Peptides and their medical uses

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 a peptide, a composition containing the peptide, and its use as an inhibitor of angiogenesis and / or neovascularization. Further, the present invention relates to the use of the peptide and the composition for the treatment of medical conditions correlated with inappropriate angiogenesis and / or neovascularization. In particular, angiogenesis and / or neovascularization correlated with VEGFR1 are referred to herein.

Background Art

[0002] Considering the high severity and wide range of medical conditions to which the inhibition of VEGFR-1 activation can be applied, there is a strong demand for synthetic compounds that can bind to VEGFR-1 and interfere with the interaction between VEGFR-1 and VEGF-A, PlGF, VEGF-B ligands, and VEGF-A / PlGF heterodimers. In fact, advantageously, synthetic compounds are essentially free of contaminants of biological origin and can also be produced at a much lower cost than recombinant biological therapeutics.

[0003] In many therapeutic approaches, monoclonal antibodies are used to neutralize ligands because they are molecules characterized by high specificity and affinity. However, synthetic molecules also have the advantages of being easier and cheaper to manufacture, more stable, and more easily deliverable.

[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, J.P. 1988. Proc. Natl. Acad. Sci. USA 85:5409-5413).

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

Chemical Formula

[0006] Scientific evidence reported by Ponticelli et al. shows that the tetrameric peptide described above binds to VEGFR1 and can inhibit the interaction of PlGF, VEGF-A, and VEGF-B in vitro with an IC50 of approximately 10 μM. Furthermore, this peptide cannot bind to VEGFR-2 and does not interfere with its activation by VEGF-A.

[0007] Finally, this peptide is 1) It exhibits anti-angiogenic activity in vitro, inhibiting the angiogenic activity of PlGF and VEGF-A. 2) It can replace the VEGF-A-sFlt1 binding in the cornea (it is not vascularized under physiological conditions), so VEGF-A is no longer present, and angiogenesis can be promoted. 3) When administered intraperitoneally, it reduces tumor growth, angiogenesis, neoarthritis and metastasis, and 4) When administered intravitreously, it reduces choroidal angiogenesis (Cicatiello et al. 2015).

[0008] The anti-angiogenic activity of this peptide is due to both its ability to inhibit the formation of new blood vessels and its ability to inhibit the recruitment of inflammatory cells, preferably monocyte-macrophage, at angiogenesis sites.

[0009] Anti-angiogenic activity is based on the ability to inhibit the recruitment of smooth muscle cells at the site of angiogenesis.

[0010] Objective of the present invention In this invention, the authors have surprisingly found that inserting an amino acid, characterized by a side chain having steric hindrance comparable to that of a chemical group, particularly a thiol or thioether group, into the C-terminus of a peptide significantly improves the activity of the molecule.

[0011] In fact, the above modifications do not impair the selective binding to VEGFR1, nor the ability to dose-dependently compete with VEGF-A and / or PlGF for binding to VEGFR1. On the contrary, these modifications can inhibit the interaction between PlGF or VEGF and VEGFR1 by 50% (IC50) at concentrations below 1000 nM. This is a completely unexpected result, considering that the affinity of the peptide reported by Ponticelli et al. for VEGFR1 is 10000 nM or higher (expressed as IC50). In other words, the peptide of the present invention has an inhibitory capacity approximately an order of magnitude greater than that of the peptide reported by Ponticelli et al.

[0012] Furthermore, the authors of this invention have surprisingly found that both the peptide described by Ponticelli et al. and the peptide of this invention exhibit a significant ability to inhibit choroidal angiogenesis when administered orally or by force feeding. Therefore, these molecules are therapeutically effective, preferably by oral administration, for treating conditions that correlate with, or are caused by, alteration of angiogenesis, preferably VEGFR1-dependent angiogenesis.

[0013] A detailed description of the present invention follows, along with non-limiting and illustrative examples that refer to the following figures and definitions. [Brief explanation of the drawing]

[0014] [Figure 1] This study demonstrates the inhibitory activity of iVR1 and iVR1-Cys against an anti-PlGF monoclonal antibody in relation to PlGF-induced phosphorylation of VEGFR-1. Analysis of VEGFR-1 phosphorylation induced by 20 ng / ml PlGF was performed on 293-VEGFR-1 cells by Western blotting. iVR1-Cys and iVR1 were simultaneously added to PlGF at a concentration of 5 μM. A human anti-PlGF neutralizing monoclonal antibody was used as an inhibitory control at a concentration of 3.3 nM. PBS was used as a negative control. [Figure 2]This study demonstrates that intravitreal administration of iVRI-Cys dose-dependently inhibits laser-induced choroidal angiogenesis. Intravitreal injection of 10 or 50 μg of iVRI-Cys resulted in dose-dependent reductions of choroidal angiogenesis equal to 48.9% and 75.9% compared to injection of the vehicle (DMSO). The same amount of iVR1 resulted in CNV inhibition equal to 37.8% and 73.9%. The control peptide (PC) showed no inhibitory ability. The amount of angiogenesis was quantified at 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. For PC and DMSO, #p<0.05, *p>0.0002, ¶p<0.02, §p>0.002. Below is an image representing CNV. The bars represent 100 μm. [Figure 3] This study demonstrates that orally administered iVRI-Cys inhibits laser-induced choroidal angiogenesis. Oral administration of iVR1-Cys at 50 mg / kg twice daily for 7 days resulted in a 45.9% reduction in choroidal angiogenesis compared to the vehicle. The same amount of iVR1 yielded similar CNV inhibition (49.7%). The amount of angiogenesis was quantified at n=18 spots for iVRI-Cys, n=20 spots for iVRI, and n=10 spots for the vehicle. Data are presented as mean ± SEM compared to the control. *p=0.001 and §p=0.007 compared to DMSO. Below is an image representing CNV. Bars represent 100 μm.

[0015] definition In this specification, the term "VEGF" means vascular endothelial growth factor. Humans have five distinct 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] In this specification, the term "VEGF-A" refers to vascular endothelial growth factor-A, formerly known as VPF (vascular permeability factor). VEGF-A is the most potent factor in 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 can interact with two receptors called VEGFR-1 and VEGFR-2.

[0017] In this specification, the term "PlGF" refers to placental growth factor, and its role is limited to the state of angiogenesis associated with pathological conditions. Four different isoforms are described in humans. All can specifically bind to VEGFR-1. Both VEGF-A and PlGF interact with VEGFR-1, and when both genes are expressed in the same cell, they can either interact with VEGFR-1 or produce a VEGF-A / PlGF heterodimer that can induce VEGFR-1 / VEGFR-2 heterodimerization, thus exhibiting potent synergistic effects in pathological conditions.

[0018] In this specification, 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 result in receptor dimerization, which in turn leads to activation by autophosphorylation of the tyrosine kinase domain. In addition to being expressed in endothelial cells, VEGFR-1 is expressed in many other cell types, 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. In this specification, the term "soluble VEGFR-1" (sVEGFR-1) refers to the soluble form of VEGF receptor 1, also known as sFlt-1. This consists of the first six IgG-like extracellular domains and tail of VEGFR-1 and is generated from the VEGFR-1 gene by alternative splicing. Normally, VEGFR-1 is expressed in the same cells that express the full-length form, but the soluble form is preferentially expressed in the cornea to maintain 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 preferably SEQ ID NOs: 3 and 4, respectively. Sequences characterized by having an identity with the sequences described herein in the range of 80-99.9% shall be considered part of this specification.

[0019] In this specification, 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 organizing domain and activation mechanism similar to that described for VEGFR-1. Unlike receptor 1, it is essentially expressed in endothelial cells. It plays a fundamental role in stimulating the proliferation, migration, and differentiation of endothelial cells.

[0020] As used herein, the term "angiogenesis" means the process by which new blood vessels are formed from existing blood vessels. As used herein, angiogenesis is preferably referred to as the process of formation of new blood vessels associated with various types of pathological conditions selected from the following: - Neovascular eye diseases preferably selected from macular edema, wet form 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, wherein the tumors are preferably selected from leukemia and lymphoma, preferably acute lymphoblastic leukemia, acute non-lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, Hodgkin lymphoma, Hodgkin 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 tumors, pancreatic tumors, skin melanoma and tumors, gastric tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors, solid tumors and / or tumor metastases, and / or - Bone or joint diseases preferably selected from rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, hypertrophy and / or hyperplasia of synovial tissue, formation of bone spurs, neoplasms and / or metastases, and combinations thereof, and / or - Vascular conditions preferably selected from atherosclerosis, hemangioma, angiosarcoma, and combinations thereof, and / or - Skin diseases preferably selected from psoriasis, fibroma, pyogenic granuloma, hair growth, Kaposi sarcoma, keloid of wound, allergic edema, neoplasms, and combinations thereof, and / or - Angiogenesis observed in conditions of adipose tissue, preferably obesity, and / or - Diabetes and / or its consequences, preferably retinopathy and / or diabetic foot, and / or - Hematopoietic diseases, preferably AIDS and / or Kaposi sarcoma.

[0021] As used herein, the term "angiogenesis" means the formation of new blood vessels, preferably associated with the formation of new blood vessels in tissues where there were no blood vessels previously and / or the formation of new blood vessels associated with an increase in the number of blood vessels in already vascularized tissues. As used herein, angiogenesis preferably depends on the activity of VEGFR-1.

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

[0023] As used herein, the term "neovascularization" means angiogenesis, preferably angiogenesis that depends on the activity of VEGFR-1.

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

[0025] As used herein, an "inhibitor" means 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, and thus prevent their interaction.

[0026] In this specification, the term “effective dose” means the administration interval at 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 may vary depending on the health status, the physical condition of the individual requiring treatment, age, the prescription of the active substance, the assessment of the physician caring for the patient, the ability of a single individual’s body (system) to respond effectively, the degree of the desired response, the taxonomic group (e.g., human, non-human primate, primate, etc.), and other relevant factors. The effective amount of the active substance described in the present invention is expected to be within a sufficiently wide interval to be determined by routine testing. Generally, 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 to 2000 mg / dose when administered systemically, preferably by enteral route, and more preferably orally, sublingually, or rectally. Alternatively, when preferably administered intravitreously, the effective dose administered is in the range of 1 to 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 program provides a single dose or multiple doses.

[0027] Detailed description of preferred embodiments of the present invention A first aspect of the present invention relates to peptides, and more preferably to isolated polymeric peptides 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 the peptide (NH2), or preferably at least one chemical group selected in Table I. This list is understood to also include side chains in the case of amino acids that mimic and / or have sterically hindered and / or chemical properties, preferably amino acids, in particular chemical groups listed in Table I, preferably amino acids, and / or preferably at least 70% similarity (this similarity is determined by methods known to those skilled in the art, e.g., by methods described, but not limited to, Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862).

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

[0031] [Table 1]

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

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

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

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

[0036] This list is also understood to include side chains in the case of amino acids that mimic sterically hindered and / or chemically impaired chemical groups, preferably amino acids, in particular those listed in Table II, preferably amino acids, and preferably with at least 70% similarity (this similarity is determined by methods known to those skilled in the art, e.g., by methods described, but not limited to, Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862).

[0037] [Table 2]

[0038] -Y3 is preferably selected from a carboxyl group, a carboxyamide group, an N-methyl-substituted carboxyamide 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] In the formula, k is an integer, preferably between 1 and 4, and B is preferably an amino group or a hydroxyl group. This trifunctional molecule is preferably in an R or S absolute configuration.

[0040] Preferably, Z1, Z2, and Z3 are used to obtain a branched structure. In fact, this type of structure is commonly used to polymerize peptides according to known methods for this purpose. For example, if 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 a structure of formula (II) having 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 amide bonds to form a branched structure. Alternatively, for example, if B is preferably a hydroxyl group, they may be linked to each other by ester bonds.

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

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

[0045] -z is preferably 1 or 0.

[0046] A preferred embodiment is the case where i=4, j=2, and z=1. A further preferred embodiment is the case where i=2, j=1, and z=0.

[0047] A more preferred embodiment is the case where i=1 and j=z=0.

[0048] If j=0, Z2 units are omitted, and if z=0, Z3 units are omitted.

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

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

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

[0052] According to a particularly preferred embodiment of the present 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 either equal to zero or does not exist.

[0054] For convenience, a particularly preferred embodiment of the peptide characterized by formula IIb will hereafter be referred to as iVR1-Cys.

[0055] The above peptides exhibit biological activity, preferably regulatory activity, more preferably activity that inhibits angiogenesis and / or angiogenesis, and this activity is improved compared to the activity of the peptide described by Ponticelli et al., as reported and described in the following experimental results (intended to be non-limiting in this specification). The angiogenesis and / or angiogenesis referred to herein is preferably VEGFR1-dependent, as previously defined.

[0056] The peptide described by Ponticelli et al. is also a tetrameric peptide characterized by 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 the peptide characterized by formula IIc will hereafter be referred to as iVR1.

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

[0060] Indeed, as demonstrated and discussed in more detail in the examples, iVR1-Cys demonstrated the ability to dose-dependently inhibit the interaction between both PlGF and VEGF-A and VEGFR-1 compared to iVR1. In particular, the concentration at which iVR1-Cys can inhibit the interaction between PlGF and VEGFR-1 by 50% (IC50) is less than 1000 nM, while the IC50 for VEGF-A / VEGFR-1 inhibition is close to or slightly above 1000 nM. On the other hand, iVR1 can inhibit the interaction between PlGF and VEGFR-1 by 50% (IC50) at concentrations close to 10000 nM. Similarly, the IC50 for VEGF-A / VEGFR-1 inhibition by iVR1 is close to or slightly above 10000 nM.

[0061] Therefore, iVR1-Cys exhibits 10 times the inhibitory capacity reported for iVR1 alone.

[0062] Furthermore, the authors showed that in in vivo assays, iVR1 inhibited choroidal angiogenesis by 37.8% and 39.3% compared to vehicle and PC (p<0.05), while iVR1-Cys inhibited it by 48.9% and 51.0% compared to vehicle and PC (p<0.02). Therefore, iVR1-Cys shows a superior inhibitory effect compared to peptide iVR1, further reducing angiogenesis by approximately 19.3%.

[0063] Finally, when administered orally or by force-feeding, both peptides, as tested as examples, can induce significant inhibition of angiogenesis compared to the vehicle.

[0064] The latter fact is important because, although the ability of iVR1 to inhibit choroidal angiogenesis and neovascularization by intravitreal injection had already been demonstrated by Ponticelli et al. and Cicatiello et al. (2015), it was not anticipated that administering the peptide via a different route, particularly by force feeding, could maintain or even improve therapeutic effects in highly complex organs such as the eye, and in cases of conditions that affect it, caused or correlated with unregulated, preferably enlarged, angiogenesis / neovascularization. In particular, the neovascular eye diseases mentioned are preferably selected from macular edema, the wet form 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 present invention via oral routes or by force feeding is therapeutically effective, for example, in the treatment of medical conditions such as cancer, which generally correlate with angiogenesis / angiogenesis. The angiogenesis or angiogenesis referred to is preferably VEGFR1-dependent.

[0066] According to one embodiment of the present invention, peptides may be modified to facilitate or improve delivery, preferably by pegylation, or by using a container / shuttle / carrier system, preferably liposomes, micelles, capsules, emulsions, matrices, gels, etc.

[0067] Further aspects of the present invention relate to compositions comprising the peptides described in detail and at least one further pharmaceutically acceptable component.

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

[0069] In this specification, pharmaceutically acceptable components mean compounds selected from excipients, diluents, carriers, adjuvants, preservatives, antibiotics, anti-inflammatory agents, oils, vitamins, antioxidants, chelating agents, solubilizers, viscous agents, inert gases, surfactants, emulsifiers, buffers, immunosuppressants, antitumor agents, and combinations thereof.

[0070] For example, according to one embodiment, the compositions include 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 the peptide of the present invention combined with a combination thereof.

[0071] Further aspects of the present invention relate to the above-mentioned peptide, preferably a peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, i.e., iVR1-Cys, for use as a pharmaceutical.

[0072] Further aspects of the present invention relate to the above-mentioned peptide, preferably a peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, i.e., iVR1-Cys, or a composition comprising the above-mentioned peptide, for use in the treatment of pathological conditions associated with or caused by inadequate angiogenesis / angiogenesis, i.e., conditions in which angiogenesis / angiogenesis is not regulated. This condition is preferably aggravated and therefore needs to be inhibited.

[0073] In addition to being useful in treating this condition, the above-mentioned peptide, preferably the peptide characterized by formula IIa, more preferably the peptide characterized by formula IIb, i.e., iVR1-Cys, or compositions containing the above-mentioned peptide, can also be used for follow-up of further alternative therapies for this condition.

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

[0075] This medical condition / state is preferably selected from the following: - Macular edema, wet form of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and neovascular eye diseases preferably selected from combinations thereof, and / or -Solid tumors and / or tumor metastases, wherein the tumor is preferably selected from leukemia and lymphoma, preferably acute lymphoblastic leukemia, acute nonlymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, Hodgkin 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, urinary tract cancer, bladder cancer, oral tumors, pancreatic tumors, melanoma and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, larynx tumors, liver tumors, testicular tumors, solid tumors and / or tumor metastases, and / or - Diseases of bone or joints 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 - Vascular conditions preferably selected from atherosclerosis, hemangiomas, hemangioendotheliomas, and combinations thereof, and / or - Skin diseases preferably selected from psoriasis, warts, pyogenic granulomas, trichomes, Kaposi's sarcoma, keloids of wounds, allergic edema, neoplasms, and combinations thereof, as well as / or - Angiogenesis observed in adipose tissue pathology, preferably obesity, and / or - Diabetes and / or resulting in, preferably retinopathy and / or diabetic foot, and / or - Hematopoietic disorders, preferably AIDS and / or Kaposi's sarcoma.

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

[0077] Furthermore, the peptides or compositions of the present invention can be associated with known surgical, radiotherapy, or chemotherapy treatments used to treat the above-mentioned medical conditions.

[0078] The peptide of the present invention or a composition containing the peptide can be formulated to be administered via any route. The route of administration is preferably selected from systemic routes, preferably oral routes, forced feeding, sublingual or rectal routes, local, subcutaneous, intramuscular, intravenous, intra-arterial, intraperitoneal, intradermal, and intraepidermal routes.

[0079] The peptides or compositions of the present invention can be formulated as solids, for example, in the form of pills, tablets, granules, soluble granules, pellets, beads, lozenges, etc. Alternatively, the peptides or compositions of the present invention can be formulated as liquid solutions administered, for example, by injection, inhalation, or spray, or as droplets or sprays.

[0080] The peptide of the present invention described above, or a composition containing this peptide, can be administered as a bolus.

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

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

[0083] Oral administration or administration by force feeding is particularly preferred. In fact, as previously described, the peptides of the present invention (including iVR1) have been shown to be effective in inhibiting angiogenesis / angiogenesis even when administered by force feeding. They have also been shown to be effective in inhibiting ocular angiogenesis / angiogenesis. In other words, it was surprisingly observed that when the peptides of the present invention (including iVR1) are administered by force feeding, they inhibit angiogenesis / angiogenesis in the eye. The angiogenesis / angiogenesis referred to is preferably VEGFR1-dependent.

[0084] In light of this scientific evidence, further aspects of the present invention relate to the peptides of the present invention, preferably at least one peptide characterized by Figure IIa, more preferably a peptide characterized by formula IIb, i.e., iVR1-Cys, and / or a peptide characterized by formula IIc, i.e., iVR1, or compositions comprising these peptides administered orally or by force feeding, for use in the treatment of inadequate, preferably enlarged angiogenesis / angiogenesis (preferably VEGFR1-dependent), or any related medical conditions.

[0085] This medical condition / state is preferably selected from the following: - Macular edema, wet form of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and neovascular eye diseases preferably selected from combinations thereof, and / or - Solid tumors and / or tumor metastases, wherein the tumor is preferably selected from leukemia and lymphoma, preferably acute lymphoblastic leukemia, acute non-lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, Hodgkin 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, urinary tract cancer, bladder cancer, oral tumors, pancreatic tumors, melanoma and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, larynx tumors, liver tumors, testicular tumors, solid tumors and / or tumor metastases, and / or - Diseases of bone or joints 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 - Vascular conditions preferably selected from atherosclerosis, hemangiomas, hemangioendotheliomas, and combinations thereof, and / or - Skin diseases preferably selected from psoriasis, warts, pyogenic granulomas, trichomes, Kaposi's sarcoma, keloids of wounds, allergic edema, neoplasms, and combinations thereof, as well as / or - Angiogenesis observed in adipose tissue pathology, preferably obesity, and / or - Diabetes and / or resulting in, preferably retinopathy and / or diabetic foot, and / or - Hematopoietic disorders, preferably AIDS and / or Kaposi's sarcoma.

[0086] The peptide or composition of the present invention is administered to any animal that requires it, preferably an animal that needs to have its VEGFR-1-dependent angiogenesis inhibited.

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

[0088] The effective amount of the peptide or composition described above to be administered is preferably within the following range: - Preferably administered systemically, preferably via a systemic enteral route, more preferably orally, sublingually, or rectally, in doses of 10 to 2000 mg / dose, - Preferably administered intravitreously, 1 to 100 mg per dose.

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

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

[0091] The sequence of this invention is annotated in accordance with the international standard WIPO ST.25, and its description is shown in Program Patent-In 3.5. The description of the sequence is attached herein.

[0092] In this specification, sequences identified in Table III and sequences having 80–99.9% identity should also be considered as being included.

[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 Assays for testing the binding of PlGF or VEGF-A to the VEGFR-1 receptor were based on the ELISA method [Ponticelli et al., JBC. 2008 Dec 5;283(49):34250-9] and performed using reagents obtained from R&D Systems.

[0095] Human recombinant receptor VEGFR-1, specifically a form consisting of seven extracellular domains of the receptor fused to the Fc domain of human IgG (R&D Systems, catalog number 321-FL), was prepared and deposited into the wells of a 96-well microplate at a concentration of 0.5 μg / ml in PBS pH 7.5 (100 μl / well) at room temperature (RT) for 16 hours.

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

[0097] Ligands, i.e., PlGF or VEGF-A, were added simultaneously with stepwise doses of iVR1, iVR1-Cys, or a control peptide (PC-[a tripeptide having the same tetrameric structure as the iVR1 peptide]) at concentrations ranging from 780 to 50,000 nM. The binding reaction was carried out at 37°C for 1 hour, followed by 1 hour at room temperature.

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

[0099] Peptide inhibitory activity was expressed as the percentage of residual binding, comparing data obtained for binding of PlGF or VEGF-A to the receptor in the presence of the tetrameric peptide with data obtained 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. iVR1-Cys demonstrated the ability to dose-dependently inhibit the interaction between both PlGF and VEGF-A and VEGFR-1. This indicates.

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

[0102] Therefore, since iVR1-Cys has approximately 10 times the inhibitory capacity of iVR1, it is expected that it can be used at one-tenth the dose in the same in vitro and in vivo experimental protocols for inhibiting angiogenesis / angiogenesis to achieve the same effect as iVR1.

[0103] The PC will not interfere.

[0104] [Table 4]

[0105] [Table 5]

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

[0107] [Table 6]

[0108] Inhibition of PlGF-induced phosphorylation of VEGFR-1 We performed an assay for PlGF-induced phosphorylation of the receptor VEGFR-1 to evaluate the inhibitory ability of the peptide iVR1-Cys, and compared its activity with that of iVR1.

[0109] To activate VEGFR-1, we used a cell line that overexpresses a receptor called 293-VEGFR-1, obtained by stable transfection from HEK-293 cells (Errico, M. et al. 2004 JBC, 279:43929-43939).

[0110] For this purpose, 293-VEGFR-1 cells were cultured until subconfluence was reached, and then the cells were “starved” by holding / incubating them in serum-free medium for at least 16 hours.

[0111] At the end of the starvation process, the culture medium was removed and the cell monolayer was incubated with 100 μM Na3VO4 for 5 minutes to inhibit the activity of endogenous phosphatase.

[0112] Next, the cells were stimulated (1) with 20 ng / ml of PlGF alone in a medium that had been starved at 37°C for 10 minutes, and (2) in the presence of a 5 μM peptide.

[0113] Anti-human PlGF neutralizing monoclonal antibodies (thrombiogenics) were used as an inhibitory control at a concentration of 3.3 nM. PBS was used as a negative control.

[0114] At the end of incubation, the cells were washed with 100 μM cold Na3VO4 and lysed in a buffer consisting of a mixture 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 protease inhibitor. The mixture was incubated at 4°C for 1 hour with gentle agitation. Finally, the cell lysates were centrifuged at 12000 x g for 15 minutes to remove cell debris. Extract quantification was performed using the Bradford method with Bio-Rad reagents. 100 μg of all protein extract was loaded onto an SDS-PAGE to reduce it to 8.5%, and then Western blotting was performed using standard methods for protein analysis.

[0115] Normalization was performed by detecting phosphorylated VEGFR-1 using a 1:500 diluted anti-p-VEGFR-1 antibody (R&D Systems, catalog number AF4170), while detecting the unphosphorylated form of the receptor using a 1:500 diluted anti-VEGFR-1 antibody (Sigma-Aldrich, catalog number V4262).

[0116] As shown in Figure 1, peptide iVR1-Cys was determined by binding assay. 50 It is used at approximately five times the concentration (5000 nM) (see Example 1) and strongly inhibits receptor phosphorylation. This inhibition is similar to that obtained with neutralizing antibodies and is significantly greater than that obtained with iVR1 at the same concentration.

[0117] Inhibition of choroidal angiogenesis by intravitreous and oral (forced feeding) administration of iVR1 and iVR1-Cys. In an experimental model of laser-induced choroidal neovascularization, it is necessary to damage Bruch's membrane, which separates the choroid from the retinal pigment epithelium (RPE). This damage is induced by laser-induced burning, which leads to perforation of Bruch's membrane and activates chorioretinal neovascularization. The proliferation of new blood vessels begins in the choroid and invades the retinal tissue above. This mouse model summarizes the main features of the exudative morphology of human age-related macular degeneration (AMD) and is actually commonly used as a preclinical model of AMD. This allows for the evaluation of the anti-angiogenic activity of the target molecule.

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

[0119] First, pupillary dilation was induced in the animals by applying 0.5% tropicamide eye drops. Next, the animals were anesthetized by intraperitoneal injection of ketamine and xylazine solutions (80 mg / kg and 10 mg / kg, respectively). Once sedated, the animals were placed on a stand and a 2.5% aqueous solution of hydroxypropyl methylcellulose was applied to both eyes. By bringing the Micron IV camera lens into contact with the solution (similar to the procedure used in microscopy with an immersion objective lens), a dual function was achieved: preventing corneal dehydration and improving fundus visualization.

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

[0121] Based on the data available in the literature, this is adequately summarized in the paper by Lambert et al. (Nature Protocols, 2013, 8:2197). It is known that the maximum angiogenesis in this experimental model is achieved 7 days after injury.

[0122] C57Bl6 / J mice were used (n=5 per group). Immediately after the laser injury induction procedure, intravitreous injection was performed, 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. DMSO alone was administered as a control.

[0123] Seven days later, the animals were sacrificed, the eyes were enucleated, and fixed with 4% paraformaldehyde. Subsequently, the anterior part of the eye, consisting of the cornea, iris, and crystals, 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 hours in the presence of 0.7% FITC-Griffonia simplicifolia Isolectin B4 (Vector Laboratories, Burlingame, California). After a series of washes, the retina was removed, and four cuts were made in the RPE / choroid. These could then be placed on slides and observed under a fluorescence microscope. Quantification of choroidal angiogenesis was performed in terms of volume. A series of images (Z-Sats, approximately 20-25 images) were acquired to assess the volume of all spots. Each image was 1 μm thick and acquired at the RPE cell level, from the top to the deepest focal plane. The fluorescence volume was measured using the ImageJ program (NIH, Bethesda, Maryland), and the sum of the fluorescence areas of all single planes was taken.

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

[0125] At a dose of 10 μg, iVR1 resulted in 37.8% and 39.3% inhibition of angiogenesis in vehicles and PCs (p<0.05), while iVR1-Cys resulted in 48.9% and 51.0% inhibition of angiogenesis in vehicles and PCs (p<0.02). Therefore, at low concentrations, the peptide iVR1-Cys shows superior inhibitory effects compared to peptide iVR1, further reducing angiogenesis by 19.3%. Thus, it is possible that the maximum threshold of inhibitory capacity of the peptide was reached at higher doses used.

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

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

[0128] The substance was prepared in a concentration suitable for single-dose administration in 200 μl of a 9:1 Nutilis / DMSO mixture and administered directly to the stomach of animals using a force-feeding-appropriate needle 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, their eyes removed, and dissected to isolate the RPE choroid, and the volume of the CNV was determined by immunofluorescence analysis, as described below.

[0130] CNV quantization was performed with 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. The peptide iVR1-Cys can induce significant inhibition of angiogenesis at a level similar to that observed with iVR1 (-49.7%, p=0.001), compared to the 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 bovine serum, FCS) in 50 mM phosphate buffer (pH 7.3) for 168 hours was determined based on the RP-HPLC chromatography method described by Ponticelli et al. [Ponticelli et al., JBC. 2008 Dec 5;283(49):34250-9], as described therein.

[0133] Reference curves were prepared by dissolving the compound in DMSO at concentrations ranging from 0.1 μmol / L to 1000 μmol / L to ensure complete dissolution. The concentration of the molecule remaining in contact with 10% FCS at an initial concentration of 10 μmol / L was then determined by plotting three aliquots at time t=0, then every hour for the first 12 hours, and subsequently at 24, 72, 120, and 168 hours. To separate any peptides bound to albumin, aliquots were diluted 1:1 with 0.1 M acetic acid, centrifuged to remove precipitates, 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 of the initial amount and plotted as a function of time. The results are shown in Table VII as the mean ± standard deviation (SD) of three measurements.

[0134] [Table 7]

Claims

1. The following equation (IIa): 【Chemistry 1】 During the ceremony, -Y1 is an amino-terminal peptide functional group (NH 2 ) and -Y2 is, It is an α-amino acid selected from D-cysteine, L-cysteine, L-methionine, D-methionine, L-methionine sulfone, D-serine, L-serine, D-threonine, L-threonine, -Y3 is a carboxyamide group, -Z1 and Z2 are selected from S-lysine and R-lysine, respectively. An isolated peptide characterized by [specific feature].

2. Equation (IIb): 【Chemistry 2】 The peptide according to claim 1, characterized by the above.

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

4. A peptide according to claim 1 or 2, or a composition according to claim 3, for use as a pharmaceutical.

5. A peptide according to claim 1 or 2, or a composition according to claim 3, for use in the treatment of pathological conditions related to or caused by unregulated angiogenesis / angiogenesis or VEGFR1-dependent angiogenesis / angiogenesis.

6. The aforementioned pathological condition, - Macular edema, wet form of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and neovascular eye diseases selected from combinations thereof. - Solid tumors and / or tumor metastases, wherein the tumor is selected from leukemia and lymphoma, acute lymphoblastic leukemia, acute nonlymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, Hodgkin 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, urinary tract cancer, bladder cancer, oral tumors, pancreatic tumors, melanoma and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, larynx tumors, liver tumors, testicular tumors, solid tumors and / or tumor metastases, - Diseases of bone or joints 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. - Vascular conditions selected from atherosclerosis, hemangiomas, hemangioendotheliomas, and combinations thereof. - Skin diseases selected from psoriasis, warts, pyogenic granulomas, hives, Kaposi's sarcoma, keloids from wounds, allergic edema, neoplasms, and combinations thereof. - Angiogenesis observed in adipose tissue pathology, obesity, - Diabetes and / or as a result, retinopathy and / or diabetic foot, and / or - Hematopoietic disorders, AIDS and / or Kaposi's sarcoma A peptide or composition for use according to claim 5, selected from the above.

7. A peptide according to claim 1 or 2, or a composition according to claim 3, for use as an inhibitor of VEGFR1 or an inhibitor of VEGFR1 activation.

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