Use of arginine polyesteramides in the manufacture of a medicament for the treatment of dry age-related macular degeneration

Abstract

The application discloses application of arginine polyester amide in preparation of a drug for treating dry age-related macular degeneration. It is found for the first time that arginine polyester amide 2A6S can be used for preventing and treating dry age-related macular degeneration (AMD), specifically, 2A6S can effectively restore the ERG amplitude of dry AMD patients, reduce the pathological damage of the fundus and maintain the morphological integrity of the RPE, reduce the expression of inflammatory factors by inhibiting the NF-kappa B pathway, and the curative effect is better than that of existing complement C3 inhibitor peptide drugs and NF-kappa B signal pathway inhibitors, has potential clinical application value for dry AMD, and is helpful to expand the treatment strategy of dry AMD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of arginine polyesteramide in the preparation of drugs for treating dry age-related macular degeneration. Background Technology

[0002] Age-related macular degeneration (AMD) is one of the leading causes of irreversible central vision loss in the elderly. Based on clinicopathological features, AMD can be divided into atrophic (dry AMD) and exudative (wet AMD), with dry AMD accounting for approximately 85%-90% of all cases. In the early and progressive stages, dry AMD is mainly characterized by drusen deposition in the macular area, abnormal retinal pigment epithelium (RPE) function, and thickening of Bruch's membrane (also known as vitrectomy). In the late stages, focal degeneration of the RPE and loss of photoreceptor cells may occur, forming geographic atrophy (GA). Some patients may also develop choroidal neovascularization, ultimately leading to severe impairment or even loss of central vision.

[0003] For a long time, dry AMD has lacked effective specific treatments, and clinical interventions have mainly been limited to nutritional supplementation and risk factor management, which are insufficient to fundamentally slow disease progression. In recent years, the U.S. Food and Drug Administration (FDA) has approved therapeutics targeting the complement pathway, such as inhibitors targeting complement C3 or C5, providing new directions for the treatment of dry AMD. However, the pathogenesis of AMD involves multiple pathological processes, including inflammatory responses, innate immune activation, oxidative stress, RPE dysfunction, and neurodegeneration. Single complement pathway inhibition strategies still have limitations in terms of efficacy stability, individual variability, and long-term safety. Meanwhile, as of now, there are no specifically approved treatments for dry AMD or GA in China, and domestic patients still face significant unmet clinical needs, urgently requiring the development of new treatment options.

[0004] Arginine-based polyesteramide materials have previously been primarily used in nucleic acid and drug delivery, and are considered to possess good biocompatibility, low toxicity, and biodegradability. However, whether they have the potential to directly intervene in the treatment of retinal degenerative diseases, especially dry AMD, remains unclear and lacks systematic research and definitive reports. Therefore, exploring the biological role of these materials in dry AMD and screening for specific structures with preferred therapeutic effects is of significant scientific and clinical value for expanding treatment strategies for dry AMD, discovering new drug applications, and promoting the development of related innovative drugs. Summary of the Invention

[0005] Addressing the current lack of effective treatments for dry age-related macular degeneration (dry AMD) and the limited efficacy of existing complement inhibition strategies, this invention aims to provide the use of arginine polyesteramide in the preparation of drugs for treating dry AMD, thereby improving visual function and delaying retinal degenerative damage. Specifically, in a sodium iodate-induced dry AMD mouse model, treatment with 2A6S significantly improved retinal electrophysiological function, reduced fundus pathological damage, and protected retinal pigment epithelium structure.

[0006] The first objective of this invention is to provide a drug capable of preventing and treating dry age-related macular degeneration, which contains arginine polyesteramide 2A6S; wherein the arginine polyesteramide 2A6S is obtained by polycondensation reaction of monomer N2 and monomer Arg-6-S, as shown in Formula 3: Formula 3.

[0007] Preferably, the monomer N2 is prepared by the following method: triethylamine and p-nitrophenol are added to acetone, the mixed solution is maintained at 0°C, succinyl chloride diluted with acetone is added dropwise to the mixed solution, the mixture is stirred at 0°C for 2 h, and then the reaction is continued overnight at room temperature to generate di-p-nitrophenyl succinate. Di-p-nitrophenyl succinate is precipitated in distilled water, thoroughly washed, vacuum dried, and recrystallized three times in a system of ethyl acetate and DMF with a volume ratio of 4:1 to obtain monomer N2; the preparation reaction formula is shown in Formula 1: Formula 1; The monomer Arg-6-S was prepared by the following method: L-arginine was mixed with 1,6-hexanediol, and then toluene and p-toluenesulfonic acid monohydrate were added. The mixture was heated to 120-130°C and stirred under reflux for 24 h. After cooling to room temperature, the supernatant was precipitated by toluene precipitation. The product was dissolved in isopropanol at 75°C and then precipitated at 4°C. The precipitation was repeated 3 times. The white viscous product was dried under vacuum to obtain the monomer Arg-6-S. The preparation reaction formula is shown in Formula 2. Formula 2.

[0008] Preferably, the dosage of the drug is 20 mg / kg.

[0009] Preferably, the drug is administered by injection.

[0010] Preferably, the drug is administered once every 48 hours.

[0011] Preferably, the drug prevents and treats dry age-related macular degeneration through at least one of the functions of (1)-(4): (1) Improves retinal structural damage; (2) Protect the structural integrity of the retinal pigment epithelium; (3) Protects retinal neurons and inhibits glial reaction and apoptosis; (4) Relieve inflammatory response by inhibiting the NF-κB signaling pathway.

[0012] Preferably, the inhibition of the NF-κB signaling pathway involves inhibiting the expression levels of genes related to the NF-κB signaling pathway and reducing the phosphorylation levels of proteins related to the NF-κB signaling pathway.

[0013] Preferably, the inhibition of the expression level of genes related to the NF-κB signaling pathway is an inhibition of... Rel , Relb , Nfkb1 , Nfkb2 、Nfkbia、 Nfkbiz , Ikbkb , Ikbke The expression level of inflammatory factors is thus inhibited, thereby suppressing the expression level of inflammatory factors. Il1b , Tnf The level of expression.

[0014] A second objective of this invention is to provide the application of arginine polyesteramide 2A6S in the preparation of drugs for the prevention and treatment of dry age-related macular degeneration.

[0015] The beneficial effects of this invention are: This invention is the first to discover that arginine polyesteramide 2A6S can (1) significantly improve visual function: effectively restore ERG amplitude in dry AMD mice to near normal levels; (2) protect retinal and RPE structures: reduce fundus pathological damage and maintain RPE morphological integrity; (3) inhibit inflammatory response: reduce the expression of inflammatory factors by inhibiting the NF-κB pathway; and (4) outperform complement or single inflammatory suppression strategies: the overall therapeutic effect is closer to that of normal controls. In conclusion, arginine polyesteramide 2A6S has potential clinical application value in dry AMD. Attached Figure Description

[0016] Figure 1 This section compares visual function in dry AMD models using different Arg-PEA treatments; A shows representative waveforms of the a and b waves in WT, SI, and different Arg-PEA treatment groups using retinal retinograms (ERG); B shows representative waveforms of the c wave in WT, SI, and different Arg-PEA treatment groups using ERG; (CE) is a quantitative statistical analysis of the amplitudes of the a, b, and c waves, using two-way ANOVA, where ns indicates P>0.05, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0017] Figure 2 The images show fundus color photography and fluorescein fundus angiography results demonstrating how 2A6S improves fundus pathological changes in a SI-induced dry AMD mouse model; A is the fundus color photography result, and B is the fluorescein fundus angiography result.

[0018] Figure 3 This section showcases the results of 2A6S improving retinal structural damage in dry AMD. A represents full-thickness HE staining images of the retina in WT, SI, and SI + 2A6S mice, scale bar: 80 µm; B is a magnified HE staining image of the outer layers, from top to bottom: RPE, OS, IS, ONL, OPL, INL, IPL, GCL, scale bar: 20 µm; C is a high-magnification HE image focused on the RPE-photoreceptor interface, from top to bottom: RPE, OS, scale bar: 10 µm; D is a quantitative analysis of the total retinal thickness and the thickness of each sublayer (RPE, OS / IS, ONL, OPL), using two-way ANOVA, where ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0019] Figure 4 This is a whole-mount retinal image showing the protection of RPE structural integrity by 2A6S; Central region: Central, Intermediate, Peripheral; Whole-mount retina; The green area is the cytoskeleton after Phalloidin staining, and the blue area is the cell nucleus after DAPI staining.

[0020] Figure 5 The diagram shows the results of 2A6S protecting retinal neurons and inhibiting glial reactions and apoptosis; A is the result of Arrestin labeling photoreceptor cells, B is the result of Recoverin labeling the inner layer of photoreceptors, C is the result of GFAP labeling Müller glial cell reaction, and D is the result of TUNEL staining to detect apoptosis.

[0021] Figure 6 The transcriptomics results reveal the 2A6S inhibitory pathway of inflammation; A is a heatmap showing the expression levels of inflammation and immune-related genes in different groups, B is a bar chart of differential expression of key inflammatory genes, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.

[0022] Figure 7This confirms that 2A6S alleviates the inflammatory response by inhibiting the NF-κB signaling pathway; A is the result of qRT-PCR detection of the relative mRNA expression levels of key genes and inflammatory factors in the NF-κB pathway. The differential analysis method was two-way ANOVA, where * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001; B is the result of Western blot analysis of NF-κB pathway-related proteins and their phosphorylation levels.

[0023] Figure 8 This study compares the ERG efficacy of 2A6S with NF-κB inhibitors and C3 complement inhibitors; A compares the waveforms of wave a and wave b in ERG tests of different treatment groups; B compares the waveforms of wave c in ERG tests of different treatment groups; C and D represent the quantitative statistical analysis of the amplitudes of waves a, b, and c, respectively. The difference analysis method is two-way ANOVA, where * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Example 1: Preparation method of arginine polyesteramide materials 1. This embodiment provides a method for preparing arginine polyester amide (Arg-PEA), primarily referencing the following reference (doi:10.1016 / j.biomaterials.2008.04.026). The synthesis of Arg-PEA generally includes three steps: ① Monomer I (Nx): preparation of p-nitrophenol ester from dicarboxylic acid monomers; ② Monomer II (Arg-yR): preparation of L-arginine diester monomer salt (taking p-toluenesulfonate as an example); ③ Obtaining the Arg-PEA polymer through solution polycondensation of monomer I and monomer II. The following example uses 2A6S.

[0026] 1.1 Synthesis of Monomer I (N2): p-Nitrophenol Dicarboxylic Acid 200 mL of acetone was added to a round-bottom flask, followed by 0.32 mol of triethylamine and 0.31 mol of p-nitrophenol. The system was maintained at 0°C under ice bath conditions. 0.15 mol of succinyl chloride was diluted with 100 mL of cold acetone and added dropwise to the above system. The mixture was stirred at 0°C for 2 h, then cooled to room temperature and reacted overnight. After the reaction was completed, the generated di-p-nitrophenyl succinate was precipitated in distilled water, thoroughly washed, dried under vacuum, and recrystallized three times in an ethyl acetate:DMF = 4:1 (v / v) system to obtain needle-like colorless crystal monomer I (N2). The specific reaction is shown in Formula 1.

[0027] Formula 1.

[0028] 1.2 Synthesis of Monomer II (Arg-6-S): L-Arginine Diester p-Toluenesulfonate 0.04 mol of L-arginine and 0.02 mol of 1,6-hexanediol were added to a three-necked round-bottom flask and mixed. Then, 400 mL of toluene (boiling point 110 °C) and 0.082 mol of p-toluenesulfonic acid monohydrate were added. The mixture was heated to 120–130 °C and stirred under reflux for 24 h. During the reaction, approximately 2.16 mL (0.12 mol) of water was collected. After the reaction was completed, the mixture was cooled to room temperature, and the supernatant was decanted after toluene precipitation. The product was then purified by dissolving it in isopropanol: after dissolving in isopropanol at 75 °C with stirring, precipitation was carried out at 4 °C, and the precipitation was repeated three times. Finally, the white viscous product was dried under vacuum to obtain a white powdery monomer II (Arg-6-S). The specific reaction is shown in Formula 2.

[0029] Formula 2.

[0030] 1.3 Synthesis of polymer 2A6S: Solution polycondensation Take 1.5 mL of dry DMSO and add monomer I (N2, 1.0 mmol) and monomer II (Arg-6-S, 1.0 mmol) under nitrogen protection, and vortex thoroughly. Since the Arg-6-S molecule contains a relatively long methylene segment (y=6), to ensure system homogeneity, heat to 75°C under magnetic stirring until a clear, transparent solution is formed. Then, add 0.31 mL (2.2 mmol) of triethylamine dropwise to neutralize acidic byproducts generated during the reaction and promote amide bond formation. Continue stirring at 75°C until the monomers are completely dissolved. After several minutes, the solution gradually turns pale yellow, indicating the start of the polycondensation reaction. The reaction system is kept at a constant temperature of 75°C for 48 h to complete the stepwise polycondensation process. To avoid the increased viscosity of the high molecular weight product affecting chain growth in the later stages of the reaction, no further stirring is performed during the reaction stage. After the reaction is complete, the reaction solution is slowly poured into excess cold ethyl acetate to precipitate the polymer. The supernatant is decanted and the precipitate is dried. The crude product was dissolved in a small amount of methanol, and then precipitated again with cold ethyl acetate to remove unreacted monomers and low-molecular-weight byproducts. This purification step was repeated twice. Finally, the precipitate was dried under vacuum at 60°C to constant weight to obtain a yellow or pale yellow solid powder polymer 2-Arg-6-S (abbreviated as 2A6S). The specific reaction is shown in Equation 3, where n=1.

[0031] Formula 3.

[0032] 2. Preparation of other Arg-PEA (2-Arg-yS, i.e., 2A2S, 2A3S, 2A4S) Without altering the above reaction route and key conditions, by replacing 1,6-hexanediol in the synthesis of monomer II with ethylene glycol, 1,3-propanediol, or 1,4-butanediol, the corresponding 2-Arg-2-S, 2-Arg-3-S, and 2-Arg-4-S (abbreviated as 2A2S, 2A3S, and 2A4S, respectively) can be prepared. In the polymerization step, monomer I and monomer II are still fed in a molar ratio of 1:1 and polycondensation reaction is carried out in DMSO at 75°C. The post-treatment and purification process is the same as above.

[0033] Example 2: Therapeutic efficacy screening of different Arg-PEA in dry AMD animal models 1. Establishment of a dry AMD mouse model (1) Mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (strain: C57BL / 6J, grade: SPF, 6-10 weeks old, weight 18-25 g, male).

[0034] (2) Animals were acclimatized under standard conditions for no less than 7 days and were randomly grouped before the experiment. All animal experimental procedures complied with the ethical guidelines for laboratory animals.

[0035] (3) Preparation of sodium iodate solution: Weigh sodium iodate (SI, NaIO3), dissolve it in sterile 0.9% physiological saline and prepare a working solution of the required concentration. Prepare and use immediately. The solution is filtered through a 0.22 μm microporous membrane for sterilization and stored under light-protected conditions until use.

[0036] (4) Anesthesia: General anesthesia was performed by intraperitoneal injection of sodium pentobarbital. The recommended dose was 50 mg / kg. After the anesthesia took effect, it was confirmed that the animal had no pain reflex.

[0037] (5) Sodium iodate modeling administration: Sodium iodate was administered via tail vein injection to induce selective damage to the retinal pigment epithelium (RPE). The dosage of sodium iodate was 25 mg / kg, and the injection volume was controlled at 5-10 mL / kg (e.g., the injection volume for a 20 g mouse was approximately 100-200 μL). The control group received an equal volume of sterile saline. During the injection, the needle was kept in the vein lumen and the injection was administered slowly. After injection, local pressure was applied to stop bleeding, and extravasation or abnormal reactions were observed.

[0038] (6) Post-modeling observation and exclusion criteria: After injection, mice were placed in a constant temperature environment to awaken and their mental state, activity level, and food and water intake were continuously observed. If obvious non-specific severe reactions occurred (such as persistent coma, convulsions, or extravasation of the drug due to administration failure), the mice were excluded in accordance with animal ethics requirements.

[0039] (7) Model formation time window and confirmation: Early manifestations of RPE damage and visual function decline can appear 3-7 days after sodium iodate injection, and a relatively stable dry AMD / map-like atrophy-like degenerative phenotype can be formed 7-14 days.

[0040] The success of the model can be confirmed by the following methods: (i) fundus photography to observe yellowish-white lesions or degenerative changes; (ii) fundus autofluorescence (FAF) to detect abnormal fluorescence signal enhancement; (iii) electroretinography (ERG) to detect decreased amplitude of waves a, b, and c; and (iv) histological staining or RPE whole-mount to observe RPE and retinal structural damage. Mice conforming to the above phenotypes were included in subsequent efficacy evaluation experiments.

[0041] 2. Arg-PEA administration and treatment methods in a dry AMD mouse model Based on the establishment of a dry AMD sodium iodate induction model, mice were treated with arginine polyester amide (Arg-PEA) material to evaluate the therapeutic effects of different Arg-PEA materials.

[0042] (1) Grouping of experimental animals: Male C57BL / 6J mice aged 8-10 weeks were randomly divided into the following experimental groups: normal control group (WT), model control group (SI), and different Arg-PEA treatment groups (treated with 2A2S, 2A3S, 2A4S, and 2A6S, respectively). Each group contained no fewer than 5 mice, and at least 3 independent biological replicates were performed to ensure the reliability and statistical validity of the data.

[0043] (2) Administration method, dosage and timing: Arg-PEA material is administered via intraperitoneal injection. The dosage is calculated based on the mouse's body weight, preferably 20 mg / kg, and is prepared as an injection solution by dissolving in sterile physiological saline or PBS, and should be used immediately after preparation.

[0044] (3) Administration time: The first Arg-PEA pretreatment injection was performed 12 h before sodium iodate modeling; after sodium iodate induction modeling was completed, Arg-PEA was injected again every 48 h to maintain effective drug exposure in vivo; the administration continued until day 7 after modeling, and then the mice were analyzed for endpoint. The model control group was injected with an equal volume of sterile saline or PBS as solvent control.

[0045] (4) Endpoint detection and sample collection time: A systematic evaluation of each group of mice was carried out on the 7th day after modeling.

[0046] (5) Technical points and implementation advantages: The above-mentioned drug administration regimen, through the combined strategy of pretreatment before modeling and maintenance drug administration after modeling, enables Arg-PEA to maintain an effective window of action before retinal damage occurs and during the damage progression stage, thereby more fully evaluating its protective and therapeutic potential in dry AMD.

[0047] 3. Visual function assessment after Arg-PEA treatment in a mouse model of dry AMD To evaluate the visual function protective effects of different Arg-PEA formulations in a dry AMD mouse model, electroretinography (ERG) was performed on mice in each experimental group using the Diagnosys CELERIS animal electrophysiology platform after model establishment and drug administration to assess changes in photoreceptors, inner retinal neurons, and retinal pigment epithelium. Each group consisted of at least five mice, and at least three independent biological replicates were performed.

[0048] (1) Dark adaptation, anesthesia and mydriatic treatment Prior to ERG testing, mice were placed in a completely dark environment for at least 12 hours for dark acclimatization. The procedure was performed under dark red light conditions, and anesthesia was administered via intraperitoneal injection of sodium pentobarbital (50 mg / kg). After anesthesia, 0.5% lidocaine eye drops were applied to both eyes for surface anesthesia, and 1% tropicamide eye drops were applied for mydriasis. During the test, a constant-temperature heating pad was used to maintain the animal's body temperature at approximately 37°C, and artificial tears were applied to the corneal surface to prevent dryness.

[0049] (2) Electrode placement and equipment setup (Diagnosys CELERIS) The Diagnosys CELERIS full-field flash ERG system was used for detection. The recording electrode was placed on the corneal surface, the reference electrode was placed subcutaneously around the mouth or cheekbone, and the ground electrode was placed subcutaneously at the tail or back. Before detection, the electrode impedance was confirmed to be within a reasonable range (usually <5 kΩ), and the signal baseline was kept stable to improve the signal-to-noise ratio and repeatability.

[0050] (3) Measurement of light stimulation parameters and scotopic ERG Under dark adaptation conditions, a full-field strobe stimulation mode was adopted, and a gradient light intensity stimulation scheme was set. The light stimulation intensities of wave A and wave B were 3.0 cd·s / m², with an interval of 5.5 s between each stimulation; the light stimulation intensity of wave C was 150 cd·s / m², with an interval of 6 s between each stimulation.

[0051] The recorded and analyzed parameters include: (i) a-wave amplitude (reflecting photoreceptor function); (ii) b-wave amplitude (reflecting signal transduction function of bipolar cells and inner retina); and (iii) c-wave amplitude (reflecting RPE-related electrophysiological function).

[0052] (4) Data processing, statistical analysis and graphing.

[0053] Raw ERG data were exported using Diagnosys CELERIS software and then statistically analyzed and plotted using GraphPad Prism9 software. Data for each group are expressed as mean ± standard error (mean ± SEM). Two-way ANOVA was used to compare differences in visual function among different treatment groups and under different light stimulation intensities, with multiple comparison correction performed as necessary. The statistical significance threshold was set at P < 0.05.

[0054] (5) Results of efficacy screening ERG functional assessment results showed significant differences in the recovery of visual function in dry AMD mice among different Arg-PEA materials. Specifically, the 2A6S treatment group exhibited amplitudes of a, b, and c waves closer to those of the WT group under multi-gradient light stimulation conditions. The 2A4S treatment group also showed partial recovery of a and c wave amplitudes. However, the 2A2S and 2A3S treatment groups did not show significant therapeutic effects at this concentration of the therapeutic precursor. Figure 1 ).

[0055] The results show that 2A6S can exhibit significant visual function protection in dry AMD models, supporting its status as a preferred material for the treatment of dry AMD.

[0056] Example 3: The effect of arginine polyesteramide 2A6S on improving retinal and RPE structural damage in dry AMD 1. Fundus evaluation in mouse models To evaluate the fundus protective effect of arginine polyester amide material 2A6S in a dry AMD mouse model, fundus color photography and fundus fluorescein angiography (FFA) were used to systematically assess fundus structural changes and blood-retinal barrier integrity in each group of mice on day 7 after sodium iodate modeling and drug administration, in order to verify the ameliorative effect of 2A6S on fundus pathological damage.

[0057] (1) Animal pretreatment and fluorescein fundus angiography imaging method Fundus assessment was performed on mice in each experimental group, with at least three animals per group, and at least three independent biological replicates were conducted. Before testing, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). After confirming stable anesthesia, surface anesthesia was performed by instilling 0.5% lidocaine eye drops into both eyes, followed by full mydriasis with 1% tropicamide eye drops. Subsequently, sodium fluorescein solution (usually 100 mg / kg) was injected intraperitoneally. After a 5-minute waiting period to allow sufficient fluorescein to enter the fundus circulation, fundus imaging (FFA) was performed using a Phoenix Micron IV retinal imaging system, focusing on retinal vascular filling and fluorescein leakage distribution characteristics. The same exposure parameters and light intensity settings were maintained during imaging to ensure data comparability between different experimental groups.

[0058] (2) Analysis of fundus angiography results and evaluation of treatment effect Fundus photography revealed obvious white spot-like lesions and focal retinal edema in the fundus of the model control group (SI) mice, indicating retinal structural damage and abnormal RPE function. In contrast, the fundus structure of the 2A6S treatment group was significantly improved, and the overall morphology was closer to that of the normal control (WT) group. Figure 2 (A) Fluorescein fundus angiography results showed that the SI group had abnormally high fluorescence areas and focal fluorescein leakage, indicating impaired blood-retinal barrier function and RPE-related degenerative changes. In contrast, the 2A6S treatment group showed a significant reduction in the number of white lesions in the fundus, a smaller area of ​​fluorescein leakage, a significant decrease in the intensity of abnormally high fluorescence signals, a more uniform fluorescence distribution, and an overall phenotype closer to the WT group. Figure 2 (B in the text). The above results indicate that 2A6S can effectively alleviate sodium iodate-induced fundus pathological changes and improve blood-retinal barrier stability and RPE function, supporting its protective role in the treatment of dry AMD from the perspective of fundus imaging.

[0059] 2. Retinal structural assessment To evaluate the effect of arginine polyester amide material 2A6S on improving retinal tissue structure damage in dry AMD mice, histological analysis of the ocular tissue of mice in each group was performed on day 7 after modeling and drug administration. The changes in retinal laminar structure and thickness were observed by hematoxylin and eosin (HE) staining.

[0060] (1) Sampling, fixation, sectioning and HE staining (a) Mice in each group were euthanized on day 7 after modeling, and both eyeballs were quickly enucleated. A small incision was made near the limbus with scissors to facilitate the penetration of fixative. The eyeballs were placed in 4% paraformaldehyde (PFA) fixative and fixed at 4°C for 24 h. After fixation, residual fixative was removed by rinsing with PBS.

[0061] (b) After fixation, the eyeballs underwent routine dehydration treatment: the samples were placed in 70%, 80%, 90%, 95%, and 100% ethanol for dehydration in stages (30 min each stage); then cleared in xylene (20 min each time, repeated twice); and finally embedded in molten paraffin (60°C, 30 min each time, repeated twice). During embedding, it was preferable to maintain a uniform eyeball orientation to ensure consistent section direction.

[0062] (c) Paraffin-embedded blocks were sectioned along the direction passing through the optic nerve head (ONH), with a section thickness of 8 μm. The sections were attached to glass slides and baked at 55°C for 120 min to enhance tissue adhesion, and were then ready for use.

[0063] (d) For HE staining, the sections were dewaxed and hydrated sequentially: dewaxed with xylene (5 min each time, repeated twice), then rehydrated stepwise with 100%, 95%, 80%, and 70% ethanol (5 min each time) and then with distilled water. The sections were stained in hematoxylin solution for 8 min, rinsed with tap water, and then differentiated with 1% hydrochloric acid alcohol for a few seconds to 10 s, followed by blueing with tap water or a weakly alkaline solution for 5 min. Then counterstained with eosin solution for 2 min, and quickly rinsed with distilled water to remove the background.

[0064] (e) After counterstaining, the sections were dehydrated stepwise with 70%, 80%, 95%, and 100% ethanol (3 min per step), then cleared with xylene (5 min each time, repeated twice), and then mounted with neutral resin and air-dried for subsequent microscopic observation, image acquisition and quantitative structural analysis.

[0065] (2) Microscopic imaging and quantitative analysis of retinal thickness After HE staining, images of retinal sections were acquired using a bright-field optical microscope. To ensure the comparability of data between different experimental groups, all images were acquired under the same magnification, exposure time, and light source intensity.

[0066] (a) Criteria for selecting material sourcing areas and field of view Standard slides passing through the optic disc were selected for analysis to ensure consistency in anatomical location across all samples. Within each slide, at least two representative visual fields were selected from the retinal region approximately 200-500 μm above and below the optic disc for imaging, thus reducing bias caused by local anatomical differences. At least three independent slides were analyzed per mouse, with at least four visual fields analyzed per slide. Data were then aggregated using the animal as the statistical unit. Each group consisted of at least three animals, and at least three independent biological replicates were performed.

[0067] (b) Methods for measuring retinal thickness Quantitative measurements of retinal thickness were performed using ImageJ or equivalent image analysis software. In each visual field, a straight line was drawn perpendicular to the retinal surface to measure the following parameters: total retinal thickness (from the nerve fiber layer to the RPE layer), outer nuclear layer (ONL) thickness, inner / outer segment (IS / OS) thickness, and outer plexiform layer (OPL) thickness. At least three independent measurement lines were taken for each visual field, and the average value was used as the representative value for that field. The average value across all visual fields for each animal was then used as the structural parameter value for that animal.

[0068] (c) Data processing and statistical analysis All quantitative data were aggregated per animal and statistically analyzed using GraphPad Prism 9 software. Data are expressed as mean ± standard error (mean ± SEM), and two-way ANOVA was used to analyze the different treatment groups. The statistical significance threshold was set at P < 0.05.

[0069] (3) Results Analysis HE staining results showed that the model control group (SI) exhibited significant retinal layer disorder, damaged outer layers, significantly thinned outer nuclear layer (ONL), and broken photoreceptor inner and outer segment layers (OS / IS), accompanied by focal degenerative damage to the retinas (RPE), RPE pigment aggregation, and focal pigment deposition, suggesting typical dry AMD-like structural damage. In contrast, the 2A6S treatment group showed significant recovery of retinal layer structure, increased ONL thickness, improved OS / IS continuity, reduced pigment deposition after 2A6S treatment, and more intact RPE structure. Further quantitative analysis showed that the 2A6S treatment group was significantly superior to the model control group in terms of total retinal thickness and thickness of multiple sublayers. Figure 3 ).

[0070] The above results indicate that the arginine polyesteramide material 2A6S can effectively reduce sodium iodate-induced retinal structural damage and partially restore tissue integrity, providing clear histological evidence for its structural protective effect in the treatment of dry AMD.

[0071] 3. RPE structural assessment To evaluate the protective effect of arginine polyesteramide material 2A6S on the structural integrity of the renal pelvis (RPE) in dry AMD mice, RPE whole-mount preparation and morphological analysis were performed on mice in each group on day 7 after modeling and drug administration to observe the arrangement of RPE cells, boundary continuity, and focal defects.

[0072] (1) Obtain a high-quality RPE whole-mount (a) Rapid eyeball harvesting and gentle pre-fixation To preserve the structural integrity of the RPE to the greatest extent possible, the eyeballs were removed immediately after mouse euthanasia (ideally within ≤2 minutes) and pre-fixed briefly in freshly prepared 4% PFA (15 minutes, 4°C) to enhance RPE-Bruch membrane adhesion stability and reduce mechanical dissection damage. After pre-fixation, the eyeballs were briefly rinsed in PBS and then dissected on ice to minimize the risk of tissue softening and autolysis.

[0073] (b) Minimal anatomical damage and complete separation of the RPE-choroidal composite layer Under a stereomicroscope, the eyeball is cut along the limbus and the cornea, iris, and lens are removed. Using fine-tipped forceps, the neuroretinal layer is slowly dissected along the periphery of the optic disc, avoiding direct traction on the RPE surface. It is preferable to dissect the neuroretina from the periphery towards the center to minimize tearing of the central RPE region. The intact RPE-Bruch's membrane-choroidal complex is preserved, avoiding scraping the RPE surface. Four to six radial incisions are made outward from the optic disc, allowing the tissue to spread naturally to form a flat, whole-mount sample, with the RPE side facing upward.

[0074] (2) Fixation and staining (a) Completely fixed, permeable and sealed treatment RPE-coated samples were placed in 4% PFA for secondary fixation (30 minutes, room temperature), followed by washing three times with PBS for 5 minutes each time. Mild permeabilization was then performed for 15 minutes with 0.1% Triton X-100 in PBS to avoid over-permeabilization and cell boundary collapse. The samples were then blocked for 60 minutes in 5% BSA or normal goat serum to reduce nonspecific background signal. (b) Phalloidin fine staining Add the fluorescently labeled Phalloidin (Invitrogen, A12379) to the sample at a recommended dilution of 1:400. Incubate at room temperature for 60 minutes in the dark, avoiding vigorous shaking to maintain the integrity of the F-actin ring structure. After staining, wash three times with PBS for 10 minutes each time to remove unbound dye.

[0075] (c) DAPI nuclear counterstaining Prepare a DAPI working solution (final concentration approximately 1 μg / mL), place the sample in the DAPI solution, and incubate at room temperature for 10 minutes in the dark to label RPE cell nuclei. After staining, wash three times with PBS (5 minutes each time) to remove excess nuclear dye and avoid excessive background signal.

[0076] (d) Anti-fluorescence quenching sealing and structural protection Place the stained RPE sample flat in the center of a glass slide, RPE side up, and add anti-fluorescence quenching mounting solution (Beyotime, P0126). Gently cover with a coverslip and press down slowly from one side, avoiding air bubbles or deformation of the RPE layer. After mounting, place the sample in a dark environment at 4°C for at least 30 minutes to cure, and complete imaging within 24-48 hours to obtain the best signal-to-noise ratio.

[0077] (3) Imaging acquisition and regional quantitative analysis After fluorescence staining and mounting of whole-mount RPE samples, images were acquired using confocal microscopy or high-resolution fluorescence microscopy. To ensure data comparability between different experimental groups, all samples were acquired under the same excitation wavelength, exposure time, gain parameters, and magnification. Based on the sample's position relative to the optic disc, the RPE tiling area was divided into central, intermediate, and peripheral zones. Multiple non-overlapping fields of view were randomly selected within each zone for imaging. At least nine representative fields of view were acquired per animal, and each group included at least three animals with three independent biological replicates. Image acquisition and subsequent analysis were performed under blinded conditions to minimize human selection bias.

[0078] The acquired images were subjected to semi-automatic quantitative analysis using ImageJ or equivalent image analysis software to objectively measure the morphology and arrangement of RPE cells. Quantitative parameters included single-cell area and its coefficient of variation, hexagonal cell proportion (reflecting honeycomb regularity), F-actin boundary continuity (reflecting cytoskeleton integrity), cell density, and the proportion of focal defect areas. Multiple cells were measured within each field of view, and the average value was calculated. Data were then aggregated using each animal as the statistical unit. The obtained data were statistically analyzed using GraphPad Prism 9 software and expressed as mean ± standard error. One-way ANOVA with multiple comparison correction was used to compare different experimental groups. The statistical significance threshold was set at P < 0.05.

[0079] (4) Results Analysis The results showed that RPE cells in the WT group exhibited a typical, regular honeycomb arrangement, with continuous F-actin signal, clear boundaries, and uniform cell area distribution; while the SI group showed significant RPE degenerative changes, including disordered cell arrangement, broken boundaries, irregular arrangement, abnormal increase or decrease in cell area, and the formation of focal defect areas. After 2A6S treatment, the regularity of RPE cell arrangement was significantly improved, the proportion of hexagonal structures increased, the continuity of the cytoskeleton was enhanced, the cell area distribution tended to be stable, and the area of ​​focal defects was significantly reduced. Figure 4 ).

[0080] The above results indicate that the arginine polyester amide material 2A6S can effectively maintain the integrity of RPE cell structure, reduce RPE degenerative damage and promote the recovery of honeycomb tissue structure. It provides direct experimental evidence for its application in the treatment of dry AMD from the perspective of cell morphology and tissue structure, and forms a multi-level technical evidence chain consistent with the aforementioned results of improvement in visual function and retinal layer structure.

[0081] 4. Retinal cell assessment To evaluate the effects of arginine polyesteramide material 2A6S on retinal cell damage, glial response, and apoptosis in dry AMD mice, immunofluorescence staining and TUNEL assays were performed on the retinal tissues of mice in each group on day 7 after modeling and drug administration to systematically assess photoreceptor survival status, glial cell response level, and degree of retinal cell apoptosis.

[0082] (1) Retinal sampling and processing (a) Sampling: After the sampling time, adult mice were euthanized, and both eyeballs were quickly removed and immediately placed in 4% PFA fixative prepared in PBS. They were then gently shaken at 4°C for about 20 minutes for initial fixation. (b) Fixation: After initial fixation, carefully peel off the retinal tissue and place the separated retina in 4% PFA / PBS fixative for a second fixation for 10 minutes to ensure tissue stability.

[0083] (c) Dehydration: After fixation, the retinal samples were transferred to 30% sucrose prepared in PBS and gently shaken overnight at 4°C for dehydration protection.

[0084] (d) Embedding: After dehydration, the sample was placed in OCT embedding medium (Sakura, 4583) and incubated at 4°C for 1 hour to promote full penetration of the embedding medium. Then, it was quickly frozen with dry ice and stored at -80°C for later use.

[0085] (e) Sectioning: The sample was sectioned using a cryostat to a thickness of approximately 14 μm. After the sections were attached to a detachable glass slide, they were dried at room temperature for 1 hour and then stored at -80°C for later use.

[0086] (2) Immunofluorescence staining (a) Drying: Warm the slides to room temperature, wipe off the water droplets on the surface of the coverslip, draw a closed circle around the sample on the coverslip with an immunohistochemistry pen (Hydrophobic Barrier (PAP) Pen), and then place the coverslip horizontally in the slide staining box (Beyotime, FSR960) with the sample side facing up, and dry for 10 minutes.

[0087] (b) Washing: Use a pipette to draw 500 μL of pre-cooled 1 × PBST (100 μL Triton-100 dissolved in 100 mL PBS) into the circle, let stand at room temperature for 5 minutes, and repeat 3 times.

[0088] (c) Blocking: Add 5% (v / v) of normal donkey serum (Jackson immunoResearch, 017-000-121) to 1 × PBST to prepare a 5% blocking solution. Add 200 μL to each coverslip and block at room temperature for 1 hour.

[0089] (d) Primary antibody incubation: Dilute the following primary antibodies with 1% blocking buffer (containing 1% normal donkey serum) prepared with 1×PBST: Arrestin (Millipore, catalog number: AB15282, dilution: 1:2000), Recoveryin (Millipore, catalog number: AB5585, dilution: 1:1000), and GFAP (Millipore, catalog number: AB5541, dilution: 1:2000) overnight (12-18 hours) in the dark at 4°C.

[0090] (e) Secondary antibody incubation: After primary antibody incubation, wash three times with 1 × PBST for 5 minutes each time, then add fluorescently labeled secondary antibody (dilution ratio of about 1:1000) and DAPI (final concentration of about 1 μg / mL), and incubate at room temperature in the dark for 1 hour.

[0091] (f) Mounting: After the secondary antibody incubation is completed, wash the slides three times with 1 × PBST for 5 minutes each time, then add anti-fluorescence quenching mounting solution to mount the slides, and use a confocal microscope to acquire images of the retinal sections.

[0092] (2) TUNEL staining This experiment mainly used the Click-iT™ Plus TUNEL Assay (C10618) kit from ThermoFisher. The experimental procedure was mainly performed according to the Fix and permeabilize tissue sections step in the kit for sample processing and staining.

[0093] (3) Results Analysis Immunofluorescence results showed that the SI group exhibited significantly weakened photoreceptor marker signals (Arrestin, Recoverin), indicating reduced photoreceptor cell survival; significantly enhanced GFAP expression indicated abnormal activation of Müller glial cells; and a significantly increased number of TUNEL-positive cells suggested elevated retinal cell apoptosis. In contrast, the 2A6S-treated group showed significantly enhanced photoreceptor marker signals, decreased GFAP expression, and a significantly reduced number of TUNEL-positive cells, indicating that 2A6S can effectively protect photoreceptor cells, inhibit excessive glial cell response, and reduce retinal cell apoptosis. Figure 5 ).

[0094] The above results, from the perspectives of cell survival, glial reaction, and apoptosis inhibition, indicate that the arginine polyesteramide material 2A6S has a clear neuroprotective effect in the dry AMD mouse model, further supporting its application potential as a candidate material for the treatment of dry AMD, and forming a multi-level technical evidence chain consistent with the aforementioned results of visual function improvement and tissue structure protection.

[0095] Example 4: Verification of the molecular mechanism by which 2A6S improves dry AMD by inhibiting inflammatory signaling pathways 1. RNA transcriptome sequencing analysis To systematically elucidate the molecular regulatory mechanism of arginine polyesteramide material 2A6S in a dry AMD mouse model, and to focus on evaluating its impact on inflammation-related signaling pathways, RNA transcriptome sequencing (RNA-seq) analysis was performed on retinal tissue after modeling and drug administration.

[0096] (1) Collection of materials (a) The experimental groups included the normal control group (WT), the sodium iodate-induced dry AMD model group (SI), and the 2A6S treatment group (SI+2A6S). After modeling and drug administration, the mice in each group were euthanized on day 7 post-modeling, and the eyeballs of both eyes were quickly harvested.

[0097] (b) Microscopic dissection was performed on ice to preserve the complex tissue consisting of the neuroretina and the outer layer of the retinal pigment epithelium, while removing only the sclera and choroidal layer to reduce interference from non-target tissues and preserve the RPE-retinal joint transcription signal that is closely related to the pathological process of dry AMD to the greatest extent.

[0098] (c) Collect retinal-RPE composite tissue samples from no fewer than 3-5 mice in each group. Samples can be processed using individual independent sample library construction or equal-volume pooling. After collection, all tissue samples should be immediately immersed in liquid nitrogen for rapid freezing and stored in liquid nitrogen until subsequent RNA extraction, transcriptome library construction, and sequencing analysis are performed.

[0099] (2) RNA sequencing was commissioned to Beijing Biomarker Biotechnology Co., Ltd., and paired-end sequencing was performed using the Illumina HiSeq 2500 platform.

[0100] (3) Bioinformatics analysis The raw reads obtained from high-throughput sequencing underwent quality control and adapter sequence removal to obtain high-quality, clean data. The clean reads were then aligned to the mouse reference genome (mm10 version) using the STAR alignment algorithm to achieve highly accurate transcript localization. Based on the alignment results, the expression levels of each gene were quantified, and the DESeq2 algorithm was used to perform statistical analysis on differentially expressed genes (DEGs) among different experimental groups (WT, SI, 2A6S) to identify significantly upregulated or downregulated gene sets. Multidimensional statistical and visualization analysis of the gene expression matrix was performed in the R language environment.

[0101] (4) Results Analysis Transcriptome analysis results as follows Figure 6 As shown. Heatmap results ( Figure 6As shown in A), in the sodium iodate-induced dry AMD model group (SI), compared with the normal control group (WT), multiple inflammation and immune-related genes were significantly upregulated and highly expressed. These genes were mainly concentrated in the following functional modules: chemotaxis, NF-κB signaling, Type I interferon production, and NLRP3 inflammasome. In the 2A6S treatment group (SI + 2A6S), the expression levels of these genes were significantly reduced overall, showing a trend from high to low expression, indicating that 2A6S can reverse the expression of these key inflammation-related genes. Further quantitative expression analysis of key inflammation-related genes was performed (…). Figure 6 B) shows that in group SI Il1b , Tnf , Ccl4 , Ccl5 , Cxcl10 The transcriptional levels of representative inflammation-related genes were significantly increased in the 2A6S treatment group, while the expression levels of these genes were significantly decreased in the 2A6S treatment group, with statistically significant differences. The bar chart results are consistent with the heatmap trend, indicating that the transcriptional activation levels of inflammation-related genes were significantly reduced after 2A6S treatment.

[0102] In summary, inflammation and immune-related pathway genes were significantly upregulated in the dry AMD model. Treatment with 2A6S significantly reduced the overall expression levels of these inflammation-related gene modules, suggesting that 2A6S can significantly inhibit inflammation-related transcriptional activation in the dry AMD model. These results validate the inhibitory effect of 2A6S on inflammatory pathways at the transcriptomic level, providing molecular-level experimental evidence for its ability to improve the pathological state of dry AMD.

[0103] 2. RT-qPCR and Western blot validation (1) RNA reverse transcription Total RNA (derived from retinal-RPE complex tissue) obtained and passed quality control in this example was processed under RNase-free conditions. A reaction system was prepared for each RNA sample at a fixed input amount (0.5 μg). Reverse transcription was performed using Novizan's HiScript III RT SuperMix for qPCR, R323-01 kit. After the reaction, the resulting cDNA was diluted to a uniform working concentration and stored at -20 ℃ for subsequent qPCR amplification and detection.

[0104] (2) RT-qPCR Using cDNA obtained from reverse transcription as a template, real-time quantitative PCR was performed using the KAPA SYBR® FAST qPCR Master Mix (2X) Kit (Roche, KK4601). The experiment was set up in three groups: WT, SI, and SI+2A6S, with each group containing at least three biological replicates, and technical replicate wells were provided for each sample. The target gene for amplification was a key gene in the NF-κB pathway. Rel , Relb , Nfkb1 , Nfkb2 , Nfkbia , Nfkbiz , Ikbkb , Ikbke and inflammatory factors Il1b , Tnf ; using internal reference genes Gapdh Normalization was performed, and the primers used are shown in Table 1. The RT-qPCR reaction system (10 µL) consisted of: 5 µL 2× KAPASYBR® FAST qPCR Master Mix, 0.25 µL primer F, 0.25 µL primer R, 1 µL cDNA, and the remainder being deionized water. The RT-qPCR reaction program was: 95℃ for 3 min, 95℃ for 10 s, 60℃ for 30 s, for 40 cycles. After the RT-qPCR reaction, the relative mRNA expression level was calculated using the ΔΔCt method, and statistical analysis was performed on the data for each group.

[0105] Table 1 qRT-PCR primer sequences (3) Protein extraction (a) Take the retinal-RPE complex tissue of mice in the WT group, SI model group and SI+2A6S treatment group, add pre-cooled RIPA lysis buffer (Beyotime, catalog number P0013B), and add a mixture of protease phosphatase inhibitors (Beyotime, catalog number P1050). Incubate at 4°C for about 30 minutes to fully lyse the cells and protect the proteins and their phosphorylation modification state.

[0106] (b) After lysis, the sample was centrifuged at 4°C and 14,000 × g for 15 minutes to remove cell debris and insoluble impurities. The supernatant was collected as the total protein sample, and the protein concentration was determined using the BCA Protein Quantitative Kit (Beyotime, catalog number P0011).

[0107] (c) After unifying the protein concentration of each sample to the same level, add protein loading buffer, heat at 95°C for 10 minutes to denature, and then store at -80°C for short-term use.

[0108] (4) Western blot immunoblotting (a) Protein electrophoresis and transfer: An equal amount of total protein sample (10 μg / lane) was taken for SDS-PAGE gel (Beyotime, catalog number P0012AC) electrophoresis separation, and the separated proteins were transferred to a 0.45 μm polyvinylidene fluoride (PVDF, Millipore Immobilon-P, IPVH00010) membrane.

[0109] (b) Blocking treatment: After the transfer was completed, the PVDF membrane was placed in TBST buffer (TBS + 0.1% Tween-20) containing 5% skim milk powder and blocked at room temperature for 1 hour to reduce nonspecific binding background.

[0110] (c) Primary antibody incubation: After blocking, place the membrane in blocking buffer containing the primary antibody and incubate overnight (12-18 hours) at 4°C. The primary antibody is preferably targeted at proteins related to inflammation and the NF-κB signaling pathway, specifically: phosphorylated NF-κB p65 (Ser536) antibody (Cell Signaling Technology, catalog number 3033), total NF-κB p65 antibody (Cell Signaling Technology, catalog number 8242), and β-actin internal control antibody (Cell Signaling Technology, catalog number 4967).

[0111] (d) Secondary antibody incubation and washing: The membrane was washed three times with TBST buffer the following day, followed by the addition of HRP-labeled secondary antibody matching the source of the primary antibody, and incubated at room temperature for 1 hour. After incubation, the membrane was washed three more times with TBST buffer, each time for about 10 minutes, to reduce background signal.

[0112] (e) Chemiluminescence imaging The target protein bands were developed using a chemiluminescent substrate (ECL luminescent reagent, Beyotime, catalog number P0018FS), and images were acquired using a chemiluminescent imaging system (G:BOX Chemi XT4).

[0113] (5) Results Analysis qRT-PCR results showed that in the SI group, key genes related to the NF-κB signaling pathway ( Rel , Relb , Nfkb1 , Nfkb2 , Nfkbia , Nfkbiz , Ikbkb , Ikbke ) and inflammatory factor genes ( Il1b , TnfThe relative mRNA expression levels of these genes were significantly higher in the SI group than in the normal control group (WT), indicating that the NF-κB pathway and inflammation-related transcriptional activity were significantly enhanced in the SI group. Compared with the SI group, the mRNA expression levels of the above genes were significantly decreased in the 2A6S treatment group (SI+2A6S), and the abnormal upregulation trend of most genes was significantly inhibited, suggesting that 2A6S can reduce the transcriptional activation levels of NF-κB pathway-related genes and inflammatory factors. Figure 7 (A) Western blot results further showed that the phosphorylation level of NF-κB protein (p-NF-κB) was significantly enhanced in the SI group, while the p-NF-κB band intensity was significantly reduced in the 2A6S treatment group. Figure 7 (B in the original text); the total NF-κB protein level remained relatively stable, with β-actin used as an internal reference for normalization correction. These results indicate that the activation level of the NF-κB signaling pathway significantly decreased after 2A6S treatment.

[0114] In summary, the NF-κB signaling pathway and inflammation-related genes are activated in the dry AMD model, and 2A6S treatment can significantly reduce the transcriptional and protein phosphorylation levels of the above pathways, thus showing an inhibitory effect on the activation state of the NF-κB pathway at the molecular level.

[0115] Example 5: Comparison of the therapeutic effects of 2A6S with NF-κB inhibitors and complement C3 inhibitory peptides 1. Selection and grouping of laboratory animals The complement system is considered to play a crucial role in the development and progression of dry age-related macular degeneration (AMD) and geographic atrophy. In recent years, complement inhibitors have been used as targeted interventions against the immune and inflammatory cascade to slow the spread of AMD lesions. Among them, pegcetacoplan acetate (MedChemExpress, catalog number HY-P3252A), a complement C3 inhibitory peptide, reduces complement-related inflammatory signals and tissue damage by blocking the complement C3-mediated cascade activation response. It is one of the representative drugs currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of dry AMD (GA), representing a current clinical treatment strategy targeting the complement single immune pathway.

[0116] Based on the aforementioned fact that 2A6S can significantly inhibit the activation of NF-κB-mediated inflammation-related signaling pathways, suggesting that the NF-κB signaling axis may be involved in the retinal protection mediated by 2A6S, this embodiment further selected the NF-κB signaling pathway inhibitor Bay 65-1942 (MedChemExpress, catalog number HY-50949) as a representative control drug for targeted intervention of the inflammation pathway.

[0117] Based on the above background, this embodiment used 8-week-old male mice, which were randomly divided into: a normal control group (WT), a dry AMD model group (SI), a 2A6S treatment group, a Bay 65-1942 treatment group, and a pegcetacoplan acetate treatment group, with no fewer than 5 mice in each group, and at least 3 independent biological replicates were set up. All animals were managed under the same breeding conditions and ethical guidelines to reduce bias caused by environmental and individual differences.

[0118] (2) Establishment of dry AMD model and administration method The establishment of dry AMD models and the administration of 2A6S in the remaining groups were the same as in Example 2.

[0119] The Bay 65-1942 (CmpdA, IKKβ inhibitor) group was administered via intraperitoneal injection, with the dosage converted based on mouse body weight, preferably 10 mg / kg; the pegcetacoplan acetate group was administered via intravitreal injection at a concentration of 150 mg / mL, with a dosage of 2 μL / eye.

[0120] (3) Visual function testing methods On day 7 post-modeling, mice in each group underwent dark adaptation, anesthesia, and mydriasis, and ERG (electroreductive retinal tract) analysis was performed using the same animal retinal electrophysiological platform as in Example 2. Under dark adaptation conditions, multiple levels of light intensity stimulation were applied, and the amplitude changes of waves a, b, and c were recorded and analyzed to systematically evaluate the impact of different treatment strategies on visual function recovery. The stimulation parameters, data acquisition methods, and statistical methods for ERG were consistent with those in Example 2 to ensure the comparability of functional results.

[0121] (4) Results Analysis like Figure 8 As shown, compared with the normal control group (WT), the amplitudes of waves a, b, and c in the dry AMD model group (SI) were significantly reduced, indicating that the function of retinal photoreceptors, nuclear layer cells, and retinal pigment epithelium in mice was significantly damaged after sodium iodate induction.

[0122] Among the three treatment strategies, the 2A6S treatment group showed a similar level of improvement in a-wave amplitude as the complement C3 inhibitory peptide group, and its overall recovery was closer to that of the WT group and superior to that of the NF-κB inhibitor group, suggesting that 2A6S has a good protective effect on photoreceptor-related functions. Figure 8 (A and C in the text). Since the a wave mainly reflects the electrophysiological functional state of retinal photoreceptors, the above results indicate that 2A6S has a strong potential protective effect in maintaining or improving photoreceptor cell function.

[0123] Regarding the amplitude of the b-wave, no significant differences were observed among the 2A6S group, the complement C3 inhibitory peptide group, and the NF-κB inhibitor group, and all three showed a recovery trend similar to that of the WT group. Figure 8 The results (A and D in the text) suggest that different treatment strategies can produce a certain degree of functional recovery in terms of improving the visual signal transmission function related to the kernel layer.

[0124] In contrast, in the C-wave amplitude analysis, only the 2A6S treatment group showed a significant improvement compared to the SI group, while the complement C3 inhibitory peptide group and the NF-κB inhibitor group did not show the same degree of recovery. Figure 8 (B and E in the text). Given that the c wave mainly reflects the functional status of the RPE and the metabolic support capacity of the outer retinal layer, this result suggests that 2A6S has a more significant protective advantage in improving RPE function and maintaining the homeostasis of the outer retinal layer.

[0125] Based on the aforementioned functional characteristics, it can be inferred that the therapeutic effect of 2A6S in the dry AMD model is not only reflected in the improvement of photoreceptor and core layer function, but also exhibits a more prominent protective effect at the RPE-related functional level. This result suggests that 2A6S may exert its therapeutic effect through multi-level retinal structural and functional protection mechanisms, rather than being limited to a single complement inhibition or NF-κB inflammatory pathway inhibition strategy, thus supporting its technological advantages as a potential therapeutic material for dry AMD from a functional perspective.

Claims

1. The application of arginine polyesteramide 2A6S in the preparation of drugs for the prevention and treatment of dry age-related macular degeneration, characterized in that, The structural formula of the arginine polyester amide 2A6S is shown below, where n=1: 。 2. The application according to claim 1, characterized in that, The arginine polyesteramide 2A6S is obtained by polycondensation reaction of monomer N2 and monomer Arg-6-S, as shown in Formula 3, where n=1: Formula 3.

3. The application according to claim 2, characterized in that, The monomer N2 was prepared by the following method: Triethylamine and p-nitrophenol were added to acetone, and the mixed solution was kept at 0°C. Succinyl chloride diluted with acetone was added dropwise to the mixed solution. After stirring at 0°C for 2 h, the mixture was cooled to room temperature and reacted overnight to generate di-p-nitrophenyl succinate. Di-p-nitrophenyl succinate was precipitated in distilled water, washed thoroughly, dried under vacuum, and recrystallized three times in a system of ethyl acetate and DMF at a volume ratio of 4:1 to obtain monomer N2. The preparation reaction formula is shown in Formula 1. Formula 1; The monomer Arg-6-S was prepared by the following method: L-arginine was mixed with 1,6-hexanediol, and then toluene and p-toluenesulfonic acid monohydrate were added. The mixture was heated to 120-130°C and stirred under reflux for 24 h. After cooling to room temperature, the supernatant was precipitated by toluene precipitation. The product was dissolved in isopropanol at 75°C and then precipitated at 4°C. The precipitation was repeated 3 times. The white viscous product was dried under vacuum to obtain the monomer Arg-6-S. The preparation reaction formula is shown in Formula 2. Formula 2.

4. The application according to claim 1, characterized in that, The dosage of arginine polyesteramide 2A6S in the drug is 20 mg / kg per dose.

5. The application according to claim 1, characterized in that, The medication is administered by injection.

6. The application according to claim 1, characterized in that, The drug is administered once every 48 hours.

7. The application according to claim 1, characterized in that, The drug prevents and treats dry age-related macular degeneration through at least one of the functions of (1)-(4): (1) Improves retinal structural damage; (2) Protect the structural integrity of the retinal pigment epithelium; (3) Protects retinal neurons and inhibits glial reaction and apoptosis; (4) Relieve inflammatory response by inhibiting the NF-κB signaling pathway.

8. The application according to claim 7, characterized in that, The inhibition of the NF-κB signaling pathway refers to suppressing the expression levels of genes related to the NF-κB signaling pathway and reducing the phosphorylation levels of proteins related to the NF-κB signaling pathway.

9. The application according to claim 8, characterized in that, The inhibition of the expression level of genes related to the NF-κB signaling pathway is described as inhibition. Rel , Relb , Nfkb1 , Nfkb2 , Nfkbia , Nfkbiz , Ikbkb , Ikbke The expression level of inflammatory factors is thus inhibited, thereby suppressing the expression level of inflammatory factors. Il1b , Tnf The level of expression.

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