WO2023138066A1 - 抗egfr抗体的用途 - Google Patents

抗egfr抗体的用途 Download PDF

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WO2023138066A1
WO2023138066A1 PCT/CN2022/117106 CN2022117106W WO2023138066A1 WO 2023138066 A1 WO2023138066 A1 WO 2023138066A1 CN 2022117106 W CN2022117106 W CN 2022117106W WO 2023138066 A1 WO2023138066 A1 WO 2023138066A1
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neovascularization
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retinal
days
cetuximab
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French (fr)
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罗德伦
刘巾男
罗第祥
彭红卫
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景泽生物医药(合肥)有限公司
上海景泽生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention belongs to the field of medicine and relates to a new application of anti-EGFR antibody.
  • Neovascularization is a common pathological change in many eye diseases. It often occurs with the cornea, iris, choroid, and retina. The resulting diseases include corneal neovascularization, choroidal neovascularization, and retinal neovascularization. eration, AMD) (including wet AMD and dry AMD, and the pathological change of wet AMD is choroidal neovascularization), neovascular glaucoma, etc. New blood vessels can appear in almost all tissues in the eye, such as the cornea, iris ciliary body, choroid, retina, macula, and optic disc.
  • VEGF Vascular endothelial growth factor
  • Ranibizumab (trade name Lucentis) can specifically bind to VEGF-A, and has been approved by the US FDA for the treatment of wet age-related macular degeneration and diabetic macular edema.
  • VEGF Trap-Eye (Aflibercept or Eylea) is an anti-VEGF recombinant protein developed by Regeneron Pharmaceuticals in the United States.
  • DME diabetic macular edema
  • CFDA State Food and Drug Administration
  • Eylea for the treatment of adult diabetic macular edema
  • AMD age-related macular degeneration
  • AMD age-related macular degeneration
  • Epidermal growth factor receptor is widely distributed in mammalian epithelial cells and is a transmembrane glycoprotein with a total relative molecular weight of 170kDa.
  • EGFR is a member of the ErbB (ErbB 1-4) family, also known as ErbB1. Studies have shown that there are high or abnormal expressions of EGFR in many solid tumors. EGFR is related to tumor cell proliferation, angiogenesis, tumor invasion, metastasis and inhibition of apoptosis.
  • EGFR consists of three parts: the extracellular ligand-binding region, the hydrophobic transmembrane domain and the intracellular kinase region. Its intracellular structure contains a tyrosine kinase domain and a carboxyl-terminal tail with multiple autophosphorylation sites, belonging to the receptor tyrosine kinase family (receptor tyrosine kinase, RTKs); the extramembrane region is composed of a ligand binding site and two cysteine-rich regions, which can bind a variety of ligands with agonistic functions.
  • RTKs receptor tyrosine kinase
  • EGF or TGF- ⁇ After the extracellular EGF or TGF- ⁇ binds to EGFR, it forms a dimer, binds an ATP molecule, and activates EGFR's own tyrosine kinase activity causes autophosphorylation of several tyrosine sites in the intracellular kinase domain.
  • the downstream ras/raf/MAPK (mitogen activated protein kinase) cascade system and phosphoinositol kinase (phosphoinositolkinase) system are activated, becoming the affinity site for downstream signal transduction, and various signals involved in mitosis are transmitted from extracellular to intracellular, thereby effectively regulating the response of cells to external stimuli, cell proliferation, survival, adhesion, migration and differentiation, cell survival and growth.
  • the complex of EGFR and ligand enters the cell through pinocytosis, is degraded or recycled to the cell surface, and completes signal transduction. Therefore, once the function of this tyrosine kinase is blocked, it will lead to various diseases.
  • EGFR monoclonal antibodies compete with endogenous ligands to bind to EGFR, and produce anti-tumor effects by inhibiting the activation of tyrosine kinases and promoting the internalization of EGFR.
  • anti-EGFR monoclonal antibodies that have been approved for marketing include: Cetuximab, Nimotuzomab, Panitumumab, and Necitumumab. It is used to treat colorectal cancer, head and neck cancer and non-small cell lung cancer respectively.
  • Cetuximab is the leader.
  • cetuximab On February 12, 2004, the U.S. FDA first approved cetuximab (Cetuximab, trade name ), its indications are: combined with irinotecan for the treatment of advanced and refractory metastatic colorectal cancer; combined with radiotherapy and chemotherapy for the treatment of locally or locally advanced squamous cell carcinoma of the head and neck.
  • Cetuximab specifically binds to EGF receptors expressed on the surface of normal cells and various cancer cells, and competitively blocks the binding of EGF and other ligands, such as transforming growth factor- ⁇ (TGF- ⁇ ).
  • TGF- ⁇ transforming growth factor- ⁇
  • Cetuximab is an IgG1 monoclonal antibody against the EGF receptor.
  • TK tyrosine kinase
  • the purpose of the present invention is to provide a new application of anti-EGFR antibody.
  • the monoclonal antibody targeting EGFR has a good inhibitory effect on ocular neovascularization, especially fundus neovascularization, such as choroidal neovascularization and retinal neovascularization, and such antibodies can be used to treat diseases related to eye or retinal neovascularization, especially choroidal neovascularization and retinal neovascularization.
  • the present invention provides the application of anti-EGFR antibody in the preparation of drugs for preventing and/or treating diseases related to ocular neovascularization, especially diseases related to fundus neovascularization.
  • the fundus neovascularization-related diseases are preferably: choroidal neovascularization-related diseases or retinal neovascularization-related diseases.
  • the fundus neovascularization-related diseases include, but are not limited to: diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, periretinal phlebitis, age-related macular degeneration, high myopia macular hemorrhage, and central exudative retinopathy.
  • the anti-EGFR antibody is a monoclonal antibody targeting EGFR, including various anti-EGFR antibodies currently on the market or applying for marketing, such as cetuximab (Cetuximab), nimotuzumab (Nimotuzomab), panitumumab (Panitumumab) and necitumumab (Necitumumab), preferably cetuximab.
  • cetuximab cetuximab
  • nimotuzumab Nemotuzomab
  • panitumumab Panitumumab
  • necitumumab Necitumumab
  • the present invention also provides the application of anti-EGFR antibody in the preparation of medicine, wherein the medicine is used to reduce the area of retinal avascular area, inhibit ocular neovascularization and/or cause ocular neovascularization to subside.
  • the present invention also provides a treatment method for diseases related to ocular neovascularization, comprising administering a prophylactically and/or therapeutically effective amount of anti-EGFR antibody to an individual in need.
  • the anti-EGFR antibody or the pharmaceutical composition containing the anti-EGFR antibody can be administered to the whole, part or vitreous of the eye or the affected eye at a preventive and/or therapeutic effective dose.
  • the formulation may be any topical formulation suitable for ophthalmic administration.
  • anti-EGFR antibodies especially cetuximab
  • Fig. 1 is the administration of rats on the day of CNV modeling, and the FFA detection results of the PBS group 7 days and 14 days after modeling,
  • Figure 2 shows the FFA test results of the aflibercept group administered on the day of CNV modeling in rats, 7 days and 14 days after modeling;
  • Fig. 3 is administration on the same day of rat CNV modeling, and the FFA detection results of JZB09-5mg/ml group 7 days and 14 days after modeling;
  • Fig. 4 is the PBS group, aflibercept and JZB09-5mg/ml group, 7 days and 14 days fluorescent spot grading histogram;
  • Figure 5 PBS group, aflibercept and JZB09-5mg/ml group 7 days and 14 days FFA neovascular leakage area histogram;
  • Fig. 6 is a histogram of PBS group, aflibercept group, JZB09-5mg/ml group, JZB09-40mg/ml group, JZB09-70mg/ml group before administration (0d), after administration 7d, after administration 10d, after administration 14d;
  • Fig. 7 is a histogram of leakage area of FFA neovascularization in PBS group, aflibercept group, JZB09-5mg/ml group, JZB09-40mg/ml group, JZB09-70mg/ml group before administration (0d), after administration 7d, after administration 10d, after administration 14 days;
  • Figure 8 shows that 5 days after administration, that is, on the 17th day (D17) after the birth of the mice, the mice in the PBS group, the Aflibercept group, the JZB09-5mg/ml group, the JZB09-40mg/ml group, and the JZB09-70mg/ml group were killed, and the eyeballs were taken for retinal smears, stained with IB4, and taken with an automatic upright microscope (Axio imager Z2);
  • Figure 9 is a histogram of the percentage of retinal avascular area in the PBS group, aflibercept group, JZB09-5mg/ml group, JZB09-40mg/ml group, JZB09-70mg/ml group in the total retinal area in Figure 8.
  • anti-EGFR antibody refers to a monoclonal antibody targeting EGFR, which can bind to, compete with, and block the binding of ligands such as EGF and TGF- ⁇ .
  • the term "Cetuximab (Cetuximab)" trade name A recombinant human/mouse chimeric monoclonal antibody that can specifically bind to human epidermal growth factor receptor (EGFR) and competitively inhibit the binding of EGF and other ligands (such as transforming growth factor ⁇ ) to the receptor.
  • EGFR epidermal growth factor receptor
  • EGF and other ligands such as transforming growth factor ⁇
  • the indications for this drug approved worldwide are metastatic head and neck cancer, non-metastatic head and neck cancer, metastatic colorectal cancer, non-small cell lung cancer, etc.
  • the amino acid sequences of its heavy chain and light chain are shown in SEQ ID NO.1 and SEQ ID NO.2, and the preparation method is described in detail in WO96/40210.
  • the antibody described in the present invention includes the antibody itself, the analog of the antibody, and the pharmaceutical composition containing the antibody.
  • diseases related to ocular neovascularization or “diseases related to fundus neovascularization” refers to diseases that occur in the eye or fundus, and the formation of new blood vessels leads to the destruction of ocular structure and function, resulting in serious visual function damage. Neovascularization is a common pathological change in many important eye diseases.
  • choroidal neovascularization also known as subretinal neovascularization
  • CNV choroidal neovascularization
  • choroidal neovascularization is a proliferative blood vessel from the choroidal capillaries, which expands through the breach of Bruch's membrane, and proliferates between Bruch's membrane and retinal pigment epithelium, or between the neural retina and retinal pigment epithelium, or between the retinal pigment epithelium and the choroid.
  • Numerous diseases involving the RPE-Bruch's membrane-choriocapillary complex can lead to the formation of CNV.
  • Degeneration, inflammation, and trauma may cause retinal ischemia and hypoxia in the outer layer of the macula, thereby producing angiogenic factors to stimulate choroidal capillary neovascularization and form subretinal neovascularization.
  • angiogenic factors to stimulate choroidal capillary neovascularization and form subretinal neovascularization.
  • the symptoms recur, the macula is severely damaged, which can cause permanent visual impairment. Diagnosis is confirmed by fundus fluorescein angiography and morphology.
  • diseases related to choroidal neovascularization include age-related macular degeneration, high myopia macular hemorrhage, central exudative retinopathy, and the like.
  • Age-related macular degeneration is a pathological aging change in the structure of the macula. Can be divided into dry (non-exudative) or wet (exudative or neovascular) 2 types. Wet AMD is characterized by choroidal neovascularization. In the middle and late stages of disease development, after the pathological changes continue to aggravate, Bruch's membrane is ruptured, and choroidal capillaries enter under the RPE or subretinal neuroepithelium through the ruptured Bruch's membrane, forming choroidal neovascularization (CNV), which is also the most direct factor affecting vision.
  • CNV choroidal neovascularization
  • High myopia macular hemorrhage High myopia refers to myopia with a diopter greater than -6.00D. It is a common eye disease. Because of the pathological changes in the fundus, it is also called pathological myopia or degenerative myopia. Among them, macular hemorrhage is one of the important complications of high myopia, which is more common in clinical practice and often leads to severe damage to central vision.
  • Central exudative chorioretinopathy also known as idiopathic choroidal neovascularization
  • CEC Central exudative chorioretinopathy
  • Retinal neovascularization refers to retinal neovascularization on the inner surface of the inner limiting membrane of the retina and in the outer layer of the retina without “vessels" between the deep retinal capillary bed and the choroidal capillary bed. It originates from the vascular proliferation of the retinal capillary bed, is often accompanied by fibrous proliferation and degeneration, and can break through the vitreous membrane inward and proliferate into the vitreous, becoming the most common cause of vitreous hemorrhage.
  • diseases related to retinal neovascularization include diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal periphlebitis, and the like.
  • Diabetic retinopathy is a relatively serious microvascular complication, and its pathological features are mainly the formation of new blood vessels and the destruction of retinal blood-retinal barrier (BRB).
  • Diabetic retinopathy is one of the most common microvascular complications of diabetes. DR can be divided into proliferative diabetic retinopathy and non-proliferative diabetic retinopathy based on the presence or absence of abnormal new blood vessels emanating from the retina.
  • ROP Retinopathy of prematurity
  • Retinal vein occlusion is a common retinal vascular disease with a long course of disease, long-term retinal ischemia, and neovascularization induced after ischemia.
  • Eales disease also known as Eales disease
  • Eales disease initially manifests as retinal periphlebitis, and then manifests as vascular changes and neovascularization caused by retinal ischemia, and the final common outcome is recurrent vitreous hemorrhage, with or without retinal detachment.
  • prophylactically and/or therapeutically effective and “effective amount” refer to the amount of an agent effective to produce the desired pharmacological, therapeutic or prophylactic result.
  • a pharmacologically effective amount results in amelioration of one or more symptoms of the disorder, or prevents progression of the disorder, or causes regression of the disorder, or prevents the disorder.
  • a therapeutically effective dose in the methods described herein can be determined by the treating physician.
  • a physician can start treatment with the manufacturer's recommended dose of an anti-EGFR antibody and adjust it based on the physician's observations of the effect of treatment.
  • the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO.1
  • the light chain amino acid sequence is shown in SEQ ID NO.2.
  • the concentrations of 10mg/ml, 20mg/ml, 30mg/ml, 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, and 80mg/ml can be obtained by preparing or concentrating the stock solution.
  • Aflibercept injection purchased from Bayer Healthcare Co., Ltd., the specification is 40mg/ml/bottle
  • the rats were instilled with Medolid (compound tropicamide eye drops) to fully dilate the pupils, anesthetized with pentobarbital sodium, and the head of the rat was fixed in front of an ophthalmic laser photocoagulation instrument. Laser photocoagulation was used to build the CNV model. Each eye was irradiated with 6 laser points. Seeing bubbles in the fundus indicated that the Bruch’s membrane was broken, indicating that the photocoagulation was successful. Modeling and testing were performed on the right eye.
  • Medolid compound tropicamide eye drops
  • Negative control group PBS group
  • experimental group cetuximab, JZB group
  • JZB09-5mg/ml group positive control group (Aflibercept injection, Aflibercept);
  • Detection time after FFA detection, that is, 7 days (d) and 14 days (d) after modeling, a total of 2 times;
  • Grade 3 hyperfluorescence of the spot, mild fluorescein leakage, leakage does not exceed the edge of the spot; Grade 4: hyperfluorescence of the spot, significant leakage of fluorescein, leakage beyond the edge of the spot.
  • Detection time after FFA detection, 7 days and 14 days after modeling, a total of 2 times;
  • Leakage area of new blood vessels measure the leakage area of each new blood vessel, and calculate the total leakage area of each animal for comparison.
  • Rats were given drugs on the day of CNV modeling, and FFA was detected 7 days after modeling.
  • Figure 1 obvious laser spots and neovascular leakage were seen in the PBS group, indicating that the model was successfully established; 14 days after modeling, FFA was detected, as shown in Figures 1, 4 and 5.
  • the laser spot grading score increased, and neovascular leakage increased significantly, suggesting that 14 days after modeling compared with 7 days, the degree of laser-induced lesions continued to progress and had a tendency to aggravate.
  • BN rats Laser photocoagulation on the fundus of brown Norwegian rats (BN rats) induces choroidal neovascularization (Choroidal neovascularization, CNV).
  • CNV choroidal neovascularization
  • An animal model similar to human choroidal neovascularization can be established, and the effect of cetuximab on inhibiting choroidal neovascularization was tested under this model.
  • Rats were dripped with Medoli (compound tropicamide eye drops) to fully dilate the pupils, anesthetized with pentobarbital sodium, and the rats were
  • the head was fixed in front of the ophthalmic laser photocoagulation instrument, laser photocoagulation was used to make CNV models, and 6 laser points were irradiated to each eye. Seeing the formation of bubbles in the fundus indicated that the Bruch’s membrane was broken, indicating that the photocoagulation was successful. Modeling and testing were performed on the right eye.
  • Negative control group PBS group
  • experimental group cetuximab, JZB group
  • JZB09-5mg/ml group JZB09-40mg/ml group
  • JZB09-70mg/ml group positive control group (Aflibercept, Aflibercept);
  • Detection time after FFA detection, 7 days after modeling (administration 0 days), 7 days, 10 days, and 14 days after drug administration, a total of 4 times/eye;
  • Fluorescence spot grading method Level 1: No hyperfluorescence in the spot; Level 2: High fluorescence in the spot but no fluorescein leakage;
  • Grade 3 hyperfluorescence of the spot, mild fluorescein leakage, leakage does not exceed the edge of the spot; Grade 4: hyperfluorescence of the spot, significant leakage of fluorescein, leakage beyond the edge of the spot.
  • Detection time after FFA detection, 7 days after modeling (administration 0 days), 7 days, 10 days, and 14 days after drug administration, a total of 4 times/eye;
  • Leakage area of new blood vessels measure the leakage area of each new blood vessel, and calculate the total leakage area of each animal for comparison.
  • fluorescent spot grading new blood vessel leakage area observes the influence of test product on laser-induced choroidal angiogenesis in rats.
  • Rats were administered 7 days after CNV modeling, before administration (0d), 7 days after administration, 10 days after administration, and 14 days after administration, FFA detection was performed. Obvious laser spots and neovascular leakage were seen in the PBS group 7 days after modeling (before administration), indicating that the model was successfully established; 14 days after modeling (administration 7 days) FFA detection, as shown in Figure 6 and Figure 7, the laser spot grading score in the PBS group increased, and neovascular leakage increased, suggesting that the degree of choroidal neovascularization caused by laser was higher at 14 days after modeling compared with 7 days It continues to progress and tends to worsen.
  • the C57BL/6J mice on the 7th day after birth were placed in a closed oxygen box with the nursing mothers, and the oxygen concentration was maintained at 75% ⁇ 2%; after 5 days of breastfeeding, the 12th day (D12) mice and the nursing mothers were put back into the indoor standard environment (oxygen concentration 21%).
  • Dosing time the mice were put back into the indoor standard environment (oxygen concentration 21%) and then administered.
  • Mode of administration intravitreal injection.
  • Dosage 1 ⁇ l/eye.
  • Dosing frequency single dose.
  • mice Five days after the administration, that is, on the 17th day after the birth of the mice, the mice were sacrificed, and the eyeballs were taken for retinal smearing, stained with IB4, and taken with an automatic upright microscope (Axio imager Z2), as shown in Figure 8.
  • the aflibercept (IVA) administration group showed a significant decrease in the area of the avascular area, and there were no new blood vessels, and the difference was statistically significant, suggesting that aflibercept inhibited the formation of new blood vessels and could reduce the area of the avascular area.
  • each dose group of JZB09 showed a significant decrease in the area of the avascular area and no new blood vessels; compared with the aflibercept (IVA) administration group, the area of the avascular area in each dose group of JZB09 (IVJ) decreased significantly, and the difference was statistically significant, suggesting that the effect of reducing the avascular area of JZB09 may be stronger than that of aflibercept.
  • Erlotinib is a small molecule inhibitor acting on epidermal growth factor receptor (EGFR) tyrosine kinase, which inhibits the phosphorylation of intracellular tyrosine kinase associated with epidermal growth factor receptor (EGFR);
  • EGFR epidermal growth factor receptor
  • AG1478 See Xin J, Yang X, Tao Y, et al. EGFR inhibitor, AG1478, inhibits inflammatory infiltration and angiogenesis in mice with diabetic retinopathy [J].
  • Clinical and Experimental Pharmacology and Physiology, 2019, 46 Experimental results and Figs A-D it can be seen that the administration of AG1478 has no effect on avascular revascularization compared with the untreated fossa with OIR model, that is, there is no significant change in the area of the avascular area before and after administration; the neovascular clusters are significantly reduced compared with the control, and the neovascular clusters account for about 1.8% of the retinal area, while the control is about 4.4%.
  • Cetuximab see Example 3, the area of the avascular area decreased significantly after the administration of cetuximab; the area of the avascular area in the cetuximab group accounted for less than 10% of the retina, and the area of the avascular area in the PBS group was about 20%; the neovascular clusters were close to 0. In the PBS group, obvious new blood vessel clusters could be seen.
  • the application of anti-EGFR antibody can significantly reduce the area of retinal avascular area, while the application of erlotinib and AG1478 has no effect on the area of retinal avascular area;
  • retinal neovascularization after the administration of anti-EGFR antibody is close to 0, while the application of erlotinib and AG1478 can reduce neovascularization compared with the control group, but the neovascularization rate is 1.7% ⁇ 0.3, 1.8%, respectively.
  • Cetuximab is more effective in inhibiting the formation of new blood vessels.

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Abstract

本发明提供了一种抗EGFR抗体在制备预防和/或治疗与眼新生血管相关的疾病药物中的应用。

Description

抗EGFR抗体的用途 技术领域
本发明属于医药领域，涉及抗EGFR抗体的新用途。
背景技术
随着人年龄的增长、近视的加剧或是相关代谢性疾病的发展，当出现例如病理性近视、年龄相关性黄斑变性、糖尿病视网膜病变等眼底疾病时，眼底会新生一些不必要的血管。这些新生血管会侵犯脉络膜、视网膜感光细胞层等原本正常的眼底组织，使视功能受损，严重的还会导致失明。
新生血管形成是很多眼部疾病的共同病理改变，常发生与角膜、虹膜、脉络膜和视网膜，导致的疾病包括角膜新生血管、脉络膜新生血管、视网膜新生血管等病变，常见的由此导致的眼部疾患包括例如：糖尿病性视网膜病变(diabetic retinopathy，DR)、早产儿视网膜病变(retinopathy ofprematurity，ROP)、年龄相关性黄斑变性(age-related macular degeneration，AMD)(包括湿性AMD和干性AMD，湿性AMD的病理改变为脉络膜新生血管)、新生血管性青光眼等。新生血管可以在角膜、虹膜睫状体、脉络膜、视网膜、黄斑及视盘等几乎眼内所有组织中出现，它能够引起这些部位的组织出血、渗出及增生等一系列病理改变，因而造成眼球结构和功能的破坏，严重损害视功能。眼底新生血管性疾病患者人数在4000万以上，且随着人口老龄化程度加重，患者人数还在不断上升。眼底新生血管性疾病的患者几乎每月都要复诊或注射相关药物，否则视力的受损会严重影响生活。
血管内皮生长因子(VEGF)对于新生血管的形成起着重要作用。研究表明，VEGF是目前所知对血管新生最特异、最有效的生长因子。1990年代以来，以VEGF为靶标，通过阻断VEGF信号通路抑制眼底血管新生的药物成为开发的热点。雷珠单抗(Ranibizumab，商品名Lucentis)可特异性地结合VEGF-A，已获美国FDA批准用于治疗湿性老年黄斑变性和糖尿病性黄斑水肿。VEGF Trap-Eye(阿柏西普或Eylea)是美国Regeneron制药公司开发的抗VEGF重组蛋白，临床研究结果显示对DME(糖尿病性黄斑水肿)的疗效令人满意，2018年，国家食品药品监督管理总局(CFDA)已批准Eylea用于治疗成人糖尿病性 黄斑水肿(DME)。KH902(康柏西普，Conbercept)是成都康弘公司开发的针对老年黄斑变性(AMD)的VEGFR-Fc重组蛋白，2013年12月批准上市用于治疗AMD。尽管针对VEGF靶标的药物在临床上取得了很大的进展，由于血管新生受多种因子的调节，血管新生的调控是一个十分复杂的动态平衡过程。传统的单一抗VEGF治疗仍有局限性，因此开发新的治疗手段具有重要的临床意义。
表皮生长因子受体(EGFR)广泛分布于哺乳动物的上皮细胞，是一种总相对分子量为170kDa的跨膜糖蛋白，EGFR是ErbB(ErbB 1～4)家族的一员，又称作ErbB1。研究表明在许多实体肿瘤中存在EGFR的高表达或异常表达。EGFR与肿瘤细胞的增殖、血管生成、肿瘤侵袭、转移及细胞凋亡的抑制有关。
EGFR由细胞外的配体结合区、疏水跨膜结构域和细胞内的激酶区3个部分组成。其胞内结构包含1个酪氨酸激酶域(tyrosine kinase domain)和具有多个自身磷酸化位点的羧基末端尾，属于受体酪氨酸激酶家族(receptor tyrosine kinase，RTKs)；膜外区域由配体结合位点和2个富含半胱氨酸区域所构成，能结合具有激动功能的多种配体，细胞外的EGF或TGF-α与EGFR结合后，形成二聚体，结合1个ATP分子，激活EGFR自身酪氨酸激酶活性，使胞内激酶区的数个酪氨酸位点发生自身磷酸化。EGFR二聚体化和磷酸化后，激活下游的ras/raf/MAPK(mitogen activated protein kinase)级联系统和磷酸肌醇激酶(phosphoinositolkinase)系统，成为下游信号转导的亲和位点，与多种参与有丝分裂信号从细胞外传递到细胞内，从而有效调节细胞对外界刺激的反应、细胞增生、存活、黏附、迁移和分化、细胞存活以及生长情况等。最后EGFR与配体的复合物通过胞饮作用进入细胞，被降解或再循环到细胞表面，完成信号转导。因此，一旦这种酪氨酸激酶的功能受阻，将会导致各种疾病产生。
随着对信号转导及其异常与肿瘤关系研究的不断深入，通过对信号转导干预治疗，即通过单克隆抗体、免疫毒素、酪氨酸激酶抑制剂、反义核酸等方式，针对信号转导通路发生异常的环节干预不正常的信号转导，从而达到抑制肿瘤生长的目的。目前最常见的治疗方式是：1、与EGFR结合、竞争和阻断EGF、TGF-α等配体的结合的单克隆抗体；2、酪氨酸激酶抑制剂，如吉非替尼、厄洛替尼等。EGFR单克隆抗体是与内源性配体竞争结合EGFR，通过抑制酪氨酸激酶的激活，促进EGFR内化等作用产生抗肿瘤效应，目前已批准上市的抗EGFR单克隆抗体有：西妥昔单抗(Cetuximab)、尼妥珠单抗(Nimotuzomab)、帕 尼单抗(Panitumumab)和耐昔妥珠单抗(Necitumumab)。分别用于治疗结直肠癌、头颈癌和非小细胞肺癌。在抗体类EGFR靶向药物中，西妥昔单抗(Cetuximab)独占鳌头。
美国FDA于2004年2月12日最早批准西妥昔单抗(Cetuximab，商品名为
Figure PCTCN2022117106-appb-000001
)，其适应症为：联合伊立替康治疗晚期和难治性转移性结直肠癌；与放化疗结合治疗局部或局部晚期的头部和颈部鳞状细胞癌。西妥昔单抗与表达于正常细胞和多种癌细胞表面的EGF受体特异性结合，并竞争性阻断EGF和其他配体，如α转化生长因子(TGF-α)的结合。西妥昔单克隆抗体是针对EGF受体的IgG1单克隆抗体，两者特异性结合后，通过对与EGF受体结合的酪氨酸激酶(TK)的抑制作用，阻断细胞内信号转导途径，从而抑制癌细胞的增殖，诱导癌细胞的凋亡，减少基质金属蛋白酶和血管内皮生长因子的产生。
一些研究(Hewing N J,Weskamp G,Vermaat J,et al.Intravitreal injection of TIMP3or the EGFR inhibitor erlotinib offers protection from oxygen-induced retinopathy in mice.[J].Invest Ophthalmol Vis,2013,54(1):864-870)表明，EGFR酪氨酸激酶抑制剂厄洛替尼的施用可以抑制小鼠视网膜的病理新生血管，这很可能是由于ADAM17和EGFR的失活；Xin J,Yang X,Tao Y,et al.EGFR inhibitor,AG1478,inhibits inflammatory infiltration and angiogenesis in mice with diabetic retinopathy[J].Clinical and Experimental Pharmacology and Physiology,2019,46通过注射链脲佐菌素建立了1型糖尿病早期视网膜病变小鼠模型，并建立了以缺氧诱导血管重建为特征的氧诱导终末期糖尿病视网膜病变(OIR)模型，在小鼠模型中验证了经典EGFR抑制剂AG1478(Tyrphostin AG 1478)可以降低DR发病机制的假设。数据表明，AG1478治疗可预防糖尿病小鼠视网膜功能障碍，减少视网膜结构和视网膜血管中线粒体结构的损害。
发明内容
本发明的目的是提供抗EGFR抗体的新用途。
根据本发明的一方面，不受任何理论束缚，以EGFR为靶点的单克隆抗体对眼新生血管，特别是眼底新生血管，例如脉络膜新生血管和视网膜新生血管具有很好的抑制作用，可将这类抗体用于治疗与眼或眼底新生血管相关的疾病，特别是脉络膜新生血管疾病和视网膜新生血管疾病。
因此，本发明提供抗EGFR抗体在制备预防和/或治疗与眼新生血管相关的疾病，特别是与眼底新生血管相关的疾病，的药物中的应用。
根据本发明，其中所述的眼底新生血管相关疾病优选的是：脉络膜新生血管相关疾病或视网膜新生血管相关疾病。
所述的眼底新生血管相关疾病包括但不限于:糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎、年龄相关性黄斑变性、高度近视黄斑出血、中心性渗出性视网膜脉络膜病变。
根据本发明，其中所述抗EGFR抗体为以EGFR为靶点的单克隆抗体，包括目前已上市的或正在申请上市的各种抗EGFR抗体，例如西妥昔单抗(Cetuximab)、尼妥珠单抗(Nimotuzomab)、帕尼单抗(Panitumumab)和耐昔妥珠单抗(Necitumumab)，优选的是西妥昔单抗。
根据本发明的另一方面，本发明也提供抗EGFR抗体在制备药物中的应用，其中所述药物用于减小视网膜无血管区面积、抑制眼新生血管形成和/或使眼新生血管消退。
根据本发明的再一方面，本发明也提供与眼新生血管相关的疾病的治疗方法，包括将预防和/或治疗有效量的抗EGFR抗体施用于有需要的个体。
根据本发明，可以将抗EGFR抗体或含有抗EGFR抗体的药物组合物以预防和/或治疗的有效剂量施用于眼部或患眼的整体、局部或玻璃体。制剂方式可以是任何适于眼部施用的局部制剂。
本发明首次证实抗EGFR抗体，特别是西妥昔单抗能有效抑制眼新生血管的形成，使视网膜无血管区域面积明显减小，新生血管消退，可用于治疗与眼或眼底新生血管形成相关的疾病。其效果优于VEGF靶点的单抗，也优于小分子酪氨酸激酶抑制剂。
附图说明
图1为大鼠CNV造模当天给药，造模后7天和14天PBS组FFA检测结果，
图2为大鼠CNV造模当天给药，造模后7天和14天阿柏西普组FFA检测结果；
图3为大鼠CNV造模当天给药，造模后7天和14天JZB09-5mg/ml组FFA 检测结果；
图4为PBS组、阿柏西普和JZB09-5mg/ml组，7天和14天荧光斑分级柱状图；
图5PBS组、阿柏西普和JZB09-5mg/ml组7天和14天FFA新生血管渗漏面积柱状图；
图6为PBS组、阿柏西普组、JZB09-5mg/ml组、JZB09-40mg/ml组、JZB09-70mg/ml组在给药前(0d)、给药后7d、给药后10d、给药后14dFFA荧光斑分级柱状图；
图7为PBS组、阿柏西普组、JZB09-5mg/ml组、JZB09-40mg/ml组、JZB09-70mg/ml组在给药前(0d)、给药后7d、给药后10d、给药后14天FFA新生血管渗漏面积柱状图；
图8为给药后5天，即小鼠出生后第17天(D17)时，PBS组、阿柏西普组、JZB09-5mg/ml组、JZB09-40mg/ml组、JZB09-70mg/ml组处死小鼠，取眼球行视网膜铺片，IB4染色，全自动正置显微镜(Axio imager Z2)拍片；
图9为图8中PBS组、阿柏西普组、JZB09-5mg/ml组、JZB09-40mg/ml组、JZB09-70mg/ml组视网膜无血管区域面积占视网膜总面积百分比柱状图。
具体实施方式
本发明中，术语“抗EGFR抗体”，指以EGFR为靶点的单克隆抗体，所述抗体可与EGFR结合、竞争和阻断EGF、TGF-α等配体的结合。
本发明中，术语“西妥昔单抗(Cetuximab)”，商品名为
Figure PCTCN2022117106-appb-000002
Figure PCTCN2022117106-appb-000003
一种重组人/鼠嵌合单克隆抗体，能够特异性结合人表皮生长因子受体(EGFR)，并竞争性抑制EGF及其它配体(如转化生长因子α)与该受体的结合，目前该药在全球范围内批准的适应症为转移性头颈癌，非转移性头颈癌，转移性结直肠癌，非小细胞肺癌等。其重链和轻链的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示，制备方法在WO96/40210中有详细描述。
本发明所述的抗体包括抗体本身、抗体的类似物，含有该抗体的药物组合物。
术语“与眼新生血管相关的疾病”,或”与眼底新生血管相关的疾病”,指发生在 眼部的或眼底的，由新生血管形成导致眼结构和功能的破坏,造成严重的视功能损害。新生血管形成是很多重要眼部疾病的共同病理改变。
本发明中，“脉络膜新生血管(CNV)”，又称视网膜下新生血管，是来自脉络膜毛细血管的增殖血管，通过Bruch膜的裂口而扩展，于Bruch膜与视网膜色素上皮之间、或神经视网膜与视网膜色素上皮之间、或位于视网膜色素上皮与脉络膜之间增殖形成。许多累及RPE-Bruch膜-脉络膜毛细血管复合体的疾病均可导致CNV的形成。变性、炎症和外伤等有可能造成黄斑部外层视网膜缺血缺氧，从而产生血管生成因子刺激脉络膜毛细血管新生，形成视网膜下新生血管。视网膜下新生血管出现的早期，可无自觉症状。随着其逐渐扩大、渗漏和破裂出血，可致视力减退、视物变形，出现中心或旁中心暗点。症状反复发作者，黄斑部受到严重破坏，可造成永久性视力障碍。通过眼底荧光血管造影和形态可确定诊断。
本发明中，“与脉络膜新生血管相关的疾病”包括年龄相关性黄斑变性、高度近视黄斑出血、中心性渗出性视网膜脉络膜病变等。
年龄相关性黄斑变性(age-related macular degeneration，AMD)是黄斑区结构的病理性衰老改变。可分为干性(非渗出性)或湿性(渗出性或新生血管性)2种类型。湿性AMD以脉络膜新生血管为突出特征，其是在疾病发展中后期，病理改变不断加重后，引起Bruch膜断裂，脉络膜毛细血管通过破裂的Bruch膜进入RPE下或视网膜神经上皮下，形成脉络膜新生血管(CNV)，这也是对视力影响的最直接因素。由于新生血管管壁的结构异常，常发生血管的渗漏和出血，进而导致黄斑部出血、水肿等引发一系列的改变，加重视力的下降甚至是突然视力大幅下降，是AMD导致失明的主要原因。
高度近视黄斑出血：高度近视指屈光度数大于-6.00D的近视，是一种常见眼病，因其眼底多有病理性改变，临床上又称为病理性近视或变性型近视，高度近视的病理改变主要包括：玻璃体变性，后巩膜葡萄肿，黄斑部漆裂纹，黄斑出血，黄斑部Fuchs斑等。其中，黄斑出血是高度近视的重要并发症之一，临床上较多见，常导致中心视力的严重损伤。
中心性渗出性脉络膜视网膜病变(central exudative chorioretinopathy,CEC)，也称为特发性脉络膜新生血管，是中青年人群中较为常见的眼科疾病之一，多单眼发病，患者在发病后会导致黄斑区脉络膜生成新生血管(表现为黄斑区孤 立的脉络膜新生血管(CNV))，新生血管区会出现反复性液体渗出和出血，引发黄斑部水肿，反复出血、渗出、瘢痕形成,严重影响患者的中心视力
本发明中，“视网膜新生血管(Retinal neo-vascularization,RNV)”，又称视网膜新生血管形成。视网膜内界膜的内面和视网膜深层毛细血管床与脉络膜毛细血管床之间无“血管”区域的视网膜外层组织有血管生长者,指视网膜新生血管。它来源于视网膜毛细血管床的血管增生,常伴随纤维增生和变性,并可向内突破玻璃体膜向玻璃体内增生,成为玻璃体出血的最常见原因。
本发明中，“与视网膜新生血管相关的疾病”包括糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎等。
糖尿病视网膜病变(DR)为一项比较严重的微血管并发症，其病理特征主要为新生血管形成以及视网膜血-视网膜屏障(BRB)被破坏等，糖尿病视网膜病变(DR)是糖尿病最常见的微血管并发症之一，是慢性进行性糖尿病导致的视网膜微血管渗漏和阻塞从而引起一系列的眼底病变，如微血管瘤、硬性渗出、棉絮斑、新生血管、玻璃体增殖、黄斑水肿甚至视网膜脱离。DR以是否有从视网膜发出的异常新生血管作为判断标准，可分为增殖性糖尿病视网膜病变和非增殖性糖尿病视网膜病变。
早产儿视网膜病变(ROP)是指在孕36周以下、低出生体重、长时间吸氧的早产儿，其未血管化的视网膜发生纤维血管瘤增生、收缩，并进一步引起牵拉性视网膜脱离和失明，一种增殖性视网膜病变，新生血管的形成在其发病中起主导作用。
视网膜静脉阻塞是常见的眼底血管病，其病程长，视网膜长期缺血，缺血后诱发新生血管形成。
视网膜静脉周围炎,又名Eales病,Eales病最初表现为视网膜静脉周围炎,继而表现为视网膜缺血所致的血管改变和新生血管化,最终的共同结局为反复发生的玻璃体出血,伴或不伴视网膜脱离。
本发明中，术语“预防和/或治疗有效”和“有效量”指试剂有效产生所期望的药理学、治疗或预防结果的量。药理学有效量使得病症的一种或多种症状改善，或防止病症进展，或引起病症消退，或预防病症。
本申请所述方法中的治疗有效剂量可以由治疗医师确定。例如，医师可以 使用抗EGFR抗体的生产厂商推荐剂量开始治疗，并根据医师对治疗效果的观察来进行调整。
本申请和实施例中提供了进一步的指导。此外，可以进行临床试验以确定在治疗患者群体时有效产生统计学上显著的治疗作用的剂量。
实施例
以下通过对本发明较佳实施方式的描述，详细说明但不限制本发明。
材料来源：
除非特别说明，本发明实施例中所述材料均由商业购买。
西妥昔单抗：德国默克里昂制药公司
商品名：爱必妥，规格100毫克/20毫升/瓶
或参考WO96/40210制备，抗体的重链氨基酸序列如SEQ ID NO.1所示，轻链氨基酸酸序列如SEQ ID NO.2所示。
Figure PCTCN2022117106-appb-000004
下述实施例中，浓度为10mg/ml、20mg/ml、30mg/ml、40mg/ml、50mg/ml、60mg/ml、 70mg/ml、80mg/ml可通过制备或将原液浓缩获得。
阿柏西普注射液：购自拜耳医药保健有限公司，规格为40mg/ml/瓶
复方托吡卡胺滴眼液，其商品名：美丽多(参天制药株式会社)
棕色挪威大鼠(BN大鼠)；斯贝福(北京)生物技术有限公司，
C57BL/6J小鼠：斯贝福(北京)生物技术有限公司
实施例1西妥昔单抗对激光诱发大鼠脉络膜血管新生的影响
一、实验方法
激光在棕色挪威大鼠(BN大鼠)眼底光凝，诱导眼底脉络膜血管新生(Choroidal neovascularization，CNV)，可建立与人类脉络膜血管新生类似的动物模型，因此，建立该动物模型并在该模型下进行相关实验。
1、造模
大鼠滴美多丽(复方托吡卡胺滴眼液)充分散瞳，使用戊巴比妥钠进行麻醉，将大鼠头部固定于眼科激光光凝仪前，激光光凝进行CNV造模，每眼照射6个激光点，见到眼底有气泡产生提示Bruch’s膜被击破，提示光凝成功。造模及检测均在右眼进行。
2、组别设计
阴性对照组(PBS组)，实验组(西妥昔单抗，JZB组)JZB09-5mg/ml组，阳性对照组(阿柏西普注射液，Aflibercept)；
动物数量：入组4只/组，3组共12只，所有造模及检测均在大鼠右眼进行。
3、给药
造模当天给药
给药方式：玻璃体注射
给药剂量：4μl/眼
给药频率：单次给药
4、检测
检测时间：FFA检测后，即造模后7天(d)，14天(d)，共2次；
荧光斑分级
荧光斑分级方法：1级：光斑没有出现高荧光；2级：光斑高荧光但没有 荧光素渗漏；
3级：光斑高荧光，轻度荧光素渗漏，渗漏不超过光斑边缘；4级：光斑高荧光，显著荧光素渗漏，渗漏超过光斑边缘。
新生血管渗漏面积
检测时间：FFA检测后即造模后7d，14d，共2次；
新生血管渗漏面积：测定各新生血管渗漏面积，计算每只动物渗漏总面积进行比较。
根据FFA检测、荧光斑分级、新生血管渗漏面积观察供试品对激光诱发大鼠脉络膜血管新生的影响。
二、实验结果
大鼠CNV造模当天给药，造模后7d FFA检测，如图1所示，PBS组可见明显激光斑及新生血管渗漏，提示模型成功建立；造模后14d FFA检测，如图1、4和5所示，PBS组可见激光斑分级评分升高，新生血管渗漏明显增加，提示造模后14d与7d相比，激光导致的病变程度持续进展且有加重趋势。
如图2、4、5所示，给药后7d，与PBS组相比，可见阿柏西普给药组激光斑分级评分及新生血管渗漏面积均明显下降，与PBS组相比差异有统计学意义，提示阿柏西普对激光导致的血管新生有较明显的抑制作用；给药后14d，阿柏西普给药组激光斑分级评分与PBS组相比未见统计学差异，但新生血管渗漏面积明显低于PBS组，且差异有显著统计学意义，阿柏西普对激光导致的血管新生有较明显的抑制作用。
如图3、4、5所示，给药后7d，与PBS组相比，JZB09组激光斑分级评分及新生血管渗漏面积略有下降；给药后14d，与PBS组相比，JZB09激光斑分级评分及新生血管渗漏面积均有明显下降，提示给药后14d，JZB09对激光导致的血管新生有较明显的抑制作用。
实施例2不同浓度西妥昔单抗对激光诱发大鼠脉络膜血管新生(CNV)的影响
一、实验方法
1、造模
激光在棕色挪威大鼠(BN大鼠)眼底光凝，诱导眼底脉络膜血管新生(Choroidal neovascularization，CNV)，可建立与人类脉络膜血管新生类似的动 物模型，并在该模型下进行西妥昔单抗抑制脉络膜新生血管作用实验。
大鼠滴美多丽(复方托吡卡胺滴眼液)充分散瞳，使用戊巴比妥钠进行麻醉，将大鼠
头部固定于眼科激光光凝仪前，激光光凝进行CNV造模，每眼照射6个激光点，见到眼底有气泡产生提示Bruch’s膜被击破，提示光凝成功。造模及检测均在右眼进行。
2、组别设计
阴性对照组(PBS组)，实验组(西妥昔单抗，JZB组)JZB09-5mg/ml组，JZB09-40mg/ml组，JZB09-70mg/ml组，阳性对照组(阿柏西普，Aflibercept)；
动物数量：入组8只/组，5组共40只，所有造模及检测均在大鼠右眼进行。
3、给药
造模后7d给药
给药方式：玻璃体注射
给药剂量：4μl/眼
给药频率：单次给药
4、检测
检测时间：FFA检测后即造模后7d(给药0d)，给药后7d、10d、14d，共4次/眼；
荧光斑分级
荧光斑分级方法：1级：光斑没有出现高荧光；2级：光斑高荧光但没有荧光素渗漏；
3级：光斑高荧光，轻度荧光素渗漏，渗漏不超过光斑边缘；4级：光斑高荧光，显著荧光素渗漏，渗漏超过光斑边缘。
新生血管渗漏面积
检测时间：FFA检测后即造模后7d(给药0d)，给药后7d、10d、14d，共4次/眼；
新生血管渗漏面积：测定各新生血管渗漏面积，计算每只动物渗漏总面积进行比较。
根据FFA检测、荧光斑分级、新生血管渗漏面积观察供试品对激光诱发大 鼠脉络膜血管新生的影响。
二、实验结果
大鼠CNV造模后7d给药，给药前(0d)、给药7d、给药10d、给药14d行FFA检测，PBS组造模后7d(给药前)见明显激光斑及新生血管渗漏，提示模型成功建立；造模后14d(给药7d)FFA检测，由图6及图7可见，PBS组激光斑分级评分升高，新生血管渗漏增加，提示造模后14d与7d相比，激光导致的脉络膜血管新生程度持续进展且有加重趋势。
给药后7d、10d、14d，与PBS组相比，由图6及图7可见，阿柏西普给药组激光斑分级评分及新生血管渗漏面积均明显下降，且差异有统计学意义，提示阿柏西普对激光导致的脉络膜血管新生有较明显的消退作用。
给药后7d、10d、14d，与PBS组相比，由图6及图7可见，供试品JZB09给药组激光斑分级评分及新生血管渗漏面积均明显下降，其中JZB09-40mg/ml剂量组、JZB09-70mg/ml剂量组荧光斑分级、新生血管渗漏面积差异有统计学意义，提示供试品西妥昔单抗-40mg/ml剂量组、西妥昔单抗-70mg/ml剂量组对激光导致的脉络膜血管新生有较明显的消退作用。
实施例3西妥昔单抗对小鼠视网膜新生血管的影响
一、实验方法
1.造模(OIR小鼠模型)
将出生后第7天(D 7)的C57BL/6J小鼠与哺乳母鼠一起放置于密闭氧箱中，氧浓度维持在75％±2％；哺乳5d后，将第12天(D12)小鼠与哺乳母鼠放回室内标准环境下(氧浓度21％)。
2、组别设计：
包括PBS阴性对照组，阿柏西普阳性对照组，实验组(西妥昔单抗，JZB09组，JZB09-5mg/ml组，JZB09-40mg/ml组，JZB09-70mg/ml组)，共5组，每组6只。
3.给药
给药时间：小鼠放回室内标准环境下(氧浓度21％)后给药。
给药方式：玻璃体注射。
给药剂量：1μl/眼。
给药频率：单次给药。
4.检测
给药后5天，即小鼠出生后第17天时，处死小鼠，取眼球行视网膜铺片，IB4染色，全自动正置显微镜(Axio imager Z2)拍片，如图8所示。
二、实验结果
如图8所示，PBS组造模后可见明显无血管区及新生血管，提示模型成功建立。
如图8、9所示，与PBS(CTRL)组相比，阿柏西普(IVA)给药组可见无血管区面积明显下降，且没有新生血管，且差异有统计学意义，提示阿柏西普抑制新生血管的生成，且可缩小无血管区面积。
如图8、9所示，与PBS(CTRL)组相比，JZB09(IVJ)各剂量组均可见无血管区面积明显下降且无新生血管；与阿柏西普(IVA)给药组相比，JZB09(IVJ)各剂量组无血管区面积明显下降，且差异具有统计学意义，提示JZB09缩小无血管区的作用可能强于阿柏西普。
对比例1西妥昔单抗与其他酪氨酸激酶抑制剂，对视网膜中心无血管区域及新生血管的作用效果比较。
厄洛替尼为作用于表皮生长因子受体(EGFR)酪氨酸激酶的小分子抑制剂，抑制与表皮生长因子受体(EGFR)相关的细胞内酪氨酸激酶的磷酸化；
AG1478((Tyrphostin AG-1478)是一种选择性的EGFR酪氨酸激酶抑制剂。
厄洛替尼，参见Hewing N J,Weskamp G,Vermaat J,et al.Intravitreal injection of TIMP3 or the EGFR inhibitor erlotinib offers protection from oxygen-induced retinopathy in mice.[J].Invest Ophthalmol Vis,2013,54(1):864-870)的实验结果及图3，a-f，EGFR抑制剂厄洛替尼的施用可以抑制小鼠视网膜的病理新生血管生成，在OIR后注射了厄洛替尼的野生型小鼠的Isolectin B4染色视网膜铺片显示，OIR后P17无血管区域面积没有差异，厄洛替尼：无血管区视网膜面积比例为22.0％±3.6，对照19.0％±1.7，其无血管区域面积给药前后无改变。新生血管簇比对照组显著减少,厄洛替尼1.7％±0.3，对照组：4.6％±1.1。
AG1478：参见Xin J,Yang X,Tao Y,et al.EGFR inhibitor,AG1478, inhibits inflammatory infiltration and angiogenesis in mice with diabetic retinopathy[J].Clinical and Experimental Pharmacology and Physiology,2019,46实验结果及FigA-D，可知，施用AG1478后，与未经治疗的窝伴OIR模型相比对无血管血运重建没有影响，即，给药前后无血管区域面积无显著改变；新生血管簇比对照明显减少，新生血管簇占视网膜的面积约1.8％，而对照约为4.4％。
西妥昔单抗：参见实施例3，西妥昔单抗施用后无血管区域面积明显下降；西妥昔单抗组无血管区域面积占视网膜比例小于10％，PBS组无血管区域面积约为20％；其新生血管簇接近于0。PBS组可见明显新生血管簇。
结论，对视网膜无血管区域，抗EGFR抗体(西妥昔单抗)的施用能显著降低视网膜无血管区域的面积，而厄洛替尼及AG1478的施用对视网膜无血管区面积无影响；对于新生血管，抗EGFR抗体(西妥昔单抗)的施用后视网膜新生血管生成接近于0，而厄洛替尼及AG1478的施用虽然较对照组新生血管有所减少，但新生血管分别为1.7％±0.3、1.8％，对抑制新生血管的生成明显西妥昔单抗更有效。

Claims (10)

  1. 抗EGFR抗体在制备预防和/或治疗与眼新生血管相关的疾病药物中的应用。
  2. 根据权利要求1所述的应用，其中所述的抗EGFR抗体为西妥昔单抗。
  3. 根据权利要求1-2任一项所述的应用，其中所述的与眼新生血管相关的疾病为与眼底新生血管相关的疾病。
  4. 跟权利要求3所述的应用，其中所述的与眼底新生血管相关的疾病为与脉络膜新生血管相关的疾病和/或与视网膜新生血管相关的疾病。
  5. 根据权利要求3所述的应用，其中所述的与眼底新生血管相关的疾病选自糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎、年龄相关性黄斑变性、高度近视黄斑出血和中心性渗出性视网膜脉络膜病变。
  6. 抗EGFR抗体在制备眼科药物中的应用，其中所述眼科药物用于减小视网膜无血管区面积、抑制眼新生血管形成和/或使眼新生血管消退。
  7. 根据权利要求6所述的应用，其中所述的抗EGFR抗体为西妥昔单抗。
  8. 根据权利要求1-7任一项所述的应用，其中所述抗EGFR抗体以局部眼用制剂施用于眼部或患眼。
  9. 根据权利要求8所述的应用，其中所述制剂施用于眼部或患眼的玻璃体。
  10. 根据权利要求1-9中任一项所述的应用，其中，所述西妥昔单抗的浓度为5-80mg/ml。
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