Medicine composition for treating wet age-related macular degeneration and application thereof

Abstract

The invention particularly discloses a medicine composition for treating wet age-related macular degeneration and application thereof, and relates to the technical field of biological medicine. The invention provides application of a neuropeptide VF (NPVF), a nucleic acid molecule encoding the NPVF or an accelerant of the NPVF to preparation of a medicine for preventing and / or treating neovascular eye diseases and / or related eye diseases induced by inflammation, and the inflammatory reaction and the progress of the disease course of wet age-related macular degeneration are effectively inhibited. The invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a drug combination for treating wet age-related macular degeneration and an application thereof. Background Art

[0002] At present, anti-vascular endothelial growth factor (VEGF) drugs are mainly used to treat diseases related to neovascularization, such as age-related macular degeneration (AMD) and macular edema (ME), to inhibit neovascularization, and ultimately inhibit the process of retinal scarring caused by neovascular leakage and bleeding, thereby protecting visual function. However, such drugs are antibody drugs with a limited duration of action. At the same time, long-term use of drugs will produce drug resistance, which will continuously reduce the effectiveness of the drugs. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of this, one of the main purposes of the present invention is to provide a use of a neuropeptide VF (NPVF), a nucleic acid molecule encoding the polypeptide or a promoter thereof in the preparation of a drug for preventing and / or treating neovascular eye diseases and / or related ophthalmic diseases induced by inflammation. By injecting the NPVF peptide segment in a wet age-related macular degeneration model, the activation of Iba1-positive microglia can be effectively inhibited, the inflammatory response of wet age-related macular degeneration can be inhibited, and the formation of new blood vessels can be effectively inhibited, the occurrence of vascular leakage can be reduced, and the progression of wet age-related macular degeneration can be effectively inhibited.

[0005] (II) Technical solution

[0006] In order to solve the above problems, the present invention provides a use of a neuropeptide VF (NPVF), a nucleic acid molecule encoding the polypeptide or a promoter thereof in the preparation of a drug for preventing and / or treating neovascular eye diseases and / or related ophthalmic diseases induced by inflammation.

[0007] In one embodiment, the NPVF is selected from:

[0008] (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0009] (2) a polypeptide that is homologous to or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0010] (3) A polypeptide derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

[0011] In one embodiment, the amino acid sequence of SEQ ID No.1 is:

[0012] MEIISSKLFILLTLATSSLLTSNIFCADELVMSNLHSKENYDKYSEPRGYPKGERSLNFEELKDWGPKNVIKMSTPAVNKMPHSFANLPLRFGRNVQEERSAGATANLPLRSGRNMEVSLVRRVPNLPQRFGRTTTAKSVCRMLSDLCQGSMHSPCANDLFYSMTCQHQEIQNPDQKQSRRLLFKKIDDAELKQEK.

[0013] In one embodiment, the amino acid sequence of SEQ ID No.3 is:

[0014] MEIISLKRFILLTVATSSFLTSNTFCTDEFMMPHFHSKEGDGKYSQLRGIPKGEKERSVSFQELKDWGAKNVIKMSPAPANKVPHSAANLPLRFGRTIDEKRSPAARVNMEAGTRSHFPSLPQRFGRTTARSPKTPADLPQKPLHSLGSSELLYVMICQHQEIQSPGGKRTRRGAFVETD.

[0015] In one embodiment, the polypeptide shown by SEQ ID No. 1 is cleaved into mature NPVF, and the mature NPVF is selected from:

[0016] (1) a polypeptide having the amino acid sequence shown in SEQ ID No. 2; or

[0017] (2) a polypeptide that is homologous to the amino acid sequence of SEQ ID NO: 2 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; or

[0018] (3) A polypeptide derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

[0019] In one embodiment, the amino acid sequence of SEQ ID No.2 is:

[0020] VPNLPQRF.

[0021] In one embodiment, the polypeptide shown by SEQ ID No. 3 is cleaved into mature NPVF, and the mature NPVF is selected from:

[0022] (1) a polypeptide having the amino acid sequence shown in SEQ ID No. 4; or

[0023] (2) a polypeptide that is homologous to the amino acid sequence of SEQ ID NO:4 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; or

[0024] (3) A polypeptide derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

[0025] In one embodiment, the amino acid sequence of SEQ ID No.4 is:

[0026] SHFPSLPQRF.

[0027] In one embodiment, the promoter comprises one or a combination of an NPVF expression vector, an exogenous NPVF, a naked nucleic acid molecule of an NPVF coding sequence, a liposome-encapsulated nucleic acid molecule of an NPVF coding sequence, or an NPVF precursor protein or conjugate or complex that can be converted into NPVF in vivo.

[0028] In one embodiment, the NPVF expression vector comprises a viral vector and / or a non-viral vector.

[0029] In one embodiment, the viral vector comprises one or a combination of an adeno-associated virus vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector or a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, a herpes virus vector, a poxvirus vector or a lentivirus vector.

[0030] In one embodiment, the non-viral vector comprises one or a combination of a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle or a liposome.

[0031] In one embodiment, the DNA vector comprises one or a combination of a plasmid vector, a cosmid vector, a phagemid vector or a human artificial chromosome.

[0032] In one embodiment, the neovascular eye disease includes one or a combination of age-related macular degeneration, diabetic retinopathy, retinal vascular occlusive disease, retinopathy of prematurity, corneal infection or neovascular glaucoma.

[0033] In one embodiment, the inflammation-induced related ophthalmic diseases include one or a combination of corneal infection, uveitis, retinitis, inflammation-related glaucoma, neovascular glaucoma, diabetic retinopathy, retinopathy of prematurity, retinal peripheral phlebitis, and age-related macular degeneration.

[0034] In one embodiment, the neovascular eye disease or inflammation-induced related ophthalmic disease includes one or a combination of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal infection, neovascular glaucoma, uveitis or retinitis.

[0035] In one embodiment, the neovascular eye disease is age-related macular degeneration.

[0036] In one embodiment, the age-related macular degeneration includes dry age-related macular degeneration (Dry AMD) and wet age-related macular degeneration (Wet AMD).

[0037] In one embodiment, the age-related macular degeneration is Wet AMD.

[0038] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0039] (1) a therapeutically effective amount of neuropeptide VF (NPVF), a nucleic acid molecule encoding the polypeptide, or a promoter thereof;

[0040] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0041] In one embodiment, the dosage form of the pharmaceutical composition includes one or a combination of granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, metered sprays.

[0042] In one embodiment, the dosage form of the pharmaceutical composition is a liquid solution.

[0043] In one embodiment, the concentration of NPVF in the liquid solution is 0.05-5 mg / mL. Preferably, the concentration is 5 mg / mL.

[0044] In one embodiment, the administration route of the pharmaceutical composition includes one or a combination of intranasal, intrathecal, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, ocular, sublingual, oral, intracerebral or transdermal.

[0045] In one embodiment, the pharmaceutical composition is administered to the eye.

[0046] In one embodiment, the pharmaceutical composition is administered by subretinal injection.

[0047] In one embodiment, the injection volume is 0.1-2 μL / eye. Preferably, the injection volume is 2 μL / eye.

[0048] In another aspect, the present invention provides a pharmaceutical preparation, which comprises the above-mentioned pharmaceutical composition.

[0049] In another aspect, the present invention provides a use of the above-mentioned pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for preventing and / or treating neovascular eye diseases and / or related ophthalmic diseases induced by inflammation.

[0050] (III) Beneficial effects

[0051] The present invention provides a use of a neuropeptide VF (NPVF), a nucleic acid molecule encoding the polypeptide, or a promoter thereof in the preparation of a product for preventing and / or treating neovascular eye disease and / or diseases and / or symptoms associated with neovascular eye disease. Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. Injection of AAV-CAG-NPVF virus into the wet age-related macular degeneration model of Rho-VEGF165 transgenic mice can effectively inhibit the activation of Iba1-positive microglia and suppress the inflammatory response of wet age-related macular degeneration.

[0053] 2. Injection of NPVF peptide into the laser-induced choroidal neovascularization model (CNV), a wet age-related macular degeneration mouse model, can effectively inhibit the formation of new blood vessels, reduce the occurrence of vascular leakage, and inhibit the progression of wet age-related macular degeneration.

[0054] 3. Since NPVF is a short peptide expressed by the human body, it is secreted by cells into the extracellular environment. Therefore, its injection is highly safe and the possibility of the human body developing resistance to it is low.

[0055] (IV) Terms and Definitions

[0056] As used herein, the terms "NPVF polypeptide", "NPVF", and "NPVF peptide fragment" are used interchangeably to refer to neuropeptide VF.

[0057] As used herein, the terms "NPVF polypeptide", "NPVF", and "NPVF peptide" are used interchangeably and refer to neuropeptide VF. The NPVF disclosed in the present invention may be a polypeptide encoded by the sequence of SEQ ID NO: 1 (human sequence) or SEQ ID NO: 3 (mouse sequence) or a homologous sequence (for example, a homologous sequence of a polypeptide can be obtained through a database or comparison software known in the art), a variant or a modified form of these polypeptides. For example, the polypeptide may be selected from:

[0058] (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0059] (2) a polypeptide that is homologous to or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0060] (3) A polypeptide derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

[0061] The polypeptide disclosed in the present invention can be a naturally purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animals, insects and mammalian cells) using recombinant technology. The polypeptide disclosed in the present invention is preferably encoded by a human NPVF gene or its homologous gene or family gene.

[0062] The variant forms of the polypeptide disclosed in the present invention include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid deletion, insertion and / or substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein or polypeptide is usually not changed. For another example, the addition of one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein or polypeptide. For example, the polypeptide disclosed in the present invention may or may not include the initial residue and still have the activity of the action.

[0063] Random mutagenesis can be produced by irradiation or exposure to mutagens, or site-directed mutagenesis or other known molecular biological techniques can be used to obtain the polypeptide in (3). The coding sequence encoding the polypeptide can be used to construct transgenic animals.

[0064] Depending on the host used in the recombinant production protocol, the polypeptides disclosed in the present invention may be glycosylated, or may be non-glycosylated. The term also includes active fragments and active derivatives of the polypeptides.

[0065] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, polypeptides encoded by sequences that can hybridize with polypeptide encoding sequences under high or low stringency conditions, and polypeptides obtained using antiserum against proteins. The present invention also discloses other polypeptides, such as fusion proteins comprising polypeptides or fragments thereof. In addition to almost full-length polypeptides, the present disclosure also includes soluble fragments of polypeptides. Typically, the fragment has at least about 10 consecutive amino acids of the polypeptide sequence, typically at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids. For example, the fragment is selected from:

[0066] (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4; or

[0067] (2) a polypeptide that is homologous to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity; or

[0068] (3) A polypeptide derived from (1) or (2) in which one or more amino acids are substituted, deleted or added in the amino acid sequence of (1) or (2).

[0069] As used herein, the terms "NPVF gene", "NPVF encoding gene", "NPVF polypeptide encoding gene" or "nucleic acid molecule encoding NPVF" are used interchangeably and refer to a nucleotide sequence encoding the NPVF disclosed in the present invention.

[0070] The nucleic acid molecules include DNA and RNA.

[0071] As used herein, the term "promoter" or "promoter of NPVF or its coding sequence" is used interchangeably and refers to a substance that can increase the level or activity of NPVF or its encoding nucleic acid molecule. The promoter that can be used in the present disclosure includes, but is not limited to: NPVF expression vector, exogenous NPVF, naked nucleic acid molecule of NPVF coding sequence, liposome-encapsulated nucleic acid molecule of NPVF coding sequence, NPVF precursor protein or conjugate or complex that can be converted into NPVF in vivo.

[0072] The NPVF or NPVF coding sequence or promoter thereof disclosed in the present invention can inhibit neovascular ocular diseases, and thus can be further used to prevent or treat diseases associated with neovascular ocular diseases, and / or related symptoms caused by neovascular ocular diseases.

[0073] As used herein, the term "neovascular eye disease" includes (but is not limited to): involvement of the choroid, retina, cornea or iris, including age-related macular degeneration, proliferative diabetic retinopathy, retinal vascular occlusive disease, retinopathy of prematurity, corneal infection, neovascular glaucoma, etc.

[0074] As used herein, the term "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as they can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0075] In the present disclosure, the expression vector can be a viral vector or a non-viral vector, and the viral vector can be an adeno-associated virus (AAV) vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector or a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, a herpes virus vector, a poxvirus vector or a lentivirus vector. In this method, the non-viral vector can be a DNA vector, a nanoparticle, a cationic polymer, an exosome, an extracellular vesicle or a liposome, and the DNA vector can be a plasmid vector, a cosmid vector, a phagemid vector or a human artificial chromosome.

[0076] Methods well known to those skilled in the art can be used to construct expression vectors containing the NPVF coding sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the construction method refers to Dali Tong, Mei Zhang, Yunru Yang, Han Xia, Haiyang Tong, Huajun Zhang, Weihong Zeng, Muziying Liu, Yan Wu, Huan Ma, Xue Hu, Weiyong Liu, Yuan Cai, Yanfeng Yao, Yichuan Yao, Kunpeng Liu, Shifang Shan, Yajuan Li, Ge Gao, Weiwei Guo, Yun Peng, Shaohong Chen, Juhong Rao, Jiaxuan Zhao, Juan Min, Qingjun Zhu, Yanmin Zheng, Lianxin Liu, Chao Shan, Kai Zhong, Zilong Qiu, Tengchuan Jin, Sandra Chiu, Zhiming Yuan, Tian Xue, Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques, Protein & Cell, Volume 14, Issue 1, January 2023, Pages 69–73.

[0077] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0078] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the NPVF polypeptide. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell. Representative examples include: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc.

[0079] When the polynucleotide disclosed in the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, which acts on the promoter to enhance the transcription of the gene. Those skilled in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells.

[0080] As used herein, the term "pharmaceutical composition" refers to a composition comprising NPVF formulated together with one or more pharmaceutically acceptable carriers.

[0081] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated using methods known in the art to provide rapid, continuous or delayed release of the active ingredient after administration to a mammal. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, lemonades, perfumed waters, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcoholic agents, capsules, creams, lozenges, tinctures, pastes, pills and soft or hard gelatin capsules.

[0082] As used herein, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or animals without excessive adverse reactions (such as toxicity, irritation, and allergic response), ie, at a reasonable benefit / risk ratio.

[0083] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art, and a full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0084] In the composition, pharmaceutically acceptable carriers include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, adhesives, diluents, lubricants, glidants, pH regulators, buffers, reinforcing agents, wetting agents, solubilizing agents, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may include other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch or talc, or liquid carriers, such as water, fatty oils or liquid paraffin.

[0085] Any known method can be used to administer the pharmaceutical composition of the present invention. The term "administering" or "applying" NPVF, nucleic acid molecules encoding the polypeptide, promoters thereof, pharmaceutical compositions or pharmaceutical preparations to a subject can be implemented using one of a variety of methods known to those skilled in the art.

[0086] For example, the pharmaceutical composition or pharmaceutical formulation can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). The compound or agent can also be suitably introduced by a rechargeable or biodegradable polymeric device or other device (e.g., a patch and a pump or formulation) that provides extended, slowed, or controlled release of the compound or agent. It can also be administered, for example, once, multiple times, and / or over one or more extended periods of time.

[0087] The administration routes include: (1) direct naked DNA or NPVF injection; (2) linking NPVF cDNA, mRNA and protein to transferrin / poly-L-lysine complex to enhance its biological effect; (3) forming complexes between cDNA, mRNA and protein and positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating cDNA, mRNA and protein in liposomes and mediating their entry into cells, which is beneficial for the smooth entry of large molecules and protects them from hydrolysis by various extracellular enzymes; (5) combining cDNA, mRNA and protein with cholesterol to increase their cytoplasmic retention time by 10 times; (6) transporting cDNA, mRNA and protein with immunoliposomes to enable their specific transport to target tissues and target cells; (7) in vitro transfection of cDNA, mRNA and protein into transfer cells (such as fibroblasts) can also better load NPVF-related drugs into target cells; (8) electroporation, that is, introducing cDNA, mRNA and protein into target cells with the help of electric current.

[0088] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level includes the type and severity of the subject, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors well known in the medical field.

[0089] As used herein, the term "treating" a condition or patient means taking steps to obtain a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, or eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, condition, or disorder, substantially improving or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition, or disorder, and avoiding harmful or annoying symptoms. Treatment also refers to accomplishing one or more of the following: (a) lessening the severity of the condition; (b) limiting the development of symptoms characteristic of the condition being treated; (c) limiting the worsening of symptoms characteristic of the condition being treated; (d) limiting the recurrence of the condition in patients who previously had the condition; and / or (e) limiting the recurrence of symptoms in patients who were previously free of symptoms of the condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0091] Figure 1 NPVF can effectively inhibit the progression of damage in the CNV mouse model. Figure 1 A is the angiography results 3, 7, and 14 days after CNV modeling and AAV subretinal injection. Figure 1 B is a statistical diagram of vascular leakage in mice 3 days after CNV modeling and AAV subretinal injection. Figure 1 C is the vascular leakage of the experimental group mice 7 days after CNV modeling and AAV subretinal injection was significantly lower than that of the control group mice. Figure 1 D is the vascular leakage of the experimental group mice 14 days after CNV modeling and AAV subretinal injection was significantly lower than that of the control group mice. Figure 1 E is the severe vascular leakage in the control group mice after CNV modeling and AAV subretinal injection. Figure 1 F is the vascular leakage of the experimental group mice gradually improved over time after CNV modeling and AAV subretinal injection. Figure 1G is the result of IB4 immunofluorescence staining of mouse choroid 14 days after CNV modeling and AAV subretinal injection. Figure 1 H is that 14 days after CNV modeling and AAV subretinal injection, the vascular proliferation in the experimental group mice was significantly lower than that in the control group mice.

[0092] Figure 2 This is the immunofluorescence staining image of Rho-hVEGF165 model mice.

[0093] Figure 3 Injection of NPVF can effectively inhibit the inflammation level of Rho-hVEGF165 transgenic mice. A. Retinal Iba1 staining results of Rho-hVEGF165 transgenic mice injected with PBS and NPVF peptide 14 days later. B. Statistical results of Q-PCR detection of retinal NPVF expression in Rho-hVEGF165 transgenic mice and wild-type mice. C. Statistical graph of retinal Iba1 positive cell density of Rho-hVEG F165 transgenic mice injected with PBS and NPVF peptide 14 days later. DETAILED DESCRIPTION

[0094] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0095] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0096] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0097] Example 1

[0098] Preparation of NPVF adeno-associated virus:

[0099] NPVF adeno-associated virus (AVV) was packaged in HEK-293T cells. In brief, 10 micrograms of pHelper vector, 5 micrograms of AAV2 / 2 vector and 5 micrograms of pITR vector (AAV-CAG-NPVF and AAV-CAG-empty) were transfected into a 10 cm diameter culture dish containing HEK-293T cells using polyethyleneimine (PEI) (PolyScience, Niles, USA). HEK-293T cells were cultured at 37°C in DMEM medium (Gibco, 11965-092) containing 10% fetal bovine serum (FBS) (Gibco, 16000-044). The supernatant of HEK-293T cells was collected on days 3 and 6 after transfection. The supernatant was concentrated using an Ultra-15 centrifugal filter (Millipore, UFC905024), and then gradient purified using 15% to 60% Optiprep (Sigma, D1556). NPVF adeno-associated virus was collected and washed six times with phosphate buffered saline (PBS), then diluted in 150 microliters of PBS. The titer of NPVF adeno-associated virus was calculated by quantitative real-time polymerase chain reaction (qRT-PCR). The prepared NPVF adeno-associated virus was stored at -80°C.

[0100] Example 2

[0101] Injection of NPVF adeno-associated virus can effectively inhibit the formation of new blood vessels and reduce the occurrence of vascular leakage:

[0102] C57BL / 6J mice were used for laser-induced choroidal neovascularization modeling. The Phoenix MicronIV retinal imaging system was used for modeling. The modeling parameters were 66mw, 532nm laser, irradiation time of 100ms, and laser spot size of 50μm. The specific steps are as follows:

[0103] 1. Anesthetize C57BL / 6J mice with isoflurane.

[0104] 2. Use compound tropicamide eye drops to dilate the pupil twice. After the pupil of the mouse is dilated, use the PhoenixMicron IV retinal imaging system and laser system to stimulate the upper, lower, left and right parts of the mouse eye once (see above for parameters) until bubbles are seen.

[0105] 3. The mice in the experimental group were injected with AAV-CAG-NPVF virus (1E12Vg / mL, 1μL / eye) by subretinal injection, and the mice in the control group were injected with an equal volume of AAV-CAG empty virus (number: 11 in the control group and 11 in the experimental group).

[0106] 4. After the injection, apply erythromycin eye ointment once, and place the mouse on a heating pad to keep it warm and observe until it wakes up.

[0107] 5. The mice were anesthetized and mydriasis was performed 3, 7, and 14 days after injection, and 50 μL of 100 g / L fluorescein sodium injection was injected intraperitoneally for fundus fluorescein angiography. (Leakage rating reference: The pharmacology study of a new recombinant human VEGF receptor-fc fusion protein on experimental choroidal neovasc ularization)

[0108] 6. 14 days after injection, the mice were perfused and the choroids were taken for immunohistochemical analysis to count the choroidal angiogenesis.

[0109] The experimental results are as follows Figure 1 The results showed that after NPVF injection, the vascular leakage of the experimental group mice was significantly reduced compared with the control group mice ( Figure 1 AD). At the same time, the disease progression of the experimental group mice gradually improved over time, while the disease progression of the control group mice did not improve significantly ( Figure 1 E, F). On the other hand, the angiogenesis of the experimental group mice 14 days after the subretinal injection of AAV-CAG-NPVF virus was significantly reduced compared with the control group mice ( Figure 1 G, H). The above results indicate that NPVF has a significant visual protection ability in CNV model mice.

[0110] Example 3

[0111] Injection of NPVF peptide can effectively inhibit the activation of Iba1-positive microglia and suppress the inflammatory response of wet age-related macular degeneration:

[0112] 1. Construction of wet age-related macular degeneration model: Rho-hVEGF165 model mouse is a transgenic wet age-related macular degeneration model, which expresses exogenous hVEGF165 in the RPE cells of mice, promotes choroidal angiogenesis, and thus leads to wet age-related macular degeneration. Rho-hVEGF165 model mouse was constructed and relevant analysis was performed.

[0113] The results of immunofluorescence staining showed that the number of microglia (Iba1 positive cells) and blood vessels (IB4 positive) increased in Rho-hVEGF165 model mice at P14 and P28 ( Figure 2). This indicates that the inflammatory response and neovascularization in the retina of Rho-hVEGF165 mice are increased compared with those of wild-type mice, and the changes are more obvious over time, showing the core phenotype of wet age-related macular degeneration.

[0114] 2. Injection of NPVF peptide into the Rho-hVEGF165 model: Inject the NPVF peptide shown in SEQ ID No.2 (5 mg / mL, 2 μL / eye) into the retina of Rho-hVEGF 165 transgenic mice at P0 to verify its protective effect in the wet AMD mouse model of Rho-hVEGF165 transgenic mice. The control group was an equal volume of PBS solution. The experimental results showed that the inflammation level of Rho-hVEGF165 transgenic mice after NPVF injection was significantly reduced at P14, indicating that the retinal inflammation level of mice in the NPVF protection group was lower than that of mice in the control group ( Figure 3 ).

[0115] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a neuropeptide VF (NPVF), a nucleic acid molecule encoding the polypeptide or a promoter thereof in the preparation of a medicament for preventing and / or treating neovascular eye diseases and / or related ophthalmic diseases induced by inflammation.

2. The use according to claim 1, characterized in that The promoter includes one or a combination of NPVF expression vector, exogenous NPVF, naked nucleic acid molecules of NPVF coding sequence, liposome-encapsulated nucleic acid molecules of NPVF coding sequence, or NPVF precursor protein or conjugate or complex that can be converted into NPVF in vivo.

3. The use according to claim 2, characterized in that: The NPVF expression vector includes a viral vector and / or a non-viral vector.

4. The use according to claim 3, characterized in that The viral vector includes one or a combination of an adeno-associated virus vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector or a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, a herpes virus vector, a poxvirus vector or a lentivirus vector.

5. The use according to claim 4, characterized in that The non-viral vector includes one or a combination of DNA vectors, nanoparticles, cationic polymers, exosomes, extracellular vesicles or liposomes.

6. The use according to claim 5, characterized in that The DNA vector comprises one or a combination of a plasmid vector, a cosmid vector, a phagemid vector or a human artificial chromosome.

7. The use according to claim 1, characterized in that The neovascular eye diseases and inflammation-induced related ophthalmic diseases include one or a combination of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal infection, neovascular glaucoma, uveitis or retinitis.

8. Use of a pharmaceutical composition in the preparation of a medicament for preventing and / or treating neovascular eye diseases and / or related ophthalmic diseases induced by inflammation, characterized in that: The pharmaceutical composition comprises: (1) a therapeutically effective amount of neuropeptide VF (NPVF), a nucleic acid molecule encoding the polypeptide, or a promoter thereof; (2) A pharmaceutically or immunologically acceptable carrier or excipient.

9. The use according to claim 8, characterized in that The administration routes of the pharmaceutical composition include one or a combination of intranasal, intrathecal, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, ocular, sublingual, oral, intracerebral or transdermal.

10. Use of a pharmaceutical preparation in the preparation of a drug for preventing and / or treating neovascular eye diseases and / or related ophthalmic diseases induced by inflammation, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to claim 8.

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