Anti-retinal neovascularization pharmaceutical composition targeting slc11a1, screening method and application

Abstract

The present application relates to a kind of anti-retinal neovascularization drug composition, screening method and application of targeting SLC11A1, drug composition includes expression inhibitor or function inhibitor of targeting SLC11A1, and delivery carrier;Expression inhibitor and function inhibitor are respectively used to inhibit the expression or function of SLC11A1 in microglial cell;Delivery carrier is used to load and deliver expression inhibitor or function inhibitor;By inhibiting the expression or function of SLC11A1 in microglial cell, reduce microglial cell pro-vascular phenotype, inhibit abnormal proliferation, migration and lumen formation of retinal endothelial cell, thereby effectively inhibit the formation of retinal neovascularization, can be used to prepare the drug for treating or preventing retinal neovascularization related diseases.

Description

Technical Field

[0001] This invention relates to the fields of ophthalmic disease treatment targets, nucleic acid drugs, drug screening and anti-angiogenic drug development technology, specifically to an anti-retinal neovascularization drug composition targeting SLC11A1 (solute carrier family 11 member 1), screening method and application. Background Technology

[0002] Retinal neovascularization is a core pathological feature of various ischemic retinal diseases, such as retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. Abnormal neovascularization can lead to retinal hemorrhage, exudation, fibrovascular proliferation, and even tractional retinal detachment, and is one of the main causes of irreversible vision impairment in working-age individuals and children.

[0003] Currently, clinical treatment mainly involves intravitreal injection of anti-vascular endothelial growth factor drugs, laser photocoagulation, and surgery. However, anti-VEGF therapy has problems such as limited response rate, need for repeated injections, and unstable long-term efficacy. In addition, some patients experience adverse reactions such as drug resistance or worsening of fibrosis. Therefore, there is an urgent need to develop anti-retinal neovascularization treatment strategies based on novel targets and mechanisms. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an anti-retinal angiogenesis drug composition targeting SLC11A1, a method for screening anti-retinal angiogenesis drugs targeting SLC11A1, and the application of an anti-retinal angiogenesis drug composition targeting SLC11A1, in view of the above-mentioned defects of the prior art.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] A drug composition targeting SLC11A1 to combat retinal neovascularization is constructed, wherein the drug composition comprises:

[0007] An expression inhibitor or a function inhibitor targeting SLC11A1, wherein the expression inhibitor and the function inhibitor are used to inhibit the expression or function of SLC11A1 in microglia, respectively.

[0008] Delivery vectors for loading and delivering the expression inhibitors or functional inhibitors.

[0009] The anti-retinal angiogenesis drug composition targeting SLC11A1 of the present invention includes, wherein the expression inhibitor comprises one or more of siRNA, shRNA, antisense oligonucleotide, miRNA mimic, CRISPR / Cas system and CRISPRi system.

[0010] The present invention describes an anti-retinal neovascularization pharmaceutical composition targeting SLC11A1, wherein the functional inhibitor comprises a small molecule compound, peptide, antibody, antibody fragment, or pharmaceutically acceptable salt thereof capable of inhibiting SLC11A1-mediated iron homeostasis disturbances, free iron accumulation, oxidative stress, lipid peroxidation, or glycolytic metabolic reprogramming.

[0011] The anti-retinal neovascularization drug composition targeting SLC11A1 of the present invention, wherein the delivery carrier comprises one or more of lipid nanoparticles, polymer nanoparticles, cationic liposomes, exosomes, engineered extracellular vesicles, adeno-associated virus vectors, hydrogels, microspheres, or ophthalmic sustained-release carriers.

[0012] A method for screening anti-retinal angiogenesis drugs targeting SLC11A1, used to obtain an expression inhibitor or functional inhibitor of SLC11A1 in the anti-retinal angiogenesis drug composition targeting SLC11A1 as described above, characterized in that the method includes the following steps:

[0013] Establish a microglial cell model induced by hypoxia or ischemia stress;

[0014] The candidate drug was administered to a microglial cell model.

[0015] Detect changes in SLC11A1 expression or function in a microglia model;

[0016] Detect indicators related to iron homeostasis, oxidative stress, lipid peroxidation, or glycolysis in a microglia model;

[0017] Collect microglial cell conditioned medium and apply it to retinal endothelial cells;

[0018] Detecting the proliferation, migration, or lumen formation capacity of retinal endothelial cells;

[0019] If a drug reduces the expression or function of SLC11A1 and inhibits endothelial cell angiogenesis, it is judged to have an anti-retinal neovascularization effect and can be used as an expression inhibitor or function inhibitor targeting SLC11A1; otherwise, it is judged not to have an anti-retinal neovascularization effect.

[0020] The method for screening anti-retinal neovascularization drugs targeting SLC11A1 described in this invention includes one or more of the following methods for detecting SLC11A1 expression: qPCR, Western blot, immunofluorescence, flow cytometry, in situ hybridization, or single-cell transcriptome analysis.

[0021] The method for screening anti-retinal neovascularization drugs targeting SLC11A1 described in this invention includes one or more of the following indicators related to iron homeostasis: free iron level, FTH1, FTL, TFRC, SLC40A1, or iron ion fluorescent probe signal.

[0022] The method for screening anti-retinal neovascularization drugs targeting SLC11A1 described in this invention includes one or more of the following indicators related to oxidative stress or lipid peroxidation: ROS, 4-HNE, MDA, C11-BODIPY signal, GPX4, SLC7A11, or ACSL4.

[0023] The method for screening anti-retinal neovascularization drugs targeting SLC11A1 described in this invention includes one or more of the following glycolytic metabolism-related indicators: lactate release, glucose consumption, ATP level, ECAR, HK2, PFKFB3, LDHA, or GLUT1.

[0024] An anti-retinal angiogenesis drug composition targeting SLC11A1 is used in the manufacture of drugs that inhibit retinal angiogenesis.

[0025] The beneficial effects of this invention are as follows: the pharmaceutical composition of this invention targets SLC11A1 by means of expression inhibitors or function inhibitors, which can effectively inhibit the expression or function of SLC11A1 in microglia, thereby reducing the pro-angiogenic phenotype of microglia; by loading and delivering expression inhibitors or function inhibitors by delivery carriers, the targeting and therapeutic effect of the drug can be improved; by inhibiting the expression or function of SLC11A1 in microglia, the abnormal proliferation, migration and lumen formation of retinal endothelial cells can be inhibited, thereby effectively inhibiting the formation of retinal neovascularization. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. The drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort:

[0027] Figure 1 This is a flowchart of a preferred embodiment of the present invention for screening anti-retinal angiogenesis drugs targeting SLC11A1;

[0028] Figure 2 This is a schematic diagram illustrating the upregulation of SLC11A1 expression in in vitro and in vivo models according to Embodiment 1 of the present invention;

[0029] Figure 3 This is a schematic diagram of ferroptosis after inhibiting SLC11A1 in Embodiment Example 3 of the present invention;

[0030] Figure 4 This is a schematic diagram of the changes in the expression of ACSL4, SLC7A11 and GPX4 proteins in the OIR retina of Example 4 of the present invention;

[0031] Figure 5 This is a schematic diagram of the changes in the expression of ferroptosis-related molecules in ROP tears in Example 4 of this invention;

[0032] Figure 6 This is a schematic diagram of the retinal vascular morphology after Ferrostatin-1 treatment in Example 4 of this invention.

[0033] Figure 7 This is a schematic diagram illustrating the changes in the expression of glycolysis-related molecules in OIR retina and hypoxic microglia in Example 5 of this invention;

[0034] Figure 8 This is a schematic diagram of the changes in endothelial cell proliferation after inhibiting SLC11A1 in Example 6 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, a clear and complete description will be provided below in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0036] A preferred embodiment of the present invention is an anti-retinal angiogenesis pharmaceutical composition targeting SLC11A1, the pharmaceutical composition comprising:

[0037] Inhibitors of SLC11A1 expression or function, which are used to inhibit the expression or function of SLC11A1 in microglia, respectively.

[0038] Delivery vectors are used to load and deliver expression inhibitors or functional inhibitors.

[0039] Microglia are innate immune cells in the retina. They can be activated and undergo functional remodeling in hypoxic and ischemic retinopathy, and influence endothelial cell behavior by secreting inflammatory factors, metabolic factors, and pro-angiogenic factors. In recent years, iron homeostasis disorder, oxidative stress, and immune metabolic reprogramming have been considered as important mechanisms regulating the pro-angiogenic phenotype of microglia.

[0040] SLC11A1 is a solute transporter involved in metal ion transport, iron homeostasis, and immune regulation. Previous studies by the applicant have found that under pathological conditions of retinal neovascularization, SLC11A1 expression is increased in microglia and may be involved in microglia iron homeostasis disorder, enhanced oxidative stress, and glycolytic metabolic reprogramming. Inhibiting SLC11A1 is expected to reduce the pro-angiogenic phenotype of microglia, thereby inhibiting retinal neovascularization.

[0041] Currently, there is a lack of anti-retinal angiogenesis drug compositions targeting SLC11A1 and candidate drug screening methods based on the SLC11A1-iron homeostasis-metabolic reprogramming axis. Therefore, developing anti-retinal angiogenesis strategies targeting SLC11A1 has significant application value.

[0042] The pharmaceutical composition of this invention targets SLC11A1 by means of expression inhibitors or function inhibitors, which can effectively inhibit the expression or function of SLC11A1 in microglia, thereby reducing the pro-angiogenic phenotype of microglia; by loading and delivering expression inhibitors or function inhibitors by delivery carriers, the targeting and therapeutic effect of the drug can be improved; by inhibiting the expression or function of SLC11A1 in microglia, the abnormal proliferation, migration and lumen formation of retinal endothelial cells can be inhibited, thereby effectively inhibiting retinal neovascularization.

[0043] A method for screening anti-retinal angiogenesis drugs targeting SLC11A1, used to obtain an expression inhibitor or functional inhibitor of SLC11A1 in the anti-retinal angiogenesis drug composition targeting SLC11A1 as described above, such as... Figure 1 As shown, the method includes the following steps:

[0044] S1: Establish a microglial cell model induced by hypoxia or ischemia stress;

[0045] S2: Administer the candidate drug to the microglia model;

[0046] S3: Detect changes in SLC11A1 expression or function in a microglia model;

[0047] S4: Detect indicators related to iron homeostasis, oxidative stress, lipid peroxidation, or glycolysis in a microglia model;

[0048] S5: Collect microglial cells conditioned medium and apply it to retinal endothelial cells;

[0049] S6: Detects the ability of retinal endothelial cells to proliferate, migrate, or form lumens;

[0050] S7: If a drug reduces the expression or function of SLC11A1 and inhibits endothelial cell angiogenesis, it is judged to have an anti-retinal neovascularization effect and can be used as an expression inhibitor or function inhibitor targeting SLC11A1; otherwise, it is judged not to have an anti-retinal neovascularization effect.

[0051] An anti-retinal angiogenesis drug composition targeting SLC11A1 is used in the manufacture of drugs that inhibit retinal angiogenesis.

[0052] The following is a verification implementation example:

[0053] Step 1: Identify targets and intervention tools;

[0054] First, it is necessary to confirm whether SLC11A1 is related to the pathological conditions of retinal neovascularization. Example 1: Verification of increased SLC11A1 expression in microglia induced by hypoxia.

[0055] Microglial cell lines or primary microglia were used to set up normoxic and hypoxic groups.

[0056] After hypoxia treatment, changes in SLC11A1 expression were detected by qPCR, Western blot, or immunofluorescence.

[0057] like Figure 2 As shown, Figure 2 A / B assay showed increased SLC11A1 expression in the OIR retina; CD assay showed upregulated SLC11A1 expression in HMC3 cells and primary microglia after hypoxia treatment; HMC3 cells were cultured in a hypoxic chamber for 6H, 12H, 24H, 36H, and 48H, respectively, and proteins were extracted, quantified, and denatured together with the normoxic group, followed by Western blotting. The results showed that, compared with the normoxic group, the expression of SLC11A1 in microglia was most significantly increased after 12H of hypoxia treatment, suggesting that SLC11A1 is involved in the microglial response under hypoxic stress. This result indicates that SLC11A1 is a potential intervention target.

[0058] After identifying SLC11A1 as a potential target, effective gene silencing tools are needed for functional studies; Example 2: siRNA intervention targeting SLC11A1;

[0059] We designed siRNA sequences targeting SLC11A1 and transfected them into microglia.

[0060] The siRNA sequence is as follows:

[0061] SEQ ID NO:1:

[0062] Chain of Justice: 5′-GGUCAAGUCUCGAGAGAUA[dT][dT]-3′

[0063] Antonym chain: 5′-UAUCUCUCGAGACUUGACC[dT][dT]-3′

[0064] SEQ ID NO:2:

[0065] Chain of Justice: 5′-CCUUGAAGAGGACCAGAAAG[dT][dT]-3′

[0066] Antonym chain: 5′-CUUUCUGGUCCUCUUCAAGG[dT][dT]-3′

[0067] The knockdown efficiency of SLC11A1 was detected by qPCR and Western blot after transfection.

[0068] The siRNA with the highest knockdown efficiency and lowest cytotoxicity was selected for subsequent experiments.

[0069] Step 2: Explore the downstream molecular mechanisms of SLC11A1;

[0070] Using the siRNA tool mentioned above, we first explored the regulatory role of SLC11A1 in its most direct biological function—iron homeostasis; Example 3: The effect of SLC11A1 intervention on iron homeostasis in microglia;

[0071] Microglia were subjected to SLC11A1 knockdown or functional inhibition treatment under normoxic or hypoxic conditions.

[0072] Intracellular free iron levels were evaluated using iron ion fluorescent probes, FerroOrange, Calcein-AM quenching assays, or other iron homeostasis detection methods.

[0073] Simultaneously, the expression changes of iron-stability-related molecules such as FTH1, FTL, TFRC, and SLC40A1 were detected.

[0074] like Figure 3 As shown, Figure 3The results showed that siRNA knockdown of SLC11A1 altered the expression of GPX4, SLC7A11, and LDHA proteins. Human retinal microvenous endothelial cells and HMC3 cells were co-cultured in transwell chambers, and the proliferation of endothelial cells was observed under different conditions. The results showed that SLC11A1-targeted intervention reduced hypoxia-induced iron homeostasis disorder in microglia.

[0075] One of the downstream consequences of iron homeostasis disorder is oxidative stress and lipid peroxidation; Implementation Example 4: The effect of SLC11A1 intervention on oxidative stress and lipid peroxidation;

[0076] Under hypoxic conditions, microglia were subjected to SLC11A1 knockdown or functional inhibition treatment, and indicators such as ROS, 4-HNE, MDA, C11-BODIPY, GPX4, SLC7A11, and ACSL4 were detected.

[0077] like Figures 4-6 As shown, Figure 4 The AD study showed changes in the expression of ACSL4, SLC7A11, and GPX4 proteins in the OIR retina; Figure 5 EG data showed dynamic expression of ACSL4 and SLC7A11 at different stages of the disease; Figure 5 The H-sample showed a decrease in GPX4 levels in the tears of children with ROP; Figure 5 I shows the expression of ferroptosis molecules in microglia; Figure 6 JK showed the vascular morphology of retinal slices after Ferrostatin-1 treatment (bar=500μm). After the retina was fixed with a special fixative FAS, it was removed, routinely stained with antibodies, cut into a four-leaf clover shape, and laid flat on a glass slide. The experimental results showed that targeted intervention for SLC11A1 can reduce the levels of oxidative stress and lipid peroxidation in microglia and improve ferroptosis-related stress.

[0078] In addition to oxidative stress, changes in iron homeostasis may also affect cellular energy metabolism; Example 5: The effect of SLC11A1 intervention on the reprogramming of glycolytic metabolism in microglia;

[0079] Under hypoxic conditions, microglia were treated with SLC11A1 knockdown or functional inhibition, and lactate release, glucose consumption, ATP levels, ECAR, and the expression of glycolysis-related molecules such as HK2, PFKFB3, LDHA, and GLUT1 were detected.

[0080] like Figure 7 As shown, Figure 7The AC (Acoustic Acid) assay was used to analyze the expression levels of LDHA and PFKFB3 proteins in the OIR retina using Western blot analysis. The DF (Polydioxanone) assay was used to analyze the changes in LDHA and PFKFB3 protein expression in microglia at different time points under hypoxic conditions using Western blot analysis. After grinding retinal tissue, proteins were extracted and quantitatively denatured, separated by SDS-PAGE electrophoresis, and transferred to a PVDF membrane. After blocking, the membrane was incubated with specific primary antibody and enzyme-labeled secondary antibody in sequence. Finally, the expression level of the target protein was detected by chemiluminescence imaging. The experimental results showed that targeted intervention of SLC11A1 can reduce hypoxia-induced glycolytic metabolic reprogramming in microglia.

[0081] Step 3: Verify the functional effects of SLC11A1 intervention on angiogenesis;

[0082] The above mechanistic studies have shown that inhibiting SLC11A1 can improve iron homeostasis, oxidative stress and metabolic status of microglia. The next step is to verify whether these changes can ultimately affect the angiogenesis function of endothelial cells. Example 6: The effect of SLC11A1 intervention on the conditioned medium of microglia on the angiogenesis behavior of endothelial cells.

[0083] Microglia from normoxic, hypoxic, hypoxic plus negative control intervention, and hypoxic plus SLC11A1 intervention groups were cultured and conditioned medium was collected.

[0084] The above-mentioned conditioned medium was applied to retinal microvascular endothelial cells, and the endothelial cell proliferation, migration, and lumen formation capabilities were detected.

[0085] The detection methods include EdU proliferation assay, Transwell migration assay, scratch assay, and Matrigel lumen formation assay.

[0086] like Figure 8 As shown, Figure 8 The AD model shows the proliferation of endothelial cells under co-culture conditions (D. bar=50μm; F. bar=250μm). The results showed that compared with the hypoxia group or the negative control group, the SLC11A1 intervention group had a decreased ability of microglia conditioned medium to induce endothelial cell proliferation, migration and lumen formation, suggesting that targeting SLC11A1 can reduce the pro-angiogenic function of microglia.

[0087] Step 4: Establish a drug screening method based on SLC11A1;

[0088] Based on the above research findings, a systematic drug screening method can be established; Implementation Example 7: Screening of anti-retinal neovascularization candidate drugs based on SLC11A1;

[0089] A hypoxic microglia screening system was established, and candidate drugs were added to the hypoxic microglia model to detect SLC11A1 expression, free iron level, lipid peroxidation level, glycolytic metabolism indicators, and pro-angiogenic factor expression.

[0090] Subsequently, microglia were collected and conditioned in culture medium was applied to retinal endothelial cells to detect endothelial cell lumen formation, migration, and proliferation.

[0091] If a candidate drug can reduce SLC11A1 expression or function and reduce endothelial cell angiogenesis behavior, then the candidate drug is judged to have anti-retinal neovascularization development value.

[0092] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A drug composition targeting SLC11A1 to combat retinal neovascularization, characterized in that, The pharmaceutical composition comprises: An expression inhibitor or a function inhibitor targeting SLC11A1, wherein the expression inhibitor and the function inhibitor are used to inhibit the expression or function of SLC11A1 in microglia, respectively. Delivery vectors for loading and delivering the expression inhibitors or functional inhibitors.

2. The anti-retinal angiogenesis drug composition targeting SLC11A1 according to claim 1, characterized in that, The expression inhibitors include one or more of siRNA, shRNA, antisense oligonucleotides, miRNA mimics, CRISPR / Cas systems, and CRISPRi systems.

3. The anti-retinal angiogenesis drug composition targeting SLC11A1 according to claim 1, characterized in that, The functional inhibitors include small molecule compounds, peptides, antibodies, antibody fragments, or pharmaceutically acceptable salts thereof capable of inhibiting SLC11A1-mediated iron homeostasis disturbances, free iron accumulation, oxidative stress, lipid peroxidation, or glycolytic metabolic reprogramming.

4. The anti-retinal angiogenesis drug composition targeting SLC11A1 according to claim 1, characterized in that, The delivery carrier includes one or more of the following: lipid nanoparticles, polymer nanoparticles, cationic liposomes, exosomes, engineered extracellular vesicles, adeno-associated virus vectors, hydrogels, microspheres, or ophthalmic sustained-release carriers.

5. A method for screening anti-retinal angiogenesis drugs targeting SLC11A1, used to obtain an expression inhibitor or functional inhibitor of SLC11A1 in the anti-retinal angiogenesis drug composition targeting SLC11A1 as described in any one of claims 1-4, characterized in that, The method includes the following steps: Establish a microglial cell model induced by hypoxia or ischemia stress; The candidate drug was administered to a microglia model. Detect changes in SLC11A1 expression or function in a microglia model; Detect indicators related to iron homeostasis, oxidative stress, lipid peroxidation, or glycolysis in a microglia model; Collect microglial cell conditioned medium and apply it to retinal endothelial cells; Detecting the proliferation, migration, or lumen formation capacity of retinal endothelial cells; If a drug reduces the expression or function of SLC11A1 and inhibits endothelial cell angiogenesis, it is judged to have an anti-retinal neovascularization effect and can be used as an expression inhibitor or function inhibitor targeting SLC11A1; otherwise, it is judged not to have an anti-retinal neovascularization effect.

6. The method for screening anti-retinal angiogenesis drugs targeting SLC11A1 according to claim 5, characterized in that, Detection of SLC11A1 expression includes one or more of the following methods: qPCR, Western blot, immunofluorescence, flow cytometry, in situ hybridization, or single-cell transcriptome analysis.

7. The method for screening anti-retinal neovascularization drugs targeting SLC11A1 according to claim 5, characterized in that, Relevant indicators of iron steady state include one or more of the following: free iron level, FTH1, FTL, TFRC, SLC40A1, or iron ion fluorescent probe signal.

8. The method for screening anti-retinal neovascularization drugs targeting SLC11A1 according to claim 5, characterized in that, Oxidative stress or lipid peroxidation-related indicators include one or more of ROS, 4-HNE, MDA, C11-BODIPY signaling, GPX4, SLC7A11, or ACSL4.

9. The method for screening anti-retinal neovascularization drugs targeting SLC11A1 according to claim 5, characterized in that, Glycolytic metabolism-related indicators include lactate release, glucose consumption, ATP levels, ECAR, HK2, PFKFB3, LDHA, or GLUT1, or one or more of these parameters.

10. The use of an anti-retinal angiogenesis drug composition targeting SLC11A1 in the manufacture of a drug for inhibiting retinal angiogenesis.

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