Polypeptide having Anti-angiogenic activity and use thereof

Abstract

Provided in the present application is a polypeptide having an anti-angiogenic activity. An amino acid sequence thereof is selected from one of sequences set forth in SEQ ID No. 03, SEQ ID No. 09, SEQ ID No. 10 and SEQ ID No. 13. The active polypeptide can be prepared by chemical synthesis or genetic engineering expression. Further provided in the present application is the use of the active polypeptide in the preparation of a drug for treating ocular neovascular diseases and other angiogenesis-related diseases.

Description

A polypeptide with anti-angiogenic activity and its application

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202411639021.8, filed on November 15, 2024, entitled "A polypeptide with anti-angiogenic activity and its application thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of peptide technology, specifically relating to a peptide with anti-angiogenic activity, its pharmaceutically acceptable salt, and their use in the preparation of medicaments for the treatment or prevention of angiogenesis-related diseases. Background Technology

[0004] Age-related macular degeneration (AMD) is a group of diseases characterized by progressive degenerative changes in the macula, the central region of the retina. It is a typical age-related eye disease and the leading cause of blindness in people over 50. With the aging population, the prevalence of AMD is increasing year by year, currently exceeding 15 million patients worldwide, and is projected to double by 2050.

[0005] Age-related macular degeneration (AMD) is mainly divided into two types: dry (non-neovascular) AMD and wet (neovascular) AMD. Dry AMD is more common, accounting for approximately 90% of all AMD cases. Its progression is relatively slow, and pathologically, it presents as small, round, whitish-yellow drusen deposits in the macula and submacula. Although dry AMD usually progresses mildly, in later stages it can develop into geographic atrophy, leading to the loss of retinal photoreceptor cells, thinning of the retinal layer, and destruction of the macular structure, ultimately causing severe vision loss or even blindness.

[0006] The main pathological feature of wet macular degeneration is abnormal angiogenesis, namely choroidal neovascularization (CNV). In this process, abnormal blood vessels grow beneath the retinal pigment epithelium (RPE) layer and can penetrate the subretinal space through gaps in the Bruch's membrane (the extracellular matrix located between the choroid and RPE), causing subretinal hemorrhage and / or fibrous scarring, ultimately leading to visual impairment.

[0007] Neovascular eye diseases, represented by AMD, are a group of blinding eye diseases characterized by pathological neovascularization, often leading to decreased vision or even irreversible vision loss. This category encompasses a variety of eye conditions, including not only age-related macular degeneration, but also diabetic retinopathy (DR), retinal vein occlusion, neovascular glaucoma, idiopathic choroidal neovascularization, retinal edema, retinopathy of prematurity (ROP), and corneal neovascularization caused by trauma or inflammation.

[0008] Besides the typical pathological feature of neovascularization, the pathogenesis of age-related macular degeneration (AMD) is extremely complex, encompassing multiple pathological changes, including: loss of rod photoreceptor cells, thinning of the choroidal layer, lipid deposition in the retinal pigment epithelium (RPE) and its basal layer, complement-mediated chronic inflammatory response, infiltration of circulating macrophages, and activation of inflammasomes and microglia. These pathological processes can collectively lead to dysfunction or even death of RPE cells, Bruch's membrane (BrM), and choroidal capillary endothelial cells, ultimately causing atrophy of the outer retina and promoting the occurrence and development of AMD.

[0009] In the early stages of AMD, the most prominent pathological changes include thickening of the Bruch's membrane, drusen formation, and pigmentary abnormalities (such as hyperpigmentation or depigmentation of the rudimentary dermal papillae). Rudimentosa are primarily composed of cellular lipids, lipoprotein deposits, and aggregates of various proteins, with common components including albumin, apolipoprotein E (APOE), complement components, immunoglobulins, and amyloid-β. The formation of drusen may be closely related to local chronic inflammatory responses, such as complement system activation, rudimentary dermal papillae (RPE) cell lysis, or RPE cell homeostasis dysregulation, thereby further exacerbating the inflammatory process.

[0010] The formation of lipid walls and abnormal lipid deposition can not only induce chronic inflammatory responses but also cause dysproutational dysfunction of the Bruch's membrane (BrM), leading to loss of extracellular matrix (ECM) homeostasis and altering the physiological function of the Bruch's membrane, thereby further promoting the formation of drusen and exacerbating the inflammatory response. With the oxidation of lipid components, protein modification, and the fusion and calcification of drusen, the Bruch's membrane may rupture, triggering more significant pathological changes. Simultaneously, lipid deposition can also promote the formation of choroidal neovascularization, which can extend into the subretinal space—the gap between photoreceptor cells and the RPE layer. This series of pathological changes will hinder the delivery of key nutrients such as vitamin A to retinal photoreceptors (rod cells and cone cells), ultimately leading to photoreceptor atrophy, degeneration, and death.

[0011] As can be seen from the brief description of the disease process above, the pathogenesis of age-related macular degeneration (AMD) is extremely complex, involving multiple pathophysiological processes such as cellular, biochemical, metabolic, and immune processes. For specific mechanisms, please refer to the relevant published literature and research data, such as Christine A. Curcio et al.'s "The oil spill in ageing Bruch membrane" (Br J Ophthalmol. 2011; 95(12):1638-45) and Joan W. Miller's "Age-related macular degeneration revisited—piecing the puzzle: the LXIX Edward Jackson memorial lecture" (Am J Ophthalmol. 2013; 155(1):1-35).

[0012] Neovascular eye diseases such as wet age-related macular degeneration (AMD) severely impact patients' visual function and quality of life. Current clinical treatment primarily relies on anti-vascular endothelial growth factor (VEGF) drugs; however, these therapies suffer from short-lived efficacy and significant individual variability in response. Repeated administration can also lead to retinal and choroidal complications, and the long-term, high treatment costs impose a significant economic burden on patients. Furthermore, a single anti-VEGF strategy is insufficient for long-term effective disease control, making the development of alternative treatment strategies particularly urgent.

[0013] Through long-term clinical practice and research, the applicant discovered that artificial short peptides designed based on the sequence of thrombospondin 1 (THBS1, Thrombospondin 1, TSP-1, NM_003246.4, NP_003237.2) exhibit significant anti-angiogenic activity. Building on this, the applicant further conducted related research for this application. Summary of the Invention

[0014] Purpose of the invention

[0015] The purpose of this application is to provide a polypeptide with anti-angiogenic activity to address the therapeutic limitations and clinical needs of existing technologies, and to develop its application in the treatment of neovascular eye diseases.

[0016] Solution

[0017] To achieve the above objectives, this application provides a polypeptide with anti-angiogenic activity obtained through screening, and provides a method for preparing the polypeptide, a composition comprising the polypeptide or a pharmaceutically acceptable salt thereof, and the application of the polypeptide and the composition.

[0018] Specifically, the technical solution of this application is as follows:

[0019] In a first aspect, this application provides a polypeptide with anti-angiogenic activity (also referred to in this application as an anti-angiogenic active polypeptide, or simply as an active polypeptide, active polypeptide molecule, active peptide, or active peptide molecule) or a pharmaceutically acceptable salt thereof; the active polypeptide sequence comprises an amino acid sequence selected from any one of SEQ ID No. 03, SEQ ID No. 09, SEQ ID No. 10, and SEQ ID No. 13.

[0020] SEQ ID No. 03: VTCGVITRI

[0021] SEQ ID No. 09: SVTCGVITRIR

[0022] SEQ ID No. 10: CSVTCGVITRIR

[0023] SEQ ID No. 13: CSVTCGGVITRIR

[0024] Preferably, the polypeptide with anti-angiogenic activity is:

[0025] (1)VRR-3, whose sequence is VTCGVITRI;

[0026] (2)VRR-9, whose sequence is SVTCGVITRIR;

[0027] (3)VRR-10, whose sequence is CSVTCGVITRIR;

[0028] (4)VRR-13, whose sequence is CSVTCGGVITRIR.

[0029] The active peptide molecules can be prepared by chemical synthesis or genetic engineering. When prepared by chemical synthesis, the purity should be no less than 50%; preferably, no less than 55%; more preferably, no less than 60%; even more preferably, no less than 65%; even more preferably, no less than 70%; even more preferably, no less than 75%; even more preferably, no less than 80%; even more preferably, no less than 85%; even more preferably, no less than 90%; even more preferably, no less than 95%; even more preferably, no less than 99%.

[0030] This application provides the salt form of the above-mentioned active peptide molecules. The polypeptide molecules described in this application may exist and be used in a non-salt form or a pharmaceutically acceptable salt form. For example, the polypeptide may form an addition salt with an inorganic acid or an organic acid, wherein the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, or sulfuric acid; and the organic acid includes carboxylic acids or sulfonic acids. Acids suitable for forming pharmaceutically acceptable salts include (but are not limited to) the following: acetic acid, 2,2-dichloroacetic acid, adipic acid, capric acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclohexanediol, dodecyl sulfate, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, lauric acid, maleic acid, malonic acid, oleic acid, oxalic acid, palmitic acid, propionic acid, stearic acid, succinic acid, undecanoic acid, valeric acid; ascorbic acid, L-aspartic acid, gentian acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, α-oxoglutarate, glycolic acid, hippuric acid, (±)-DL-lactic acid, (+)-L-lactic acid, lactobionic acid, (-)-L-malic acid, (±)-DL-lactic acid, etc. - Mandelic acid, orotic acid, L-pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, (+)-camphoric acid, (+)-(1S)-camphor-10-sulfonic acid; benzenesulfonic acid, benzoic acid, 4-acetaminobenzoic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, dihydroxynaphthoic acid, tannic acid, (±)-DL-tartaric acid, (+)-L-tartaric acid; naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, camphorsulfonic acid, methanesulfonic acid, p-toluenesulfonic acid; alginic acid, galactopyric acid, glucoheponic acid, glycolic acid; acylated amino acids, cyclohexanesulfonic acid, ethane-1,2-disulfonic acid, perchloric acid, phosphoric acid, sulfuric acid, boric acid, hydrobromic acid, hydroiodic acid, nitric acid, thiocyanate.

[0031] Secondly, the active peptide molecule or its pharmaceutically acceptable salt provided in this application exhibits significant cell proliferation and migration inhibition activities in in vitro experiments. Regarding cell proliferation inhibition, the active peptide can reduce cell survival rate by at least 5%; preferably, by at least 10%; more preferably, by at least 15%; even more preferably, by at least 20%; and most preferably, by at least 25%. The term "reduction in cell survival rate" can be understood as an inhibitory effect on cell proliferation.

[0032] Regarding cell migration inhibition, the active peptide can reduce the cell scratch healing rate by at least 5%; preferably, by at least 10%; more preferably, by at least 15%; even more preferably, by at least 20%; even more preferably, by at least 25%; even more preferably, by at least 30%; even more preferably, by at least 35%; and most preferably, by at least 40%. The term "reducing the cell scratch healing rate" can be understood as inhibiting cell migration ability.

[0033] The active peptide molecule or its pharmaceutically acceptable salt provided in this application has apoptosis-inducing activity, capable of increasing the apoptosis level by at least 1%; preferably, increasing the apoptosis level by at least 2%; more preferably, increasing the apoptosis level by at least 3%; even more preferably, increasing the apoptosis level by at least 4%; and most preferably, increasing the apoptosis level by at least 5%.

[0034] Thirdly, this application provides a composition comprising the above-described active peptide molecule and / or its pharmaceutically acceptable salt, and optionally a pharmaceutically acceptable excipient. In the composition, the concentration of the active peptide molecule or its pharmaceutically acceptable salt is not less than 10 μg / mL, for example, it may be at least 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, or 100 μg / mL. In some embodiments, the useful concentration range of the active ingredient in the composition may be any range comprised of the aforementioned values, including but not limited to: 10–15 μg / mL, 15–20 μg / mL, 20–25 μg / mL, 25–30 μg / mL, 30–35 μg / mL, 35–40 μg / mL, 40–45 μg / mL, 45–50 μg / mL, 50–55 μg / mL, 55–60 μg / mL, 60–65 μg / mL, 65–70 μg / mL, 70 –75μg / mL, 75–80μg / mL, 80–85μg / mL, 85–90μg / mL, 90–95μg / mL, 95–100μg / mL, 100–200μg / mL, 200–300μg / mL, 300–400μg / mL, 400–500μg / mL, 500–600μg / mL, 600–700μg / mL, 700–800μg / mL, 800–900μg / mL or 900–1000μg / mL.

[0035] The "pharmaceuticalally acceptable excipients" mentioned in this application refer to excipients and additives used in the preparation and formulation of pharmaceuticals; these are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical formulation. In addition to their basic functions such as excipient formation, carrier construction, and stability enhancement, these excipients also play crucial roles such as solubilization, co-solubilization, and sustained-release, and are important components that may affect the quality, safety, and efficacy of the formulation. Based on their function and use, pharmaceutical excipients can be categorized as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors, etc.

[0036] Furthermore, the composition of the third aspect of this application may further comprise an effective amount of an anti-vascular endothelial growth factor (VEGF) agent, preferably, the concentration of the anti-VEGF agent in the composition is from 10 μg / mL to 5000 μg / mL. In one specific embodiment, the composition comprises the polypeptide or a pharmaceutically acceptable salt thereof, in a content of 0.001 μg to 1 μg; and an anti-VEGF agent, in a content of 10 μg to 10 mg.

[0037] The "anti-vascular endothelial growth factor agent" as described herein refers to a molecule or ion capable of specifically reducing VEGF protein expression levels and / or inhibiting VEGF binding to VEGFR and its downstream functions. The anti-VEGF agent may be selected from the following categories: antigen-binding fragments (such as Fab, F(ab')2, scFv, Fv fragments, sdAb / VHH, Fc fusion proteins, bispecific antibodies, or multivalent antibodies), monoclonal antibodies, fusion proteins, oligonucleotides (such as siRNA, aptamers, microRNA / microRNA inhibitors / microRNA mimics), nucleic acid constructs, and any combination of one or more of the above. Preferably, the anti-vascular endothelial growth factor agent is selected from aflibercept, ranibizumab, faricimab, brolucizumab, and conbercept.

[0038] Furthermore, the composition of the third aspect of this application may further comprise an effective amount of a glucocorticoid drug or molecule, preferably selected from triamcinolone acetonide, dexamethasone, fluocinolone acetonide, hydrocortisone, prednisone, and methylprednisolone.

[0039] Fourthly, the compositions described in this application can be prepared as liquid formulations, semi-solid formulations, or solid formulations. The liquid formulations are selected from eye drops and ophthalmic injections; and / or the semi-solid formulations are selected from ophthalmic gels and ophthalmic ointments; and / or the solid formulations are selected from ophthalmic tablets and ophthalmic implants.

[0040] Fifthly, this application provides for the use of the said active peptide molecules, their pharmaceutically acceptable salts, or compositions comprising them in any of the following aspects: preparation of formulations for inhibiting cell migration, inhibiting cell proliferation, and / or inducing apoptosis; preparation of medicaments or compositions for treating ocular neovascular diseases. The ocular neovascular diseases include, but are not limited to: diabetic retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), ischemic retinal vein occlusion, neovascular glaucoma, choroidal neovascularization, retinal edema, geographic atrophy, retinal vein occlusion, uveitis-associated intraocular neovascularization, Behcet's disease, tuberculous uveitis, pathological myopia with choroidal neovascularization, choroidal tumors, cytomegalovirus retinitis, intraocular malignancies (including retinoblastoma, choroidal melanoma, and intraocular lymphoma), adnexal tumors (such as optic nerve tumors), and corneal neovascularization caused by trauma or inflammation.

[0041] Sixthly, the method of using the active peptide molecule, its pharmaceutically acceptable salt, or a combination thereof provided in this application includes applying it to corneal tissue requiring prevention or treatment. Feasible routes of administration include topical administration, such as eye drops, subconjunctival injection, or intravitreal injection; alternative dosage forms include contact lens formulations, ophthalmic ointments, injections, or eye drops.

[0042] The seventh aspect relates to the administration regimens of the active peptide molecules described in this application, their pharmaceutically acceptable salts, or combinations thereof. The dosage can be appropriately adjusted based on factors such as patient symptoms, age, weight, and route of administration, and is not strictly limited. In one specific embodiment, the daily dose for intravitreal injection can be from about 0.001 ng to 10 ng, preferably from about 0.05 ng to 5 ng. The concentration range of the injection solution can be from about 10 ng / mL to 1000 ng / mL, for example, about 10 ng / mL, about 100 ng / mL, about 300 ng / mL, or about 1000 ng / mL. Subconjunctival injection can be administered in a single or divided dose, preferably periodically according to symptom improvement, such as once or multiple times daily, once every few days, for several days to several months.

[0043] Furthermore, the active peptide molecule, its salt, or composition thereof described in this application can be used in combination with an anti-vascular endothelial growth factor agent, and the two can be administered simultaneously or sequentially. For example, the active peptide, its salt, or composition thereof of this application can be administered first, followed by an anti-vascular endothelial growth factor agent after a certain interval; or the anti-vascular endothelial growth factor agent can be administered first, followed by an active peptide, its salt, or composition thereof of this application after a certain interval.

[0044] Furthermore, the active peptide molecule, its salt, or combination thereof described in this application can be used in combination with glucocorticoid drugs or molecules, and the two can be administered simultaneously or sequentially. For example, the active peptide or its combination thereof of this application can be administered first, followed by a glucocorticoid drug or molecule after a certain interval; or the glucocorticoid drug or molecule can be administered first, followed by the active peptide or its salt or combination thereof of this application after a certain interval.

[0045] Furthermore, the active peptide molecule, its salt, or combination thereof described in this application can be used in combination with an anti-vascular endothelial growth factor agent and a glucocorticoid drug or molecule. The three can be administered simultaneously or sequentially. For example, the active peptide or its combination thereof can be administered first, followed by a glucocorticoid drug or molecule after a certain interval, and then the anti-vascular endothelial growth factor agent after another certain interval; or the glucocorticoid drug or molecule can be administered first, followed by an anti-vascular endothelial growth factor agent after a certain interval, and then the active peptide molecule, its salt, or combination thereof described in this application after another certain interval.

[0046] Eighthly, this application provides a nucleic acid molecule encoding the said active peptide molecule, and a vector containing the nucleic acid molecule. The vector may be a prokaryotic or eukaryotic expression vector, including conventional expression vectors, viral vectors, and vectors suitable for gene therapy. Examples of viral vectors include, but are not limited to: herpes simplex virus vectors, poxvirus vectors, adenovirus vectors, retroviral vectors, or insect virus vectors.

[0047] Beneficial effects of this application

[0048] The anti-angiogenic peptides provided in this application not only effectively circumvent the pro-angiogenic effects that may exist in wild-type TSP-1 protein or its natural peptide sequences, but also significantly improve the limitations faced by proteins and macromolecular peptides in clinical applications, providing a new strategy and application basis for the treatment of neovascular eye diseases.

[0049] In addition, the active peptide molecules have the characteristics of simple preparation process, controllable production cost, high product purity and excellent biological activity, showing good prospects in the development and industrial application of drugs for the treatment of eye diseases. Attached Figure Description

[0050] One or more embodiments of this specification are illustrated and explained by way of example with reference to the accompanying drawings. It should be noted that the term "exemplary" as used herein means "as an example, embodiment, or illustration" and does not imply that the described embodiments are superior to other embodiments in terms of performance or effect.

[0051] Figure 1 is a line graph showing the statistical analysis results obtained after screening the cell proliferation inhibitory activity of various peptides using the CCK-8 assay in Example 2. The results show that peptides VRR-3, VRR-7, VRR-9, VRR-10, and VRR-13 have inhibitory effects on cell proliferation. With the increase of peptide concentration, their inhibitory efficiency on cell proliferation gradually increases, exhibiting a typical dose-response relationship.

[0052] Figure 2 shows the effect of different concentrations of peptides on cell migration in the scratch assay in Example 3.

[0053] Figure 3 shows the statistical results of the scratch healing rates of peptides VRR-3, VRR-7, VRR-9, VRR-10, and VRR-13 in Example 3 (corresponding to Figures 3A-E, respectively). The statistical significance is expressed as: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0054] Figure 4 shows the statistical results of the effects of different polypeptide molecules (VRR-3, VRR-7, VRR-9, VRR-10, VRR-13) on the relative migration rate of cells at the same concentration in Example 3.

[0055] Figure 5 illustrates the in vivo therapeutic effect of the peptide assessed by fluorescein fundus angiography (FFA) in Example 4, showing the in vivo angiography results. The figures are as follows: A, control group; B, VRR-3 treatment group; C, VRR-7 treatment group; D, VRR-9 treatment group; E, VRR-10 treatment group; F, VRR-13 treatment group.

[0056] Figure 6 shows the expression of angiogenesis-related proteins in cultured cells and model animal tissues detected by Western blotting in Example 5.

[0057] Figure 7 illustrates the changes in cell viability in Example 6. Figure A shows that VEGF intervention can improve cell viability; Figure B shows that VRR-13 treatment can reverse the VEGF-induced increase in cell viability.

[0058] Figure 8 shows the changes in cell migration rate in Example 7. The results show that VEGF intervention can increase cell migration rate, while VRR-13 treatment can reverse the VEGF-induced increase in migration rate.

[0059] Figure 9 shows the changes in cell proliferation rate under different experimental conditions as assessed by EdU-488 in Example 8. The results showed that, compared with the control group, treatment with 1 ng / mL VEGF significantly increased cell proliferation rate; however, this promoting effect was reversed after co-treatment with 10 μg / mL VRR-13, indicating that VRR-13 can effectively inhibit VEGF-induced cell proliferation.

[0060] Figure 10 shows the flow cytometry detection in Example 9.

[0061] Figure 11 shows the statistical results of apoptosis rate detected by flow cytometry in Example 9.

[0062] Figure 12 shows the dose-response curve of VRR-13 in Example 11, which was used to calculate its half-maximal inhibitory concentration (IC50). 50 .

[0063] Figure 13 shows the HE staining results of the retina in the laser-induced CNV mouse model after VRR-13 treatment in Example 11. The results show that the retinal layers in the VRR-13 treatment group are clear and well-arranged, with no obvious pathological changes.

[0064] Figure 14 shows the HE staining results of the retina of the Matrigel-induced CNV model mice in Example 11 after VRR-13 treatment. The results show that the retinal layers in the VRR-13 treatment group are clear, well-arranged, and morphologically intact, with no obvious pathological changes.

[0065] Figure 15 shows the condition of the eye cup after injecting 100 times the conventional dose of VRR-13 into the vitreous cavity in Example 11.

[0066] Figure 16 shows the HE staining results of retinal tissue from Matrigel-induced CNV model mice after VRR-3 treatment in Example 12. As shown in the figure, in the CNV model group, disordered outer retinal structure, discontinuity of the RPE layer, and RPE cell proliferation, free cells, and irregular luminal structures (i.e., CNV, indicated by white arrows) can be observed at the laser photocoagulation site. These lesions pull on the retina, forming localized depressions. Scale bar = 50 μm; Abbreviations: GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

[0067] Figure 17 illustrates the effect of VRR-13 treatment on angiogenesis in laser-induced CNV model mice at the end of modeling (day 21). CD31 (green) positive cells were observed by retinal immunofluorescence staining, and DAPI (blue fluorescence) was used for nuclear counterstaining. Results showed that, compared to the control group, the CNV group exhibited disordered retinal structure, with abnormal angiogenesis and retinal traction leading to localized depressions. Compared to the CNV group, the VRR-13 treatment group showed a significantly reduced density of abnormal vessels in the outer nuclear layer. Scale bar = 50 μm. The figure labels the retinal layers: GCL (Gangiocyte Layer); IPL (Inner Pleural Layer); INL (Inner Nuclear Layer); OPL (Outer Pleural Layer); ONL (Outer Nuclear Layer); OS+IS (Inner and Outer Ganglia of Photoreceptor Cells); RPE (Retinal Pigment Epithelium).

[0068] Figure 18 shows the effect of VRR-13 treatment on cell death in the laser-induced CNV model in Example 14. On day 21 after modeling, TUNEL staining was performed on the retinas of mice in the normal group, CNV model group, and VRR-13 treatment group. TUNEL-positive cells were labeled with CY3 (yellow fluorescence), and nuclear counterstaining was performed with DAPI (gray fluorescence). The results showed that, compared with the normal group, a large number of TUNEL-positive cells were observed in the CNV formation area in both the CNV group and the VRR-13 treatment group. Scale bar = 50 μm. The figure shows the retinal layer structure. Statistical significance is expressed as: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0069] Figure 19 shows the colocalization analysis results of the effect of VRR-13 treatment on laser-induced CNV model vascular endothelial cell death in Example 14. TUNEL and CD31 immunofluorescence co-staining was performed on the retinas of mice in each group 21 days after modeling. The results showed significant colocalization between CD31-positive cells (green) and TUNEL-positive cells (CY3, yellow), indicating apoptosis of vascular endothelial cells. Cell nuclei were counterstained with DAPI (blue). Scale bar = 50 μm.

[0070] Figure 20 shows the observation results of VRR-13 on retinal patches in a Matrigel-induced CNV rat model in Example 15. The experiment involved retinal patching after cardiac perfusion with FITC-dextran; green fluorescence indicated areas of vascular leakage. All retinal patch images were acquired and recorded under a 10x microscope.

[0071] Figure 21 shows the HE staining results of the effect of VRR-13 treatment on the development of choroidal neovascularization in a Matrigel-induced CNV rat model in Example 15. (A) Retinal structure of normal SD rats; (B, C) Retinal structure of the model group 10 weeks after injection, showing the migration of RPE cells from the RPE layer to the ONL layer, and disordered structure of the inner and outer nuclear layers. White arrows indicate Matrigel injection sites; green arrow (C) indicates cell infiltration below the Matrigel deposition area. Scale bar = 1000 μm / 50 μm. The figure shows the retinal layers: GCL (Gangellal CL); IPL (Inner plexiform layer); INL (Inner nuclear layer); ONL (Outer nuclear layer); RPE (Retinal pigment epithelium).

[0072] Figure 22 shows the immunofluorescence staining results of the VRR-13 peptide inhibiting CNV development in the Matrigel-induced rat choroidal neovascularization (CNV) model of Example 15. Figures a1 and a3 show the retinal structure of normal SD rats, with normal vascular morphology. Figures a2 and a4 show the observation results at the injection site (indicated by the white arrow in Figure a2) 10 weeks after subretinal injection of Matrigel: displacement of visual RPE cells is observed, migrating from the RPE layer to the ONL layer, forming abnormal cavities between the cells and the choroid (i.e., the injection site); and the formation of abnormal neovascular lumens is also visible at the injection site (indicated by the dark arrow in Figure a2). Furthermore, retinal structural disorder and cell infiltration are visible in Figure a4 (indicated by the white arrow). The staining markers are as follows: CD31 shows green fluorescence, VEGF shows red fluorescence, and DIAP, as a nuclear counterstain, shows blue fluorescence. The scale bar in the figures is 50 μm. Abbreviations for each layer: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Detailed Implementation

[0073] To facilitate understanding of this application, some key terms are defined below. Unless otherwise expressly stated, the technical terms used herein have the meanings commonly understood by those skilled in the art to which this application pertains.

[0074] Singular and Plural Forms: In this specification and claims, the singular forms “a,” “an,” and “the” also include the plural references, unless the context clearly indicates otherwise.

[0075] "Including" and similar terms: Terms such as "including," "comprise," or "include" should be understood as open inclusion, meaning that the presence of other elements or components not listed is permitted unless otherwise expressly limited.

[0076] Technical methods: Unless otherwise stated, the technical methods involved in the embodiments of this application, including experiments related to molecular biology, microbiology, cell biology, biochemistry and immunology, are all within the scope of conventional technology in this field.

[0077] Numerical range and the term “about”: When the term “about” is used with a numerical value, it indicates that the value may fluctuate within a certain range, including but not limited to reasonable deviations such as ±5%, ±2%, ±1%, and ±0.1%, to be applicable to the disclosed method.

[0078] "And / or" and "or": "And / or" means any one, two, or more of the listed options. The term "or" is also interpreted inclusively, meaning it includes at least one of the listed items, as well as multiple or other unlisted items, unless explicitly stated otherwise, such as "only one," "consisting of," etc.

[0079] When the terms “at least” or “greater than” are used before a series of values, they apply to each value in that series.

[0080] When the terms “not more than” or “less than” are used before a series of values, they apply to each value in the series.

[0081] All ranges relating to the same component or property include endpoints, and these endpoints can be combined independently. Since these ranges are continuous, they encompass every numerical value between a minimum and a maximum. It should also be understood that any numerical range referenced in this application is intended to include all subranges within that range.

[0082] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to those in a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, such as publicly available computer software like BLAST, BLAST 2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0083] "Pharmaceutically acceptable" means that the substances used (such as active ingredients or excipients) are suitable for contact with biological tissues without causing excessive irritation, allergy, immunogenicity, or toxicity, and have the expected therapeutic effect, provided that the benefit / risk ratio is reasonable. Pharmaceutically acceptable excipients should be compatible with other components in the formulation, including but not limited to liquid and solid fillers, diluents, binders, lubricants, flow aids, surfactants, dispersants, disintegrants, emulsifiers, wetting agents, suspending agents, thickeners, solvents, isotonic / isotonic agents, buffers, pH adjusters, absorption delay agents, sweeteners, flavoring agents, colorants, stabilizers, preservatives, antioxidants, antimicrobial agents, antibacterial agents, antifungal agents, adjuvants, encapsulating materials, and coating materials. Relevant technologies can be referenced in authoritative literature, such as: Remington, The Science and Practice of Pharmacy, 21st Ed.; Lippincott Williams & Wilkins (Philadelphia, Pennsylvania 2005); Hand book of Pharmaceutical Excipients, 5th Ed.; Rowe et al., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Hand book of Pharmaceutical Additive, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); Pharmaceutical Preformulation and Formulation, Gibson, Ed., CRC Press LLC (Boca Raton, Florida 2004). Unless there is a clear incompatibility, the use of conventional carriers or excipients is not excluded.

[0084] The "anti-vascular endothelial growth factor agent" mentioned in this article refers to a molecule or ion that can specifically reduce VEGF protein expression levels and / or inhibit VEGF binding to VEGFR and its downstream functions. The anti-VEGF agent can be selected from the following categories: antigen-binding fragments (such as Fab, F(ab')2, scFv, Fv fragments, sdAb / VHH, Fc fusion proteins, bispecific antibodies, or multivalent antibodies), monoclonal antibodies, fusion proteins, oligonucleotides (such as siRNA, aptamers, microRNA / microRNA inhibitors / microRNA mimics), nucleic acid constructs, and any combination of one or more of the above.

[0085] Glucocorticoids are a class of steroid hormones primarily secreted by the zona fasciculata of the adrenal cortex, but they can also be synthesized artificially. Their main function is to regulate the metabolism of carbohydrates, fats, and proteins in the body, and they possess potent anti-inflammatory and immunosuppressive effects. In this article, "glucocorticoid drugs or molecules" refers to marketed drugs or their active ingredients, candidate and investigational drugs or molecules, or other molecules that possess the above-mentioned functions.

[0086] The meaning of “treatment”: This term includes the prevention, relief or elimination of medical conditions and their associated symptoms or causes, specifically covering the following aspects: preventing the occurrence of conditions (e.g., AMD) or associated symptoms; reducing the risk of the development of conditions or associated symptoms; delaying the onset of conditions or associated symptoms; and slowing the progression of conditions or associated symptoms.

[0087] In addition, the English abbreviations used in this article and their corresponding English and Chinese names are shown in Table 1.

[0088] Table 1 List of Abbreviations

[0089] The preferred embodiments of this application will be described in detail below with reference to specific examples. It should be understood that the embodiments described herein are only used to further illustrate the principles and features of this application and are not intended to limit the scope of protection of this application. Without departing from the concept of this application, those skilled in the art can make several adjustments and improvements to this application, and these modifications or substitutions should also be considered to fall within the scope of protection of this application.

[0090] Example 1: Main reagent sources and routine experimental methods

[0091] (1) Main reagents and sources

[0092] Table 2. Main Reagents and Sources

[0093] (2) Cell Culture

[0094] The RF / 6A cell line used in this application was purchased from Cekoo Biotechnology. Cells were cultured in MEM basal medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics, and placed in a cell culture incubator at 37°C and 5% carbon dioxide. Cell growth was observed regularly, and the medium was changed or passaged as needed based on the growth density.

[0095] (3) Laser-induced mouse CNV model

[0096] Anesthesia and pretreatment:

[0097] ① Mice were anesthetized by intraperitoneal injection of 1.25% aphthylamine (0.2 ml / 10 g);

[0098] ② Use compound tropicamide to dilate the pupil multiple times until the pupil diameter is enlarged to about 1.5 mm, then instill oxybuprocaine hydrochloride eye drops to anesthetize the ocular surface in order to maintain eye position stability during the operation;

[0099] ③ Apply gatifloxacin ophthalmic gel to keep the cornea moist and to act as a light-transmitting medium for laser conduction.

[0100] Laser parameters and operating procedures:

[0101] ① A CNV model was established by using a laser with a wavelength of 577nm to penetrate the Bruch film.

[0102] ② After anesthetizing, the mice were fixed to the operating platform and placed in front of a slit lamp. A contact ophthalmoscope or coverslip was placed in front of the cornea, and the laser was introduced into the fundus. Given the absence of a macular region in mice, 2–4 points of laser photocoagulation were performed around the optic nerve in the left eye of each mouse. Specific laser parameters were: spot diameter 50 μm, intensity 250 mW, and exposure time 50 ms. Successful laser photocoagulation was indicated by the visible formation of a small number of bubbles at the photocoagulation spot.

[0103] ③ After the laser operation, place the anesthetized mouse on an electric blanket to maintain its body temperature until it wakes up, and apply moisturizing eye ointment to both eyes to prevent corneal dryness.

[0104] (4) Establishing a rat CNV model by subretinal injection of Matrigel

[0105] Anesthesia and Material Preparation:

[0106] ① SD rats were anesthetized by intraperitoneal injection of 1.25% aphthylamine (0.2 mL / 10 g);

[0107] ② Thaw Matrigel overnight at 4°C, avoiding repeated freeze-thaw cycles to maintain its gelling properties. Keep the entire process on ice to prevent premature gelation.

[0108] Injection procedure and postoperative care:

[0109] ① Select a point 1-2 mm posterior to the limbus as the injection point, avoiding the retinal vascular area. Use a 30G needle to insert into the subretinal space through the vitreous cavity and slowly inject Matrigel. Observing local retinal bulging indicates successful injection;

[0110] ② After the injection, place the rat on an electric blanket to maintain its body temperature until it wakes up, and apply moisturizing eye ointment to both eyes to prevent corneal dryness.

[0111] (5) Sampling and preparation of frozen sections of mouse and rat eyeballs

[0112] Eyeball harvesting procedure:

[0113] ① At the set time point, mice or rats were euthanized by cervical dislocation, and the eyeballs were removed with toothless forceps and washed in PBS solution;

[0114] ② Under a stereomicroscope, the cornea and iris are removed along the limbus, and the lens is removed, while the "cup" structure formed by the retina-choroid-sclera complex is completely preserved. Care should be taken to avoid damaging the retina during the operation.

[0115] Frozen section preparation steps:

[0116] ① Fixation: Place the prepared "eye cup" in FAS eye fixative for 50–60 minutes, then rinse with PBS 3 times, 2–3 minutes each time;

[0117] ② Dehydration: Gradual dehydration treatment was carried out by sequentially dehydrating with 10% sucrose solution for 10 minutes, 20% sucrose solution for 20 minutes, and 30% sucrose solution for 30 minutes;

[0118] ③Embedding: Place the eye cup with the rim facing up in the embedding box, flash freeze in liquid nitrogen, and then store the sample at -20℃.

[0119] ④ Sectioning: Section the sections using a cryostat, setting the section thickness to 12 μm. Bake the sections at 37°C for 30–40 minutes, then store them at -20°C for later use.

[0120] (6) HE staining

[0121] ① Pretreatment: Place the slides in high-resolution constant staining pretreatment solution for 1 minute;

[0122] ② Hematoxylin staining: Stain with hematoxylin solution for 3–5 minutes, rinse with tap water for 3–5 minutes, differentiate with differentiation solution, rinse again with tap water for 3–5 minutes, use blue solution to blue, and rinse with running water for 3–5 minutes.

[0123] ③ Eosin staining: The sections were placed in 85% alcohol for 5 minutes and 95% alcohol for 5 minutes in turn, and then stained with eosin staining solution for 5 minutes.

[0124] ④ Dehydration, clearing and mounting: Place the sections in three anhydrous ethanol tanks for 5 minutes each for dehydration, then transfer them to two xylene tanks for clearing for 5 minutes each, and finally mount them with neutral resin.

[0125] ⑤ Microscopic examination and analysis: Observe and acquire images under an optical microscope, and perform structural analysis.

[0126] The retinal tissue structure of mice was observed using HE staining. Using normal mice as a control, the changes in tissue structure in the VRR-13 treatment group mice 21 days after laser-induced modeling were analyzed. The results showed that the retinal layers of the VRR-13 group mice were clearly defined and neatly arranged, with no obvious pathological changes, indicating that retinal toxicity was not induced under this condition.

[0127] (7) Intravitreal injection method in mice

[0128] Anesthesia and injection sites:

[0129] ① Mice were anesthetized by intraperitoneal injection of 1.25% aphthylamine (0.2ml / 10g);

[0130] ② Apply oxybuprocaine hydrochloride eye drops to administer ocular surface anesthesia in order to suppress reflexive eye movements caused by the procedure and maintain ocular stability;

[0131] ③ The injection site should be the flat part of the ciliary body to avoid damage to the lens and retinal tissue.

[0132] Injection procedure and postoperative care:

[0133] ① First, use a 30G needle to establish an insertion channel, then switch to a microinjector and insert the needle along the tunnel into the vitreous cavity, slowly injecting the medication. After injection, leave the needle in place for 10–15 seconds before withdrawing it to prevent drug reflux;

[0134] ②After the injection, place the mouse on an electric blanket to maintain normal body temperature until it wakes up, and apply moisturizing eye ointment to both eyes to prevent corneal dryness.

[0135] (8) TUNEL staining

[0136] Detection principle:

[0137] ① Apoptosis was detected using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling assay (TUNEL) with a commercially available detection kit.

[0138] Staining steps:

[0139] ① Washing: Add PBS to the slices and incubate for 5–10 minutes to wash away the OCT embedding agent;

[0140] ②Circling: Circle pattern: Wipe the liquid off the surface of the slide and draw a circle around the tissue sample with an immunohistochemical pen; Permeability: Incubate with PBS solution containing 0.5% Triton X-100 at room temperature for 5 minutes to enhance cell membrane permeability;

[0141] ③ Prepare TUNEL test solution, taking 100μl as an example: Test solution preparation: Prepare TUNEL reaction solution according to the kit instructions (taking 100μL system as an example);

[0142] Table 3 TUNEL test solution

[0143] ④ Washing: Add PBS to cover the tissue, let stand for 5–10 minutes, and repeat washing 3 times;

[0144] ⑤ Colorimetric reaction: Add 70–100 μL of TUNEL test solution to each tablet and incubate at 37°C for 1 hour in a light-proof, humidified box;

[0145] ⑥ Washing: Rinse with PBS for 7 minutes each time, for a total of 3 washes;

[0146] ⑦ Mounting: Wipe the liquid off the surface of the slide and mount it with a DAPI-containing anti-fluorescence quenching mounting medium;

[0147] ⑧ Microscopic examination and image acquisition: Observe the sections and acquire images under a confocal microscope. DAPI shows blue fluorescence at UV excitation wavelengths of 330–380 nm and emission wavelengths of 420 nm; TUNEL-positive signals show red fluorescence in the CY3 channel (excitation wavelength 550 nm, emission wavelength 570 nm). Cell nuclei appear blue, and TUNEL-positive cells appear red.

[0148] (9) TUNEL / immunofluorescence co-staining

[0149] ① Washing: Add PBS to cover the tissue for 5–10 minutes to wash away the OCT embedding agent;

[0150] ②Circle pattern: Wipe the liquid off the surface of the slide and use an immunohistochemical pen to draw circles around the tissue;

[0151] ③ Blocking: Add blocking solution containing 10% normal goat serum (NGS), 1% BSA and 0.3% PBST, and block at room temperature for 1.5 hours;

[0152] ④ Primary antibody incubation: Wipe off the blocking solution, add the primary antibody diluted with the blocking solution in an appropriate ratio, and place the slide in a humidified chamber and incubate overnight at 4°C;

[0153] ⑤ Washing: Rinse the sections with PBS for 7 minutes each time, for a total of 3 times;

[0154] ⑥ Permeability: Incubate with PBS containing 0.5% Triton X-100 at room temperature for 5 minutes, followed by washing with PBS 3 times for 7 minutes each time;

[0155] ⑦ Secondary antibody reaction with TUNEL: Add 70–100 μL of TUNEL detection solution containing fluorescent secondary antibody per slide and incubate at 37°C for 1 hour in the dark;

[0156] ⑧ Washing: Rinse the sections with PBS for 7 minutes each time, for a total of 3 times;

[0157] ⑨ Mounting: Wipe the liquid off the surface of the slide and mount it with a DAPI-containing anti-fluorescence quenching mounting medium;

[0158] ⑩ Microscopic examination and image acquisition: Images were observed and acquired under a confocal microscope. DAPI (excitation wavelength 330–380 nm, emission wavelength 420 nm) labeled cell nuclei appeared blue; TUNEL positive signals appeared red in the CY3 channel (excitation wavelength 550 nm, emission wavelength 570 nm); target antigens appeared green in the FITC channel (excitation wavelength 465–495 nm, emission wavelength 515–555 nm).

[0159] Example 2: Screening of anti-angiogenic peptides

[0160] To identify peptides with anti-angiogenic activity, the applicant designed and synthesized 14 candidate peptide sequences, including:

[0161] VRR-1: The amino acid sequence is GVITRIR (SEQ ID No. 01);

[0162] VRR-2: The amino acid sequence is VTCGVIT (SEQ ID No. 02);

[0163] VRR-3: The amino acid sequence is VTCGVITRI (SEQ ID No. 03);

[0164] VRR-4: The amino acid sequence is VTCGVITRT (SEQ ID No. 04);

[0165] VRR-5: The amino acid sequence is VTCGVITR (SEQ ID No. 05);

[0166] VRR-6: The amino acid sequence is VTCGVITRC (SEQ ID No. 06);

[0167] VRR-7: The amino acid sequence is VTCGVITRV (SEQ ID No. 07);

[0168] VRR-8: The amino acid sequence is VTCGVI (SEQ ID No. 08);

[0169] VRR-9: The amino acid sequence is SVTCGVITRIR (SEQ ID No. 09);

[0170] VRR-10: The amino acid sequence is CSVTCGVITRIR (SEQ ID No. 10);

[0171] VRR-11: The amino acid sequence is CSVTCGVIITRIRVT (SEQ ID No. 11);

[0172] VRR-12: The amino acid sequence is CSVTCGGVIICIRV (SEQ ID No. 12);

[0173] VRR-13: The amino acid sequence is CSVTCGGVITRIR (SEQ ID No. 13);

[0174] VRR-14: The amino acid sequence is VCSVTCGGVIVICRVT (SEQ ID No. 14).

[0175] In the screening experiment, RF / 6A cells were used as a model, and the CCK-8 assay was used to detect the inhibitory effect of each peptide on cell proliferation. Five dose groups were set up for each peptide. For example, for the VRR-1 peptide, the following dose groups were set up: 10 μg VRR-1 peptide (final concentration of VRR-1 peptide in culture medium: 10 μg / mL); 25 μg VRR-1 peptide (final concentration of VRR-1 peptide in culture medium: 25 μg / mL); 50 μg VRR-1 peptide (final concentration of VRR-1 peptide in culture medium: 50 μg / mL); 75 μg VRR-1 peptide (final concentration of VRR-1 peptide in culture medium: 75 μg / mL); and 100 μg VRR-1 peptide (final concentration of VRR-1 peptide in culture medium: 100 μg / mL). The dose groups for other peptides were set up in a similar manner. Each dose group had six replicates. Meanwhile, each plate was set with three replicates of a blank cell control (containing only cells) and three replicates of a cell-free control for calculating cell viability.

[0176] The experimental steps are as follows: 1) Obtain the chemically synthesized polypeptide molecules, dissolve and dilute the polypeptides, and prepare a high-concentration polypeptide storage solution; 2) Seed RF / 6A cultured cells into 96-well cell culture plates, with 0.8 × 10⁴ cells per well (100 μL / well); 3) After the cells adhere, add a certain amount of polypeptide storage solution to make the final concentrations of the polypeptides 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL, respectively; 4) Incubate the culture plates at 37℃ in a 5% CO₂ incubator for 24 hours; 5) Remove the culture plates, add 10 μL of CCK-8 reagent to each well, and then incubate for another 4 hours; 6) After 4 hours of incubation, measure the absorbance at 450 nm using a microplate reader, and calculate the cell viability of each well; 7) Statistically analyze the cell viability of each polypeptide at each concentration using SPSS statistical software, and calculate the mean and standard deviation.

[0177] Cell viability 100% = (OD value of experimental group cells in wells - OD value of cell-free control wells) / (OD value of blank control cells in wells - OD value of cell-free control wells) × 100%.

[0178] Table 4 shows a set of experimental results on cell viability (inhibition of cell proliferation).

[0179] Table 4 Cell viability

[0180] The above experiment was repeated three times, and the data from the three independent experiments were statistically analyzed using SPSS. The results showed that among the tested peptides, VRR-3, VRR-7, VRR-9, VRR-10, and VRR-13 significantly inhibited the proliferation activity of RF / 6A cells, while the remaining peptides did not show a significant effect on cell proliferation. The cell viability rates treated with VRR-3, VRR-7, VRR-9, VRR-10, and VRR-13 peptides were plotted, and the results are shown in Figure 1. As can be seen from Figure 1 and Table 4, all five peptides effectively inhibited the proliferation of RF / 6A cells, and the inhibitory effect was dose-dependent; that is, as the peptide concentration increased, the cell viability gradually decreased, exhibiting a typical dose-response relationship. Among the five peptides, VRR-13 showed the most significant inhibitory effect, followed by VRR-3, VRR-7, VRR-10, and VRR-9. Statistical analysis showed that VRR-13 had a significantly higher inhibitory efficiency on cell proliferation than VRR-7, VRR-10 and VRR-9, with statistically significant differences (p<0.001).

[0181] Example 3: Cell Scratch Assay

[0182] Based on the research results of the above embodiments, the applicant selected VRR-3, VRR-7, VRR-9, VRR-10 and VRR-13 peptides for further functional verification.

[0183] Using a standard cell scratch assay with RF / 6A cells as a model, the effects of different peptides on cell migration ability were evaluated. For each peptide, one control group and five dosage groups were set up. Taking VRR-3 peptide as an example, the following groups were set up: VRR-3 peptide control group (no VRR-3 peptide in cell culture medium); VRR-3 peptide 10 μg dosage group (VRR-3 peptide concentration of 10 μg / mL in cell culture medium); VRR-3 peptide 25 μg dosage group (VRR-3 peptide concentration of 25 μg / mL in cell culture medium); VRR-3 peptide 50 μg dosage group (VRR-3 peptide concentration of 50 μg / mL in cell culture medium); VRR-3 peptide 75 μg dosage group (VRR-3 peptide concentration of 75 μg / mL in cell culture medium); VRR-3 peptide 100 μg dosage group (VRR-3 peptide concentration of 100 μg / mL in cell culture medium). The control and dosage groups for other peptides were set up in a similar manner. Each dose group was tested with three replicates to ensure the reliability of the experimental results.

[0184] The experimental steps are as follows: 1) Prepare high-concentration stock solutions of 5 polypeptides for later use; 2) Draw 3 parallel reference lines across the bottom back of the 6-well cell culture plate using a ruler; 3) Inoculate each well with 2 × 10⁶ peptides. 5 4) Using the tip of a 200μL micropipette, create scratches on the cell layer along a direction perpendicular to the reference line, then gently wash three times with PBS to remove detached cells; 5) Replace the culture medium with low serum medium containing 1% FBS, and add the appropriate volume of peptide storage medium according to the experimental design to achieve the preset final peptide concentration for each experimental group; 6) Continue culturing at 37℃ and 5% CO2, observe the cells and acquire images under a 10× microscope at 0h and 24h, calculate the scratch healing rate of each culture well, and perform statistical analysis.

[0185] Scratch healing rate = ((0h scratch distance - 24h scratch distance) / 0h scratch distance) × 100%

[0186] Figure 2 shows a representative set of scratch assay results. Based on the statistical analysis data in Tables 5 and 6, Figures 3 and 4, and using a cell proliferation and migration rate lower than 80% of the control group as the criterion for VVR inhibitory activity, the inhibitory effects of each peptide were evaluated as follows: VRR-3 peptide showed inhibitory activity against cell proliferation and migration at concentrations of 75 μg / mL and 100 μg / mL; VRR-7 peptide showed inhibitory effects at concentrations of 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL; VRR-9 peptide showed inhibitory effects at concentrations of 50 μg / mL, 75 μg / mL, and 100 μg / mL; and VRR-10 and VRR-13 peptides showed inhibitory activity against cell proliferation and migration at concentrations of 10 μg / mL, 25 μg / mL, 50 μg / mL, 75 μg / mL, and 100 μg / mL.

[0187] Table 5 Results of the scratch test

[0188] Table 6 Statistical Analysis of Scratch Test Results

[0189] Example 4: In vivo efficacy evaluation of a laser-induced choroidal neovascularization model

[0190] Choroidal neovascularization (CNV) is a major pathological feature of age-related macular degeneration (AMD). The 577nm laser-induced CNV model in mice effectively simulates this pathological process and is widely used in AMD and related research. In this embodiment, the applicant uses this model to evaluate the in vivo anti-angiogenic effect of the active peptide provided in this application.

[0191] Animal grouping and administration. Male C57BL / 6J mice (19-24g) aged 6-8 weeks were selected and acclimatized for one week in an SPF-grade environment at the Experimental Animal Center of Kunming Medical University. After stratification by weight, they were randomly divided into one control group and five treatment groups (VRR-3, VRR-7, VRR-9, VRR-10, VRR-13), with 6 mice in each group.

[0192] CNV Model Preparation and Drug Intervention. A mouse CNV model was prepared according to standard procedures for this study. Specifically: 1) On day 1 of the experiment, mice were anesthetized by intraperitoneal injection of ready-to-use tribromoethanol solution / Aphthyl (1.25% Aphthyl) at a dose of 0.2 ml / 10 g mouse body weight; 2) The pupils were dilated multiple times using compound tropicamide until the pupil diameter increased to approximately 1.5 mm; 3) The mice were fixed on the operating platform and placed in front of a slit lamp, with a contact ophthalmoscope placed in front of the cornea to guide the laser into the fundus; 4) 3–4 points of laser photocoagulation (spot diameter 100 μm, spot intensity 250 mW, exposure time 50 ms) were performed around the optic nerve in the left eye of each mouse. Successful laser photocoagulation was indicated by the appearance of a small number of bubbles at the photocoagulation spot; 5) The anesthetized mice were placed on an electric blanket to maintain body temperature until awakening, and antibiotic moisturizing eye ointment was applied to both eyes to prevent corneal dryness.

[0193] On day 7 after modeling, intravitreal injections were administered. Each treatment group received 0.2 μL (1 mg / mL) of the corresponding peptide solution, while the control group received an equal volume of balanced salt solution (BSS). Specifically, mice in the VRR-3 treatment group received an intravitreal injection of 0.2 μL (1 mg / mL) of VRR-3 peptide solution; mice in the VRR-7 treatment group received an intravitreal injection of 0.2 μL (1 mg / mL) of VRR-7 peptide solution; mice in the VRR-9 treatment group received an intravitreal injection of 0.2 μL (1 mg / mL) of VRR-9 peptide solution; mice in the VRR-10 treatment group received an intravitreal injection of 0.2 μL (1 mg / mL) of VRR-10 peptide solution; mice in the VRR-13 treatment group received an intravitreal injection of 0.2 μL (1 mg / mL) of VRR-13 peptide solution; and mice in the control group received an intravitreal injection of 0.2 μL (BSS).

[0194] Efficacy evaluation. On days 7 and 14 after laser modeling, fundus fluorescein angiography (FFA) was performed on mice in each group to obtain in vivo imaging. Representative images are shown in Figure 5. ImageJ software was used to quantify the laser spot area of ​​each mouse on days 7 and 14 after modeling, and the CNV area change rate was calculated using the following formula:

[0195] CNV area change rate = ((laser spot area on day 14 - laser spot area on day 7) / laser spot area on day 7) × 100%

[0196] The CNV area change rate in mice of the control group and each treatment group was statistically analyzed using SPSS software, and the results are shown in Table 7. Compared with day 7, the laser spot area of ​​mice in the control group was significantly increased on day 14, indicating that the CNV model was successfully established. Compared with the control group, each peptide treatment group showed significant therapeutic effects. Specifically, although the laser spot area of ​​the VRR-3, VRR-9, and VRR-10 treatment groups increased slightly on day 14 compared with day 7, the increase was much lower than that of the control group. The laser spot area of ​​the VRR-7 and VRR-13 treatment groups decreased significantly on day 14 compared with day 7, showing a stronger effect in inhibiting CNV progression. Intergroup comparisons further showed that the efficacy of the VRR-13 treatment group was significantly better than that of the VRR-7 treatment group (P<0.001). No obvious toxic side effects were observed in any treatment group throughout the experiment.

[0197] Table 7. Change rate of CNV area in mice of the control and treatment groups.

[0198] Using the control group data as a reference, the significance of the differences between the treatment groups was calculated, and is expressed as follows: ***, P < 0.001.

[0199] To further evaluate the in vivo activity of VRR-7 and VRR-13 peptides, the applicant conducted a dose-response study to systematically evaluate the inhibitory effects of the two peptides on angiogenesis at different drug concentrations. Following the protocol described above, male C57BL / 6J mice aged 6-8 weeks were used as experimental subjects. After acclimatization, mice were stratified by body weight and randomly assigned to one control group and eight treatment dose groups, with six mice in each group. The eight treatment trial groups were: VRR-7 0.2 mg (0.2 mg / mL), VRR-7 0.7 mg (0.7 mg / mL), VRR-7 1.2 mg (1.2 mg / mL), VRR-7 2.0 mg (2.0 mg / mL), VRR-13 0.2 mg (0.2 mg / mL), VRR-13 0.7 mg (0.7 mg / mL), VRR-13 1.2 mg (1.2 mg / mL), and VRR-13 2.0 mg (2.0 mg / mL).

[0200] Seven days after successfully establishing the CNV model, mice were administered intravitreal injections of a uniform 0.2 μL. For example, in the VRR-7 0.2 mg experimental group, 0.2 μL of a 0.2 mg / mL VRR-7 peptide solution was injected intravitreally; in the VRR-1 2.0 mg experimental group, 0.2 μL of a 2.0 mg / mL VRR-7 peptide solution was injected intravitreally; and the dosage for other groups followed the same pattern. In the control group, 0.2 μL of BSS was injected intravitreally.

[0201] On days 7 and 14 after laser-induced modeling, fluorescein fundus angiography was performed on mice in each group to obtain in vivo imaging. Subsequently, ImageJ software was used for quantitative analysis of the images, and the laser spot area was measured on days 7 and 14 after laser modeling. SPSS statistical software was used to statistically analyze the CNV area change rate between the control group and each treatment group to evaluate the efficacy. Specific results are shown in Table 8.

[0202] Table 8. Comparative study of the anti-angiogenic effects of VRR-7 and VRR-13 peptides (n=6)

[0203] Using the data from the VRR-7 group as a reference, the significance of the differences in the corresponding VRR-13 group was calculated, as shown below: ***, P < 0.001.

[0204] The results showed that, compared with the VRR-7 group, VRR-13 exhibited stronger inhibitory effects at all dose concentrations (all p < 0.001), and this effect showed a concentration-dependent trend, indicating that VRR-13 administration produced a more significant therapeutic effect. Conclusion: VRR-13 peptides demonstrated significantly superior anti-CNV efficacy to VRR-7 in vivo, with a clear dose-response relationship.

[0205] Example 5: Western Blot detection of angiogenesis-related protein expression

[0206] To further explore the mechanism of action of VRR-13 peptide in anti-angiogenesis, this study used Western blotting to detect changes in the expression of angiogenesis-related proteins in RF / 6A cultured cells and retinal tissue.

[0207] In the cellular level study, three experimental groups were set up: a control group, a VEGF intervention group, and a VEGF / VRR-13 co-intervention group, with three replicates in each group. In the VEGF intervention group, recombinant human VEGF protein was added to the culture medium at a final concentration of 2 ng / mL; in the VEGF / VRR-13 co-intervention group, VEGF protein and VRR-13 peptide were added to the culture medium at a final concentration of 2 ng / mL and a final concentration of 10 μg / mL. The expression levels of angiogenesis-related proteins VEGFA and CD31 in cells of each group were detected using Western blotting. Figure 6A shows the expression bands of VEGFA and CD31 proteins in cells of each group. Statistical analysis results in Figures 6B and 6C show that, compared with the control group, VEGF treatment significantly upregulated the expression levels of VEGFA and CD31; while after combined intervention with VRR-13 peptide, the expression of both proteins was significantly downregulated. Figures 6B and 6C show that the use of recombinant human VEGF protein significantly upregulated the expression of VEGFA and CD31; while intervention with VRR-13 at a concentration of 10 μg / mL downregulated the expression levels of VEGFA and CD31. These results suggest that at the cultured cell level, the VRR-13 peptide can effectively antagonize the expression of VEGF-induced angiogenesis-related proteins, further validating its potential role in inhibiting angiogenesis.

[0208] Table 9. Relative protein expression levels of CD31 and VEGF

[0209] Furthermore, we conducted animal-level studies. Ten healthy male SD rats aged 6–8 weeks were selected and randomly divided into a CNV control group and a VRR-13 peptide treatment group after stratification by body weight, with 5 rats in each group. A choroidal neovascularization (CNV) model was established by subretinal injection of Matrigel. The specific procedure was as follows: rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital at a dose of 50 mg / kg. Matrigel was then injected subretinally into the equatorial region of both eyes using a 30G needle. Immediately after model establishment, intravitreal injection was performed. The VRR-13 treatment group received 1 μL of a 1 mg / mL VRR-13 peptide solution intravitreally in both eyes, while the control group received an equal volume of 1 μL of BSS solution.

[0210] Rats' optic cup tissues were collected at different time points (2, 4, 6, 8, and 10 weeks) after modeling, and total protein was extracted using RIPA lysis buffer. Protein samples were separated by 10% SDS-PAGE electrophoresis, transferred to PVDF membranes (Millipore, USA), and blocked with 5% skim milk for 2 hours. Subsequently, the corresponding primary antibody was added and incubated overnight at 4°C, followed by three washes with TBST, and then incubated with the corresponding secondary antibody at room temperature for 90 minutes, followed by three more washes with TBST. Finally, the images were developed using an ECL chemiluminescence system, and the experimental results were analyzed using ImageJ software.

[0211] Table 10. Relative protein expression levels of CD31 and VEGF at different time points.

[0212] Figure 6 (D–F) shows the Western blot results and statistical analysis of VEGF and CD31 protein expression at different time points in the animal experiment. The results showed that with the extension of the modeling time, the expression levels of VEGF and CD31 proteins in the CNV control group gradually increased. VEGF protein expression peaked at 4 weeks and then gradually decreased, while CD31 protein expression peaked at 6 weeks and then declined. Compared with the CNV control group, the VRR-13 treatment group showed significantly lower VEGF and CD31 protein expression at 2, 4, and 6 weeks after modeling (statistical significance is expressed as: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0213] The above results indicate that the VRR-13 peptide can effectively inhibit the temporal expression of key angiogenesis-related proteins in the process of CNV formation in animal models, further verifying its anti-angiogenic effect.

[0214] Example 6: Inhibitory effect of VRR-13 peptide on VEGF-induced proliferation of RF / 6A cells

[0215] To explore the mechanism of the anti-angiogenic effect of VRR-13 peptide, this example further evaluated the effect of the peptide on the proliferation of RF / 6A cells induced by VEGF.

[0216] Specifically, RF / 6A cells were spaced at 0.8 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL of culture medium per well in 96-well plates. After cell attachment, the cells were divided into the following groups:

[0217] Two control groups were set up: a cell-free control group and a blank control group, with three replicates in each group. The cell-free control group received culture medium only, without cell seeding; the blank control group received both culture medium and cells, but no VEGF or peptides were added (no drug treatment was performed).

[0218] Two intervention groups were set up: a VEGF intervention group and a VEGF / VRR-13 intervention group, with three replicates in each group. In the VEGF intervention group, VEGF was added to the culture medium at a final concentration of 2 ng / mL; in the VEGF / VRR-13 intervention group, VEGF and VRR-13 peptide were added to the culture medium at a final concentration of 2 ng / mL and 10 μg / mL, respectively.

[0219] 24 hours after drug intervention, 10 μL of CCK-8 reagent was added to each well, and the 96-well plate was incubated in an incubator (37℃, 5% CO2) for another 4 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula:

[0220] Cell viability 100% = (OD value of experimental group cells - OD value of cell-free control group) / (OD value of blank control group cells - OD value of cell-free control group) × 100%.

[0221] The above experiments were independently repeated three times, and the data were statistically analyzed using statistical software. The results are shown in Figure 7. As shown in Figure 7A and Table 11, the addition of VEGF significantly promoted the proliferation of RF / 6A cells, especially at VEGF concentrations of 0.5 ng / mL and 1 ng / mL, where the cell number was significantly increased by about 20% compared to the blank control group, indicating that VEGF effectively promoted the proliferation of RF / 6A cells. Figure 7B and Table 12 show that, in the presence of 2 ng / mL VEGF, the addition of VRR-13 peptide at a final concentration of 10 μg / mL reduced the cell number by 13% compared to the blank control group and by 29% compared to the VEGF-only treatment group, indicating that VRR-13 can significantly inhibit the cell proliferation enhancement effect induced by VEGF.

[0222] Table 11 Cell viability at different exogenous VEGF concentrations

[0223] Table 12 Cell viability after VRR-13 treatment

[0224] Example 7: Inhibitory effect of VRR-13 peptide on VEGF-induced cell migration

[0225] To further investigate the role of VRR-13 peptide in angiogenesis, the applicant evaluated its effect on VEGF-induced migration behavior of RF / 6A cells.

[0226] Experimental grouping and drug administration. This study set up one control group and two intervention groups: a VEGF intervention group and a VEGF / VRR-13 intervention group, with three replicates in each group. Control group cells: no drug administration treatment; VEGF intervention group cells: VEGF was added to the culture medium at a final concentration of 2 ng / mL; VEGF / VRR-13 intervention group cells: VEGF and VRR-13 peptide were added to the culture medium at a final concentration of 2 ng / mL and 10 μg / mL, respectively.

[0227] Cell scratch assay procedure: 1) Draw three parallel lines across the culture wells on the back of a 6-well plate using a ruler; 2) Place RF / 6A cells at a density of 2 × 10⁶ cells per well. 5 1) Seed cells at a density of 1,000, and cultured at 37°C and 5% CO2 until cell confluence reached 80%–90%; 2) Using a 200 μL sterile pipette tip, create scratches on the cell layer along a direction perpendicular to the reference line. Then gently wash three times with PBS to remove detached cells; 3) Replace the culture medium with low serum medium containing 1% FBS, and add the corresponding drugs or peptide solutions according to the above groupings, and continue to culture at 37°C and 5% CO2; 4) Observe and photograph the scratched areas under a 10x microscope at 0 hours and 24 hours of culture, respectively, measure the scratch width using image analysis software, and perform statistical analysis (Figure 8).

[0228] Scratch healing rate = ((0h scratch distance - 24h scratch distance) / 0h scratch distance) × 100%

[0229] The above experiments were independently repeated three times, and the experimental data were statistically analyzed to determine whether the differences between groups were statistically significant. The experimental results shown in Figure 8 indicate that, compared with the control group, the relative cell migration rate in the VEGF intervention group increased by 5%, reaching 29.15%. In the VEGF / VRR-13 co-treatment group, the relative cell migration rate decreased by 9% compared with the control group and by 14% compared with the VEGF-only treatment group, indicating that the VRR-13 peptide can significantly inhibit VEGF-induced cell migration.

[0230] Example 8: Inhibitory effect of VRR-13 peptide on VEGF-induced cell proliferation

[0231] To investigate the regulatory role of VRR-13 peptide in angiogenesis, the applicant further evaluated its effect on VEGF-induced RF / 6A cell proliferation using the conventional EdU assay (BeyoClick). TM EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, China).

[0232] Experimental grouping and drug administration. This study set up one control group and two intervention groups: a VEGF intervention group and a VEGF / VRR-13 intervention group, with three replicates in each group. The control group cells received no drug treatment; the VEGF intervention group cells received VEGF at a final concentration of 1 ng / mL in the culture medium; and the VEGF / VRR-13 intervention group cells received VEGF and VRR-13 peptide at a final concentration of 1 ng / mL and 10 μg / mL in the culture medium.

[0233] Detection steps: 1) Spread RF / 6A cells at a density of 0.8 × 10⁶ cells per well. 4 1) Seed cells at a density of 1000 μL each in 24-well plates, with 500 μL of culture medium added to each well; 2) After cell adhesion, administer drug intervention according to the above groups for 24 hours; 3) After intervention, add 10 μM EdU working solution to each well and incubate at 37°C and 5% CO2 for 2 hours; 4) Discard the culture medium, fix the cells with 4% paraformaldehyde, and then follow the BeyoClick protocol. TM EdU labeling and detection were performed according to the instructions of the EdU cell proliferation assay kit; 5) Cell nuclear staining was performed using Hoechst 33342 (maximum excitation / emission wavelength: 346nm / 460nm), and Azide labeled with Alexa Fluor 488 was used to detect EdU incorporation (maximum excitation / emission wavelength: 495nm / 519nm). Finally, the cells were observed and images were acquired under a fluorescence microscope.

[0234] The above experiments were independently repeated three times. Image analysis software was used to statistically analyze the proportion of EdU-positive cells and calculate the cell proliferation rate. Data are expressed as mean ± standard deviation. Statistical analysis was performed to analyze differences between groups, and the results are shown in Figure 9 and Table 13. Compared with the control group, treatment with 1 ng / mL VEGF significantly promoted RF / 6A cell proliferation; however, the addition of 10 μg / mL VRR-13 peptide decreased the cell proliferation rate by approximately 9% compared to the VEGF-only treatment group, indicating that VRR-13 peptide can effectively inhibit VEGF-induced cell proliferation.

[0235] Table 13 EdU-positive cell rate (%)

[0236] Example 9: Induction of apoptosis by VRR-13 peptide

[0237] To further investigate the biological function of the VRR-13 peptide, the applicant used flow cytometry to analyze the effect of VRR-13 on apoptosis in RF / 6A cells.

[0238] Experimental Design and Grouping. This study included one control group and two intervention groups: a VEGF intervention group and a VEGF / VRR-13 intervention group, with three replicates in each group. Cells in the control group received no drug treatment; the VEGF intervention group received VEGF at a final concentration of 2 ng / mL in the culture medium; and the VEGF / VRR-13 intervention group received VEGF at a final concentration of 2 ng / mL and VRR-13 peptide at a final concentration of 10 μg / mL in the culture medium.

[0239] Cell seeding and drug administration. RF / 6A cells were seeded at a rate of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 50%–60% in 6-well plates, and then appropriate drug interventions were performed according to the above grouping.

[0240] Cell processing and staining. Twenty-four hours after drug intervention, cells were washed once with PBS and digested with 1 mL of 0.25% EDTA-free trypsin. When the cells became rounded and some began to float, complete culture medium was added to stop the digestion. Cells were gently resuspended using a micropipette, and the cell suspension was collected. The cells were centrifuged at 1000–1500 rpm for 5 minutes, and the supernatant was discarded. The cells were then washed twice with pre-cooled PBS, and the cell pellet was collected after centrifugation.

[0241] Resuspend the cell pellet in 100 μL of 1× Binding Buffer, then add 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide (PI) in sequence. Mix gently and react at room temperature in the dark for 15 minutes.

[0242] Flow cytometry analysis. After staining, add 400 μL of 1×Binding Buffer to each tube, mix well, and immediately perform flow cytometry analysis. The parameters are set as follows: excitation wavelength 488 nm, FITC channel (FL1) for detecting Annexin V-FITC green fluorescence (emission wavelength 525±20 nm), and PI channel (FL2) for detecting PI red fluorescence (emission wavelength 585±21 nm).

[0243] Data analysis. FlowJo software was used to analyze flow cytometry data, and cells were divided into four regions based on fluorescence signals: Q1: cell debris or mechanically damaged cells; Q2: late-stage apoptotic and necrotic cells; Q3: early-stage apoptotic cells; Q4: normal cells. The apoptosis rate was expressed as the sum of the proportions of cells in regions Q2 and Q3.

[0244] The experimental results are shown in Figures 10 and 11 and Table 14. The apoptosis rate of the VEGF / VRR-13 intervention group was significantly higher than that of the control group and the VEGF intervention group, with an increase of 5%, indicating that VRR-13 peptide can effectively induce apoptosis.

[0245] Table 14 Cell apoptosis rate (%)

[0246] Example 10: In vivo preventive effect study of VRR-13 peptide

[0247] Animal grouping and administration. Healthy male B6N mice (weighing 180–200 g) aged 6–8 weeks were selected as experimental subjects. After one week of acclimatization, the mice were stratified by weight and randomly divided into one control group and three prevention test groups, with 6 mice in each group. The three prevention test groups were the low-dose VRR-13 prevention group, the medium-dose VRR-13 prevention group, and the high-dose VRR-13 prevention group.

[0248] For prophylactic administration, mice in each group were injected intravitreally three days before the establishment of the laser-induced CNV model. Mice in the low-dose VRR-13 prophylactic group received an intravitreal injection of 0.2 μL (0.5 mg / mL) of VRR-13 peptide solution; mice in the medium-dose VRR-13 prophylactic group received an intravitreal injection of 0.2 μL (1 mg / mL) of VRR-13 peptide solution; mice in the high-dose VRR-13 prophylactic group received an intravitreal injection of 0.2 μL (2 mg / mL) of VRR-13 peptide solution; and mice in the control group received only the injection procedure without any drug administration.

[0249] CNV Model Preparation: 1) Anesthesia. On day 1 of the experiment, mice were anesthetized by intraperitoneal injection of 1.25% aphthylamine solution (0.2 mL / 10 g body weight). 2) Laser Photocoagulation Modeling. After anesthesia, mice were fixed on a platform and placed in front of a slit lamp. After full pupil dilation, 3–4 points of laser photocoagulation were performed around the optic nerve of the left eye of each mouse using a 577 nm laser. The parameters were set as follows: spot diameter 100 μm, intensity 250 mW, and exposure time 50 ms. The appearance of bubbles in front of the laser spot was used as a sign of successful photocoagulation. 3) Postoperative Care. After modeling, the mice were kept warm with an electric blanket until they woke up, and antibiotic eye ointment was applied to both eyes to prevent infection and keep them moist.

[0250] Efficacy evaluation methods. On days 2 and 8 after laser modeling, in vivo imaging and contrast-enhanced imaging (FFA) were performed on mice in each group. ImageJ software was used to analyze and measure the laser spot area, and the CNV area change rate was calculated using the following formula:

[0251] CNV area change rate = ((laser spot area on day 8 - laser spot area on day 2) / laser spot area on day 2) × 100%

[0252] Table 15 Results of Prophylactic Dosing Trials

[0253] Using the control group data as a reference, the significance of the differences between the prevention groups was calculated, and is expressed as follows: ***, P < 0.001.

[0254] Statistical analysis of data. SPSS software was used for statistical analysis of the data, and the results of inter-group comparisons are shown in Table 16. Compared with the control group, the CNV area change rate of mice in each VRR-13 prevention group was significantly reduced, and this improvement effect was dose-dependent, that is, the inhibitory effect was more significant with the increase of VRR-13 concentration.

[0255] Example 11: Safety evaluation of VRR-13 peptide

[0256] 1) In vitro cytotoxicity evaluation – half-maximal inhibitory concentration (IC50) determination

[0257] Using the RF / 6A cell line as the research subject, the effect of different concentrations of VRR-13 peptide on cell viability was evaluated. The following concentration gradients of VRR-13 peptide were established: 10 μg / ml, 25 μg / ml, 50 μg / ml, 75 μg / ml, 100 μg / ml, 200 μg / ml, 800 μg / ml, and 1000 μg / ml. Based on the cell viability data corresponding to each concentration, concentration-cell viability curves were plotted (Figure 12). The calculated IC50 values ​​of VRR-13 in RF / 6A cells ranged from 268.6 μg / ml to 342.6 μg / ml.

[0258] Table 16 shows the calculation of the IC50 value of VRR-13 for different concentrations using logarithmic cell viability assays.

[0259] 2) Safety observation and tolerance evaluation of laser-induced CNV model

[0260] Using BN rats as a model, the safety and tolerability of VRR-13 peptide after intravitreal injection were evaluated.

[0261] A rat choroidal neovascularization (CNV) model was established using laser-induced induction. On day 7 post-modeling, rats received an intravitreal injection of 1 μL of a 1 mg / mL VRR-13 polypeptide solution. Healthy BN rats without any treatment served as the control group. Eye tissue was harvested on day 21 post-modeling for HE staining, and the results are shown in Figure 13. Compared to the control group, the retinal layers in the VRR-13-treated group showed clear and well-organized structures with no obvious pathological changes, suggesting that VRR-13 did not induce retinal toxicity under these conditions.

[0262] 3) Long-term safety evaluation of the subretinal Matrigel injection model

[0263] A rat model of CNV was established by subretinal injection of Matrigel. On day 7 post-modeling, 1.0 μL of VRR-13 peptide solution (1 mg / mL) was injected intravitreally for treatment, with the observation period extended to 10 weeks. Healthy BN rats without any treatment served as the control group. At week 10, eyeballs from both the normal and VRR-13 treatment groups were harvested for HE staining analysis, as shown in Figure 14. Compared with the normal group, the retinal structure of the VRR-13 treatment group was intact and clearly layered, with no obvious histopathological changes, indicating that VRR-13 maintains good safety during long-term observation.

[0264] 4) Safety evaluation of dose escalation

[0265] Healthy adult BN rats were selected and divided into a control group and three experimental groups (low-dose, medium-dose, and high-dose VRR-13), with three animals in each group. The low-dose group received an intravitreal injection of 1.0 μL (1 mg / mL) of VRR-13 peptide solution, the medium-dose group received 1.0 μL (10 mg / mL) of VRR-13 peptide solution, and the high-dose group received 1.0 μL (100 mg / mL) of VRR-13 peptide solution. The control group received only the injection procedure and no drug administration.

[0266] During the observation period, no abnormalities were observed in the behavior, eating, and sleep patterns of the rats in any group, consistent with the control group. Regarding ocular examination, evaluation using a microscope, slit lamp, and contact lens system revealed no significant abnormalities in the cornea, lens, vitreous body, or retina. Blood samples were collected from the rat tails for liver and kidney function tests, and the results were all within the normal range. Histopathological examination of the isolated ocular tissue further confirmed that no significant abnormal changes were observed in the ocular structures of any dosage group. Figure 15 shows the morphology of the eye cup after high-dose VRR-13 injection, and no abnormalities were observed.

[0267] In summary, in vitro and in vivo studies showed that VRR-13 peptide treatment did not induce cytotoxicity, and intravitreal injection in vivo did not cause significant pathological changes in the retina and other ocular structures. Furthermore, it demonstrated good safety and tolerability at doses up to 100 mg / mL.

[0268] Example 12 Study on the combined therapeutic effect of VRR-13 / aflibercept / dexamethasone

[0269] 1) Therapeutic effect of VRR-13 on Matrigel-induced CNV in rats

[0270] Experimental Animals and Grouping: Thirty healthy male SD rats aged 6-8 weeks were selected and randomly divided into three groups (n=10 per group) according to body weight using a stratified randomization method: a normal control group, a CNV model control group, and a VRR-13 treatment group. Normal Control Group: No modeling or intervention was performed. VRR-13 Treatment Group: A CNV model was established by subretinal injection of Matrigel, and 1 μL of a 1 mg / mL VRR-13 peptide solution was injected intravitreally on day 7 post-modeling. CNV Model Control Group: A CNV model was established by subretinal injection of Matrigel, and an equal volume of BSS solution was injected intravitreally on day 7 post-modeling.

[0271] Experimental Procedure: Eye samples were collected from each group of animals at weeks 2, 4, 6, 8, and 10 after modeling, with two animals from each group sampled at each time point. During the peak of CNV formation, the area of ​​neovascularization leakage was assessed using FITC-dextran retinal smears to investigate the duration of VRR-13's inhibitory effect on CNV. At week 10 after modeling, HE staining of retinal tissue was performed to observe structural changes and morphological alterations at the injection site.

[0272] Figure 16 shows representative retinal tissue sections on day 21 after modeling. The results are as follows: Normal control group: The retinal structure is intact and orderly arranged, and the retinal pigment epithelium is continuous and morphologically normal. CNV model control group: Significant pathological changes were observed at the Matrigel injection site, including disordered outer retinal structure and interruption of the continuity of the retinal pigment epithelium. Simultaneously, retinal pigment epithelial cell proliferation and detachment were observed, accompanied by irregular luminal structure proliferation, confirming successful CNV formation. These lesions pulled on the retinal tissue, forming localized depressions. VRR-13 treatment group: Compared with the CNV model control group, the overall structural integrity of the retina was significantly maintained, and the retinal pigment epithelium still maintained partial continuity. Furthermore, the degree of CNV proliferation and its traction on the retina were significantly reduced.

[0273] 2) Study on the combined therapeutic effects of VRR-13 with aflibercept and dexamethasone

[0274] Experimental Animals and Grouping: Male C57BL / 6J mice aged 6-8 weeks were selected and randomly divided into 16 groups (n=6 per group) after stratification by body weight. The grouping design was as follows: CNV model control group (NO.1201), VRR-13 peptide 0.1× dose test group (NO.1202), aflibercept 0.1× dose test group (NO.1203), VRR-13 / aflibercept 0.1× dose combined test group (NO.1204), VRR-13 peptide 0.2× dose test group (NO.1205), aflibercept 0.2× dose test group (NO.1206), VRR-13 / aflibercept 0.2× dose combined test group (NO.1207), VRR-13 peptide 0.3× dose test group (NO.1208), aflibercept 0.2× dose combined test group (NO.1207), and aflibercept 0.3× dose test group (NO.1208). The following groups were tested: VRR-13 0.3× dose group (NO.1209), VRR-13 / aflibercept 0.3× dose combined group (NO.1210), dexamethasone 0.1× dose group (NO.1211), VRR-13 / dexamethasone 0.1× dose combined group (NO.1212), dexamethasone 0.2× dose group (NO.1213), VRR-13 / dexamethasone 0.2× dose combined group (NO.1214), dexamethasone 0.3× dose group (NO.1215), and VRR-13 / dexamethasone 0.3× dose combined group (NO.1216). Dosage: A 1× dose of VRR-13 peptide was 0.2 μg per eye, a 1× dose of aflibercept was 0.8 μg, and a 1× dose of dexamethasone was 1 μg.

[0275] Following the protocol described in the foregoing embodiments, all mice underwent laser-induced CNV modeling. On day 7 post-modeling, mice in each group were administered intravitreal injections according to the following specific administration regimen.

[0276] CNV model control group (NO.1201): 0.2 μL of balanced salt solution (BSS) was injected into the vitreous cavity.

[0277] VRR-13 peptide 0.1× dose test group (NO.1202): 0.2 μL of VRR-13 solution was injected intravitreally, containing 0.02 micrograms of VRR-13 peptide (equivalent to 10% of the standard dose).

[0278] Aflibercept 0.1× dose trial group (NO.1203): 0.2 μL of aflibercept solution was injected intravitreally, containing 0.08 micrograms of aflibercept (equivalent to 10% of the standard dose).

[0279] VRR-13 / aflibercept 0.1× dose combined test group (NO.1204): 0.2 μL of VRR-13 / aflibercept mixed solution was injected intravitreally, in which the amount of VRR-13 peptide was 0.02 μg and the amount of aflibercept was 0.08 μg.

[0280] VRR-13 peptide 0.2× dose test group (NO.1205): 0.2 μL of VRR-13 solution was injected intravitreally, containing 0.04 micrograms of VRR-13 peptide (equivalent to 20% of the standard dose).

[0281] Aflibercept 0.2× dose trial group (NO.1206): 0.2 μL of aflibercept solution was injected intravitreally, containing 0.16 micrograms of aflibercept (equivalent to 20% of the standard dose).

[0282] VRR-13 / Aflibercept 0.2× dose combined test group (NO.1207): 0.2 μL of VRR-13 / Aflibercept mixed solution was injected intravitreally, in which the amount of VRR-13 peptide was 0.04 μg and the amount of Aflibercept was 0.16 μg.

[0283] VRR-13 peptide 0.3× dose test group (NO.1208): 0.2 μL of VRR-13 solution was injected intravitreally, containing 0.06 micrograms of VRR-13 peptide (equivalent to 30% of the standard dose).

[0284] Aflibercept 0.3× dose trial group (NO.1209): 0.2 μL of aflibercept solution was injected intravitreally, containing 0.24 micrograms of aflibercept (equivalent to 30% of the standard dose).

[0285] VRR-13 / Aflibercept 0.3× dose combined test group (NO.1210): 0.2 μL of VRR-13 / Aflibercept mixed solution was injected intravitreally, in which the amount of VRR-13 peptide was 0.06 μg and the amount of Aflibercept was 0.24 μg.

[0286] Dexamethasone 0.1× dose test group (NO.1211): 0.2 μL of dexamethasone solution was injected intravitreally, containing 0.1 micrograms of dexamethasone (equivalent to 10% of the standard dose).

[0287] VRR-13 / dexamethasone 0.1× dose combined test group (NO.1212): 0.2 μL of VRR-13 / dexamethasone mixed solution was injected intravitreally, in which the amount of VRR-13 peptide was 0.02 μg and the amount of dexamethasone was 0.1 μg.

[0288] Dexamethasone 0.2× dose test group (NO.1213): 0.2 μL of dexamethasone solution was injected intravitreally, containing 0.2 micrograms of dexamethasone (equivalent to 20% of the standard dose).

[0289] VRR-13 / dexamethasone 0.2× dose combined test group (NO.1214): 0.2 μL of VRR-13 / dexamethasone mixed solution was injected intravitreally, in which the amount of VRR-13 peptide was 0.04 μg and the amount of dexamethasone was 0.2 μg.

[0290] Dexamethasone 0.3× dose test group (NO.1215): 0.2 μL of dexamethasone solution was injected intravitreally, containing 0.3 micrograms of dexamethasone (equivalent to 30% of the standard dose).

[0291] VRR-13 / dexamethasone 0.3× dose combined test group (NO.1216): 0.2 μL of VRR-13 / dexamethasone mixed solution was injected intravitreally, in which the amount of VRR-13 peptide was 0.06 μg and the amount of dexamethasone was 0.3 μg.

[0292] On days 7 and 14 after laser modeling, fundus fluorescein angiography (FFA) was performed on mice in each group to obtain in vivo images. The laser spot area of ​​each mouse on days 7 and 14 was quantified using ImageJ software, and the CNV area change rate was calculated using the following formula. SPSS software was used to statistically analyze the CNV area change rates of the control group and each treatment group; the results are shown in the table below.

[0293] CNV area change rate after 14 days of modeling = (Area of ​​laser spot on day 14 - Area of ​​laser spot on day 7) / Area of ​​laser spot on day 7 × 100%

[0294] Table 17 Results of the study on the combined treatment effect

[0295] The results and conclusions are as follows: 1) Except for the 0.1× dose group, under the same dose conditions (0.2× dose group, 0.3× dose group), the VRR-13 experimental group showed statistically significant superiority over the aflibercept experimental group in reducing the fluorescence leakage area (p<0.0001), indicating that VRR-13 has stronger efficacy in inhibiting CNV. 2) Except for the 0.1× dose group, under the same dose conditions (0.2× dose group, 0.3× dose group), compared with the VRR-13 monotherapy experimental group and the aflibercept monotherapy experimental group, the combination experimental group had a statistically significant CNV inhibition effect, proving that the combination treatment regimen has a synergistic and enhanced therapeutic effect. 3) The combination study of VRR-13 peptide and dexamethasone showed that in the 0.2× dose combination group (p<0.01) and the 0.3× dose combination group (p<0.05), the combination with dexamethasone significantly improved the anti-CNV effect of VRR-13 peptide.

[0296] Example 13 Inhibitory effect of VRR-13 on angiogenesis in the late stage of a disease model

[0297] To evaluate the inhibitory effect of VRR-13 peptide on pathological angiogenesis at the end of the modeling period, the applicant conducted this study. After one week of acclimatization, 6-8 week old male C57BL / 6J mice were stratified by body weight and randomly assigned to one control group and two treatment groups: a normal control group (Control), a CNV model group, and a VRR-13 treatment group, with 6 animals in each group. Following the protocol described in the above embodiment, laser-induced CNV modeling was performed on mice in the CNV model group and the VRR-13 treatment group. Seven days after successful modeling, intravitreal injection of 0.2 μL was performed on mice in the CNV model group and the VRR-13 treatment group. Mice in the VRR-13 treatment group received an intravitreal injection of 0.2 μL of a 1 mg / mL VRR-13 peptide solution; mice in the CNV model group received an intravitreal injection of 0.2 μL of BSS.

[0298] On day 21 after modeling, ocular tissues from mice in each group were obtained and subjected to immunofluorescence staining. CD31 antibody (green fluorescence) was used to label vascular structures, and angiogenesis in the retinal model area was observed. The results are shown in Figure 17 (representative immunofluorescence staining image): In the normal control group (Control), CD31 positive signals were only observed in the inner 5 layers of the retina, while no blood vessels were observed in the outer 5 layers from the outer omentum (OPL) to the retinal pigment epithelium (RPE). Compared with the normal control group, the CNV model group showed disordered retinal structure, with obvious abnormal angiogenesis and localized retinal indentation due to vascular traction. In contrast, although some abnormal blood vessels were still visible in the outer nuclear layer (ONL) of the VRR-13 treatment group, their density was significantly lower than that of the CNV model group.

[0299] The above results indicate that the VRR-13 peptide can effectively inhibit pathological angiogenesis in the late stage of the disease model and has the potential for anti-angiogenic therapy.

[0300] Example 14: Study on the promotion of retinal vascular endothelial cell apoptosis by VRR-13 in a laser-induced CNV model.

[0301] To investigate whether VRR-13 can specifically induce vascular endothelial cell death (apoptosis) in a laser-induced mouse CNV model, the applicant conducted this study. After a week of acclimatization, 6-8 week old male C57BL / 6J mice were stratified by body weight and randomly assigned to one control group and two treatment groups: a normal control group, a CNV model group, and a VRR-13 treatment group, with 6 animals in each group. Following the protocol described in the above embodiment, laser-induced CNV modeling was performed on mice in the CNV model group and the VRR-13 treatment group. Seven days after successful modeling, intravitreal injections of 0.2 μL were administered to mice in both groups. For the VRR-13 treatment group, 0.2 μL of a 1 mg / mL VRR-13 peptide solution was injected intravitreally; for the CNV model group, 0.2 μL of BSS was injected intravitreally.

[0302] On day 21 after modeling, eye tissues were obtained from mice in each group, and TUNEL staining analysis was performed. The percentage of TUNEL-positive cells in the outer retinal nucleus (ONL) layer was calculated using the following formula:

[0303] TUNEL-positive cell rate in ONL layer = Number of TUNEL-positive cells / (ONL area / 20) * 100%

[0304] The results are shown in Figure 18 and Table 18: Compared with the normal group (Control), both the CNV model group and the VRR-13 treatment group showed a large number of TUNEL-positive cells in the CNV formation area. Notably, the number of TUNEL-positive cells in the VRR-13 treatment group was significantly higher than that in the CNV model group, and this difference was statistically significant.

[0305] Table 18. Percentage of TUNEL-positive cells in ONL layer (%)

[0306] Given that TUNEL staining can label various types of dead cells, further analysis was performed using TUNEL and CD31 immunofluorescence co-staining technology to clarify the cell type. The results, shown in Figure 19, indicate significant co-localization between TUNEL and CD31 positive signals, confirming that the observed dead cells were indeed vascular endothelial cells.

[0307] The above results indicate that VRR-13 can specifically induce apoptosis in vascular endothelial cells within the CNV region, which may be one of the key mechanisms by which it inhibits the development of choroidal neovascularization.

[0308] Example 15: Evaluation of the therapeutic effect of VRR-13 in a Matrigel-induced rat CNV model induced by subretinal injection

[0309] Following the protocol described in the above embodiments, a rat choroidal neovascularization (CNV) model was established by subretinal injection of Matrigel. After one week of acclimatization, healthy male SD rats aged 6–8 weeks were stratified by weight and randomly divided into a normal control group (Control), a CNV model group, and a VRR-13 treatment group, with 6 rats in each group. Following the protocol described above, Matrigel was injected subretinally into the CNV model group and the VRR-13 treatment group to establish the CNV rat model. On day 7 post-injection modeling, intravitreal injections were performed: the VRR-13 treatment group received 1 μL of VRR-13 peptide solution (concentration 1 mg / mL) into both eyes, while the control group received an equal volume of 1 μL of BSS solution. The normal control group rats received no treatment, including CNV modeling and drug administration.

[0310] At week 4 post-modeling, retinal vascular leakage was evaluated in some rats (n=3 per group). At week 10 post-modeling, the remaining rats (n=3 per group) were sacrificed, and ocular tissue was separated. Histological morphology was observed by HE staining, and injection site, RPE displacement, and angiogenesis were observed by immunofluorescence staining.

[0311] 1) Evaluation of retinal vascular leakage

[0312] The area of ​​CNV leakage was quantitatively assessed using FITC-dextran cardiac perfusion and retinal smear techniques. The specific method was as follows: After anesthetizing rats, FITC-dextran solution was perfused intracardiacly until fluorescence appeared in the lips and limbs. The eyeballs were then harvested. The eyeballs were fixed in FAS ocular fixative for 40 minutes. Under a microscope, the cornea, lens, and vitreous humor were removed. The RPE-retina-sclera complex was then radially cut in 5-6 lines and smeared. The occurrence and development of retinal neovascularization were observed under a confocal microscope.

[0313] The formulas for calculating the fluorescence leakage area and the rate of change of the fluorescence leakage area are as follows:

[0314] Fluorescent leakage area = Measured leakage area / 1 PD (viewing disc area)

[0315] The results of representative FITC retinal patch studies are shown in Figure 20 and Table 19. Compared with the CNV model group, the VRR-13 treatment group showed a significant reduction in CNV leakage area after 4 weeks of intervention, and the difference was statistically significant.

[0316] Table 19 CNV area (PD) of retinal patch after FITC injection into the retina and cardiac perfusion in rats.

[0317] 2) Histological observation (HE staining)

[0318] The HE staining results are shown in Figure 21. It can be observed that in the normal control group rats, the retinal structure is intact, with each layer neatly arranged. The RPE layer is continuous and intact, the photoreceptor cell layer is tightly connected to the RPE layer, and a clear Bruch membrane is visible beneath the RPE (Figure 21A). In the CNV model group rats, 10 weeks after subretinal Matrigel injection, RPE cell migration and near-new RPE layer formation were still observed (Figure 21B). The injection site is clearly visible (white arrow in Figure 21C), and significant cell infiltration is observed beneath the Matrigel deposition area (green arrow in Figure 21C).

[0319] 3) Immunofluorescence staining analysis

[0320] The immunofluorescence staining results are shown in Figure 22. It can be observed that in the retina of normal control rats, CD31 and VEGF co-labeling revealed clearly defined normal retinal vessels (Figure 22a1). In the CNV model group rats, 10 weeks after Matrigel injection, RPE cells still migrated from their original location to the outer nuclear layer, forming abnormal cavities (i.e., the injection site), consistent with the HE staining results. Abnormal neovascularization (green arrow in Figure 22a2) and retinal structural disorder accompanied by cell infiltration were observed in this area (white arrow in Figure 22a4).

[0321] The above results indicate that VRR-13 can effectively inhibit Matrigel-induced CNV development, reduce pathological vascular leakage, and protect retinal structures.

[0322] It should be understood that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications or equivalent substitutions based on this application within the spirit and principles of the technical solutions disclosed in this application should not be considered as departing from the scope of protection of this application. The scope of protection of this application shall be determined by the appended claims. Industrial applicability

[0323] The synthetic peptide provided in this application effectively overcomes the angiogenesis-promoting effect of wild-type TSP-1 protein or peptides, while exhibiting excellent anti-angiogenic activity. This peptide has a small molecular weight and well-defined structure, solving common problems in clinical applications of proteins or large peptides such as high immunogenicity, poor stability, and limited tissue permeability, thus providing a novel candidate molecule for the treatment of neovascular eye diseases.

[0324] Furthermore, the artificial active peptides can be prepared by chemical synthesis, which is simple, cost-controllable, produces high-purity products with stable biological activity, and has good industrialization prospects and application potential in the development of drugs for the treatment of eye diseases and corresponding pharmaceutical preparations.

Claims

1. A polypeptide having antiangiogenic activity or a pharmaceutically acceptable salt thereof, characterized in that, The polypeptide has an amino acid sequence shown in any one of SEQ ID No. 03, SEQ ID No. 09, SEQ ID No. 10, or SEQ ID No.

13.

2. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, The polypeptide or its pharmaceutically acceptable salt can reduce cell scratch healing rate by at least 5%, preferably at least 10%; and / or the polypeptide or its pharmaceutically acceptable salt can reduce cell viability by at least 5%, preferably at least 10%; and / or the polypeptide or its pharmaceutically acceptable salt can increase cell apoptosis level by at least 1%.

3. The polypeptide of any one of claims 1 to 2, characterized in that, The polypeptide is a chemically synthesized polypeptide; preferably, the purity of the polypeptide is not less than 50%; more preferably, the purity of the polypeptide is not less than 90%.

4. A composition characterized in that, The composition comprises any one of the polypeptides of claims 1 to 3 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

5. The composition according to claim 4, characterized in that, The concentration of the polypeptide or its pharmaceutically acceptable salt in the composition is from 10 μg / mL to 5000 μg / mL.

6. The composition according to any one of claims 4 or 5, characterized in that, The composition is a liquid formulation, a semi-solid formulation, or a solid formulation.

7. The composition of claim 6, wherein, The liquid formulation is selected from eye drops and ophthalmic injections; And / or the semi-solid formulation is selected from ophthalmic gels and ointments; And / or solid dosage forms are selected from ophthalmic tablets and ophthalmic implants.

8. The composition according to any one of claims 4 to 7, characterized in that, The composition further comprises an effective amount of an anti-vascular endothelial growth factor (VEGF) agent; preferably, the concentration of the anti-vascular endothelial growth factor agent in the composition is from 10 μg / mL to 5000 μg / mL.

9. The composition of claim 8, wherein, The anti-vascular endothelial growth factor agent is selected from antigen-binding fragments, antibodies, fusion proteins, oligonucleotides, nucleic acid constructs, or combinations thereof; preferably, the anti-vascular endothelial growth factor agent is selected from aflibercept, ranibizumab, faricimab, brolucizumab, and conbercept.

10. The composition according to any one of claims 4 to 9, characterized in that, The composition further comprises an effective amount of a glucocorticoid drug or molecule; preferably, the glucocorticoid drug or molecule is selected from triamcinolone acetonide, dexamethasone, fluocinolone acetonide, hydrocortisone, prednisone, and methylprednisolone.

11. The use of the polypeptide of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition of any one of claims 4 to 10, in the preparation of an agent for inhibiting cell migration, inhibiting cell proliferation and / or inducing apoptosis.

12. The use of the polypeptide of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition of any one of claims 4 to 10, in the preparation of an agent for the prevention and / or treatment of neovascular eye diseases.

13. Use according to claim 12, characterized in that, The neovascular eye diseases mentioned are selected from diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, ischemic retinal vein occlusion, choroidal neovascularization, retinal edema, neovascular glaucoma, and corneal neovascularization caused by trauma or inflammation.

14. A method of inhibiting proliferation and / or migration of retinal cells, characterized in that, The method comprises administering to cells an effective amount of the polypeptide of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition of any one of claims 4 to 10.

15. A method of treating a neovascular ocular condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-14. The method comprises administering to the patient's eye an effective amount of any one of claims 1 to 3, the polypeptide or a pharmaceutically acceptable salt thereof, or any one of claims 4 to 10.

16. A method of treating a neovascular ocular condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-15. The method comprises administering, simultaneously or sequentially, an effective amount of the polypeptide or a pharmaceutically acceptable salt thereof as described in claims 1 to 3, or the composition as described in any one of claims 4 to 7, and an effective amount of an anti-vascular endothelial growth factor agent and / or a glucocorticoid drug or molecule to the fundus of a patient.

Images