Preparation method and application of non-vegf-dependent cnv secondary subretinal fibrovascular membrane animal model

Abstract

The present application relates to the field of biotechnology, and particularly relates to a preparation method of an animal model of non-VEGF-dependent CNV secondary subretinal fibrovascular membrane and application thereof. The present application provides a method for evaluating the effectiveness of non-VEGF-targeted treatment of retinal choroidal neovascularization diseases based on a secondary laser-induced choroidal neovascularization subretinal fibrovascular membrane mouse model. The method can be used to evaluate the anti-neovascularization effect of other therapeutic drugs or treatment methods other than anti-VEGF drugs, explore the pathogenesis of refractory ocular fundus neovascularization diseases, and screen effective intervention methods.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing an animal model of subretinal fibrovascular membrane secondary to non-VEGF-dependent CNV and its application. Background Technology

[0002] Choroidal neovascularization (CNV) is a fundus disease affecting all ages and is one of the most common vision-threatening diseases. CNV frequently occurs in age-related macular degeneration (AMD), pathological myopia, retinal vein occlusion, retinal choroiditis, and diabetic retinopathy. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is the first-line clinical treatment, including monoclonal antibodies (ranibizumab, bevacizumab), fusion proteins (aflibercept, conbercept), and dual-target antibodies (fareximab). However, 30-50% of patients do not respond well to anti-VEGF treatment or even do not respond, eventually developing macular fibrosis, which severely affects the patient's visual quality.

[0003] While various anti-VEGF drugs are widely used clinically, different concentrations and intervention frequencies can alleviate some CNV disease progression to a certain extent. However, they also increase the risk of intraocular infection, increase the economic burden, and long-term follow-up studies have found that more than 50% of treated eyes eventually develop macular fibrosis and lose vision. Due to individual differences, the complexity of disease type and severity among patients, and the lack of response or poor response to anti-VEGF treatment, there are currently no better treatment options for these patients. Therefore, finding anti-angiogenic treatments beyond the VEGF target is one of the current focuses in the ophthalmic biomedical field. Establishing a model of choroidal neovascularization disease that is not dependent on VEGF can be used to evaluate the effectiveness of drugs / treatments beyond the VEGF target, providing a suitable tool for seeking effective treatments for patients who are not suitable for anti-VEGF drug therapy. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an animal model and evaluation method for non-VEGF-dependent CNV secondary subretinal fibrovascular membrane, which can be used to evaluate the effectiveness of drugs and / or treatments for choroidal neovascularization diseases other than VEGF targets.

[0005] This invention provides a method for preparing an animal model of subretinal fibrovascular membrane secondary to non-VEGF-dependent CNV, and the application of the animal model of subretinal fibrovascular membrane secondary to non-VEGF-dependent CNV in the preparation of non-VEGF therapeutic response products;

[0006] Furthermore, the method for preparing the non-VEGF-dependent CNV-secondary subretinal fibrosis model is as follows: taking the optic disc as the center, the animal's Bruch membrane is broken by a first laser to obtain the CNV lesion, and then a second laser is performed on the corresponding part of the CNV lesion to obtain the non-VEGF-dependent CNV-secondary subretinal fibrovascular membrane animal model.

[0007] The parameters of the first laser and / or the second laser are: energy 100~250mw, pulse interval 100~150ms, and spot diameter 50~200μm;

[0008] The first laser treatment can be performed at any point in the animal's adult or old age. Specifically, the animal used in this invention is a mouse or rat, and the animal's age at the time of the first laser irradiation is 2 to 14 months (adult) or 14 to 24 months (old). In a specific embodiment of this invention, the first laser treatment is performed when the mouse is 8 weeks old. In mouse biology, 4 weeks is usually converted to 1 month.

[0009] The second laser is applied 6 to 10 days after the first laser.

[0010] The first and / or second laser beams consist of 3 to 6 laser points; specifically, when the mouse is a small mouse, the number of laser points is 3 to 5 per mouse, and when the mouse is a large mouse, the number of laser points is 3 to 6 per mouse.

[0011] The distance between each laser point and the optic disc is ≥ two optic disc diameters;

[0012] In this invention, the diameter of the optic disc varies depending on the animal being studied, as detailed below:

[0013] The diameter of the optic disc in mice is 150 μm to 200 μm;

[0014] The diameter of the optic disc in rats is 400 μm to 500 μm;

[0015] The diameter of the rabbit's optic disc is 900μm~1600μm;

[0016] The first laser and / or the second laser are followed by a sterilization and disinfection step; the sterilization and disinfection drug is levofloxacin hydrochloride eye drops.

[0017] Furthermore, the animal includes mice, rats, rabbits, and / or monkeys; in a specific embodiment of the invention, the animal is a mouse.

[0018] This invention provides the application of the non-VEGF-dependent CNV-secondary subretinal fibrovascular membrane animal model prepared by the aforementioned method in evaluating the efficacy of non-VEGF-targeted drugs and / or combinations of VEGF-targeted and non-VEGF-targeted drugs in the treatment of choroidal neovascularization. The evaluation adopts the following steps:

[0019] A control group and a treatment group were set up to treat the animal model of non-VEGF-dependent CNV secondary subretinal fibrovascular membrane. The effectiveness of the non-VEGF-targeted drug and / or the combination of VEGF-targeted and non-VEGF-targeted drugs in the treatment of choroidal neovascularization was determined based on whether the difference in the severity of pathological neovascularization between the control group and the treatment group was significant.

[0020] The control group was treated with recombinant human IgG1 protein.

[0021] The treatment group consists of treatments using non-VEGF target drugs and / or a combination of VEGF target and non-VEGF target drugs.

[0022] Furthermore,

[0023] The VEGF-targeting drugs include: aflibercept, ranibizumab, bevacizumab, busizumab, faraximab, conbercept, and / or pergaratne. The anti-VEGF drugs specifically used in this project include aflibercept (Eylea) and faraximab (Faricimab). The monoclonal antibody Ang-2 drug used in this invention is nevaximab (Nesvacumab).

[0024] The non-VEGF target drugs include: anti-Ang-2 drugs, anti-PDGF drugs and / or anti-FGF drugs; further, the anti-Ang-2 drugs include monoclonal antibody Ang-2 drugs, and the monoclonal antibody Ang-2 drugs include nevaxumab.

[0025] The combination drugs targeting VEGF and non-VEGF targets include fareximab.

[0026] Indicators of the severity of the pathological neovascularization include: the area of ​​subretinal fibrovascular membrane lesions and / or CD31. + Area of ​​newly formed blood vessels.

[0027] In the method described in this invention, the animal model of non-VEGF-dependent CNV secondary subretinal fibrovascular membrane is treated between the 7th and 20th day after the second laser treatment, preferably the 14th day.

[0028] In the method described in this invention, if the control group is compared with the treatment group, the area of ​​subretinal fibrovascular membrane lesions and / or CD31 +A significant decrease in neovascularization area indicates that non-VEGF target drugs and / or a combination of VEGF target and non-VEGF target drugs are effective in treating choroidal neovascularization.

[0029] In this invention, the choroidal neovascular diseases include diabetic retinopathy, retinal vein occlusion, age-related macular degeneration, pathological myopia, angiostriate syndrome and other degenerations, ocular inflammation, and / or ocular tumors; this invention does not limit these.

[0030] The method described in this invention for evaluating the effectiveness of non-VEGF-targeted treatments for choroidal neovascularization is based on a mouse model of subretinal fibrovascular membrane induced by secondary laser therapy. Aflibercept (representative anti-VEGF drug), fareximab (representative anti-VEGF + anti-Ang-2 dual-target drug), and nevazumab (representative anti-Ang-2 single-target drug) were administered intravitreally at different time points and concentrations, and their anti-angiogenic and anti-fibrotic effects were compared. Results showed that early, high-concentration intervention was more effective. Administration 7-13 days after the secondary laser therapy is suitable for testing the efficacy of dual (multi-)targeted treatments targeting VEGF and other non-VEGF targets for retinal choroidal neovascularization. Administration 14 days or later after the secondary laser therapy is suitable for testing the efficacy of treatments targeting targets other than VEGF for retinal choroidal neovascularization.

[0031] The present invention optimizes and experimentally verifies the timing of drug treatment, demonstrating that the method constructed after treatment at the timing described in this application is most effective against diseases treated with VEGF and other adjuvant therapies.

[0032] In this invention, other methods were used to prepare CNV-secondary subretinal fibrovascular membrane models. It was found that CNV-secondary subretinal fibrosis models prepared by other methods have the following defects in anti-VEGF drug response studies: (1) Laser-induced CNV models in adult mice cannot test resistance to anti-VEGF treatment; (2) Laser-induced CNV in aged mice (12-18 months) has certain resistance to low-dose aflibercept (2 μg / eye). Since the binding affinity of aflibercept to mouse VEGF is lower than that to human VEGF, this dose is lower than the conventional clinical dose, and therefore cannot accurately reflect the actual clinical response in humans; (3) The use of aged mouse models is time-consuming and costly.

[0033] Advantages of the method described in this invention:

[0034] (1) More obvious clinical features, closer to the fundus features of patients: The existing technology of laser-induced CNV in aged mice shows that the lesions are self-healing, and the lesions gradually disappear 3 to 4 weeks after laser treatment, which is inconsistent with the actual situation of clinical patients. The two-stage laser model provided by this invention shows that the lesions persist (>60 days, PMID: 32818091). In addition, the lesions in this model are manifested as subretinal fibrovascular membranes, which are more similar to the clinical features of wet AMD patients who do not respond well to anti-VEGF treatment. It can more accurately simulate the clinical characteristics and pathological process of human choroidal neovascularization, and provide a reliable model for the treatment research of choroidal neovascularization diseases (such as diabetic retinopathy, retinal vein occlusion, age-related macular degeneration, etc.).

[0035] (2) Wider application: Existing technologies show that laser-induced CNV in aged mice is resistant to low-dose VEGF antibodies (2 μg / eye), while the model of 8-week-old mice used in this invention shows varying degrees of VEGF resistance at different time points after the second laser treatment. On day 7 after the second laser treatment, the mice showed resistance to clinically high-dose VEGF antibodies (8 μg / eye); on day 14 after the second laser treatment, they showed strong resistance to high-concentration VEGF antibodies (40 μg / eye). Therefore, our model simulates different patient groups in clinical practice (patients who are effective, partially effective, and ineffective against clinically high-dose anti-VEGF drugs), and can be used to explore the pathogenesis and therapeutic targets of refractory choroidal neovascularization, evaluate and screen new treatment methods, etc.

[0036] (3) More precise evaluation of non-VEGF-targeted drugs: As mentioned earlier, this model showed strong resistance to high-concentration (40 μg / eye) VEGF antibodies on day 14 after the second laser treatment, simulating the clinically refractory patient population. Meanwhile, this study found that at this specific time point, drugs containing anti-non-VEGF (such as anti-Ang-2) components, such as fareximab and nevazumab, showed significant effectiveness in anti-angiogenesis and anti-fibrosis. This advantage indicates that this model can effectively eliminate interference from the VEGF pathway in the "refractory" pathological state where anti-VEGF therapy is ineffective, serving as a more ideal in vivo validation platform to highly specifically and accurately evaluate the anti-choroidal angiogenesis effects of novel drugs or combination therapies targeting non-VEGF.

[0037] (4) More convenient to operate: This evaluation method is simple to operate, low in cost and highly reproducible, and is suitable for the evaluation and testing of the effects of novel drugs against retinal choroidal neovascularization in preclinical trials.

[0038] This invention provides a method for evaluating the response to anti-angiogenic therapy in retinal choroidal neovascularization diseases based on a mouse model of subretinal fibrovascular membrane induced by secondary laser-induced choroidal neovascularization. This method can be used to evaluate the anti-angiogenic and anti-fibrotic effects of drugs targeting non-VEGF or other therapeutic agents or treatments, explore the pathogenesis of refractory fundus neovascular diseases, and screen for effective interventions. Attached Figure Description

[0039] Figure 1 The images show fundus photography and OCT results after the first and second laser treatments in a two-stage laser-induced CNV secondary subretinal fibrovascular membrane animal model. A represents fundus photography of the CNV lesion induced by the first laser treatment; B represents OCT of the CNV lesion induced by the first laser treatment; C represents fundus photography of the subretinal fibrotic lesion induced by the second laser treatment; and D represents OCT of the subretinal fibrotic lesion induced by the second laser treatment.

[0040] Figure 2 Fundus photography and fluorescein fundus angiography (FFA) were performed at 10, 20, 30, and 40 days after the first or second laser-induced CNV secondary subretinal fibrovascular model. A shows fundus photography (ascending) and FFA (descending) at different time points after the lesion in the traditional modeling method (single laser-induced CNV); B shows fundus photography (ascending) and FFA (descending) at different time points after the second laser-induced subretinal fibrosis with hemorrhage; C shows fundus photography (ascending) and FFA (descending) at different time points after the second laser-induced subretinal fibrosis without hemorrhage.

[0041] Figure 3 Confocal images of a mouse model of CNV-secondary subretinal fibrovascular membrane, showing the staining results of RPE / choroidal flaps; where A is an overlay of Collagen-1, CD31, and DAPI co-staining; B is Collagen-1 staining; and C is CD31 staining.

[0042] Figure 4 This study shows the expression of VEGFa in the retina at 3, 7, 20, and 30 days after the first or second laser-induced retinal injury. A represents the statistical analysis of VEGFa expression in the retina at different time points after the first laser treatment; B represents the statistical analysis of VEGFa expression in the retina at different time points after the second laser treatment. Mean ± SD, one-way ANOVA, n=6, *P<0.05, ***P<0.001;

[0043] Figure 5 This demonstrates the formation of large vessels (arterial type) in lesions during the late stage (20 days) of a two-stage laser-induced subretinal fibrovascular membrane model; where A represents Collagen-1. +(Pink) Subretinal fibrosis lesion; B represents CX3CR1 in the lesion. + (GFP) + (Green) infiltrating cells; C is αSMA + (Red) Vascular smooth muscle cells and myofibroblasts; arrows indicate arterial neovascularization in the lesion; D is a co-stained overlay image.

[0044] Figure 6 The experimental flowchart is shown. Immediately after the second laser treatment (S0), 7 days (S7), and 14 days (S14), IgG1 Fc, low dose 8 μg / μL / eye (Eylea (8 μg)), and high dose 40 μg / μL / eye aflibercept (Eylea (40 μg)) were injected into the vitreous cavity. After 10 days, the eyeballs were collected for RPE / choroidal smear staining to detect the reduction rate of neovascularization and fibrosis area in the lesions.

[0045] Figure 7 The control group, low-dose group, and high-dose group were compared to those treated with secondary laser at different times, showing CD31 levels. + Confocal images showing changes in the area of ​​neovascularized lesions; where A represents CD31 levels in lesions of the control group, low-dose group, and high-dose group immediately after secondary laser treatment. + Confocal images of neovascularization; B shows CD31 levels in lesions of the control group, low-dose group, and high-dose group 7 days after the second laser treatment. + Confocal images of neovascularization; C represents CD31 levels in lesions of the control group, low-dose group, and high-dose group 14 days after the second laser treatment. + Confocal images of newly formed blood vessels;

[0046] Figure 8 The control group, low-dose group, and high-dose group were compared to those treated with secondary laser at different times, showing CD31 levels. + Statistical analysis of the area of ​​neovascularized lesions; where A represents the CD31 concentration in lesions of the control group, low-dose group, and high-dose group immediately after secondary laser treatment. + Statistical chart of neovascularization area; B represents CD31 levels in lesions of the control group, low-dose group, and high-dose group 7 days after the second laser treatment. + Statistical chart of neovascularization lesion area; C represents CD31 levels in lesions of the control group, low-dose group, and high-dose group 14 days after the second laser treatment. + Statistical map of neovascularization lesion area; mean ± SD, one-way ANOVA, n = 35-47 lesions / 9-12 eyes / 8-10 mice / group, **P<0.01, ***P<0.001, ****P<0.0001; D represents CD31 at different time points in the low-dose and high-dose groups compared to the control group. + The reduction in the area of ​​neovascularization lesions;

[0047] Figure 9Collagen-1 was shown in the control group, low-dose group, and high-dose group, with different administration times of the secondary laser. + Confocal images of subretinal fibrosis; where A is the confocal image of subretinal fibrosis in the control group, low-dose group and high-dose group immediately after the second laser treatment; B is the confocal image of fibrotic lesions in the control group, low-dose group and high-dose group 7 days after the second laser treatment; C is the confocal image of fibrotic lesions in the control group, low-dose group and high-dose group 14 days after the second laser treatment.

[0048] Figure 10 Collagen-1 was shown in the control group, low-dose group, and high-dose group, with different administration times of the secondary laser. + Statistical analysis of the area of ​​subretinal fibrosis lesions; where A represents the control group, low-dose group, and high-dose group receiving Collagen-1 immediately after secondary laser treatment. + A) Statistical graph of subretinal fibrosis lesion area; B) Statistical graph of fibrosis lesion area in the control group, low-dose group and high-dose group 7 days after secondary laser treatment; C) Statistical graph of fibrosis lesion area in the control group, low-dose group and high-dose group 14 days after secondary laser treatment; Mean ± SD, one-way ANOVA, n = 35-47 lesions / 9-12 eyes / 8-10 mice / group, **P<0.01, ****P<0.0001; D) The reduction rate of subretinal fibrosis lesion area in the low-concentration and high-concentration groups at each time point compared with the control group;

[0049] Figure 11 The flowchart of the non-VEGF drug intervention experiment is shown. IgG1 Fc (6 μg / μL / eye), Faricimab (6 μg / μL / eye), and Nesvacumab (3 μg + IgG1 Fc (3 μg) / μL / eye) were injected intravitreally on days 7 (S7) and 14 (S14) after the second laser treatment. Ten days later, eyeballs were collected for RPE / choroidal smear staining to detect the reduction rate of neovascularization and fibrosis area in the lesions.

[0050] Figure 12 The control group and the Faricimab and Nesvacumab groups were shown to have CD31 levels at different times after secondary laser administration. + Confocal images showing changes in the area of ​​neovascularized lesions; where A represents the CD31 concentration in lesions of the control group treated with Faricimab and Nesvacumab 7 days after the second laser treatment. + Confocal images of neovascularization; B shows CD31 levels in lesions of the control group treated with Faricimab and Nesvacumab 14 days after the second laser treatment. + Confocal images of newly formed blood vessels;

[0051] Figure 13 The control group and the Faricimab and Nesvacumab groups were shown to have CD31 levels at different times after secondary laser administration. + Statistical analysis of the area of ​​neovascularized lesions; where A represents the CD31 concentration in lesions of different groups treated 7 days after secondary laser therapy. + Statistical chart of neovascularization lesion area; B represents CD31 levels in lesions of different groups treated 14 days after secondary laser therapy. + Statistical chart of neovascularization lesion area; mean ± SD, one-way ANOVA, n = 37-40 lesions / 10 eyes / 6-7 mice / group, **P<0.01, ***P<0.001; C represents CD31 at different time points in the Faricimab and Nesvacumab treatment groups compared to the control group. + The reduction in the area of ​​neovascularization lesions;

[0052] Figure 14 The control group and the Faricimab and Nesvacumab groups were shown to be administered Collagen-1 at different times during the second laser treatment. + Confocal images of subretinal fibrosis; where A represents the control group treated with Collagen-1 7 days after the second laser treatment, and the Faricimab and Nesvacumab groups. + Confocal images of subretinal fibrosis lesions; B shows the Collagen-1 group treated 14 days after the second laser treatment, and the Faricimab and Nesvacumab groups. + Confocal images of subretinal fibrosis lesions;

[0053] Figure 15 The control group and the Faricimab and Nesvacumab groups were shown to be administered Collagen-1 at different times during the second laser treatment. + Statistical analysis of the area of ​​subretinal fibrosis lesions; where A represents the area of ​​Collagen-1 in lesions of different groups treated 7 days after secondary laser therapy. + Statistical chart of subretinal fibrosis lesion area; B represents the Collagen-1 concentration in lesions of different groups treated 14 days after the second laser treatment. + Statistical map of subretinal fibrosis lesion area; mean ± SD, one-way ANOVA, n = 37-40 lesions / 10 eyes / 6-7 mice / group, *P<0.05, **P<0.01; C represents the difference in Collagen-1 at different time points between the Faricimab and Nesvacumab treatment groups compared to the control group. + The reduction in the area of ​​subretinal fibrosis lesions. Detailed Implementation

[0054] This invention provides an animal model and evaluation method for non-VEGF-dependent CNV secondary subretinal fibrovascular membrane. This model can be used to evaluate the effectiveness of drugs and / or treatments for choroidal neovascularization diseases other than those targeting VEGF. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0055] (1) The control group consisted of: in the anti-VEGF example: IgG1 Fc antibody (8 μg / μL / eye), or IgG1 antibody (8 μg) or IgG1 8 μg; in the anti-non-VEGF (anti-Ang-2) example: IgG1 Fc antibody (6 μg / μL / eye), or IgG1 antibody (6 μg) or IgG1 6 μg;

[0056] (2) The low-dose group was: Eylea aflibercept, 8 μg / μL / eye, or Eylea (8 μg) or Eylea 8 μg;

[0057] (3) The high-dose group was: Eylea aflibercept, 40 μg / μL / eye, or Eylea (40 μg) or Eylea 40 μg;

[0058] (4) The anti-VEGF+anti-Ang-2 dual-target group is: Faricimab, 6 μg / μL / eye, or Faricimab (6 μg) or Faricimab 6 μg;

[0059] (5) The anti-Ang-2 single target group is: Nesvacumab (3μg) + IgG1 Fc antibody (3μg) / μL / eye, or Nesvacumab + IgG1 (6μg).

[0060] The optic disc diameter refers to the maximum width of the optic nerve head (optic disc) in the horizontal or vertical direction across the fundus. It is an important anatomical parameter commonly used in ophthalmic examinations and diagnoses. Within the normal range, the optic disc diameter is divided into horizontal and vertical diameters; these diameters vary depending on individual differences, but generally the horizontal diameter is approximately 1.5–1.8 mm, and the vertical diameter is approximately 1.7–2.0 mm. The optic disc diameter referred to in this invention is the horizontal diameter.

[0061] 40μg / μL / eye means that the effective dose of 1μL of medication in one eye is 40μg.

[0062] n = 35-47 lesions / 9-12 eyes / 8-10 mice / group, meaning that each group has 8-10 mice undergoing secondary laser-induced subretinal fibrotic vascular membrane formation, of which 9-12 eyes undergo RPE / choroidal patch staining, and the number of lesions obtained after laser treatment is 35-47 (generally, the number of lesions obtained is the number of laser points); importantly, in this description, the specific number of eyes refers to the specific number of eyes that underwent RPE / choroidal patch staining, and other similar descriptions in this invention have the same meaning as here.

[0063] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0064] Example 1: Establishment of a laser-induced subretinal fibrovascular membrane animal model

[0065] I. Animal Selection and Preparation

[0066] The C57BL / 6J mice (8 weeks old, weighing 18-22g, male) used in this experiment were provided by Hunan Silek Jingda Company. All animals had quarantine certificates and were housed in individually ventilated cages (IVCs) in a specific pathogen-free (SPF) environment with a 12-hour day-night cycle and free access to food and water. The animal experimental protocol used in this experiment was approved by the Animal Welfare and Ethics Committee of Aier Eye Hospital (Reference No.: AEI20230016). The animal use statement of the Association for Research in Vision and Ophthalmology (ARVO) was followed.

[0067] II. Establishment of an animal model of laser-induced CNV secondary to subretinal fibrovascular membrane

[0068] Intraperitoneal anesthesia was administered at the following dosage: salbutamol (37.5 mg / kg) + xylazine (5 mg / kg). Before laser induction, compound tropicamide eye drops (containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride) were used to dilate the pupils, and promecaine hydrochloride was used for topical anesthesia. Sodium carboxymethyl cellulose was used to moisten the ocular surface.

[0069] A two-stage laser-induced CNV-secondary subretinal fibrotic vascular membrane mouse model was established using the IRIDEX 577 ophthalmic laser photocoagulation system.

[0070] On day D0, the mice were given the first laser treatment. After complete anesthesia, a coverslip was placed on the ocular surface to induce choroidal neovascularization (CNV).

[0071] 1. Laser parameters: energy 100mv~250mv, pulse interval 100ms~150ms, spot diameter 50μm~200μm.

[0072] 2. Laser application: Centering on the optic disc, adjust the eyepiece in the slit lamp to focus the laser beam under the retina, avoiding blood vessels. Depress the pedal to fire the laser; the immediate appearance of a bright yellow bubble indicates successful Bruch's membrane rupture. Select 3-4 laser points on the top, bottom, left, and right sides, each at least two optic disc diameters away from the optic disc tip. Figure 1 and Figure 2 ).

[0073] After the laser treatment, administer levofloxacin hydrochloride eye drops to prevent infection, and sodium carboxymethyl cellulose to keep the ocular surface moist. Place the animal on a warming blanket until it recovers from anesthesia.

[0074] Second laser treatment: 6 to 10 days after the first laser treatment, a second laser is performed on the lesion site induced by the first laser. The parameters and methods are the same as above. The appearance of bubbles or subretinal hemorrhage at the laser site is considered a successful induction.

[0075] III. Evaluation of Model Success

[0076] Following the above technical approach, a mouse model of CNV-induced subretinal fibrovascular membrane (SNV) induced by secondary laser was successfully established. A bright yellow bubble was observed to form after the first laser treatment, indicating successful Bruch's membrane rupture. Figure 1 (A and B in the text); 7 days after photocoagulation, a second laser treatment is performed on the CNV lesion. The appearance of bubbles or subretinal hemorrhage at the laser site is considered a successful induction. Figure 1 (C and D in the text).

[0077] 1. Retinal fluorescein angiography: A mouse model of subretinal fibrotic vascular membrane was induced by a single laser or by a double laser. Fluorescence angiography was performed 10, 20, 30, and 40 days after the first or second laser treatment to detect fluorescence leakage in the retinal lesions.

[0078] 2. RPE / choroidal smear staining: Mouse eyeballs were collected at 10, 20, 30, and 40 days after the second laser treatment for RPE / choroidal immunofluorescence smear staining to detect the area of ​​neovascularization (CD31) and subretinal fibrosis (Collagen-1) in the lesions. The specific methods are as follows:

[0079] ① Separation of RPE / choroidal tissue: The eyeball was fixed in 2% paraformaldehyde (PFA) at room temperature for 2 hours, washed twice with ice-cold PBS, and the eyeball was dissected in ice-cold PBS to separate the retina from the RPE / choroidal tissue. The RPE / choroid was then transferred to a 96-well plate and washed with PBS.

[0080] ② Blocking and permeabilization: Prepare PBS solution containing 10% sheep serum, 2% BSA and 0.1% Triton X-100, and add 100 μL to a 96-well plate to cover RPE / choroid tissue. Block and permeabilize on a shaker at room temperature for 2 hours, and wash 3 times with PBST for 5 minutes each time.

[0081] ③ Primary antibody incubation: Add a mixture of primary antibodies diluted with 2% BSA, including rabbit collagen-1 (1:200) and rat CD31 (1:100), incubate overnight at 4°C, wash 3 times with PBST for 5 minutes each time.

[0082] ④ Secondary antibody incubation: Add mixed secondary antibodies diluted with 2% BSA, including goat-anti-rabbit 594 (1:200) and goat-anti-rat 488 (1:200), incubate at room temperature for 2 hours, wash 3 times with PBST, 5 minutes each time.

[0083] ⑤ Nuclear staining: Add DAPI (4′, 6-diamidino-2-phenylindole, 1:5000), incubate at room temperature for 5 minutes, wash 3 times with PBST, 5 minutes each time.

[0084] ⑥ RPE / choroid preparation: Place the stained RPE / choroid onto a coverslip containing an anti-fluorescence quencher, scleral side up. Trim away muscle tissue and optic nerve, and cut the eyecup into a four-leaf clover shape along the edge. After flattening the RPE / choroid tissue, press the slide on top and seal it with nail polish. Observe and photograph using a Zeiss LSM 880 laser scanning confocal microscope. Quantitatively analyze the area of ​​subretinal fibrosis and neovascularization using ImageJ software to evaluate the anti-angiogenic and anti-fibrotic effects of drugs at different time points and concentrations.

[0085] III. Results

[0086] 1. Fluorescein angiography: In a single laser-induced CNV model, significant fluorescein leakage was observed at the lesion site 10 days after laser treatment, gradually decreasing significantly from 20 to 40 days post-laser treatment. Figure 2 (A) In the two-stage laser model, significant fluorescein leakage persisted at different time points in lesions with and without subretinal hemorrhage. Figure 2 (B and C in the text).

[0087] 2. Immunofluorescence staining of RPE / choroidal planks: Immunofluorescence staining of mouse eyeballs with RPE / choroidal planks showed that RPE / choroidal planks stained with Collagen-1... + A large number of CD31 were detected in subretinal fibrosis lesions. + blood vessel signal ( Figure 3 ).

[0088] The above results suggest that the secondary laser-induced retinal injury model we established is a more clinically relevant animal model of CNV-induced subretinal fibrovascular membrane.

[0089] Example 2: Secondary laser induction does not affect retinal expression of VEGGA in the subretinal fibrovascular membrane.

[0090] 1. Testing:

[0091] Using mouse models of subretinal fibrotic vascular membrane induced by single laser-induced CNV or double laser-induced CNV, retina were collected at 3, 7, 20 and 30 days for RT-PCR detection of vascular endothelial growth factor A (VEGFa) expression.

[0092] The method of inducing CNV using a single laser is as described in the first laser step of Example 1. After the first laser treatment, levofloxacin hydrochloride eye drops are administered to prevent infection, sodium carboxymethyl cellulose is used to maintain ocular surface moisture, and the animal is placed on a warming blanket to await anesthesia recovery.

[0093] The mouse model of subretinal fibrotic vascular membrane induced by secondary laser is the mouse model of CNV secondary subretinal fibrotic vascular membrane induced by secondary laser in Example 1.

[0094] 2. Results:

[0095] VEGFa expression in the retina of a mouse model undergoing one or two stages of laser treatment: VEGFa expression began to increase significantly 3 days after the first laser treatment, reaching its peak on day 7, and then decreased significantly on days 20 and 30, with statistically significant differences. Figure 4 A). There was no statistically significant difference in retinal expression of VEGFa at different time points after the second laser treatment (in section A). Figure 4 (B in the middle).

[0096] Example 3: Secondary laser-induced aortic-like angiogenesis in subretinal fibrovascular lesions

[0097] 1. Testing:

[0098] Using two-stage laser-induced CX3CR1 GFP / +The formation of subretinal fibrovascular membrane in mice was performed as described in Example 1. Eyeballs were collected 20 days after the second laser treatment for RPE / choroidal patch staining to detect fibrosis markers (Collagen-1) and vascular smooth muscle cells and myofibroblasts (αSMA).

[0099] 2. Results:

[0100] In the later stage of the subretinal fibrovascular membrane animal model (20 days after the second laser treatment), the lesion Collagen-1 + The abnormal vessels in subretinal fibrosis lesions are mainly of the large artery type. Figure 5 (arrow), and the blood vessel is surrounded by a large amount of infiltrated CX3CR1. + Microglia / macrophages ( Figure 5 These large artery-like vascular pathological changes do not respond well to anti-VEGF treatment (PMID: 33022379).

[0101] This result, together with the results of Example 2, indicates that secondary laser-induced retinal injury did not significantly increase retinal VEGFa expression, and that neovascularization in the late stage lesions was mainly of the large artery type. This suggests that neovascularization in the late stage lesions of the mouse model of secondary laser-induced subretinal fibrotic vascular membrane is mainly of the non-VEGF-dependent type, and can be considered as a tool for the study of non-VEGF-dependent choroidal neovascular diseases.

[0102] Example 4: Late-stage animal model of subretinal fibrovascular membrane induced by secondary laser showed no response to anti-VEGF treatment.

[0103] To further clarify whether the late stage of the secondary laser-induced subretinal fibrovascular membrane animal model can be used as a method to evaluate the effectiveness of non-VEGF-targeted treatments for choroidal neovascularization, we treated the model with different concentrations of anti-VEGF drugs at different time points and tested the effectiveness of the response.

[0104] I. Anti-VEGF drug treatment methods

[0105] To evaluate the therapeutic effects of different time points and different doses of anti-VEGF drugs on CNV.

[0106] 1. Administration method: Mice (the subretinal fibrovascular membrane mouse model established in Example 1) were intravitreally injected with the fusion protein anti-VEGF drug aflibercept (40 mg / mL, trade name EYLEA, catalog number: KTOL5FT). Aflibercept is a recombinant fusion protein formed by the fusion of human vascular endothelial growth factor receptor 1 and two extracellular domains with the Fc domain of human IgG1. As a soluble decoy receptor, aflibercept binds to VEGF-A, VEGF-B, and placental growth factor (PGF), inhibiting the binding and activation of these factors to the natural VEGF receptor.

[0107] 2. Intervention Time: Three treatment intervention time points were established: immediately after the second laser treatment (S0), 7 days after the second laser treatment (S7), and 14 days after the second laser treatment (S14). The procedure is as follows: Figure 6 As shown.

[0108] 3. Drug concentration: Based on the commonly used clinical dose of 2 mg / 0.05 mL (40 mg / mL specification) and the high dose of 8 mg / 70 μL (114.3 mg / mL specification), and the ratio of vitreous cavity volume between mice and humans being approximately 1:1000, this study divided mice into three groups at each time point: the control group injected with recombinant human IgG1-Fc protein (8 μg / μL / eye), the aflibercept low-dose group (8 μg / μL / eye—clinically high-dose drug, low-dose group (Eylea 8 μg)), and the aflibercept high-dose group (40 μg / μL / eye—clinically standard specification original drug concentration, high-dose group (Eylea 40 μg)), with 9~12 eyes / 8~10 mice / group.

[0109] 4. Sample Collection: Anti-VEGF drug injections were administered at three treatment intervention time points: the IgG Fc antibody (8 μg / μL / eye) control group, the low-dose aflibercept group (Eylea aflibercept, 8 μg / μL / eye), and the high-dose aflibercept group (Eylea aflibercept, 40 μg / μL / eye). The interventions were performed immediately after the second laser treatment (S0), 7 days after the second laser treatment (S7), and 14 days after the second laser treatment (S14). Eyeballs were collected 10 days after drug intervention for RPE / choroidal patch staining of blood vessels (CD31) and fibrosis markers (Collagen-1) to assess the degree of response to anti-choroidal neovascularization treatment.

[0110] II. Results

[0111] 1. Assessment of response to anti-VEGF therapy - changes in neovascularization area

[0112] Ten days after intravitreal injection, eyeballs were collected for RPE / choroid staining for CD31 (neovascularization) and statistical analysis was performed.

[0113] The results showed that: ① S0 (administered immediately after the second laser): Compared with the control group, the CD31 levels in the low-dose aflibercept group and the high-dose aflibercept group were significantly lower. + The area of ​​neovascularization decreased significantly in both groups, but there was no statistically significant difference between the two concentration groups. Figure 7 and Figure 8 A in the text). ② S7 (administered 7 days after the second laser): CD31 in the high-dose aflibercept group. + The area of ​​neovascularization was significantly smaller in the low-dose aflibercept group than in the control group, and there was no statistically significant difference between the low-dose aflibercept group and the control group, or between the high-dose aflibercept group and the control group. Figure 7 and Figure 8 (B) ③ S14 (administered 14 days after the second laser): CD31 in the control group, the low-dose aflibercept group, and the high-dose aflibercept group + There was no statistically significant difference in the area of ​​neovascularization. Figure 7 and Figure 8 (C in the middle).

[0114] Decrease ratio = (lesion area in the IgG Fc group - lesion area in the drug intervention group at the same time point) / lesion area in the IgG Fc group. For both immediate administration after the second laser treatment and administration 7 days later, the reduction ratio of subretinal fibrosis lesion area was higher in the high-concentration group than in the low-concentration group (S0: 52.40% vs 74.10%; S7: 34.10% vs 67.50%). In both low-concentration and high-concentration groups, it was found that the earlier the intravitreal injection of anti-VEGF drugs, the higher the CD31 concentration. + The higher the percentage reduction in neovascularization area, i.e.: S0 > S7 > S14 ( Figure 8 (D in the middle).

[0115] 2. Assessment of response to anti-VEGF therapy - changes in the area of ​​subretinal fibrosis lesions

[0116] The results showed that: ① S0 (administered immediately after the second laser): Compared with the control group, the area of ​​subretinal fibrovascular membrane lesions was significantly reduced in the high-dose aflibercept group, while there was no statistically significant difference in the low-dose aflibercept group. Figure 9 and Figure 10 A in the text). ② S7 (administration 7 days after the second laser): The area of ​​subretinal fibrovascular membrane lesions in the high-dose aflibercept group was significantly smaller than that in the control group and the low-dose aflibercept group. Figure 9 and Figure 10 B in the text). ③ S14 (administered 14 days after the second laser): There was no statistically significant difference in the area of ​​subretinal fibrovascular membrane lesions among the control group, the low-concentration drug group, and the high-concentration drug group. Figure 9 and Figure 10 (C in the middle).

[0117] The decrease ratio of lesion area was calculated as follows: (lesion area in the IgG Fc group - lesion area in the drug intervention group at the same time point) / lesion area in the IgG Fc group. Immediately after the second laser treatment (S0) and 7 days later (S7), the decrease ratio of subretinal fibrovascular membrane lesion area was higher in the high-concentration drug group than in the low-concentration group (S0: 17.50% vs 49.60%; S7: 23.30% vs 63.20%). However, the decrease was not significant when the drug was administered 14 days after the second laser treatment (S14). Figure 10 (D in the middle).

[0118] Example 5: A secondary laser-induced subretinal fibrovascular membrane animal model responded to anti-Ang-2 treatment in the late stage (non-VEGF dependent).

[0119] To verify whether a secondary laser-induced subretinal fibrovascular membrane animal model at 7 days and beyond can serve as a method for evaluating the effectiveness of non-VEGF-targeted treatment for choroidal neovascularization, the model was administered anti-angiogenic factor-2 (Ang-2) at different time points, and the degree of response was examined.

[0120] I. Anti-Ang-2 drug treatment methods

[0121] To evaluate the therapeutic effects of different time points and different doses of anti-VEGF drugs on CNV.

[0122] 1. Administration method: Mice (the subretinal fibrovascular membrane mouse model established in Example 1) were injected intravitreally with an anti-VEGF + anti-Ang-2 bispecific antibody - Faricimab (120 mg / mL, trade name Faricimab, catalog number: RG7716), or an Ang-2 monoclonal antibody - Nevaxumab (20.28 mg / mL, trade name Nesvacumab, catalog number: HY-P99036), or recombinant human IgG1 protein (6.56 mg / mL, catalog number: HY-P99001).

[0123] 2. Intervention Time: Two treatment intervention time points were established, namely 7 days after the second laser treatment (S7) and 14 days after the second laser treatment (S14), during which intravitreal medication was administered. The procedure is as follows: Figure 11 As shown.

[0124] 3. Grouping and Drug Concentration: The ratio of vitreous cavity volume between mice and humans was approximately 1:1000. At each time point, the mice were divided into three groups: ① a control group injected with recombinant human IgG1 protein (6 μg / μL / eye), ② a faricimab group (6 μg / μL / eye), and ③ a Nesvacumab + IgG1 group (3 μg + 3 μg / μL / eye – strictly controlling the molar concentration of Ang-2 binding sites and total protein load, abbreviated as Nesvacumab or Nesvacumab group). Nesvacumab has two Ang-2 binding sites, while faricimab binds to VEGF at one site and Ang-2 at the other, meaning it is monovalently bound to Ang-2. Furthermore, both have similar molecular weights (both ~150 kDa). This implies that at the same mass concentration, faricimab binds to Ang-2 at half the number of sites as Nesvacumab. To eliminate the influence of total protein on the intraocular microenvironment and ensure consistent anti-Ang-2 efficacy (number of binding sites), the aforementioned three groups were established. n = 37~40 lesions / 10 eyes / 6~7 mice / group.

[0125] 4. Sample collection: Interventions were conducted at two time points, 7 days after the second laser treatment (S7) and 14 days after the second laser treatment (S14). Eyeballs were collected 10 days after drug intervention for RPE / choroidal patch staining of blood vessels (CD31) and fibrosis markers (Collagen-1) to assess the degree of response to anti-choroidal neovascularization treatment.

[0126] II. Results

[0127] 1. Assessment of response to anti-Ang-2 therapy - changes in neovascularization area

[0128] Ten days after intravitreal injection, eyeballs were collected for RPE / choroid staining for CD31 (neovascularization) and statistical analysis was performed.

[0129] The results showed that: ①S7 (administration 7 days after the second laser): CD31 in the control group, Faricimab group, and Nesvacumab+IgG1 group + There was no statistically significant difference in the area of ​​neovascularization. Figure 12 and Figure 13 (A) ②S14 (administered 14 days after the second laser): Compared with the control group, the Faricimab group and the Nesvacumab+IgG1 group had CD31 + The area of ​​new blood vessels decreased significantly in both groups, but there was no statistically significant difference between the two drug groups. Figure 12 and Figure 13 (B in the middle).

[0130] Decrease ratio = (lesion area in the IgG Fc group - lesion area in the drug intervention group at the same time point) / lesion area in the IgG Fc group. Anti-Ang-2 drugs and CD31 were administered 7 days after the second laser treatment. + There was no statistically significant difference in neovascularization area between the treatment groups, but there was a decreasing trend. Intervention with Faricimab (which has both anti-VEGF and anti-Ang-2 properties) or Nesvacumab + IgG1 (anti-Ang-2) 14 days after the second laser treatment reduced CD31 levels. + The area of ​​neovascularization decreased significantly (S7: 7.59% vs 40.15%; S14: 52.55% vs 66.06%). In both the Faricimab and Nesvacumab+IgG1 groups, it was found that the later the stage of the disease, the more likely the anti-Ang-2 drug was injected into the vitreous cavity, the lower the CD31 concentration. + The higher the percentage reduction in neovascularization area, i.e.: S14 > S7 ( Figure 13 (C) This indicates that late-stage intervention at non-VEGF-dependent targets in a mouse model of secondary laser-induced subretinal fibrosis can effectively alleviate choroidal neovascularization, while anti-VEGF treatment has no effect.

[0131] 2. Assessment of response to anti-Ang-2 therapy - changes in the area of ​​subretinal fibrosis lesions

[0132] The results showed that: ① S7 (administered immediately after secondary laser): there was no statistically significant difference in the area of ​​subretinal fibrovascular membrane lesions among the control group, Faricimab group, and Nesvacumab+IgG1 group. Figure 14 and Figure 15 A in the text). ②S14 (administered 14 days after the second laser): Compared with the control group, the area of ​​subretinal fibrovascular membrane lesions in both the Faricimab group and the Nesvacumab+IgG1 group was significantly reduced, but there was no statistically significant difference between the two drug groups. Figure 14 and Figure 15 (B in the middle).

[0133] Decrease ratio = (lesion area in the IgG Fc group - lesion area in the drug intervention group at the same time point) / lesion area in the IgG Fc group. Fourteen days after the second laser treatment, intervention with Faricimab (which has both anti-VEGF and anti-Ang-2 properties) or Nesvacumab (anti-Ang-2) significantly reduced the area of ​​subretinal fibrovascular membrane lesions (S7: -0.21% vs 26.08%; S14: 42.96% vs 56.02%). Figure 15 (C in the middle).

[0134] The above results indicate that the dependence of neovascularization in the lesion on VEGF varies at different time points after the second laser treatment. From 0 to 6 days after the second laser treatment, the lesion shows a high dependence on VEGF, and treatment with low or high doses of anti-VEGF drugs is effective. From 7 to 13 days after the second laser treatment, the dependence on VEGF decreases, and treatment with low doses of anti-VEGF drugs is ineffective, but treatment with high doses of anti-VEGF drugs is effective. After 14 days after the second laser treatment, the lesion is no longer dependent on VEGF, and treatment with low or high doses of anti-VEGF drugs is ineffective, but treatment with drugs containing anti-non-VEGF agents (such as anti-Ang-2) can significantly inhibit choroidal neovascularization. The period from 7 to 13 days after the second laser treatment is suitable for testing the efficacy of dual (multi-)target therapy combined with VEGF and other non-VEGF targets for retinal choroidal neovascularization. After 14 days after the second laser treatment, it is suitable for testing the efficacy of treatment targeting other targets (non-VEGF dependent) for retinal choroidal neovascularization.

[0135] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an animal model of subretinal fibrovascular membrane secondary to non-VEGF-dependent CNV, characterized in that, To obtain CNV lesions by first lasering the Bruch membrane of the animal with the optic disc as the center, and then performing a second laser on the corresponding site of the CNV lesion, the non-VEGF-dependent CNV secondary subretinal fibrovascular membrane animal model was obtained. The parameters of the first laser and / or the second laser are: energy 100~250mw, pulse interval 100~150ms, and spot diameter 50~200μm; The animals were between 2 and 24 months old at the time of the first laser irradiation. The second laser is applied 6 to 10 days after the first laser.

2. The preparation method according to claim 1, characterized in that, The number of laser points for the first laser and / or the second laser is 3 to 6; The distance between each laser point and the optic disc is greater than or equal to two optic disc diameters.

3. The preparation method according to claim 2, characterized in that, The first laser and / or the second laser are followed by a sterilization and disinfection step; the sterilization and disinfection drug is levofloxacin hydrochloride eye drops.

4. The preparation method according to claim 3, characterized in that, The animals include mice, rats, rabbits, and / or monkeys.

5. The application of the non-VEGF-dependent CNV-secondary subretinal fibrovascular membrane animal model prepared by the preparation method according to any one of claims 1 to 4 in evaluating the efficacy of non-VEGF-targeted drugs and / or VEGF-targeted and non-VEGF-targeted combination drugs in the treatment of choroidal neovascularization diseases, characterized in that, The assessment employs the following steps: A control group and a treatment group were set up to treat the animal model of non-VEGF-dependent CNV secondary subretinal fibrovascular membrane. The effectiveness of the non-VEGF-targeted drug and / or the combination of VEGF-targeted and non-VEGF-targeted drugs in the treatment of choroidal neovascularization was determined based on whether the difference in the severity of pathological neovascularization between the control group and the treatment group was significant. The control group was treated with recombinant human IgG1 protein. The treatment group consists of treatments using non-VEGF target drugs and / or a combination of VEGF target and non-VEGF target drugs.

6. The application according to claim 5, characterized in that, The non-VEGF target drugs include anti-Ang-2 drugs, anti-PDGF drugs, and / or anti-FGF drugs; The combination drugs targeting VEGF and non-VEGF targets include fareximab.

7. The application according to claim 6, characterized in that, The anti-Ang-2 drugs include nevasumoab.

8. The application according to claim 5, characterized in that, Indicators of the severity of the pathological neovascularization include: the area of ​​subretinal fibrovascular membrane lesions and / or CD31. + Area of ​​newly formed blood vessels.

9. The application according to claim 5, characterized in that, The treatment time for the CNV-secondary subretinal fibrovascular membrane animal model was from the 7th to the 20th day after the second laser treatment.

10. The application according to any one of claims 5 to 9, characterized in that, The choroidal neovascular diseases include diabetic retinopathy, retinal vein occlusion, age-related macular degeneration, pathological myopia, angiostriate syndrome and other degenerations, ocular inflammation, and / or ocular tumors.

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