preferably selected from the group consisting of: leukemias and lymphomas, preferably acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infantile 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, cancers of the urinary system, bladder cancer, oral tumors, pancreatic tumors, melanoma and skin tumors, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors; and / or

[0127] - bone or joint diseases, preferably selected from: rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, synovial tissue hypertrophy and / or hyperplasia, osteophyte formation, neoplasms and / or metastases and combinations thereof; and / or

[0128] - vascular disease, preferably selected from: atherosclerosis, hemangioma, hemangioendothelioma and combinations thereof; and / or

[0129] - a skin disease, preferably selected from the group consisting of: psoriasis, warts, pyogenic granuloma, hair growth, Kaposi's sarcoma, wound keloid, allergic edema, neoplasms and combinations thereof; and / or

[0130] - angiogenesis observed in adipose tissue diseases, preferably obesity; and / or

[0131] - Diabetes and / or its consequences, preferably retinopathy and / or diabetic foot; and / or

[0132] - Hematopoietic diseases, preferably AIDS and / or Kaposi's sarcoma.

[0133] For the above-mentioned medical purposes, the peptides and compositions of the present invention may be optionally combined with known drugs for treating the above-mentioned diseases, or may also be used before or after known drugs for treating the above-mentioned diseases.

[0134] Furthermore, the peptides or compositions of the invention may be combined with known treatments of the surgical, radiotherapy or chemotherapy type for the treatment of the above mentioned diseases.

[0135] The peptides of the present invention or compositions comprising the peptides as described above can be formulated for administration by any route. The administration route is preferably selected from: systemic routes, preferably oral routes, oral gavage, sublingual or rectal routes, topical, subcutaneous, intramuscular, intravenous, intraarterial, intraperitoneal, intradermal and intraepidermal routes.

[0136] The peptide or composition of the present invention can be formulated into a solid, such as a pill, tablet, granule, soluble granule, pellet, bead, lozenge, etc. Alternatively, the peptide or composition of the present invention can be formulated into a liquid solution, such as by injection, inhalation or aerosol administration, or formulated into drops or sprays.

[0137] The peptides of the invention or the compositions comprising said peptides as described above may be administered as a bolus.

[0138] The peptides of the invention or the compositions comprising the peptides as described above may be administered via a medical device, for example via a stent, a pump or a patch.

[0139] Administration may preferably be continuous, by controlled release or by constant release, preferably using an ocular drug delivery device.

[0140] Administration by oral route or oral gavage is particularly preferred. In fact, as previously mentioned, the peptides of the present invention, including iVR1, have been shown to be effective in inhibiting angiogenesis / neoangiogenesis when administered by oral gavage. They have been shown to be effective in inhibiting angiogenesis / neoangiogenesis in the eye; in other words, when the peptides of the present invention, including iVR1, were administered by oral gavage, inhibition of angiogenesis / neoangiogenesis in the eye was surprisingly observed. The angiogenesis / neoangiogenesis in question is preferably VEGFR1-dependent.

[0141] Based on this scientific evidence, another aspect of the present invention relates to the peptides of the present invention, preferably at least one peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, i.e. iVR1-Cys, and / or a peptide characterized by formula IIc, i.e. iVR1, administered orally or intragastrically, or a composition comprising said peptides, for the treatment of diseases caused by improper (preferably increased) angiogenesis / neoangiogenesis (preferably VEGFR1-dependent angiogenesis / neoangiogenesis) or in any case associated with improper (preferably increased) angiogenesis / neoangiogenesis (preferably VEGFR1-dependent angiogenesis / neoangiogenesis).

[0142] The disease / condition is preferably selected from:

[0143] - neovascular eye disease, preferably selected from the group consisting of macular edema, wet age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinopathy due to central retinal vein occlusion, vitreous hemorrhage and retinal detachment and combinations thereof; and / or

[0144] - solid tumors and / or tumor metastases, the tumors being preferably selected from the group consisting of: leukemias and lymphomas, preferably acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infantile 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, cancers of the urinary system, bladder cancer, oral tumors, pancreatic tumors, melanoma and skin tumors, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors; and / or

[0145] - bone or joint diseases, preferably selected from: rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, synovial tissue hypertrophy and / or hyperplasia, osteophyte formation, neoplasms and / or metastases and combinations thereof; and / or

[0146] - vascular disease, preferably selected from: atherosclerosis, hemangioma, hemangioendothelioma and combinations thereof; and / or

[0147] - a skin disease, preferably selected from the group consisting of: psoriasis, warts, pyogenic granuloma, hair growth, Kaposi's sarcoma, wound keloid, allergic edema, neoplasms and combinations thereof; and / or

[0148] - angiogenesis observed in adipose tissue diseases, preferably obesity; and / or

[0149] - Diabetes and / or its consequences, preferably retinopathy and / or diabetic foot; and / or

[0150] - Hematopoietic diseases, preferably AIDS and / or Kaposi's sarcoma.

[0151] The peptide or composition of the present invention is administered to any animal in need thereof, preferably an animal in need of inhibition of VEGFR-1-dependent neoangiogenesis.

[0152] The animal is preferably a mammal, more preferably the animal is a human.

[0153] The effective dosage of the peptide or composition as described above is preferably in the range of:

[0154] - between 10 mg / dose and 2000 mg / dose, wherein preferably the administration is systemic, preferably by a systemic enteral route, more preferably orally, sublingually or rectally; or

[0155] - Between 1 mg / dose and 100 mg / dose, wherein intravitreal administration is preferred.

[0156] Alternatively, the effective dose administered is preferably within the range between 0.16 mg / Kg body weight and 33.3 mg / Kg body weight.

[0157] The treatment regimen preferably provides a single dose or multiple doses.

[0158] The sequences of the present invention are annotated according to the international standard WIPO ST.25 and their descriptions were generated with the program Patent-In 3.5. The description of the sequences is attached herewith.

[0159] In the present invention, the sequences identified in Table III and sequences having identities ranging from 80% to 99.9% are to be considered as described.

[0160] Table III

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] Example

[0167] Dose-dependent inhibition of VEGF-A / VEGFR1 and PlGF / VEGFR-1 interactions .

[0168] 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.

[0169] Human recombinant receptor VEGFR-1, specifically a version consisting of the seven extracellular domains of the receptor fused to the human IgG Fc domain (R&D Systems, catalog number 321-FL), was adhered at a concentration of 0.5 μg / ml in PBS pH 7.5 in the wells of a 96-well microplate (100 μl / well) for 16 hours at room temperature (RT).

[0170] After blocking nonspecific binding sites in the wells with a buffer solution consisting of PBS pH 7.5 containing 3% BSA, 5 ng / ml recombinant PlGF of human origin (R&D Systems, catalog number 264-PG) or 5 ng / ml recombinant VEGF-A (R&D Systems, catalog number 293-VE) in PBET (PBS pH 7.5, BSA 0.1%, EDTA 5 mM, Tween 0.004%) was added to the wells with attached receptors.

[0171] Simultaneously with the ligand (i.e., PlGF or VEGF-A), graded doses of iVR1, iVR1-Cys, or a control peptide (PC-[(S-Ser)-(S-Ala)-(S-Cha) tripeptide, having a tetrameric structure identical to that of the iVR1 peptide]) were added at concentrations comprised between 780 nM and 50000 nM. The binding reaction was performed at 37°C for 1 hour and then at room temperature for 1 hour.

[0172] At the end of the binding and / or competition step, anti-human-PlGF biotinylated polyclonal antibody (R&D Systems, catalog number BAF264) or anti-human-VEGF-A (R&D Systems, catalog number BAF293) was added to the wells at a concentration of 300ng / ml in PBET. Incubate at 37°C for 1 hour, then incubate at room temperature for 1 hour, add HRP-conjugated avidin-streptavidin system (Vectastain elite ABC kit) and HRP substrate (o-phenylenediamine-Sigma, catalog number P1526) to the wells. Quantification was performed by determining the absorbance at 490nM.

[0173] Any inhibitory activity of the peptides is expressed as % of remaining binding, comparing data obtained for PlGF or VEGF-A binding to the receptor in the presence of the tetrameric peptide with data obtained in the absence of the tetrameric peptide. iVR1 represents a positive control for inhibition of PlGF / VEGFR-1 or VEGF-A / VEGFR-1 interactions.

[0174] The results are given in Tables IV and V and show that, iVR1-Cys showed a dose-dependent inhibition of PlGF and The ability of VEGF-A to interact with VEGFR-1 .

[0175] iVR1-Cys can inhibit the interaction between PlGF and VEGFR-1 by 50% (IC 50 ) is lower than 1000nM, while IC for VEGF-A / VEGFR-1 50 Close to or slightly above 1000nM .

[0176] Thus, iVR1-Cys has an inhibitory potency that is approximately 10-fold higher than that of iVR1, and it is therefore expected that iVR1-Cys can be used at a 10-fold lower dose in the same in vitro and in vivo experimental protocols involving angiogenesis / neoangiogenesis inhibition to obtain the same effects as those obtained with iVR1.

[0177] PC provided no inhibition.

[0178] Table IV - Dose-dependent inhibition of PlGF / VEGFR-1 interaction

[0179]

[0180] Table V - Dose-dependent inhibition of VEGF-A / VEGFR-1 interaction

[0181]

[0182] The ability of tetrameric peptides having formula (II) but with Y2 other than D-cysteine ​​to inhibit VEGF-A / VEGFR-1 binding was assessed by the binding assay described above. Y2 of the peptide and the corresponding IC for inhibition of VEGF-A / VEGFR-1 interaction 50 Shown in Table VI.

[0183] Table VI - ICs for inhibition of VEGF-A / VEGFR-1 interaction 50

[0184]

[0185]

[0186] Inhibition of PlGF-induced VEGFR-1 phosphorylation .

[0187] An assay of PlGF-induced phosphorylation of the receptor VEGFR-1 was performed to evaluate the inhibitory capacity of the peptide iVR1-Cys and to compare its activity with that of iVR1.

[0188] To activate VEGFR-1, a cell line overexpressing this receptor, called 293-VEGFR-1, obtained by stable transfection of HEK-293 cells was used (Errico, M. et al. 2004 JBC, 279: 43929-43939).

[0189] For this purpose, 293-VEGFR-1 cells were cultured until subconfluence was reached and then "starved" by maintaining / incubating the cells in serum-free medium for at least 16 hours.

[0190] At the end of the starvation step, the medium was removed and the cell monolayers were incubated with 100 μM Na3VO4 for 5 min to inhibit the activity of endogenous phosphatases.

[0191] The cells were then stimulated (1) with 20 ng / ml PlGF alone in the medium used for starvation for 10 min at 37°C, and (2) with PlGF in the presence of the peptide at a concentration of 5 μM.

[0192] As an inhibition control, a neutralizing monoclonal antibody against human PlGF (Thrombogenics) was used at a concentration of 3.3 nM. PBS was used as a negative control.

[0193] At the end of the incubation, the cells were washed with cold 100 μM Na3VO4 and then lysed in a buffer containing Tris-HCl 20 mM pH 8, EDTA 5 mM, NaCl 150 mM, 1% Triton-X100, 10% glycerol, zinc acetate 10 mM, Na3VO4 100 μM and a protease inhibitor cocktail and incubated at 4 ° C for 1 hour under gentle agitation. Finally, the cell lysate was centrifuged at 12000 x g for 15 minutes to remove cell debris. The quantification of the extracts was performed using the Bradford method using Bio-Rad reagents. 100 μg of each protein extract was loaded onto 8.5% reducing SDS-PAGE and then protein analysis standard methods were performed by western blotting.

[0194] Anti-p-VEGFR-1 antibody (R&D Systems, catalog number AF4170) at a dilution of 1:500 was used to detect phosphorylated VEGFR-1, while normalization was performed by detecting the non-phosphorylated form of the receptor using anti-VEGFR-1 antibody (Sigma-Aldrich, catalog number V4262) at a dilution of 1:500.

[0195] like Figure 1 As shown in FIG. 1 , the peptide iVR1-Cys was expressed at its IC determined in the binding assay (see Example 1). 50 Use of an approximately 5-fold higher concentration (5000 nM) resulted in a potent inhibition of receptor phosphorylation similar to that obtained with the neutralizing antibody and significantly greater than that obtained with the same concentration of iVR1.

[0196] Inhibition of choroidal neovascularization by intravitreal and oral (gavage) administration of iVR1 and iVR1-Cys .

[0197] The experimental model of laser-induced choroidal neovascularization needs to damage Bruch's membrane, and the damage of Bruch's membrane separates choroid from retinal pigment epithelium (RPE). This damage is caused by laser-induced burning, and burning causes Bruch's membrane perforation, thereby activating chorioretinal vascularization, i.e., the growth of new blood vessels that invade the overlying retinal tissue from the choroid. This mouse model summarizes the main features of exudative human age-related macular degeneration (AMD), and is in fact usually used as a preclinical model of AMD. It enables the anti-angiogenic activity of molecules of interest to be assessed.

[0198] To enable visualization of the mouse fundus and to induce injury using laser light, a Micron IV integrated system was used according to the experimental procedures described below.

[0199] First, pupil dilation was induced in the animals by applying 0.5% tropicamide eye drops. The animals were then anesthetized by intraperitoneal injection of ketamine and xylazine solutions (80 mg / Kg and 10 mg / Kg, respectively). After sedation, the animals were placed on a stand and a 2.5% aqueous solution of hydroxypropylmethylcellulose was applied to both eyes. It had the dual function of preventing corneal dehydration and improving visualization of the fundus by contacting the camera lens of the Micron IV with the solution (similar to the procedure used in microscopy with an immersion objective).

[0200] In order to induce damage with laser, first the laser pointer is activated and focused so that the laser beam is applied using the RPE layer as a reference. The area where the laser beam is applied must be away from the main blood vessels of the retina to prevent possible bleeding. The burning efficiency in the Bruch membrane layer is confirmed by the bubble formed immediately after applying the laser beam. The application condition of the laser beam is that the power is 200mW for 100msec.

[0201] Based on the data presented in the literature, which was nicely summarized by Lambert et al. (Nature Protocols, 2013, 8:2197), it is known that maximal neovascularization in this experimental model is obtained 7 days after injury.

[0202] C57Bl6 / J mice were used, n=5 per group. Immediately at the end of the laser-induced injury procedure, intravitreal injections were performed using a Hamilton syringe with a 32 g needle to administer 10 μg and 50 μg of iVR1-Cys or iVR1 and 50 μg of PC in 1 μL DMSO. As a control, DMSO itself was injected.

[0203] Seven days later, animals were killed, and eyes were removed and fixed with 4% paraformaldehyde. Subsequently, the anterior segment of the eye consisting of cornea, iris and lens was removed under a stereomicroscope. The remaining part, defined as "eye cup" or posterior segment, consists of sclera, choroid, RPE and retina, and was incubated for 16 hours in the presence of 0.7% FITC- Griffonia simplicifolia isolectin B4 (Vector Laboratories, Burlingame, CA). After a series of washings, the retina was removed, and four cuts were made on RPE / choroid so that RPE / choroid could be sealed on a slide, so as to be observed under a fluorescence microscope. The volume of choroidal neovascularization was quantified. In order to evaluate the volume of each spot, a series of images (Z-Stacks, about 20-25 images) were collected from the upper surface to the deepest focal plane at the level of RPE cells, and each image had a thickness of 1 μm. The volume of fluorescence was measured by ImageJ program (NIH, Bethesda, MD) by summing the fluorescent areas of each individual plane.

[0204] Quantification of CNVs was performed on n=12 and 15 spots for iVR1 10 μg and 50 μg, n=10 and 8 spots for iVR1-Cys 10 μg and 50 μg; quantification of CNVs was performed on n=15 spots for PC and n=14 spots for DMSO. Figure 2 The results presented in show that both peptides were able to induce a dose-dependent inhibition of neovascularization. At the higher dose (50 μg), a potent, significant, and comparable inhibition of neovascularization was obtained: iVR1-Cys-75.9% and -74.6% (p>0.002) compared to vehicle and PC; iVR1-73.9% and -76.5% (p>0.0002) compared to vehicle and PC.

[0205] At a dose of 10 μg, iVR1 caused an inhibition of neovascularization of 37.8% and 39.3% compared to vehicle and PC (p<0.05), while iVR1-Cys caused an inhibition of neovascularization of 48.9% and 51.0% compared to vehicle and PC (p<0.02). Thus, at low concentrations, peptide iVR1-Cys exhibited a greater inhibitory potency than peptide iVR1, as it further reduced neovascularization by 19.3%. Thus, at the higher doses used, the inhibitory capacity of the peptides may have reached a maximum threshold.

[0206] For experiments with oral administration (gavage), choroidal neovascularization was induced in C57Bl6 / J mice according to the aforementioned experimental procedures, with n=5 animals per group. Immediately after the animals recovered from anesthesia after the injury was induced, the peptide iVR1 and iVR1-Cys and the vehicle were administered twice a day for 7 days as specified in the experimental protocol. Based on previous data obtained by intraperitoneal administration of peptide iVR1 (Cicatiello et al. 2015, Oncotarget, 6, 10563–10576), the peptide was administered at a dose of 50 mg / Kg.

[0207] To enable oral administration, the peptides were dissolved in DMSO and then mixed with Nutilis food thickener, resulting in a final mixture consisting of 9 parts Nutilis and 1 part DMSO.

[0208] The substances were prepared in concentrations such that each single administration could be administered using 200 μl of a 9:1 Nutilis / substance mixture in DMSO, directly into the stomach of the animals for gavage using a suitable needle with a 20-gauge opening. In the control group, 200 μl of a 9:1 Nutilis / DMSO mixture were administered.

[0209] At the end of the experiment, animals were sacrificed, eyes were removed and dissected to separate the RPE-choroid, and the volume of CNV was determined by immunofluorescence analysis as described below.

[0210] Quantification of CNV was performed on n=18 spots for iVR1-Cys, n=20 spots for iVR1, and n=10 spots for vehicle.

[0211] The results are Figure 3 It is given in and shows that Compared with vehicle, the peptide iVR1-Cys was able to induce neovascularization 0.007), similar to the level observed for iVR1 (-49.7%, p= 0.001) .

[0212] Serum protease stability of iVR1-Cys .

[0213] The stability of the peptide iVR1-Cys in 10% serum (fetal calf serum, FCS) in 50 mM phosphate buffer pH 7.3 at 168 h was determined as described by Ponticelli et al., relying on a method based on RP-HPLC chromatography described therein [Ponticelli et al., J Biol Chem. 2008 Dec 5; 283(49): 34250-9].

[0214] A reference curve was constructed by dissolving the compound in DMSO at increasing concentrations between 0.1 μmol / L and 1000 μmol / L to obtain complete dissolution. The residual concentration of the molecule in contact with 10% FCS at an initial concentration of 10 μmol / L was then determined by withdrawing 3 aliquots at time t=0, then 3 aliquots per hour during the first 12 hours, and then 3 aliquots at 24h, 72h, 120h and 168h. The aliquots were diluted 1:1 with 0.1 M acetic acid to separate any peptides bound to albumin, centrifuged to remove any precipitated material and analyzed by RP-HPLC under the conditions reported by Ponticelli et al. The amount of residual peptide detected in the aliquots was expressed as a percentage relative to the initial amount and plotted as a table over time. The results are shown in Table VII as the mean ± standard deviation (SD) of three determinations.

[0215] Table VII

[0216] Time (h) Residual peptide ± SD% 0 101±4 1 100±11 2 99±7 3 98±5 4 97±4 5 96±5 6 99±6 7 95±8 8 96±5 9 95±6 10 94±10 11 94±8 12 93±4 24 93±6 72 92±7 120 91±5 168 92±8

Claims

1. An isolated peptide characterized by the following formula (IIa) in -Y1 is the amino terminal peptide functional group (NH2); -Glu represents glutamate; -Cys(Bzl) represents benzylcysteine; -Cha represents cyclohexylalanine; -Y2 is an α-amino acid selected from the group consisting of D-cysteine, L-cysteine, L-methionine, D-methionine, L-methionine sulfone, D-methionine sulfone, D-serine, L-serine, D-threonine, L-threonine; -Y3 is a carboxyl group; - Z1 and Z2 are S-lysine or R-lysine.

2. The peptide according to claim 1, characterized by formula (IIb), wherein:

3. A composition comprising at least one peptide according to claim 1 or 2 and at least one additional pharmaceutically acceptable ingredient.

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