WO2022162030A1 - Peptides for inhibiting angiogenesis - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention therefore relates to peptides of synthetic origin and biologically active in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands, and use thereof as a medicament.
- the peptides and pharmaceutical compositions also find application in the prevention of tumor pathologies, angiogenesis, and neovascularization.
- VEGFR-1 also known as Flt-1
- VEGFR-2 also known as KDR
- VEGF-A endothelial vascular growth factor A
- PIGF placental growth factor
- VEGF-B endothelial vascular growth factor A
- VEGF-A blockade by using a neutralizing antibody (bevacizumab) which prevents the activation of VEGFR-2 and partly of VEGFR-1 , was the first approved anti-angiogenic therapy strategy for the treatment of cancer (Hurwitz H, et al. 2004, New England Journal of Medicine 350:2335- 2342).
- the second effective approach still for cancer therapy is represented by the use of tyrosine kinase receptors (TKI) inhibitors (for example, sorafenib and sunitinib) capable of blocking the activation of both VEGF receptors, and at the same time of other receptors, given the general low specificity of TKIs which recognize more than one receptor albeit with different affinities (Escudier B. et al. 2007, New England Journal of Medicine 356: 125-134; Motzer RJ, et al., 2006 J Clin Oncol 24:16-24).
- TKI tyrosine kinase receptors
- the Fab fragment of bevacizumab (ranibizumab) has been approved for the treatment of the wet form of age-related macular degeneration (Rosenfeld PJ, et al. 2006, New England Journal of Medicine 355:1419-1431 ). Subsequently, the whole antibody was used for the same therapeutic purpose, initially in an off-label manner, then officially approved by CATT Research Group, Martin DF, et al. 2011 , New England Journal of Medicine 364:1897-1908).
- a new drug consisting of a recombinant protein made of two domains of the VEGFR-1 and VEGFR-2 receptors extracellular portion, responsible for binding to VEGF-A, VEGF- B and PIGF ligands, fused to the Fc portion of human lgG1 , and effective in preventing the interaction of the three ligands with the two receptors, VEGFR-1 and VEGFR-2, has been approved for the therapy of the wet form of age-related macular degeneration (Heier JS, et al. 2012, Ophthalmology 119:2537-2548).
- the object of the present invention is therefore the development of synthetic peptides suitable for interfering in the interaction between VEGF-A, VEGF-B and PLGF ligands and the two receptors VEGFR-1 and VEGFR-2, which do not have the known disadvantages for currently available drugs.
- the present invention therefore relates to peptides of synthetic origin and biologically active in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands, and consequently of the reactions cascade resulting from this binding.
- the peptides of the invention are capable of preventing the interaction VEGF- A/VEGFR-2 with an IC50 of about 10 pM and the interaction between VEGF-A and VEGFR-1 , as well as that between PIGF and VEGFR-1 , with an IC50 of just under 20 pM.
- the activity of the compounds was evaluated in assays on cultures of primary endothelial cells, where they are able to prevent VEGFR-1 and VEGFR-2 phosphorylation induced by VEGF-A, as well as formation of capillary-like structures induced by VEGF-A.
- the peptides are able to inhibit angiogenesis in the in vivo murine model of laser-induced choroidal neovascularization, which recapitulates wet senile macular degeneration in humans.
- the present invention therefore relates to a peptide or salts thereof for use in the inhibition of angiogenesis or neovascularization, said peptide having the amino acid sequence of SEQ ID NO: 1 and the general formula (I):
- - Y1 is selected from the group consisting of an amino terminal group (NH2), or an amino acid selected from the group consisting of D-Alanine, D-Aspartic Acid, D- Valine, D-Glutamic Acid, L-Cyclohexyl-alanine, D-Phenylalanine, D-Threonine, D- Methionine, D-Lysine, D-Cysteine(S-acetamidomethyl), D-Tyrosine, D-Proline, D- Leucine, D-Arginine, D-Asparagine, D-lsoleucine, D-Arginine(N-Tosyl), D-Serine, L-Cysteine(S-benzyl), L-Cysteine(S-acetamidomethyl), D-Histidine, D-Glutamine, D-Tryptophane, L-Glutamic Acid-(P-allyl), [3-Alanine, L-Cysteine
- R- R-Glu indicates glutamic acid with R absolute configuration at the Ca of the amino acid (R-glutamic acid);
- - S-Cys(Bzl) indicates benzylcysteine with S absolute configuration at the Ca of the amino acid (S-benzyl-cysteine) having a benzyl group bound to the sulfur of the amino acid side-chain;
- - k is an integer number from 1 to 4,
- - A is an amine group
- - i is a number selected from the group consisting of 1 , 2 and 4;
- - j is a number selected from the group consisting of 1 and 2;
- - Y2 is selected from the group consisting of Glycine, Beta-alanine and s-amino caproic acid;
- - Y3 is selected from the group consisting of a carboxy terminal group, a carboxamide group, a N-methyl substituted or N,N-dimethyl disubstituted carboxamide, a hydroxyl group and a hydrogen, for use as a medicament.
- composition comprising one or more peptides for use in the inhibition of angiogenesis or neovascularization, either the same or different, according to the present invention and pharmacologically acceptable excipients.
- the present invention describes the use of the peptides or the pharmaceutical composition in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands.
- the present invention relates to a method for treating a neovascular disease or cancer.
- FIGURE 1 shows the results of an ELISA assay which confirms that the peptide of formula (IV) according to the present invention inhibits VEGFR-1 and VEGFR-2 phosphorylation induced by VEGF-A.
- FIGURE 2 shows representative images of the dose-dependent inhibition exerted by the peptide of formula (IV), hereinafter referred to as iVsB, on the formation of capillary-like structures obtained by inducing HUVEC grown on matrigel with VEGF- A at a concentration of 100 ng/mL.
- VEGF-A is capable of inducing structure formation at a level comparable to that of the positive control represented by complete EGM-2 medium.
- the medium free of EMB growth factors was used as a negative control.
- the iVsB peptide was used at concentrations between 10 and 50 pM.
- the control peptide (PC) was evaluated at a concentration of 50 pM, as detailed in Example 3.
- FIGURE 3 shows data confirming that the iVsB peptide inhibits laser-induced choroidal neovascularization (CNV), as described in Example 4.
- Intravitreal injection of 50 pg of the iVsB peptide of the invention results in reduction of laser-induced choroidal neovascularization by about 40% compared to the injection of PC or vehicle (DMSO).
- the images ( Figure 3B) are representative of CNV, the bar within the iVsB image represents 100 pm.
- the present invention therefore relates to peptides of synthetic origin and biologically active in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands, and consequently of the reactions cascade resulting from this binding.
- the present invention therefore concerns the use of a peptide or salts thereof for use in the inhibition of angiogenesis or neovascularization, said peptide having the amino acid sequence of SEQ ID NO: 1 and the general formula (I):
- - Y1 is selected from the group consisting of an amino terminal group (NH2), or an amino acid selected from the group consisting of D-Alanine, D-Aspartic Acid, D- Valine, D-Glutamic Acid, L-Cyclohexyl-alanine, D-Phenylalanine, D-Threonine, D- Methionine, D-Lysine, D-Cysteine(S-acetamidomethyl), D-Tyrosine, D-Proline, D- Leucine, D-Arginine, D-Asparagine, D-lsoleucine, D-Arginine(N-Tosyl), D-Serine, L-Cysteine(S-benzyl), L-Cysteine(S-acetamidomethyl), D-Histidine, D-Glutamine, D-Tryptophane, L-Glutamic Acid-(P-allyl), [3-Alanine, L-Cysteine
- R- R-Glu indicates glutamic acid with R absolute configuration at the Ca of the amino acid (R-glutamic acid);
- - S-Cys(Bzl) indicates benzylcysteine with S absolute configuration at the Ca of the amino acid (S-benzyl-cysteine) having a benzyl group bound to the sulfur of the amino acid side-chain;
- - k is an integer number from 1 to 4,
- - A is an amine group
- - i is a number selected from the group consisting of 1 , 2 and 4;
- - j is a number selected from the group consisting of 1 and 2;
- - Y2 is selected from the group consisting of Glycine, Beta-alanine and s-amino caproic acid;
- - Y3 is selected from the group consisting of a carboxy terminal group, a carboxamide group, a N-methyl substituted or N,N-dimethyl disubstituted carboxamide, a hydroxyl group and a hydrogen.
- Table 1 shows the amino acids that may be used as Y1 :
- Table 1 shows the amino acids that may be used as Y2:
- Z1 , Z2 and Z3 are joined together by amide bonds so as to form a branched structure
- - Y1 is a terminal amino group (NH2)
- - Z1 and Z2 are S-Lysine.
- the peptide having the sequence of SEQ ID NO: 2 and the general formula (III) can be represented by the simple formula:
- - Z1 and Z2 are S-Lysine
- - Y3 is a terminal carboxy group (COOH), said peptide having the sequence of SEQ ID NO: 3 and the general formula (IV)
- the peptide having the sequence of SEQ ID NO: 3 and the general formula (IV) can be represented by the simple formula:
- the peptide molecule of formula IV can conventionally be defined as iVsB, (Inhibitor of VEGFRs Binding).
- the tripeptides (R-Glu)-(S- Cys(Bzl))-(S-Cys(Bzl)) are bound through the C-terminal portion either to the a- amino group or to the s-amino group of lysine.
- S-lysine (S-Lys) is interchangeable with R-lysine (R-Lys) and the Gly-COOH group is interchangeable with the Gly-C0NH2 group or with the COOH group, meaning that glycine can be omitted and the C-terminal group can be interchangeably a CO-NH2 carboxamide group.
- the peptide of formula (IV), according to the present invention was further characterized for its ability to bind VEGFR-1 and VEGFR-2.
- the tetrameric tripeptide of formula (IV), called iVsB blocks the binding of VEGF-A to VEGFR-2 and VEGFR-1 receptors and that of PIGF to VEGFR-1 receptor. It is capable of producing a 50% inhibition of the interaction between VEGF-A and VEGFR-2 (IC50) at a concentration of just under 10 pM, and an inhibition of the interaction between VEGF-A and PIGF with VEGFR-1 , still by 50% (IC50), at a concentration slightly lower than 20 pM.
- the peptides described in the present invention were further characterized for their ability to inhibit VEGFR-1 and VEGFR-2 phosphorylation induced by growth factors, preferably by VEGF-A.
- a pharmaceutical composition comprising one or more peptides, either the same or different, according to the present invention and pharmacologically acceptable excipients, for use in the inhibition of angiogenesis or neovascularization is described.
- excipients can be, for example, at least one or more suitable diluents, carriers or adjuvants.
- a “diluent, carrier or adjuvant” is any suitable excipient, diluent and/or adjuvant which neither induce by itself the production of antibodies that are harmful to the individual receiving the composition nor cancel the effect of the composition.
- a carrier or adjuvant can improve the response stimulated by the peptides object of the invention, for example by determining a continuous release of the peptides or pharmaceutical composition object of the invention for an extended period of time (slow release formulation).
- Carriers or suitable adjuvants typically consist of one or more compounds included in the following list, although not exhaustive, lipid microparticle type carriers such as: Solid Lipid Nanoparticles (SLN), Nanostructured Lipid Carriers (NLC), Solid Lipid Microparticles (SLM), liposomes, W/O/W emulsion, or microparticles made of biodegradable and biocompatible polymers (PLGA, PLA or PHEA/PEG).
- the peptide and the pharmaceutical composition comprising one or more peptides may be used as a medicament in both humans and animals (veterinary use).
- the animal may be any animal capable of forming new blood vessels; in particular such an animal may be a mammal, such as a human being. It is expected that the effective dose of the active substance described in the invention falls within a sufficiently wide range to be determined by routine tests. Typically, the dose ranges between 4% and 0.001 %.
- the use of one or more peptides or the pharmaceutical composition according to the present invention is a use for ophthalmic, or topical ophthalmic administration.
- Said composition may be in the form of eye drops, powder, granules, cream or gel, erodible ocular inserts, or powder or in the liquid form of a suspension, emulsion, microemulsion, solution, eye spray, eye bath, or an injectable composition.
- the pharmaceutical composition when in injectable form, it can be a liquid solution or a suspension, and said injection can be carried out intravitreally.
- the present invention describes the use of the peptides or the pharmaceutical composition in inhibiting angiogenesis or neovascularization.
- the neovascularization can be dependent on VEGFR-1 and VEGFR- 2.
- in vitro and in vivo inhibition of angiogenesis and neovascularization of the eye dependent on VEGFR-1 and VEGFR-2 was analysed. For instance, as can be seen in Examples 3 and 4, the inhibition of capillary-like structures formation induced by VEGF and the inhibition of choroidal neovascularization by intravitreal administration of the peptides of the present invention was shown.
- Tetrameric Tripeptides of Formula (I), (III) and (IV), described by the present invention were further characterized for their ability to inhibit the formation of capillary-like structures by primary endothelial cells grown on extracellular matrix extract induced by growth factors, preferentially by VEGF-A.
- Tetrameric Tripeptides of formula (I), (III) and (IV) were further characterized to be modulators of neovascularization dependent on VEGFR-2 and VEGFR-1 , or angiogenesis dependent on VEGFR-2 and VEGFR-1 .
- the invention also relates to the conjugated form of the Tetrameric Tripeptides of formula (I), (III) and (IV), with a non-immunogenic hydrophilic polymer.
- This aspect tries to solve the problem of improving the solubility of the tetrameric peptide in water or in aqueous buffers.
- the hydrophilic polymers are selected from polyethyleneglycol, polyvinyl-pyrrolidones, carbohydrates with a molecular weight between 100 and 20,000 Dalton.
- said inhibition of angiogenesis and neovascularization are in the treatment of cancer (tumor growth) or a neovascular disease.
- Said neovascular disease can be a disease of the bones or joints, blood vessels, skin, angiogenesis resulting from adipose tissue diseases, diabetes and/or its consequences, or hematopoiesis diseases.
- said neovascular disease is a neovascular edema disease of the eye.
- said neovascular disease of the eye is senile macular degeneration, a neovascular disease of the eye surface resulting from infection, inflammation, hypoxia, trauma or degeneration/loss of the limbal barrier, diabetic retinopathy, central retinal vein occlusion, premature infant retinopathy, macular edema and associated inflammation, retinal vein occlusion retinopathy (e.g. central retinal vein), vitreous haemorrhage, retinal detachment, central serous chorioretinopathy, age-related macular degeneration (exudative form), and myopic macular degeneration (choroidal neovascularization) or combinations thereof.
- retinal vein occlusion retinopathy e.g. central retinal vein
- vitreous haemorrhage e.g. central retinal vein
- retinal detachment e.g. central serous chorioretinopathy
- age-related macular degeneration exudative form
- corneal neovascularization examples include bacterial, viral, fungal, or parasitic keratitis, corneal graft rejection, graft versus host disease (GvDH), atopic conjunctivitis, Turner syndrome, Terrien’s marginal degeneration, pterygium, sterile corneal ulcer, dry eye syndrome.
- tumors being selected from leukemias and lymphomas, preferably acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin’s lymphoma, Hodgkin’s disease, infant or adult solid tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms tumor, osteosarcoma and chondrosarcomas, lung cancer, colon and rectal cancer, breast cancer, prostate cancer, uterus cancer, ovarian cancer, urinary system cancer, bladder cancer, oral cavity cancer, pancreatic cancer, melanoma and skin cancers, stomach cancer, brain cancer, thyroid cancer, laryngeal cancer, liver cancer, testicular cancer
- peptides or the pharmaceutical composition according to the present invention for inhibiting angiogenesis and neovascularization can be for the treatment of:
- - diseases of the bones or joints preferably selected from: rheumatoid arthritis, synovitis, cartilage and/or bone destruction, osteomyelitis, hypertrophy and/or hyperplasia of the synovial tissue, osteophytes formation, neoplasms and/or metastases and combinations thereof; and/or
- - blood vessels diseases preferably selected from: atherosclerosis, hemangioma, hemangioendothelioma and combinations thereof; and/or
- - skin diseases preferably selected from: psoriasis, warts, pyogenic granulomas, hair growth, Kaposi’s sarcoma, wound keloids, allergic edema, neoplasms, and combinations thereof; and/or
- retinopathy and/or diabetic food preferably retinopathy and/or diabetic food
- - hematopoiesis diseases preferably AIDS and/or Kaposi’s sarcoma.
- ischemic retinal diseases the supply of blood and oxygen to the retina is reduced, the peripheral portion of the retina loses the source of nutrients and stops functioning properly.
- Common causes of retinopathy are occlusion of the central retinal vein, stenosis of the carotid artery, diabetes (diabetic retinopathy) and sickle cell retinopathy. Retinopathy is also observed in premature infants (premature infant retinopathy). Diabetic retinopathy is the leading cause of vision loss in diabetic patients.
- new blood vessels grow grow (neovascularization) occurs. These vessels often grow from the surface of the retina, invading the vitreous.
- CNV Choroidal neovascularization
- CNV the leading cause of vision loss. It is estimated that CNV is present in 5-10% of myopic people and occurs practically in all choroidal ruptures during the healing phase. In most cases it recedes spontaneously, but in 15-30% of patients it can recur and lead to bleeding and serious macular detachment with concomitant vision loss.
- the peptides and the pharmaceutical composition according to the present invention may also be used for the manufacture of a medicament that can be included in a treatment regimen for conditions associated with VEGFR-1 and VEGFR-2 dependent neovascularization.
- the aforementioned treatment regimen may include other compounds or drugs which act on VEGFR-1 and VEGFR-2 dependent neovascularization, or which determine an improvement in the side effects of said treatment.
- Other compounds or drugs may be present in said treatment regimen including a neutralizing PIGF antibody and/or an anti-VEGFR antibody and/or an anti-VEGF-A antibody.
- the present invention describes the use of peptides or the pharmaceutical composition in inhibiting the binding between the VEGFR-1 and VEGFR-2 receptors and their ligands.
- the present invention relates to a method for the treatment of cancer or a neovascular disease, by means of the step of using the peptides or the pharmaceutical composition of the invention.
- angiogenesis and neovascularization are inhibited by the use of the peptides or pharmaceutical composition, and preferably said neovascular disease is a neovascular disease of the eye.
- said neovascular disease of the eye is diabetic retinopathy.
- said neovascular disease of the eye is age- related macular degeneration or a neovascular disease of the eye surface resulting from infection, inflammation, hypoxia, trauma, or degeneration/loss of the limbal barrier.
- the present invention relates to peptides capable of interacting with the two VEGF receptors involved in the formation of blood vessels, VEGFR-1 and VEGFR-2, through the activation induced by the common ligand VEGF-A, and by PIGF which specifically binds VEGFR-1 .
- these compounds are able to compete with VEGF-A in binding the two receptors, and with PIGF in binding VEGFR-1 , in a dose-dependent manner. They are able to achieve its activation by inhibiting the VEGF-A induced phosphorylation of the two receptors (Example 2).
- Example 3 The results reported in Example 3 show how these compounds have the ability to inhibit the formation of capillary-like structures by primary endothelial cells grown on extracellular matrix extract induced by growth factors, preferably by VEGF-A. Furthermore, as described in Example 4, these compounds are capable of inhibiting laser-induced choroidal neovascularization in vivo, an experimental model that recapitulates the wet form of age-related macular degeneration.
- the set of results allows us to define the peptides of the invention as inhibitors of VEGFR-1 and VEGFR-2 dependent neovascularization or angiogenesis.
- Examples 1 -4 allow to conclude that the tetrameric peptide of formula (IV), depending on the expression levels of VEGFR-2, VEGFR-1 and its soluble variant, is able to inhibit a neovascularization process.
- This peptide can therefore be defined as an inhibitor of neovascularization or angiogenesis, or more specifically an inhibitor of VEGFR-1 and VEGFR-2 dependent neovascularization or angiogenesis.
- Example 1 Dose dependent inhibition of VEGF-AA/EGFR2, VEGF-AA/EGFR-1 and PIGFA/EGFR-1
- inhibitory peptide iVsB
- the binding assay of VEGF-A to VEGFR-1 and VEGFR-2 receptors, and PIGF to VEGR-1 receptor are based on the ELISA method and were performed using reagents acquired from R&D Systems.
- the human recombinant receptors VEGFR- 2 (R&D Systems, cat No. 357-KD) and VEGFR-1 (R&D Systems, cat No. 321 -FL) (consisting of seven extracellular domains fused to the Fc domain of human IgGs), were adhered to the wells of 96-well microplates at a concentration of 1 pg/mL.
- VEGF-A (R&D Systems, cat No. 293-VE) was added, at a concentration of 10 ng/mL. The concentration used was 10 ng/mL also for human PIGF (R&D Systems, cat No. 264-PG).
- iVsB or PC between 1 .25 and 50 pM were added at the same time as the growth factors.
- a biotinylated anti-hVEGF-A (R&D Systems, cat No. BAF293) or anti-hPIGF (R&D Systems, cat No.
- iVsB is able to inhibit in a dose dependent manner both the VEGF-AA/EGFR-2 interaction with an IC50 of just under 10 pM, and the VEGF-AA/EGFR-1 interaction, albeit with lower efficiency (IC50 of about 20pM). It is also able to inhibit the PIGFA/EGFR-1 interaction, still at a concentration of just under 20 pM. Conversely, PC is unable to inhibit the binding of the growth factors to the two receptors. Table 3 - Dose-dependent inhibition of VEGF-A/VEGFR-2 and VEGF-A/VEGFR-1 interaction
- Example 2 Inhibition of VEGF-A induced VEGFR-1 and VEGFR-2 phosphorylation To evaluate the inhibitory capacity of the iVsB peptide in functional terms, the VEGF- A induced VEGFR-1 and VEGFR-2 receptor phosphorylation assay was developed.
- Primary human endothelial cells HLIVEC were used for the activation of VEGFR-2, in which this receptor is well detectable.
- HLIVEC a cell line overexpressing the 293-VEGFR-1 receptor, obtained by stable transfection starting from HEK-293 cells, was used. For this purpose, HLIVEC and 293-VEGFR-1 cells were cultured until subconfluence.
- the cells were then starved, incubating 293-VEGFR-1 cells in serum- free culture medium while HLIVEC cells, normally cultured in the presence of 2% serum and a cocktail of growth factors, were incubated in the culture medium with 1 % serum for at least 16 hours.
- the culture medium was removed, and the cell monolayers were incubated with 100 pM NasVC for 5 minutes in order to inhibit the activity of the endogenous phosphatase.
- the cells were then stimulated with VEGF- A at 50 ng/mL in the media used for starvation for 10 minutes at 37°C.
- iVsB was added simultaneously to VEGF-A at a 25 pM concentration.
- DMSO was used as a negative control.
- the cells were washed with cold 100 pM NasVO4 and then lysed in the buffer consisting of 20 mM Tris-HCI pH 8, 5 mM EDTA, 150 mM NaCI, 1 % Triton-X100, 10% glycerol, 10 mM zinc acetate, 100 pM NasVO4 and a mixture of protease inhibitors, incubating for 1 hour at 4°C with gentle shaking.
- the cell lysates were centrifuged at 12,000xg for 15 minutes to remove cell debris.
- the extracts were quantified with the Bradford method using the BioRad reagent. 100 pg of each protein extract were loaded on 8.5% reducing SDS- PAGE, and the standard method was then used to analyse the proteins by Western blot.
- iVsB is able to determine the inhibition of phosphorylation of both receptors, confirming that the inhibition of the interaction observed in the in vitro ELISA assay corresponds to a functional inhibition of the receptor activation.
- Example 3 Inhibition of capillary-like structures formation induced by VEGF-A The formation of capillary-like structures (CTFs) by primary endothelial cells, suitably grown on an extracellular matrix support (Matrigel, BD cat. No. 354230), following stimulation with pro-angiogenic factors, is an assay commonly accepted for evaluating the anti-angiogenic activity of compounds.
- CTFs capillary-like structures
- HLIVEC cells were used, with EGM-2 culture medium, consisting of EBM base medium supplemented with 2% serum and a cocktail of growth factors.
- Matrigel was layered in the wells of a 24-well cell culture plate. 230 pL of cold matrigel were added to each well, and then the plate was incubated for 30 minutes at 37°C to allow the matrigel solidification. Exponentially growing HLIVEC cells were detached from the plates, washed, and resuspended in EBM at a density of 120,000 cells/mL. 60,000 cells in 0.5 mL of medium were used for each well containing the matrigel.
- the cells simply resuspended in EBM, were used as a negative control of the experiment.
- the cells were resuspended in complete EGM-2 medium.
- VEGF-A was added at a concentration of 100 ng/mL to the cells resuspended in EBM immediately before seeding them in the wells.
- a dose dependency experiment was performed at concentrations between 10 and 50 pM.
- the PC activity at the maximum concentration used for iVsB was also evaluated.
- the two peptides were added to the HLIVEC mixture in EBM + 100 ng/mL VEGF-A immediately prior to inoculating the cells into the wells in which the matrigel had been stratified.
- VEGF-A stimulates CTF in a manner comparable to the positive control (EGM-2), while with the negative control (EBM) the cells remain dispersed on the matrigel.
- EBM positive control
- PC is unable to inhibit CTF
- iVsB is active in a dosedependent manner.
- the cells are dispersed as in the negative control, at 20pM the inhibition is still consistent although the formation of capillary-like structures is observed, a phenomenon even more present at a peptide concentration of 10pM.
- Example 4 Inhibition of choroidal neovascularization by intravitreal administration of iVsB.
- the experimental model of laser-induced choroidal neovascularization involves damage generation at the level of the Bruch membrane that separates the choroid from the retinal pigment epithelium (RPE). The damage is caused by a laser- induced bum that causes Bruch’s membrane perforation thus activating chorioretinal vascularization, the growth of new vessels that, starting from the choroid, invade the overlying retinal tissue.
- RPE retinal pigment epithelium
- This murine model recapitulates the main characteristics of the exudative form of human senile degenerative maculopathy (AMD) and is, in fact, commonly used as a preclinical model of AMD. It allows to evaluate the anti-angiogenic activity of the molecules of interest.
- AMD human senile degenerative maculopathy
- Pupil dilation is induced before anesthetization of the animal.
- 0.5% Tropicamide eye drops were applied topically.
- the animal was then anesthetized by intraperitoneal injection of a solution of ketamine and xylazine (80 mg/kg and 10 mg/kg, respectively).
- ketamine and xylazine 80 mg/kg and 10 mg/kg, respectively.
- a 2.5% hydroxy-propyl-methylcellulose aqueous solution was applied to both eyes. It has the dual function of preventing corneal dehydration and improving the visualization of the fundus by placing the lens of the Micron IV chamber in contact with the solution (a procedure similar to that used in microscopy with immersion objectives).
- the laser pointer is first activated and focused to apply the laser beam using the RPE layer as a reference.
- the area where the laser beam is applied must be far from the retinal main vessels to prevent any bleeding.
- the efficiency of the burn at the level of the Bruch membrane is confirmed by the formation of a bubble immediately after the laser beam application.
- the experimental conditions used in the experiments were obtained with the conditions: 100 msec and 200mW.
- C57BI6/J mice, n 5 per group, were subjected to anaesthesia and then the laser damage procedure was performed as described above. At the end of the procedure, the intravitreal injection was immediately performed, using a Hamilton syringe with a 32g needle, administering 50p g of iVsB or PC in 1 pL of DMSO, in the left eye. DMSO alone was injected as a control.
- the animals were sacrificed, the eyes were enucleated and fixed in 4% paraformaldehyde. Subsequently, the anterior segment of the eye, consisting of cornea, iris and lens, was removed under the stereomicroscope. The remaining part defined ‘eye-cups’ or posterior segment, consisting of: sclera, choroid, RPE and retina, was incubated in the presence of 0.7% FITC-Griffonia simplicifolia Isolectin B4 (Vector Laboratories, Burlingame, CA) for sixteen hours. After a series of washings, the retina is removed, and four cuts are made on the RPE/choroid which allow it to be mounted on a slide for observation under a fluorescence microscope.
- FITC-Griffonia simplicifolia Isolectin B4 Vector Laboratories, Burlingame, CA
- the quantification of CNV is done in terms of volume.
- a series of images Z-Satcks, approximately 20-25 images
- the fluorescence volume is measured by the Imaged program (NIH, Bethesda, MD) by adding the fluorescence area of each individual plane.
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Abstract
The present invention therefore relates to peptides of synthetic origin andbiologically active in inhibiting the binding between VEGFR-1 and VEGFR-2receptors and their ligands, and use thereof as a medicament.The use as a medicament of a pharmaceutical composition comprising one or moreof said peptides, either the same or different, is also described.The peptides and pharmaceutical compositions also find application in theprevention of tumor pathologies, angiogenesis, and neovascularization.
Description
PEPTIDES FOR INHIBITING ANGIOGENESIS
DESCRIPTION
FIELD OF THE INVENTION
The present invention therefore relates to peptides of synthetic origin and biologically active in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands, and use thereof as a medicament.
The use as a medicament of a pharmaceutical composition comprising one or more of said peptides, either the same or different, is also described.
The peptides and pharmaceutical compositions also find application in the prevention of tumor pathologies, angiogenesis, and neovascularization.
STATE OF THE ART
Vascular endothelial growth factor receptors closely involved in the formation of new blood vessels, VEGFR-1 (also known as Flt-1 ) and VEGFR-2 (also known as KDR) are both recognized by endothelial vascular growth factor A (VEGF-A), which plays a central role in angiogenesis. The other two pro-angiogenic members of the VEGF family, placental growth factor (PIGF) and VEGF-B, specifically interact with VEGFR-1 (De Falco S. 2012, Experimental Molecular Medicine 44: 1 -9). The binding of the factors to the receptors induces dimerization of the receptor with consequent autophosphorylation and activation of intracellular signaling.
Numerous data, both of gene inactivation and use of inhibitors of ligands or receptors of the VEGF family, have shown how inhibition of the two VEGFRs activation results in a powerful inhibition of pathological neoangiogenesis in different contexts, such as cancer and neovascular diseases of the eye, in particular senile macular degeneration and diabetic retinopathy (De Falco S. 2014 The Korean Journal of Internal Medicine 29: 1 -11 ).
Specific VEGF-A blockade, by using a neutralizing antibody (bevacizumab) which prevents the activation of VEGFR-2 and partly of VEGFR-1 , was the first approved anti-angiogenic therapy strategy for the treatment of cancer (Hurwitz H, et al. 2004, New England Journal of Medicine 350:2335- 2342). The second effective approach still for cancer therapy is represented by the use of tyrosine kinase receptors (TKI) inhibitors (for example, sorafenib and sunitinib) capable of blocking the activation of
both VEGF receptors, and at the same time of other receptors, given the general low specificity of TKIs which recognize more than one receptor albeit with different affinities (Escudier B. et al. 2007, New England Journal of Medicine 356: 125-134; Motzer RJ, et al., 2006 J Clin Oncol 24:16-24).
As regards anti-angiogenic therapy in neovascular diseases of the eye, the Fab fragment of bevacizumab (ranibizumab) has been approved for the treatment of the wet form of age-related macular degeneration (Rosenfeld PJ, et al. 2006, New England Journal of Medicine 355:1419-1431 ). Subsequently, the whole antibody was used for the same therapeutic purpose, initially in an off-label manner, then officially approved by CATT Research Group, Martin DF, et al. 2011 , New England Journal of Medicine 364:1897-1908). More recently a new drug (aflibercept), consisting of a recombinant protein made of two domains of the VEGFR-1 and VEGFR-2 receptors extracellular portion, responsible for binding to VEGF-A, VEGF- B and PIGF ligands, fused to the Fc portion of human lgG1 , and effective in preventing the interaction of the three ligands with the two receptors, VEGFR-1 and VEGFR-2, has been approved for the therapy of the wet form of age-related macular degeneration (Heier JS, et al. 2012, Ophthalmology 119:2537-2548).
Furthermore, several clinical studies have highlighted the therapeutic capacity of these molecules also in other ocular disease contexts, such as retinopathy due to central vein occlusion, diabetic retinopathy, premature infant retinopathy (Diabetic Retinopathy Clinical Research Network, Wells JA, et al. 2015, New England Journal of Medicine 372:1 193-203; Mintz-Hittner HA, et al.', BEATROP Cooperative Group. 2011 , New England Journal of Medicine 364:603-615). All three drugs, bevacizumab, ranibizumab, and aflibercept are administered by intravitreal injection.
The approval of these drugs has definitively validated the three pro-angiogenic members of the VEGF family and the two related receptors as preferential targets for anti-angiogenic therapy.
Given the high number of diseases that require prevention of VEGFR-1 and VEGFR-2 receptors activation, a strong demand for inhibitors of the receptor-ligand interaction is expected. Being of biological origin, the drugs currently available may have the disadvantage of contamination resulting from the preparation steps, in
addition to having high production costs.
The object of the present invention is therefore the development of synthetic peptides suitable for interfering in the interaction between VEGF-A, VEGF-B and PLGF ligands and the two receptors VEGFR-1 and VEGFR-2, which do not have the known disadvantages for currently available drugs.
SUMMARY OF THE INVENTION
The present invention therefore relates to peptides of synthetic origin and biologically active in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands, and consequently of the reactions cascade resulting from this binding.
The peptides of the invention are capable of preventing the interaction VEGF- A/VEGFR-2 with an IC50 of about 10 pM and the interaction between VEGF-A and VEGFR-1 , as well as that between PIGF and VEGFR-1 , with an IC50 of just under 20 pM.
The activity of the compounds was evaluated in assays on cultures of primary endothelial cells, where they are able to prevent VEGFR-1 and VEGFR-2 phosphorylation induced by VEGF-A, as well as formation of capillary-like structures induced by VEGF-A. The peptides are able to inhibit angiogenesis in the in vivo murine model of laser-induced choroidal neovascularization, which recapitulates wet senile macular degeneration in humans.
In a first aspect, the present invention therefore relates to a peptide or salts thereof for use in the inhibition of angiogenesis or neovascularization, said peptide having the amino acid sequence of SEQ ID NO: 1 and the general formula (I):
{{{[Y1 - R-Glu - S-Cys(Bzl) - S- Cys(Bzl)]2 - Z1 }i - Z2 }j - Z3 }q - Y2 - Y3 (I) wherein:
- Y1 is selected from the group consisting of an amino terminal group (NH2), or an amino acid selected from the group consisting of D-Alanine, D-Aspartic Acid, D- Valine, D-Glutamic Acid, L-Cyclohexyl-alanine, D-Phenylalanine, D-Threonine, D- Methionine, D-Lysine, D-Cysteine(S-acetamidomethyl), D-Tyrosine, D-Proline, D- Leucine, D-Arginine, D-Asparagine, D-lsoleucine, D-Arginine(N-Tosyl), D-Serine, L-Cysteine(S-benzyl), L-Cysteine(S-acetamidomethyl), D-Histidine, D-Glutamine, D-Tryptophane, L-Glutamic Acid-(P-allyl), [3-Alanine, L-Cysteine(S-p-methyl-
benzyl), L-Cysteine(S-tert-butyl), L-Methionine-sulfone, L-Methionine-sulfoxide, Glycine or combinations thereof;
- R-Glu indicates glutamic acid with R absolute configuration at the Ca of the amino acid (R-glutamic acid);
- S-Cys(Bzl) indicates benzylcysteine with S absolute configuration at the Ca of the amino acid (S-benzyl-cysteine) having a benzyl group bound to the sulfur of the amino acid side-chain;
- Z1 , Z2 and Z3: when present, are independently a molecule of formula (II) with R or S absolute configuration:
A — (CH)— COOH
( ICH)k
A (||) wherein:
- k is an integer number from 1 to 4,
- A is an amine group;
- i is a number selected from the group consisting of 1 , 2 and 4;
- j is a number selected from the group consisting of 1 and 2;
- q is 1 ; with the proviso that: when i = 4, j = 2, q = 1 when i = 2, j = 1 , q = 1 , and Z3 is absent when i = 1 , j = 1 , q = 1 , and Z2 and Z3 are absent;
- Y2 is selected from the group consisting of Glycine, Beta-alanine and s-amino caproic acid;
- Y3 is selected from the group consisting of a carboxy terminal group, a carboxamide group, a N-methyl substituted or N,N-dimethyl disubstituted carboxamide, a hydroxyl group and a hydrogen, for use as a medicament.
In a second aspect, a pharmaceutical composition comprising one or more peptides
for use in the inhibition of angiogenesis or neovascularization, either the same or different, according to the present invention and pharmacologically acceptable excipients.
In a third aspect, the present invention describes the use of the peptides or the pharmaceutical composition in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands.
In yet another aspect, the present invention relates to a method for treating a neovascular disease or cancer.
DESCRIPTION OF THE FIGURES
The invention will now be described in detail and with reference to the attached Figures.
FIGURE 1 shows the results of an ELISA assay which confirms that the peptide of formula (IV) according to the present invention inhibits VEGFR-1 and VEGFR-2 phosphorylation induced by VEGF-A.
Western blot analysis of VEGFR-2 and VEGFR-1 phosphorylation induced by 50 ng/mL of VEGF-A, in HUVEC and 293-VEGFR-1 cells, respectively. iVsB was added simultaneously to VEGF-A at a concentration of 25 pM. DMSO was used as a negative control, as described in Example 2.
FIGURE 2 shows representative images of the dose-dependent inhibition exerted by the peptide of formula (IV), hereinafter referred to as iVsB, on the formation of capillary-like structures obtained by inducing HUVEC grown on matrigel with VEGF- A at a concentration of 100 ng/mL. VEGF-A is capable of inducing structure formation at a level comparable to that of the positive control represented by complete EGM-2 medium. The medium free of EMB growth factors was used as a negative control. The iVsB peptide was used at concentrations between 10 and 50 pM. The control peptide (PC) was evaluated at a concentration of 50 pM, as detailed in Example 3.
FIGURE 3 shows data confirming that the iVsB peptide inhibits laser-induced choroidal neovascularization (CNV), as described in Example 4. Intravitreal injection of 50 pg of the iVsB peptide of the invention results in reduction of laser-induced choroidal neovascularization by about 40% compared to the injection of PC or vehicle (DMSO). Quantification of the neovascularization volume was performed
based on n=9 spots for PI, n=6 spots for PC, and n=9 spots for the vehicle. The data are represented in the graph (Figure 3A) as mean ± SEM with respect to the control. *p=0.028 vs DMSO. The images (Figure 3B) are representative of CNV, the bar within the iVsB image represents 100 pm.
DETAILED DESCRIPTION OF THE INVENTION
The present invention therefore relates to peptides of synthetic origin and biologically active in inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and their ligands, and consequently of the reactions cascade resulting from this binding.
In a first aspect, the present invention therefore concerns the use of a peptide or salts thereof for use in the inhibition of angiogenesis or neovascularization, said peptide having the amino acid sequence of SEQ ID NO: 1 and the general formula (I):
{{{[Y1 - R-Glu - S-Cys(Bzl) - S- Cys(Bzl)]2 - Z1 }i - Z2 }j - Z3 }q - Y2 - Y3 (I) in cui:
- Y1 is selected from the group consisting of an amino terminal group (NH2), or an amino acid selected from the group consisting of D-Alanine, D-Aspartic Acid, D- Valine, D-Glutamic Acid, L-Cyclohexyl-alanine, D-Phenylalanine, D-Threonine, D- Methionine, D-Lysine, D-Cysteine(S-acetamidomethyl), D-Tyrosine, D-Proline, D- Leucine, D-Arginine, D-Asparagine, D-lsoleucine, D-Arginine(N-Tosyl), D-Serine, L-Cysteine(S-benzyl), L-Cysteine(S-acetamidomethyl), D-Histidine, D-Glutamine, D-Tryptophane, L-Glutamic Acid-(P-allyl), [3-Alanine, L-Cysteine(S-p-methyl- benzyl), L-Cysteine(S-tert-butyl), L-Methionine-sulfone, L-Methionine-sulfoxide, Glycine or combinations thereof;
- R-Glu indicates glutamic acid with R absolute configuration at the Ca of the amino acid (R-glutamic acid);
- S-Cys(Bzl) indicates benzylcysteine with S absolute configuration at the Ca of the amino acid (S-benzyl-cysteine) having a benzyl group bound to the sulfur of the amino acid side-chain;
- Z1 , Z2 and Z3: when present, are independently a molecule of formula (II) with R or S absolute configuration:
A - (CH)— COOH
(CH)k
A (||) wherein:
- k is an integer number from 1 to 4,
- A is an amine group;
- i is a number selected from the group consisting of 1 , 2 and 4;
- j is a number selected from the group consisting of 1 and 2;
- q is 1 with the proviso that: when i = 4, j = 2, q = 1 when i = 2, j = 1 , q = 1 , and Z3 is absent when i = 1 , j = 1 , q = 1 , and Z2 and Z3 are absent;
- Y2 is selected from the group consisting of Glycine, Beta-alanine and s-amino caproic acid;
- Y3 is selected from the group consisting of a carboxy terminal group, a carboxamide group, a N-methyl substituted or N,N-dimethyl disubstituted carboxamide, a hydroxyl group and a hydrogen.
Table 1 shows the amino acids that may be used as Y1 :
Amino acids are shown with their trivial name, which can be replaced by their systematic name according to the IIIPAC nomenclature as reported in: Nomenclature and Symbolism for Amino Acids And Peptides IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), Pure & Appl. Chem., Vol. 56, No. 5, pp. 595— 624, 1984.
According to a preferred embodiment of the invention, Z1 , Z2 and Z3 are joined together by amide bonds so as to form a branched structure;
The R/S notations that define the absolute configuration of the chiral centers present in the groups shown in the two tables are interchangeable with the L/D notation, following rules reported in the literature and as is known to experts in the field (see, for example: Rules for the nomenclature of organic chemistry section E: stereochemistry (Recommendations 1974), collators: L. C. Cross and W. Klyne; Pure & Appl. Chem., Vol. 45, pp. 11 — 30. Pergamon Press, 1976. Printed in Great
Britain.).
In a further embodiment, in the peptide according to the present invention, when:
- i is 4; j is 2; and q is 1 ; or when:
- i is 2; j is 1 ; and q is 1 and Z3 is absent or when:
- i is 1 ; j is 1 , q is 1 , and Z2 and Z3 are absent.
In one embodiment, in the peptide according to the present invention:
- i is 2;
- j is 1 ;
- q is 1 ; and
- Z3 is not present (/.e. it is absent), and said peptide having the amino acid sequence of SEQ ID NO: 2 and the general formula (III)
Y1-(R-Glu)-(S-Cys(Bzl))-(S-Cys(Bzl)
(HI).
In a further embodiment of the present invention, in the peptide according to the present invention:
- Y1 is a terminal amino group (NH2); and
- Z1 and Z2 are S-Lysine.
The peptide having the sequence of SEQ ID NO: 2 and the general formula (III) can be represented by the simple formula:
Y1-[ R-Glu - S-Cys(Bzl) - S-Cys(Bzl)]4-Z12-Z2-Y2-Y3-COOH (SEQ ID NO:3).
In yet another embodiment, in the peptide according to the present invention:
- Y1 a terminal amino group (NH2);
- Z1 and Z2 are S-Lysine;
- Y2 is Glycine; and
- Y3 is a terminal carboxy group (COOH), said peptide having the sequence of SEQ ID NO: 3 and the general formula (IV)
S-Lys
The peptide having the sequence of SEQ ID NO: 3 and the general formula (IV) can be represented by the simple formula:
H2N-[ R-GIU - S-Cys(Bzl) - S-Cys(Bzl)]4-Lys2-Lys-Gly-COOH (SEQ ID NO:3), equivalent to:
H2N-[ D-GIU - L-Cys(Bzl) - L-Cys(Bzl)]4-Lys2-Lys-Gly-COOH (SEQ ID NO:3).
The peptide molecule of formula IV can conventionally be defined as iVsB, (Inhibitor of VEGFRs Binding).
In the peptides according to the present invention, the tripeptides (R-Glu)-(S- Cys(Bzl))-(S-Cys(Bzl)) are bound through the C-terminal portion either to the a- amino group or to the s-amino group of lysine. In the peptide of formula (IV), S-lysine (S-Lys) is interchangeable with R-lysine (R-Lys) and the Gly-COOH group is
interchangeable with the Gly-C0NH2 group or with the COOH group, meaning that glycine can be omitted and the C-terminal group can be interchangeably a CO-NH2 carboxamide group.
Surprisingly, the peptide of formula (IV), according to the present invention, was further characterized for its ability to bind VEGFR-1 and VEGFR-2. In particular, the tetrameric tripeptide of formula (IV), called iVsB, blocks the binding of VEGF-A to VEGFR-2 and VEGFR-1 receptors and that of PIGF to VEGFR-1 receptor. It is capable of producing a 50% inhibition of the interaction between VEGF-A and VEGFR-2 (IC50) at a concentration of just under 10 pM, and an inhibition of the interaction between VEGF-A and PIGF with VEGFR-1 , still by 50% (IC50), at a concentration slightly lower than 20 pM.
Advantageously, the peptides described in the present invention were further characterized for their ability to inhibit VEGFR-1 and VEGFR-2 phosphorylation induced by growth factors, preferably by VEGF-A.
In a second aspect, a pharmaceutical composition comprising one or more peptides, either the same or different, according to the present invention and pharmacologically acceptable excipients, for use in the inhibition of angiogenesis or neovascularization is described. Such excipients can be, for example, at least one or more suitable diluents, carriers or adjuvants.
A “diluent, carrier or adjuvant” is any suitable excipient, diluent and/or adjuvant which neither induce by itself the production of antibodies that are harmful to the individual receiving the composition nor cancel the effect of the composition.
A carrier or adjuvant (acceptable from a pharmaceutical point of view) can improve the response stimulated by the peptides object of the invention, for example by determining a continuous release of the peptides or pharmaceutical composition object of the invention for an extended period of time (slow release formulation). Carriers or suitable adjuvants typically consist of one or more compounds included in the following list, although not exhaustive, lipid microparticle type carriers such as: Solid Lipid Nanoparticles (SLN), Nanostructured Lipid Carriers (NLC), Solid Lipid Microparticles (SLM), liposomes, W/O/W emulsion, or microparticles made of biodegradable and biocompatible polymers (PLGA, PLA or PHEA/PEG).
The peptide and the pharmaceutical composition comprising one or more peptides,
either the same or different, may be used as a medicament in both humans and animals (veterinary use). The animal may be any animal capable of forming new blood vessels; in particular such an animal may be a mammal, such as a human being. It is expected that the effective dose of the active substance described in the invention falls within a sufficiently wide range to be determined by routine tests. Typically, the dose ranges between 4% and 0.001 %.
In one embodiment, the use of one or more peptides or the pharmaceutical composition according to the present invention is a use for ophthalmic, or topical ophthalmic administration.
Said composition may be in the form of eye drops, powder, granules, cream or gel, erodible ocular inserts, or powder or in the liquid form of a suspension, emulsion, microemulsion, solution, eye spray, eye bath, or an injectable composition. When the pharmaceutical composition is in injectable form, it can be a liquid solution or a suspension, and said injection can be carried out intravitreally.
In addition, the need for a formulation for ophthalmic use that is stable, well tolerated by the eye, that improves the solubility and bioavailability of the active is strongly felt. In particular, carrier systems such as microemulsions improve the solubility of poorly soluble molecules and stabilize them thus improving their bioavailability and optimizing the possibility of reaching the back of the eye.
In a third aspect, the present invention describes the use of the peptides or the pharmaceutical composition in inhibiting angiogenesis or neovascularization. In one embodiment, the neovascularization can be dependent on VEGFR-1 and VEGFR- 2. In particular, in vitro and in vivo inhibition of angiogenesis and neovascularization of the eye dependent on VEGFR-1 and VEGFR-2 was analysed. For instance, as can be seen in Examples 3 and 4, the inhibition of capillary-like structures formation induced by VEGF and the inhibition of choroidal neovascularization by intravitreal administration of the peptides of the present invention was shown.
Surprisingly, the Tetrameric Tripeptides of Formula (I), (III) and (IV), described by the present invention were further characterized for their ability to inhibit the formation of capillary-like structures by primary endothelial cells grown on extracellular matrix extract induced by growth factors, preferentially by VEGF-A.
The Tetrameric Tripeptides of formula (I), (III) and (IV) were further characterized to
be modulators of neovascularization dependent on VEGFR-2 and VEGFR-1 , or angiogenesis dependent on VEGFR-2 and VEGFR-1 .
The invention also relates to the conjugated form of the Tetrameric Tripeptides of formula (I), (III) and (IV), with a non-immunogenic hydrophilic polymer. This aspect tries to solve the problem of improving the solubility of the tetrameric peptide in water or in aqueous buffers. The hydrophilic polymers are selected from polyethyleneglycol, polyvinyl-pyrrolidones, carbohydrates with a molecular weight between 100 and 20,000 Dalton.
In one embodiment, in the use of the peptides or the pharmaceutical composition, said inhibition of angiogenesis and neovascularization are in the treatment of cancer (tumor growth) or a neovascular disease. Said neovascular disease can be a disease of the bones or joints, blood vessels, skin, angiogenesis resulting from adipose tissue diseases, diabetes and/or its consequences, or hematopoiesis diseases.
Preferably, said neovascular disease is a neovascular edema disease of the eye.
In a more preferred embodiment, said neovascular disease of the eye is senile macular degeneration, a neovascular disease of the eye surface resulting from infection, inflammation, hypoxia, trauma or degeneration/loss of the limbal barrier, diabetic retinopathy, central retinal vein occlusion, premature infant retinopathy, macular edema and associated inflammation, retinal vein occlusion retinopathy (e.g. central retinal vein), vitreous haemorrhage, retinal detachment, central serous chorioretinopathy, age-related macular degeneration (exudative form), and myopic macular degeneration (choroidal neovascularization) or combinations thereof.
Examples of diseases that can be associated with corneal neovascularization are bacterial, viral, fungal, or parasitic keratitis, corneal graft rejection, graft versus host disease (GvDH), atopic conjunctivitis, Turner syndrome, Terrien’s marginal degeneration, pterygium, sterile corneal ulcer, dry eye syndrome.
The use of the peptides or the pharmaceutical composition for the inhibiting angiogenesis and neovascularization in the treatment of cancer, is foreseen in the treatment of solid and liquid tumors and/or tumor metastasis, preferably said tumors being selected from leukemias and lymphomas, preferably acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, multiple
myeloma, Hodgkin’s lymphoma, Hodgkin’s disease, infant or adult solid tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms tumor, osteosarcoma and chondrosarcomas, lung cancer, colon and rectal cancer, breast cancer, prostate cancer, uterus cancer, ovarian cancer, urinary system cancer, bladder cancer, oral cavity cancer, pancreatic cancer, melanoma and skin cancers, stomach cancer, brain cancer, thyroid cancer, laryngeal cancer, liver cancer, testicular cancer.
The use of peptides or the pharmaceutical composition according to the present invention for inhibiting angiogenesis and neovascularization, can be for the treatment of:
- diseases of the bones or joints, preferably selected from: rheumatoid arthritis, synovitis, cartilage and/or bone destruction, osteomyelitis, hypertrophy and/or hyperplasia of the synovial tissue, osteophytes formation, neoplasms and/or metastases and combinations thereof; and/or
- blood vessels diseases, preferably selected from: atherosclerosis, hemangioma, hemangioendothelioma and combinations thereof; and/or
- skin diseases, preferably selected from: psoriasis, warts, pyogenic granulomas, hair growth, Kaposi’s sarcoma, wound keloids, allergic edema, neoplasms, and combinations thereof; and/or
- angiogenesis observed in diseases of the adipose tissue, preferably obesity; and/or
- diabetes and/or its consequences, preferably retinopathy and/or diabetic food; and/or
- hematopoiesis diseases, preferably AIDS and/or Kaposi’s sarcoma.
In ischemic retinal diseases, the supply of blood and oxygen to the retina is reduced, the peripheral portion of the retina loses the source of nutrients and stops functioning properly. Common causes of retinopathy are occlusion of the central retinal vein, stenosis of the carotid artery, diabetes (diabetic retinopathy) and sickle cell retinopathy. Retinopathy is also observed in premature infants (premature infant retinopathy). Diabetic retinopathy is the leading cause of vision loss in diabetic patients. In the ischemic retina, new blood vessels grow (neovascularization) occurs. These vessels often grow from the surface of the retina, invading the vitreous. These new vessels are unable to replace the flow of necessary nutrients
but, on the contrary, can cause multiple issues such as vitreous hemorrhage, retinal detachment, and uncontrolled glaucoma. These issues appear because the new vessels are fragile and prone to bleeding. Other eye diseases in which angiogenesis is believed to play a fundamental role include intraocular and choroidal disorders, leukomalacia, neoplasms, and metastases. Choroidal neovascularization (CNV) is the growth of new blood vessels that originate from the choroid through a break in the Bruch membrane into the sub-retinal pigment epithelium (sub-RPE) or sub- retinal space. The location, the growth pattern, the type (1 or 2) of CNV depends on the age of the patient and the specific case. Bleeding and exudations occur causing further vessel growth and producing the negative visual symptoms. CNV is the leading cause of vision loss. It is estimated that CNV is present in 5-10% of myopic people and occurs practically in all choroidal ruptures during the healing phase. In most cases it recedes spontaneously, but in 15-30% of patients it can recur and lead to bleeding and serious macular detachment with concomitant vision loss.
The peptides and the pharmaceutical composition according to the present invention may also be used for the manufacture of a medicament that can be included in a treatment regimen for conditions associated with VEGFR-1 and VEGFR-2 dependent neovascularization. The aforementioned treatment regimen may include other compounds or drugs which act on VEGFR-1 and VEGFR-2 dependent neovascularization, or which determine an improvement in the side effects of said treatment. Other compounds or drugs may be present in said treatment regimen including a neutralizing PIGF antibody and/or an anti-VEGFR antibody and/or an anti-VEGF-A antibody.
In a fourth aspect, the present invention describes the use of peptides or the pharmaceutical composition in inhibiting the binding between the VEGFR-1 and VEGFR-2 receptors and their ligands.
In yet another aspect, the present invention relates to a method for the treatment of cancer or a neovascular disease, by means of the step of using the peptides or the pharmaceutical composition of the invention.
In one embodiment of the treatment method, angiogenesis and neovascularization are inhibited by the use of the peptides or pharmaceutical composition, and preferably said neovascular disease is a neovascular disease of the eye. In a more
preferred embodiment, said neovascular disease of the eye is diabetic retinopathy. In a further preferred embodiment, said neovascular disease of the eye is age- related macular degeneration or a neovascular disease of the eye surface resulting from infection, inflammation, hypoxia, trauma, or degeneration/loss of the limbal barrier.
The present invention relates to peptides capable of interacting with the two VEGF receptors involved in the formation of blood vessels, VEGFR-1 and VEGFR-2, through the activation induced by the common ligand VEGF-A, and by PIGF which specifically binds VEGFR-1 . As highlighted in Example 1 , these compounds are able to compete with VEGF-A in binding the two receptors, and with PIGF in binding VEGFR-1 , in a dose-dependent manner. They are able to achieve its activation by inhibiting the VEGF-A induced phosphorylation of the two receptors (Example 2). The results reported in Example 3 show how these compounds have the ability to inhibit the formation of capillary-like structures by primary endothelial cells grown on extracellular matrix extract induced by growth factors, preferably by VEGF-A. Furthermore, as described in Example 4, these compounds are capable of inhibiting laser-induced choroidal neovascularization in vivo, an experimental model that recapitulates the wet form of age-related macular degeneration.
The set of results allows us to define the peptides of the invention as inhibitors of VEGFR-1 and VEGFR-2 dependent neovascularization or angiogenesis.
In particular, the results of Examples 1 -4 allow to conclude that the tetrameric peptide of formula (IV), depending on the expression levels of VEGFR-2, VEGFR-1 and its soluble variant, is able to inhibit a neovascularization process. This peptide can therefore be defined as an inhibitor of neovascularization or angiogenesis, or more specifically an inhibitor of VEGFR-1 and VEGFR-2 dependent neovascularization or angiogenesis.
Examples of embodiments of the present invention, provided for illustrative purposes, are reported below.
EXAMPLES
Example 1 : Dose dependent inhibition of VEGF-AA/EGFR2, VEGF-AA/EGFR-1 and PIGFA/EGFR-1
The schematic formula of the peptide identified with the general formula (IV) and the
SEQ ID NO: 3, named inhibitory peptide (iVsB) is:
H2N-[ D-GIU - L-Cys(Bzl) - L-Cys(Bzl)]4-Lys2-Lys-Gly-COOH (SEQ ID NO:3).
The same peptide with a D-Ala instead of L-Cys (Bzl) at the C terminus of the tripeptide sequence was used as control peptide (PC):
H2N-[ D-GIU - L-Cys(Bzl) - D-Ala]4-Lys2-Lys-Gly-COOH (SEQ ID NO: 4).
The binding assay of VEGF-A to VEGFR-1 and VEGFR-2 receptors, and PIGF to VEGR-1 receptor, are based on the ELISA method and were performed using reagents acquired from R&D Systems. The human recombinant receptors VEGFR- 2 (R&D Systems, cat No. 357-KD) and VEGFR-1 (R&D Systems, cat No. 321 -FL) (consisting of seven extracellular domains fused to the Fc domain of human IgGs), were adhered to the wells of 96-well microplates at a concentration of 1 pg/mL. After blocking the non-specific binding sites to the wells using a buffer solution containing 3% BSA, human VEGF-A (R&D Systems, cat No. 293-VE) was added, at a concentration of 10 ng/mL. The concentration used was 10 ng/mL also for human PIGF (R&D Systems, cat No. 264-PG). Graduated doses of iVsB or PC between 1 .25 and 50 pM were added at the same time as the growth factors. At the end of the competitive phase, a biotinylated anti-hVEGF-A (R&D Systems, cat No. BAF293) or anti-hPIGF (R&D Systems, cat No. BAF264) antibody was added, followed by an avidin-streptavidin system conjugated with HRP (Vectastain elite ABC kit) and a substrate for HRP (ortho-phenylene-diamine - Sigma, cat No. P1526). The quantification was carried out by determining the absorbance at 490 nm. The possible inhibitory activity of the mixtures was expressed in terms of % binding, by comparing the data obtained on the binding of VEGF-A or PIGF to the receptors in the presence of the peptide with those in the absence of the same.
As reported in Tables 3 and 4, iVsB is able to inhibit in a dose dependent manner both the VEGF-AA/EGFR-2 interaction with an IC50 of just under 10 pM, and the VEGF-AA/EGFR-1 interaction, albeit with lower efficiency (IC50 of about 20pM). It is also able to inhibit the PIGFA/EGFR-1 interaction, still at a concentration of just under 20 pM. Conversely, PC is unable to inhibit the binding of the growth factors to the two receptors.
Table 3 - Dose-dependent inhibition of VEGF-A/VEGFR-2 and VEGF-A/VEGFR-1 interaction
Example 2: Inhibition of VEGF-A induced VEGFR-1 and VEGFR-2 phosphorylation To evaluate the inhibitory capacity of the iVsB peptide in functional terms, the VEGF- A induced VEGFR-1 and VEGFR-2 receptor phosphorylation assay was developed. Primary human endothelial cells HLIVEC were used for the activation of VEGFR-2, in which this receptor is well detectable. For VEGFR-1 , instead, a cell line overexpressing the 293-VEGFR-1 receptor, obtained by stable transfection starting from HEK-293 cells, was used.
For this purpose, HLIVEC and 293-VEGFR-1 cells were cultured until subconfluence. The cells were then starved, incubating 293-VEGFR-1 cells in serum- free culture medium while HLIVEC cells, normally cultured in the presence of 2% serum and a cocktail of growth factors, were incubated in the culture medium with 1 % serum for at least 16 hours.
At the end of the starvation, the culture medium was removed, and the cell monolayers were incubated with 100 pM NasVC for 5 minutes in order to inhibit the activity of the endogenous phosphatase. The cells were then stimulated with VEGF- A at 50 ng/mL in the media used for starvation for 10 minutes at 37°C. iVsB was added simultaneously to VEGF-A at a 25 pM concentration. DMSO was used as a negative control. At the end of the incubation, the cells were washed with cold 100 pM NasVO4 and then lysed in the buffer consisting of 20 mM Tris-HCI pH 8, 5 mM EDTA, 150 mM NaCI, 1 % Triton-X100, 10% glycerol, 10 mM zinc acetate, 100 pM NasVO4 and a mixture of protease inhibitors, incubating for 1 hour at 4°C with gentle shaking.
Upon completion, the cell lysates were centrifuged at 12,000xg for 15 minutes to remove cell debris. The extracts were quantified with the Bradford method using the BioRad reagent. 100 pg of each protein extract were loaded on 8.5% reducing SDS- PAGE, and the standard method was then used to analyse the proteins by Western blot.
Two specific anti-p-VEGFR-1 (R&D Systems, cat. No. AF4170), diluted 1 :500, and anti-p-VEGFR-2 (Cell Signaling), diluted 1 :1000, antibodies were used to detect the two phosphorylated receptors, while normalization was performed by detecting the non-phosphorylated forms of the receptors using the anti-VEGFR-1 antibody (Sigma-Aldrich, cat. No. V4262) diluted 1 :500, and the anti-VEGFR-2 antibody (Santa Cruz Biotechnology) diluted 1 :500.
As shown in Figure 1 , iVsB is able to determine the inhibition of phosphorylation of both receptors, confirming that the inhibition of the interaction observed in the in vitro ELISA assay corresponds to a functional inhibition of the receptor activation. Example 3: Inhibition of capillary-like structures formation induced by VEGF-A The formation of capillary-like structures (CTFs) by primary endothelial cells, suitably grown on an extracellular matrix support (Matrigel, BD cat. No. 354230),
following stimulation with pro-angiogenic factors, is an assay commonly accepted for evaluating the anti-angiogenic activity of compounds.
HLIVEC cells were used, with EGM-2 culture medium, consisting of EBM base medium supplemented with 2% serum and a cocktail of growth factors. Matrigel was layered in the wells of a 24-well cell culture plate. 230 pL of cold matrigel were added to each well, and then the plate was incubated for 30 minutes at 37°C to allow the matrigel solidification. Exponentially growing HLIVEC cells were detached from the plates, washed, and resuspended in EBM at a density of 120,000 cells/mL. 60,000 cells in 0.5 mL of medium were used for each well containing the matrigel.
The cells, simply resuspended in EBM, were used as a negative control of the experiment. For the positive control of the experiment, the cells were resuspended in complete EGM-2 medium. To verify the efficacy of VEGF-A in stimulating CTFs, VEGF-A was added at a concentration of 100 ng/mL to the cells resuspended in EBM immediately before seeding them in the wells. To verify the ability of the iVsB peptide to inhibit this process, a dose dependency experiment was performed at concentrations between 10 and 50 pM. The PC activity at the maximum concentration used for iVsB was also evaluated. The two peptides were added to the HLIVEC mixture in EBM + 100 ng/mL VEGF-A immediately prior to inoculating the cells into the wells in which the matrigel had been stratified.
After six hours incubation, the CTF formation was evaluated by observation under the microscope. The experiment is stopped by fixing the cells with 0.2% glutaraldehyde and 1 % paraformaldehyde in PBS. The experiment was carried out in triplicate for each sample.
As shown in Figure 2, VEGF-A stimulates CTF in a manner comparable to the positive control (EGM-2), while with the negative control (EBM) the cells remain dispersed on the matrigel. PC is unable to inhibit CTF, while iVsB is active in a dosedependent manner. At 50 pM the cells are dispersed as in the negative control, at 20pM the inhibition is still consistent although the formation of capillary-like structures is observed, a phenomenon even more present at a peptide concentration of 10pM.
Example 4: Inhibition of choroidal neovascularization by intravitreal administration of iVsB.
The experimental model of laser-induced choroidal neovascularization involves damage generation at the level of the Bruch membrane that separates the choroid from the retinal pigment epithelium (RPE). The damage is caused by a laser- induced bum that causes Bruch’s membrane perforation thus activating chorioretinal vascularization, the growth of new vessels that, starting from the choroid, invade the overlying retinal tissue. This murine model recapitulates the main characteristics of the exudative form of human senile degenerative maculopathy (AMD) and is, in fact, commonly used as a preclinical model of AMD. It allows to evaluate the anti-angiogenic activity of the molecules of interest.
In order to visualize the mouse fundus and induce damage with the laser, the Micron IV system was used, and the experimental procedure described below was performed.
Pupil dilation is induced before anesthetization of the animal. For this purpose, 0.5% Tropicamide eye drops were applied topically. The animal was then anesthetized by intraperitoneal injection of a solution of ketamine and xylazine (80 mg/kg and 10 mg/kg, respectively). Once sedated, the animal was placed on the stand and a 2.5% hydroxy-propyl-methylcellulose aqueous solution was applied to both eyes. It has the dual function of preventing corneal dehydration and improving the visualization of the fundus by placing the lens of the Micron IV chamber in contact with the solution (a procedure similar to that used in microscopy with immersion objectives). To induce damage with the laser, the laser pointer is first activated and focused to apply the laser beam using the RPE layer as a reference. The area where the laser beam is applied must be far from the retinal main vessels to prevent any bleeding. The efficiency of the burn at the level of the Bruch membrane is confirmed by the formation of a bubble immediately after the laser beam application. The experimental conditions used in the experiments were obtained with the conditions: 100 msec and 200mW.
From the literature data, well summarized in the article by Lambert et al. (Nature Protocols, 2013, 8: 2197) it is known that, in this experimental model, the maximum of neovascularization is obtained seven days after the damage.
C57BI6/J mice, n=5 per group, were subjected to anaesthesia and then the laser damage procedure was performed as described above. At the end of the procedure,
the intravitreal injection was immediately performed, using a Hamilton syringe with a 32g needle, administering 50p g of iVsB or PC in 1 pL of DMSO, in the left eye. DMSO alone was injected as a control.
After seven days, the animals were sacrificed, the eyes were enucleated and fixed in 4% paraformaldehyde. Subsequently, the anterior segment of the eye, consisting of cornea, iris and lens, was removed under the stereomicroscope. The remaining part defined ‘eye-cups’ or posterior segment, consisting of: sclera, choroid, RPE and retina, was incubated in the presence of 0.7% FITC-Griffonia simplicifolia Isolectin B4 (Vector Laboratories, Burlingame, CA) for sixteen hours. After a series of washings, the retina is removed, and four cuts are made on the RPE/choroid which allow it to be mounted on a slide for observation under a fluorescence microscope. The quantification of CNV is done in terms of volume. To evaluate the volume of each spot, a series of images (Z-Satcks, approximately 20-25 images) each 1-pm thick, starting from the upper surface to the deepest focal plane at the level of the RPE cells, are acquired. The fluorescence volume is measured by the Imaged program (NIH, Bethesda, MD) by adding the fluorescence area of each individual plane.
CNV quantification was performed on n=9 spots for iVsB, n=6 for PC, and n=9 spots for the vehicle, and the results are reported in Figure 3. The results show a significant inhibitory capacity of iVsB (~ 40%) with respect to the carrier and the control peptide PC.
From the detailed description and the Examples reported above, the advantages achieved by means of the peptides and the pharmaceutical composition of the present invention are apparent. In particular, these peptides proved to be surprisingly and advantageously suitable for the treatment of tumor pathologies, angiogenesis and neovascularization.
Claims
1. A peptide or salts thereof, for use in the inhibition of angiogenesis or neovascularization said peptide having the amino acid sequence of SEQ ID NO:1 and the general formula (I)
{{{[Y1 - R-Glu - S-Cys(Bzl) - S- Cys(Bzl)]2 - Z1 }i - Z2 }j - Z3 }q - Y2 - Y3 wherein:
- Y1 is selected from the group consisting of an amino terminal group (NH2), or an amino acid selected from the group consisting of D-Alanine, D-Aspartic Acid, D- Valine, D-Glutamic Acid, L-Cyclohexyl-alanine, D-Phenylalanine, D-Threonine, D- Methionine, D-Lysine, D-Cysteine(S-acetamidomethyl), D-Tyrosine, D-Proline, D- Leucine, D-Arginine, D-Asparagine, D-lsoleucine, D-Arginine(N-Tosyl), D-Serine, L-Cysteine(S-benzyl), L-Cysteine(S-acetamidomethyl), D-Histidine, D-Glutamine, D-Tryptophane, L-Glutamic Acid-(P-allyl), [3-Alanine, L-Cysteine(S-p-methyl- benzyl), L-Cysteine(S-tert-butyl), L-Methionine-sulfone, L-Methionine-sulfoxide, Glycine or combinations thereof;
- R-Glu indicates glutamic acid with R absolute configuration at the Ca of the amino acid (R-glutamic acid);
- S-Cys(Bzl) indicates benzylcysteine with S absolute configuration at the Ca of the amino acid (S-benzyl-cysteine) having a benzyl group bound to the sulfur of the amino acid side-chain;
- Z1 , Z2 and Z3: when present, are independently a molecule of formula (II) with R or S absolute configuration:
wherein:
- k is an integer number from 1 to 4,
- A is an amine group;
24
- i is a number selected from the group consisting of 1 , 2 and 4;
- j is a number selected from the group consisting of 1 and 2;
- q is 1 with the proviso that: when i = 4, j = 2, q = 1 ; when i = 2, j = 1 , q = 1 , and Z3 is absent; when i = 1 , j = 1 , q = 1 , and Z2 and Z3 are absent;
- Y2 is selected from the group consisting of Glycine, Beta-alanine and s-amino caproic acid;
- Y3 is selected from the group consisting of a carboxy terminal group, a carboxamide group, a N-methyl substituted or N,N-dimethyl disubstituted carboxamide, a hydroxyl group and a hydrogen.
2. The peptide for use according to claim 1 , wherein:
- i is 2;
- j is 1 ;
- q is 1 ; and
- Z3 is not present, said peptide having the amino acid sequence of SEQ ID NO:2 and the general formula (III)
Y1-(R-Glu)-(S-Cys(Bzl))-(S-Cys(Bzl)
3. The peptide for use according to any one of claims 1 or 2, wherein:
- Y1 is an amino terminal group; and
- Z1 and Z2 are S-Lysine.
4. The peptide for use according to any one of claims 1 to 3, wherein:
- Y1 is an amino terminal group;
- Z1 and Z2 are S-Lysine;
- Y2 is Glycine; and
- Y3 is a carboxy terminal group, said peptide having the sequence of SEQ ID NO: 3 and the general formula (IV)
S-Lys
5. A pharmaceutical composition comprising one or more peptides, either the same or different, according to any one of claims 1 to 4 and pharmacologically acceptable excipients, for use in the inhibition of angiogenesis or neovascularization.
6. The peptide for use according to any one of claims 1 to 4 or the pharmaceutical composition for use according to claim 5, wherein said inhibition of angiogenesis and neovascularization are for the treatment of cancer or a neovascular disease, wherein said neovascular disease is a disease of the bones or joints, blood vessels, skin, angiogenesis resulting from adipose tissue diseases, diabetes and/or its consequences, or hematopoiesis diseases.
7. The peptide for use or the pharmaceutical composition for use according to claim 6, wherein said neovascular disease is a neovascular disease of the eye.
8. The peptide for use or the pharmaceutical composition for use according to claim 7, wherein said neovascular disease of the eye is senile macular degeneration, diabetic retinopathy, central retinal vein occlusion, premature infant retinopathy, macular edema and associated inflammation, retinal vein occlusion, central serous chorioretinopathy, age-related macular degeneration (exudative form), a neovascular disease of the eye surface resulting from infection, inflammation, hypoxia, trauma or degeneration/loss of the limbal barrier, or myopic macular degeneration (choroidal neovascularization).
9. The pharmaceutical composition for use according to any one of claims 5 to 8, wherein said composition is ophthalmically administered.
10. The pharmaceutical composition for use according to any one of claims 5 or
9, wherein said composition is in the form of eye drops, powder, granules, cream or gel, erodible ocular inserts, or powder or in the liquid form of a suspension, emulsion, microemulsion, solution, eye spray, eye bath, or an injectable.
11. The pharmaceutical composition for use according to any one of claims 5 to
10, wherein said composition is in the form of an injectable liquid solution or suspension, and wherein said injection is an intravitreal injection.
12. The peptide for use according to any one of claims 1 to 4 and 6-8, or the pharmaceutical composition for use according to any one of claims 5 to 11 , wherein said use is for inhibiting the binding between VEGFR-1 and VEGFR-2 receptors and ligands thereof.
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