Methods for Treating Retinal Vasculopathy

Abstract

The present invention relates to a method for treating certain retinal vasculopathy, such as, inter alia, diabetic macular edema, diabetic retinopathy, and dry and neovascular age-related macular degeneration. In some cases, the method includes treating a patient suffering from retinal vasculopathy by administering a drug to the patient. The drug can be one or more of the drugs described herein. TIFF2024521770000002.tif118149

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 193,327, filed May 26, 2021, and U.S. Provisional Patent Application No. 63 / 220,771, filed July 12, 2021, the disclosures of each of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates to methods of treating certain retinal vasculopathies, such as diabetic macular edema, diabetic retinopathy, and age-related macular degeneration. [Background technology]

[0003] 2. Background of the Invention Pathological angiogenesis remains one of the major challenges in the treatment of certain retinal vasculopathies, such as diabetic macular edema (DME), diabetic retinopathy (DR), and age-related macular degeneration (AMD), which are serious diseases that often lead to vision loss and blindness.

[0004] DME is a complication of diabetic retinopathy (DR) following chronic, poorly controlled diabetes and is the most common form of sight-threatening retinopathy in diabetic patients (Tan et al. 2016, IDF 2019). Approximately 1 in 14 people with diabetes has some degree of DME (Coney 2019). The overall prevalence of DR in diabetic patients using retinal imaging was estimated to be 35%, with sight-threatening DR present in 12% (WHO 2015). Prevalence depends on the type of diabetes and duration of the disease. For both types of diabetes, type 1 diabetes (T1D) and type 2 diabetes (T2D), after 25 years of duration, the prevalence is close to 30% (Browning et al. 2018).

[0005] The previous standard of care to stabilize the vision of patients with such eye diseases included laser photocoagulation, surgical vitrectomy, and photodynamic therapy with verteporfin. Currently, anti-vascular endothelial growth factor (anti-VEGF) therapy and its ability to restore vision in patients suffering from such eye vascular diseases has been widely used since the approval of Lucentis® (ranibizumab) in 2006 and Eylea® (aflibercept) in 2011. The recent success of therapies that inhibit the function of VEGF demonstrates that specific targeting of growth factors involved in vascular permeability and growth is an effective means of treating DR-associated vascular dysfunction, edema, and neovascularization.

[0006] However, such anti-VEGF treatments often resulted in off-target effects on healthy blood vessels. Thus, identifying molecular mismatches between healthy and diseased blood vessels would enable targeted therapies that selectively eliminate pathological vasculature while sparing blood vessels essential for physiological tissue function.

[0007] Another drawback of standard anti-VEGF treatments is that they require frequent (e.g., monthly or even twice-monthly intravitreal injections) and long-term administration to maintain visual gains (Heier et al. Ophthalmology 2012;119:2537-48; the Comparison of Age-Related Macular Degeneration Treatment Trials [CATT] Research Group 2016 Ophthalmology 2016;123:1751-61). This has led to poor patient compliance with this frequent treatment regimen.

[0008] There is also evidence that some patients experience suboptimal responses to anti-VEGF treatment, and some patients do not respond to anti-VEGF treatment at all. See Brown DM, et al., Ophthalmology. 2013;120:2013-2022, and Nguyen-Khoa BA, et al., BMC Ophthalmol. 2012;12:11.

[0009] Recently, the presence and accumulation of senescent cells in an individual may contribute to aging and age-related dysfunctions and diseases, such as glaucoma, cataracts, diabetic pancreas, and osteoarthritis, among others (see van Deursen JM., Nature. 2014 May 22; 509(7501): 439-446; Childs, B. et al., Nat Med. 2015 December; 21(12): 1424-1435). Normally, mitotic cells can permanently withdraw from the cell cycle in response to cellular stresses, including dysfunctional telomeres, DNA damage, strong mitotic signals, and disrupted chromatin. This response, called cellular senescence, has been shown to be important in inhibiting the proliferation of dysfunctional or damaged cells, particularly in suppressing the development of cancer malignant tumor mechanisms (see Campisi J., Cell 120:513-22 (2005); Campisi J., Curr. Opin. Genet. Dev. 21:107-12 (2011)). Senescent cells are characterized by a number of cellular phenotypes, including insensitivity to mitogenic stimuli, flattened morphology, increased senescence-associated β-galactosidase activity (SA-β-gal), elevated p16 expression, shortened telomeres, elevated cyclin-dependent kinase inhibitor expression, altered chromatin structure, extensive DNA damage foci, resistance to apoptosis, and activation of the inflammation-induced senescence-associated secretory phenotype (SASP) (see Coppe, JP, et al., Annu Rev Pathol. 2010;5:99-118).

[0010] Recently, pathological vasculature in the retina has been shown to selectively participate in cellular senescence (see Crespo-Garcia et al., Cell Metabolism 2021, 33, 1-15). Given that senescent cells are likely causally involved in certain aspects of age-related decline in health and contribute to certain age-related diseases, including cancer, effective therapeutic approaches are being researched and developed. In some cases, small molecule compounds have been identified that selectively remove senescent cells accumulated in and around affected areas and alleviate the adverse signs and symptoms of the resulting condition. Several intracellular pathways that are active in senescent cells have been shown to be amenable to targeting, including the MDM2 pathway, the Bcl pathway, the Akt pathway, and the proteasome pathway, among others (see WO / 2015 / 171591: Zhou et al., WO / 2015 / 116740: Laberge et al., WO / 2019 / 133904: Hudson et al.).

[0011] Apart from or in addition to targeting senescent cells in the retina, an alternative mechanism by which VEGF expression may be increased in retinal vasculopathy involves endothelial endoplasmic reticulum (ER) stress and activating transcription factor 4 (ATF4). ATF4 is one of several transcription factors that are activated as part of the unfolded protein response (UPR) to stress. Translation of ATF4 mRNA into ATF4 protein is induced in response to several stresses that result in the inhibition of general protein synthesis via phosphorylation of eukaryotic translation initiation factor 2 (eIF2) (see Baird TD, et al., Adv Nutr. 2012;3:307-321). These stresses include nutrient deprivation, ER stress, oxidative stress, and hypoxia (see Abcouwer, Steven F., J Clin Cell Immunol,;Suppl1(11):1-12,2013). The age-related adaptive response to such stresses and the decline in hormesis provide an opportunity for therapeutic targeting this mechanism.

[0012] Thus, there is a need to develop alternative methods for treating retinal vasculopathy. Summary of the Invention

[0013] The present invention provides novel methods of treating retinal vasculopathy. The following are specifically contemplated as part of the disclosed invention:

[0014] Embodiment 1. A method of inhibiting pathogenic angiogenesis in an affected eye comprising administering an agent selected from the group consisting of an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2.

[0015] Embodiment 2. A method of inhibiting vascular leakage in an affected eye comprising administering an agent selected from the group consisting of an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2.

[0016] Embodiment 3. The method of embodiment 1 or 2, wherein the diseased eye is the result of senescent endothelial cells.

[0017] Embodiment 4. The method of embodiment 1 or 2, wherein the affected eye is the result of senescent pericytes.

[0018] Embodiment 5. The method of embodiment 1 or 2, wherein the affected eye is the result of diabetic macular edema (DME), diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR), dry age-related macular degeneration (dAMD), and neovascular age-related macular degeneration (nvAMD).

[0019] Embodiment 6. A method of removing senescent endothelial cells, comprising contacting the cells with an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2.

[0020] Embodiment 7. A method of removing senescent pericytes, comprising contacting the cells with an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2.

[0021] Embodiment 8. A method of treating a patient suffering from retinal vasculopathy, comprising administering to the patient an agent selected from the group consisting of an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2.

[0022] Embodiment 9. The method of embodiment 8, wherein the retinal vasculopathy is diabetic macular edema (DME), diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR), dry age-related macular degeneration (dAMD), and neovascular age-related macular degeneration (nvAMD).

[0023] Embodiment 10. The method of any one of embodiments 1-5, 8-9, wherein the agent is a small molecule, an antibody, a polypeptide, an antisense oligonucleotide, or a small interfering ribonucleic acid.

[0024] Embodiment 11. The method of embodiment 6 or 7, wherein the inhibitor or agonist is a small molecule, an antibody, a polypeptide, an antisense oligonucleotide, or a small interfering ribonucleic acid.

[0025] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the inhibitor of GPX4 is selected from the group consisting of RSL3, ML210, ML162, JKE-1674, DPI-7, buthionine sulfoximine (BSO), FIN56, auranofin, erastin, artemisinin, sulfasalazine, artesunate, dihydroatemicin, compound #19, compound #25, sorafenib, altretamine, almitrine, artemether, artemisone, and lanperisone.

[0026] Embodiment 13. The method of any one of embodiments 1 to 11, wherein the inhibitor of GLS1 is 6-diazo-5-oxo-L-norleucine, GK921, UPGL00004, telaglenastat, JHU395, ethyl 2-(2-amino-4-methylpentanamido)-DON, IPN60090, and BPTES.

[0027] Embodiment 14. The method of any one of embodiments 1 to 11, wherein the inhibitor of PAPP-A is an antibody.

[0028] Embodiment 15. The method of embodiment 14, wherein the antibodies are PAC-1scFv, PAC-1-D8scFv, PAC-2scFv, PAC-5scFv, and mAb-PA1 / 41.

[0029] Embodiment 16 The method of any one of embodiments 1 to 11, wherein the inhibitor of PAPP-A is a polypeptide.

[0030] Embodiment 17. The method of embodiment 16, wherein the polypeptides are pro-MBP, stanniocalcin-1 (STC1), stanniocalcin-2 (STC2), and bikunin.

[0031] Embodiment 18. The method of any one of embodiments 1 to 11, wherein the inhibitor of PAPP-A is an antisense oligonucleotide.

[0032] Embodiment 19. The inhibitor of cGAS is selected from the group consisting of 3-(1-(6,7-dichloro-1H-benzo[d]imidazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)isobenzofuran-1(3H)-one, 1-(6,7-dichloro-9-(1-methyl-1H-pyrazol-3-yl)-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)-2-hydroxyethane- 12. The method of any one of embodiments 1-11, wherein the compound is 1-one, 1-[9-(6-amino-3-pyridinyl)-6,7-dichloro-1,3,4,5-tetrahydro-2H-pyrido[4,3b]indol-2-yl]-2-hydroxy-ethanone, or (1R,2S)-2-(7-oxo-5-phenyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3carboxamide)cyclohexane-1-carboxylic acid.

[0033] Embodiment 20. The method of any one of embodiments 1 to 11, wherein the inhibitor of STING is H-151, C-178, C-176, or compound 18.

[0034] Embodiment 21. The method of any one of embodiments 1 to 11, wherein the inhibitor of mTOR is rapamycin, Palomid 529, sirolimus, everolimus, temsirolimus, ridaforolimus, umirolimus, zotarolimus, and an ATP-competitive mTOR kinase inhibitor.

[0035] Embodiment 22. The GCN2 agonist is selected from the group consisting of 4-(2-amino-4-methyl-3-(2-methylquinolin-6-yl)benzoyl)-1-methyl-2,5-diphenyl-1H-pyrazol-3(2H)-one, 1-(5-(4-amino-2,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)indolin-1-yl)-2-(3-fluoro-5-(trifluoromethyl)phenyl)-ethanone, 4- The method of any one of embodiments 1 to 11, wherein the therapeutic agent is (2-amino-4-methyl-3-(2-(methylamino)benzo[d]thiazol-6-yl)benzoyl)-1-methyl-2,5-diphenyl-1H-pyrazol-3(2H)-one, leucinol, histidinol, threoninol, SB-203207, SB-219383, dovitinib, neratinib, sunitinib, and elotinib. [Brief description of the drawings]

[0036] [Figure 1] Quantification of senescent cell burden in AMD and DR / DME as assessed by immunohistochemical staining for p16 compared to normal in postmortem retinal donor tissue from patients who had a antemortem diagnosis of AMD, DR / DME, or neither is depicted (see Example 2). [Diagram 2] Quantification of senescent cell burden in PDR as assessed by immunohistochemical staining for p16 in postmortem retinal donor tissue from patients with a antemortem diagnosis of PDR or neither, compared to normal, is depicted (see Example 2). [Figure 3A] 3A-3B depict the GPX4 inhibitor, RSL3, showing selective killing of irradiated senescent HRMEC cells versus non-senescent HRMEC cells in a dose-dependent manner (FIG. 3A), and selective killing of irradiated senescent HUVEC cells versus non-senescent HUVEC cells in a dose-dependent manner (FIG. 3B), see Example 1. [Figure 3B] See legend to Figure 3A. [Figure 4]We depict a GPX4 inhibitor, JKE-1674, which shows selective killing of irradiated senescent versus non-senescent HUVEC cells in a dose-dependent manner, see Example 1. [Diagram 5] We depict a GPX4 inhibitor, ML210, which shows selective killing of irradiated senescent HRMEC cells versus non-senescent HRMEC cells in a dose-dependent manner, see Example 1. [Figure 6A] Figures 6A and 6B depict two GPX4 inhibitors, compound #19 (Figure 6A) and compound #25 (Figure 6B), together showing selective ablation of irradiated versus non-senescent HUVEC cells in a dose-dependent manner, see Example 1. [Figure 6B] See legend to Figure 6A. [Figure 7] FIG. 1 is a schematic diagram of the GPX4 competitive target-engaged gel shift assay used to detect inhibition of GPX4 in cell lines or tissues; see Example 7. [Figure 8A] Figures 8A-8C depict target engagement of the GPX4 inhibitor, RSL3, in adult mouse retina. Gel shift assays were performed, with each lane containing 20 μL of retinal lysate labeled with RSL3 and streptavidin for gel shift (Figure 8A). For quality control, equal amounts of vehicle-treated retinal lysates were combined and treated with 100 μM RSL3 or DMSO (Figure 8B). Target engagement was quantified based on integrated band densitometry (ImageJ®) (Figure 8C). See Example 7. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 9A] 9A-9B depict the target engagement of GPX4 inhibitor compounds #19 (FIG. 9A) and #25 (FIG. 9B) by incubation with senescent HUVEC cells for 6 hours. See Example 7. [Figure 9B] See legend to Figure 9A. [Figure 10A]10A-10D depict GCN2 pathway activation using reporter genes ATF4 (FIGS. 10A and 10B) and DDIT3 (FIGS. 10C and 10D) in H2122 cells. See Example 8. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 10D] See legend to Figure 10A. [Figure 11A] Figures 11A-11C depict that systemic administration of the GCN2 activator, Compound 39, activates GCN2 in the retina. Figures 11A and 11B show that Compound 39 activated reporter genes ATF4 and DDIT3 in the retina at various doses and time points in both normoxic and oxygen-induced retinopathy (OIR) mice. Figure 11C shows that intraperitoneally administered Compound 39 activated ATF4 protein expression at 30 and 60 mpk in mouse retina. See Example 9. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 12] Figures 12A and 12B show that compound 39, a GCN2 activator, regulates disease-related gene expression by activating the expression of both the redox factor Slc7a11 (Figure 12A) and the anti-angiogenic factor Serpinf1 (Figure 12B) after intraperitoneal administration at various doses and time points in both normoxic and OIR neonatal mice. See Example 10. [Figure 13] We show that intraperitoneal administration of the GCN2 activator Compound 39 at 30 mpk activated ATF4 protein expression, increased phosphor-eIF2α levels, and downregulated HIF1α protein expression in retinas from OIR neonates as early as 4 hours after treatment. See Example 10. [Figure 14]Figures 14A-14B show that activation of GCN2 improves retinal vasculature in OIR mouse model. Specifically, compound 39, a GCN2 activator, can improve neovascularization in the retina of treated OIR mice when administered systemically (Figure 14A), but cannot improve avascular area (Figure 14B). See Example 11. [Figure 15] Figures 15A-15B show that activation of GCN2 improves angiogenesis in OIR mouse model.Compound 39 can improve angiogenesis in the retina of treated OIR mice (Figure 15B) compared with vehicle control (Figure 15A).See Example 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention I. Definition As used herein, the term "about" refers to the normal error range for the respective value, readily known to one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to the value or parameter itself.

[0038] As used herein, "administering" refers to a method of administering a dosage of the compound-meglumine to a patient suffering from a retinal vasculopathy herein. The compositions utilized in the methods described herein can be administered, for example, intravitreally (e.g., by intravitreal injection), ocularly (e.g., by ocular injection), or intraocularly (e.g., by intraocular injection). The method of administration can vary depending on a variety of factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0039] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0040] As used herein, the term "anti-VEGF treatment" or "anti-VEGF therapy" refers to any currently approved anti-VEGF drug indicated for treating retinal vasculopathy. Such approved drugs include, for example, Lucentis® (ranibizumab) or Eylea® (aflibercept), and any approved biosimilar to ranibizumab or aflibercept.

[0041] An "agonist" or "activator" or "activating" agent is one that activates, stimulates, or increases the biological or signaling activity of a target or antigen to which it binds. In some circumstances, it is contemplated that an agonist agent can act in a manner similar to that in which a ligand engages and activates its cognate receptor.

[0042] As used herein, "ferroptosis" refers to an art-recognized form of cell death involving iron-mediated production of reactive oxygen species and characterized in part by lipid peroxidation. "Ferroptosis inducer" or "ferroptosis activator" refers to an agent that induces, promotes or activates ferroptosis.

[0043] As used herein, "retinal vasculopathy" refers to ocular diseases involving pathogenic angiogenesis, such as, for example, diabetic macular edema, diabetic retinopathy, and dry or neovascular (or wet) age-related macular degeneration. Other ocular diseases may include, for example, geographic atrophy.

[0044] "Senescent cells" are generally considered to be derived from cell types that typically replicate but no longer replicate as a result of senescence or other events that cause a change in cell state. In other words, senescent cells are cells that enter an irreversible arrest of cell proliferation. Senescent cells can also become resistant to apoptosis and thus persist in a subject and accumulate as the subject ages. Furthermore, senescent cells exhibit a pleiotropic secretory phenotype or senescence-associated secretory phenotype (SASP), which is believed to be a pathological soluble factor that may play a role in the early presentation or ongoing pathology of senescence-associated cancer. For purposes of practicing aspects of the present invention, senescent cells can be identified as expressing, for example, p16, or at least one marker selected from p16, senescence-associated β-galactosidase, or p21. Senescent cells may also be identified as secreting SASP factors, such as, but not limited to, inflammatory factors (e.g., but not limited to, MMP1, MMP3, TNF-α, IL-1β, prostaglandins, etc.), and / or profibrotic factors (e.g., but not limited to, TGFβ1, TGFβ2, CTGF, TIMP-1, MCP-1, etc.) and / or growth factors (e.g., but not limited to, VEGF-α, IL-6, IL-8, Pai-1, etc.).

[0045] Candidate inhibitors or agonists are typically referred to as "senolytic" if they more selectively eliminate senescent cells compared to replicating cells of the same tissue type, or quiescent cells lacking SASP markers. In one aspect, achieving about 2-10 fold selectivity, or about 10-1000 fold selectivity, or about 100-2000 fold selectivity, or about 10-2000 fold selectivity, or about 1000-2000 fold selectivity, or greater than 1000 fold selectivity for the elimination of senescent cells over non-senescent cells is contemplated as part of the methods of the invention. Alternatively, or additionally, the methods of the invention may be effectively used if they reduce the release of pathological soluble factors or mediators as part of the senescence-associated secretory phenotype (SASP) that play a role in, or inhibit the resolution of, the initial presentation or ongoing pathology of the condition. In this regard, the term "senolytic" is exemplary, with the understanding that the compounds and methods of the invention may function primarily by selectively inhibiting rather than eliminating senescent cells (senolytic inhibitors), and as such may be used in the methods of the invention, with ensuing benefits.

[0046] As used herein, "treatment" (and "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of the therapeutic methods claimed herein include, but are not limited to, prevention of the onset or recurrence of DME, DR or AMD, alleviation of symptoms of DME, DR or AMD, reduction of any direct or indirect pathological consequences of DME, DR or AMD, reduction in the rate of disease progression in DME, DR or AMD, improvement or alleviation of the disease state in DME, DR or AMD, and remission or improved prognosis for DME, DR or AMD.

[0047] Other terms used herein have their ordinary meanings unless otherwise stated or required.

[0048] Compositions and methods The present invention provides methods for treating certain retinal vasculopathies, such as diabetic macular edema (DME), diabetic retinopathy (DR), and age-related macular degeneration (AMD).

[0049] DME is a complication of diabetic retinopathy (DR) following chronic, poorly controlled diabetes and is the most common form of sight-threatening retinopathy in diabetic patients. Approximately 1 in 14 people with diabetes have some degree of DME. The World Health Organization (WHO) reported in 2015 that the overall prevalence of DR in diabetic patients using retinal imaging is estimated to be 35%, with sight-threatening DR being present in 12%. The prevalence depends on the type of diabetes and the duration of the disease. After a duration of 25 years, the prevalence is close to 30% for both types of diabetes, type 1 diabetes and type 2 diabetes. In the United States, at least 5.5 million people over the age of 40 are estimated to have DR in the absence of DME, and an additional 800,000 to 1 million patients have DME. By some estimates, only 40% of them are diagnosed and treated, and about 5% are diagnosed and observed.

[0050] Diabetic macular edema (DME) is a macular thickening secondary to diabetic retinopathy (DR) and is due to defects in the blood-retinal barrier leading to vascular leakage and fluid accumulation. See Bhagat N, et al., Surv Ophthalmol. 2009;54:1-32. DME is associated with the expression of several inflammatory factors, including vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and leukostasis. See Funatsu H, et al., Ophthalmology. 2009;116:73-79, and Miyamoto K, et al., Proc Natl Acad Sci USA. 1999;96:10836-10841. Moreover, the expression of these factors correlates with both retinal vascular permeability and disease severity, thus supporting their important pathogenetic role.

[0051] DR: The prevalence of diabetic retinopathy increases with age and is the most common cause of blindness in people over 50 years of age. It is a multifactorial disorder, and hyperglycemia exerts toxic effects on cells and inflammatory cytokines involved in many aspects of diabetic eye disease. Diabetic retinopathy involves thickening of the capillary basement membrane, preventing pericytes from contacting the endothelial cells of the capillaries. Loss of pericytes increases capillary leakage and can lead to the breakdown of the blood-retinal barrier. As capillaries weaken, aneurysms can form and further leak. The duration and severity of hyperglycemia are factors associated with the development of diabetic retinopathy. These effects of hyperglycemia can also impair retinal neural function. This is the early stage of diabetic retinopathy, termed non-proliferative diabetic retinopathy (NPDR).

[0052] Diabetic retinopathy is also a degenerative disease of the neural retina that is associated with changes in neural function before the onset of clinical vascular disease. Retinal capillaries can become occluded in diabetes causing ischemic areas of the retina. The non-perfused tissue responds by inducing the growth of new blood vessels (i.e., neovascularization) from existing vessels. These new vessels can also cause vision loss, a condition called proliferative diabetic retinopathy (PDR), because the new vessels are fragile and prone to leaking blood into the eye. In advanced proliferative diabetic retinopathy, the neovascular VEGF-mediated response continues with retinal neovascularization, putting the eye at further risk for severe vision loss due to the development of vitreous hemorrhage or traction retinal detachment. If untreated, this can result in irreversible vascular or neural damage, highlighting the need for early intervention.

[0053] AMD: Age-related macular degeneration (AMD) is a leading cause of visual impairment and severe vision loss. It ranks third among causes of visual impairment worldwide, with a blindness rate of 8.7%.

[0054] AMD is a multifactorial disorder caused by deterioration of the central portion of the retina, with dysregulation of complement, lipid, angiogenic, inflammatory, and extracellular matrix pathways involved in its pathogenesis. AMD pathology is characterized by the progressive accumulation of characteristic yellow deposits called drusen (e.g., accumulations of extracellular proteins and lipids) between the retinal pigment epithelium of the macula and the underlying choroid, which are thought to damage the retina over time.

[0055] There are two basic types of AMD: "dry" and "wet" (also known as neovascular AMD or nvAMD). Approximately 85%-90% of AMD cases are of the "dry" (atrophic) type, and 10-15% are of the "wet" (exudative) type. Dry AMD (dAMD) is defined by the gradual loss of retinal pigment epithelium (RPE) and photoreceptor cells in the macula. Patients with dry AMD have a gradual loss of central vision due to the death of photoreceptor cells and their closely associated retinal pigment epithelium (RPE) cells, accompanied by the deposition of drusen. Wet AMD is characterized by the proliferation of abnormal blood vessels beneath the macular epithelium. Wet AMD results in vision loss due to abnormal vascular proliferation of the choriocapillaris through Bruch's membrane (e.g., choroidal neovascularization). Clinically, it is classified as early (moderate drusen and retinal pigment changes) to late (neovascular AMD and atrophic AMD).

[0056] Senolytic assay: Candidate agents of the present invention can also be evaluated for their ability to selectively kill senescent cells. Cultured cells are contacted with an agent and the degree of cytotoxicity or inhibition of cells is determined. The ability of an agent to kill or inhibit senescent cells can be compared to the effect of the agent on normal cells that are freely dividing at low density and normal cells that are quiescent at high density. As a non-limiting example, Example 1 provides an illustration of a cell-based assay that uses radiation-induced senescence in a human endothelial HUVEC cell line and a human RPE cell line to test and quantify candidate agents of the present invention. Similar protocols are known and can be developed or optimized to test the ability of candidate senolytic compounds to kill other senescent cell types.

[0057] Pathways: The invention described and claimed herein contemplates treating retinal vasculopathy using inhibitors or agonists of certain identified biological pathways. The invention described and claimed herein also contemplates removing senescent cells, such as senescent endothelial cells and / or senescent pericytes, to treat retinal vasculopathy using inhibitors or agonists of certain identified biological pathways. The biological pathways contemplated are as follows:

[0058] Glutathione peroxidase 4 (GPX4) pathway: Regulation of oxidative stress and redox systems plays a key role in carcinogenesis and cancer progression and has therefore attracted much attention as an area of ​​cancer therapeutic target. Glutathione peroxidase 4 (GPX4) belongs to a family of glutathione peroxidases consisting of eight known mammalian isoenzymes (GPX1-8), whose members catalyze the reduction of hydrogen peroxide, organic hydroperoxides and lipid hydroperoxides, thereby protecting cells from oxidative stress and signaling cell death by inhibiting peroxidation of membrane phospholipids. When GPX4 activity is impaired, lipid peroxidation can trigger ferroptosis, an oxidative iron-dependent form of non-apoptotic cell death. Ferroptosis induced by exogenous agents is selectively lethal to tumor cells addicted to GPX4 repair activity, suggesting that induction of ferroptosis may be a beneficial approach for treating some cancers. See Stockwell BR, et al., Cell. 2017;171:273-285.

[0059] Exemplary GPX4 inhibitors: Any GPX4 inhibitor currently known or developed in the art can be tested and developed for aging activity for the treatment of aging-related disease and / or age-related disease according to the present invention.In particular, the GPX4 inhibitors that can be utilized in the method of the present invention include, but are not limited to, for example, RSL3, ML210, ML162, JKE-1674, DPI-7, buthionine sulfoximine (BSO), FIN56, auranofin, erastin, artemisinin, sulfasalazine, artesunate, dihydroatemicin, sorafenib, altretamine, almitrine, artemether, artemisone, or lanperisone. Other non-limiting exemplary GPX4 inhibitors can be compounds 1-273 disclosed and claimed in WO2019 / 168999, compounds 1-102 disclosed and claimed in WO2020 / 176757, and compounds 1-12 and compounds A-1-A-19 disclosed and claimed in WO2021 / 041536, each of which is incorporated by reference in its entirety.

[0060] GPX4 Biological Assay: Studies have shown that lipophilic antioxidants such as ferrostatin can rescue cells from GPX4 inhibition-induced ferroptosis. For example, mesenchymal state GPX4 knockout cells can survive in the presence of ferrostatin, but when the supply of ferrostatin is terminated, these cells undergo ferroptosis (see, e.g., Viswanathan et al., Nature 547:453-7, 2017). It has also been experimentally determined that GPX4i can be rescued by blocking other components of the ferroptosis pathway that apoptosis inhibitors do not rescue, such as lipid ROS scavengers (ferrostatin, liproxystatin), lipoxygenase inhibitors, iron chelators, and caspase inhibitors. These findings suggest a non-apoptotic iron-dependent oxidative cell death (i.e., ferroptosis). Therefore, the ability of a molecule to induce ferroptosis cancer cell death, and such ability is flagged by the addition of ferrostatin, clearly indicates that the molecule is a GPX4 inhibitor. An exemplary assay used to test the inhibitory potential of a candidate GPX4 inhibitor is as follows. The mobility shift of GPX4 Western blot assay can be used to directly evaluate target engagement in a cell-based assay after incubation with a candidate GPX4 inhibitor. The mobility shift can be used as a pharmacodynamic marker for GPX4 irreversible inhibitors. Furthermore, candidate GPX4 inhibitors can be evaluated in a cell-based Western blot analysis of GPX4.

[0061] Glutaminase inhibitor 1 (GLS1) pathway: Glutamine is the most abundant plasma amino acid and is involved in many growth-promoting pathways. In particular, glutamine is involved in oxidation in the TCA cycle and maintaining cellular redox balance, and also provides nitrogen for nucleotide and amino acid synthesis (Curi et al., Front. Biosci. 2007, 12, 344-57; DeBerardinis and Cheng, Oncogene 2010, 313-324). Glutaminase is a mitochondrial amidohydrolase enzyme that produces glutamic acid from glutamine. It is involved in the detoxification of ROS.

[0062] Exemplary GLS1 Inhibitors: Any GLS1 inhibitor currently known in the art or developed may be tested and developed for the treatment of retinal vasculopathy as disclosed in accordance with the present invention. In particular, GLS1 inhibitors that can be utilized in the methods of the present invention include, but are not limited to, the following: 6-diazo-5-oxo-L-norleucine (diazooxonorleucine, L-6-diazo-5-oxonorleucine, DON), an antibiotic isolated from Streptomyces, is a glutaminase antagonist with an IC50 of approximately 1 mM against cKGA (kidney-type glutaminase); GK921 is a transglutaminase 2 (TGase2) inhibitor with an IC50 of 8.93 μM under modified assay conditions; UPGL00004 is a potent glutaminase C (GAC) inhibitor with an IC50 of 29 nM that shows high selectivity for GAC over GLS2; and bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) is an inhibitor with an IC50 of approximately 10 nM against cKGA (kidney-type glutaminase). 0.16 μM; telaglenastat (CB-839) is a potent, selective and orally bioavailable glutaminase inhibitor with an IC50 of 24 nM against recombinant human GAC; JHU395 is an orally bioavailable GA (glutamine antagonist) prodrug that circulates inactive in plasma but is designed to penetrate and release active GA in target tissues; JHU-083 (ethyl 2-(2-amino-4-methylpentanamide)-DON) is a novel prodrug of DON that selectively blocks glutaminase activity; and IPN-60090 dihydrochloride (also known as IACS-6274) is an orally active and highly selective inhibitor of glutaminase 1 (GLS1; IC50=31 nM) with no observed activity against GLS-2.

[0063] GLS1 Biological Assay: An exemplary assay used to test the inhibitory potential of candidate GLS1 inhibitors is as follows: Glutamate oxidase / AmplexRed® binding assay can be used to measure the ability of GLS1 inhibitor compounds to bind to purified recombinant 6His-tagged GLS1 and inhibit its activity in vitro. The binding reaction can be stopped by adding the reagents 6-(2-bromoethynyl)-2,3-dimethyl-quinazolin-4-one, Amplex Red, horseradish peroxidase, and glutamate oxidase in TRIS buffer. The reaction can be read on a Perkin Elmer EnVision® using a 535 / 590 nm optical filter, and the raw data can be analyzed using Genedata® to generate IC50 values. Another assay that can be used is the PC3 cell binding assay, which uses AmplexRed to measure cellular glutamate depletion. PC3 cells can be seeded into multi-well plates containing candidate inhibitors. After incubation, plates can be read on a Perkin Elmer EnVision® using 535 / 590 nm optical filters and the raw data analyzed using proprietary software to generate IC50 values.

[0064] Pregnancy-associated plasma protein-A (PAPP-A) or papalysin-1 pathway: PAPP-A, a zinc metalloproteinase, binds to surface glycoaminoglycans (GAGs) and cleaves IGFBP-2, -4, and -5 to release IGF1 or IGF2 for local signaling. PAPP-A in pregnancy serum is linked via disulfide bonds to the proform of eosinophil major basic protein (proMBP), forming a 2:2 complex of approximately 500 kDa designated PAPP-A / proMBP. The serum form of PAPP-A is derived from a preproprotein that contains a putative 22-residue signal peptide, a 58-residue pro-part, and a 1547-residue circulating mature polypeptide. The amino acid sequence shows no overall similarity to any known proteins, but contains two sequence motifs common to methincins, three Lin-12 / Notch repeats known from the metalloprotease superfamily, the Notch protein superfamily, and five short consensus repeats known from components of the complement system. Transgenic knockout of PAPP-A can result in extended life span in mice, possibly by reducing circulating IGF-I levels (see Hotzenberger et al., Nature (2002) Dec 4 and Conover et al., Development, 131(5):1187-1194 (2004)).

[0065] Exemplary PAPP-A Inhibitors: Any PAPP-A inhibitor currently known or developed in the art may be tested and developed for the treatment of retinal vasculopathy as disclosed in accordance with the present invention. In particular, PAPP-A inhibitors that may be utilized in the methods of the present invention include, but are not limited to, antibodies that specifically bind to PAPP-A. These can include, for example, PAC-1scFv, PAC-1-D8scFv, PAC-2scFv, PAC-5scFv, as disclosed in WO2020 / 198166, U.S. Patent No. 8,653,020, Mikkelsen et al., Oncotarget 5, 1014-1025 (2014), Mikkelsen et al., J. Biol. Chem. 283, 16772-16780 (2008), and Mohrin et al., bioRxiv, doi: https: / / doi.org / 10.1101 / 2020.02.05.936310 (this version posted on February 6, 2020), all of which are incorporated herein by reference in their entirety. Other PAPP-A inhibitors that can be used in the methods of the present invention include, but are not limited to, polypeptides that can inhibit the function of PAPP-A. These can include, for example, pro-MBP, stanniocalcin-1 (STC1), or stanniocalcin-2 (STC2). Still other PAPP-A inhibitors that can be used in the methods of the present invention include, but are not limited to, antisense oligonucleotides that target PAPP-A, such as, for example, 18-base phosphorothioate oligodeoxynucleotides that correspond to human PAPP-A mRNA and consist of the antisense sequence 5'-GCCCAACTCCTGCTGGAA-3' (AS-PAPP-A) (see Tanaka et al., Cancer Cell Biol Jan 2004). Still other PAPP-A inhibitors that can be used in the methods of the present invention include, but are not limited to, the Kunitz-type protease inhibitor Bikunin.

[0066] PAPP-A Biological Assay: An exemplary assay used to test the inhibitory potential of candidate PAPP-A inhibitors is as follows: A cellular pAKT assay can be used in which IGFBP-4 protein is mixed with IGF1 and incubated at 37°C for 30 minutes. PAPP-A protein is added to the IGFBP-4 / IGF1 mixture and incubated at 37°C for 4-5 hours. HEK293 cells are placed in EMEM medium without serum and allowed to adhere overnight. The IGF1 / IGFBP-4 / PAPP-A mixture is added to the cells at a final dilution of 1:30 and incubated at 37°C for 20 minutes. Cells are lysed in MSD Tris lysis buffer and analyzed by Phospho(Ser473) / Total Akt Whole Cell Lysate kit (MSD, K15100D) according to the manufacturer's protocol. To assess the neutralizing potency of anti-PAPP A antibodies, PAPP-A protein can be pre-incubated with various concentrations of anti-PAPP-A antibodies before being added to the IGF1 / IGFBP-4 mixture.

[0067] Another PAPP-A biological assay that can be used is the in vitro PAPP-A enzymatic cleavage of IGF binding proteins. In such an assay, human and mouse PAPP-A proteins with C-terminal C-myc and Flag tags are expressed by a stably transfected HEK293 cell line and purified by heparin column chromatography. Human and mouse IGFBP-2, IGFBP-4 and IGFBP-5 proteins are recombinantly produced by transient expression in HEK293 cells with N-terminal (for human proteins) or C-terminal (for mouse proteins) 6His tags and purified by Ni-Sepharose column chromatography. For the enzymatic cleavage reaction, IGFBP-2 and IGFBP-4 proteins are pre-incubated with IGF1 of the appropriate species (R&D Systems, 291-G1-200 for human and 791-MG-050 for mouse) for 30 minutes at 37°C. IGFBP2 / IGF1, IGFBP-4 / IGF1 or IGFBP-5 proteins are then mixed with various concentrations of PAPP-A and incubated at 37°C for 2-4 hours. The final concentrations in the cleavage reaction can be 90 nM for IGFBP and 850 nM for IGF1. Proteins are then resolved by capillary electrophoresis on a Wes instrument (ProteinSimple) using a capillary cartridge kit (ProteinSimple, Cat. No. SM-W002-1), probed with THE HisTag antibody (GeneScript, Cat. No. A00186) and visualized with an anti-mouse detection module (ProteinSimple, Cat. No. DM-002). To assess the neutralization potency of anti-PAPP A antibodies, PAPP-A proteins can be pre-incubated with various concentrations of anti-PAPP-A antibodies before adding them to the IGF1 / IGFBP-4 mixture. PAPP-A concentrations in these assays can be fixed at 0.4 nM for IGFBP-4, 3.5 nM for IGFBP-2, and 0.08 nM for IGFBP-5 cleavage.

[0068] cGAS-STING pathway: Recognition of microbial nucleic acids is a major mechanism by which the immune system detects pathogens. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytoplasmic DNA sensor that activates the innate immune response by producing the second messenger cGAMP, which activates the adaptor stimulator of interferon genes or STING. STING then recruits TANK-binding kinase 1 (TBK1) and IκB kinase to activate IFN regulatory factor 3 (IRF3) and NF-κB, respectively, resulting in the production of type I interferons and inflammatory cytokines, which can also lead to autoimmune and inflammatory diseases. cGAS has also been reported to be involved in cellular senescence, and mouse embryonic fibroblasts (MEFs) derived from cGas- / - mice showed reduced signs of senescence and underwent faster spontaneous immortalization compared to MEFs from WT mice (see Yang et al., PNAS June 2017).

[0069] Exemplary cGAS Inhibitors: Any cGAS inhibitor currently known or developed in the art may be tested and developed for the treatment of retinal vasculopathy as disclosed in accordance with the present invention. In particular, cGAS inhibitors that may be utilized in the methods of the present invention include, but are not limited to, RU.521, which is 3-(1-(6,7-dichloro-1H-benzo[d]imidazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)isobenzofuran-1(3H)-one (Vincent J. et al., 2017. Natl. Acad. Sci., 2013, 113:1311-1315, 2013). Commun. 28(1):750), or G140, which is 1-(6,7-dichloro-9-(1-methyl-1H-pyrazol-3-yl)-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)-2-hydroxyethan-1-one, or G150, which is 1-[9-(6-amino-3-pyridinyl)-6,7-dichloro-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl]-2-hydroxy-ethanone (for both G140 and G150, see L. et al. 2019. Nat. Commun. 10, 2261), or (1R,2S)-2-(7-oxo-5-phenyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carboxamide)cyclohexane-1-carboxylic acid, PF-06928215 (see Hall et al., PLOS ONE 2017, 12, e0184843).

[0070] Exemplary STING inhibitors: Any STING inhibitor currently known or developed in the art may be tested and developed for the treatment of retinal vasculopathy as disclosed in accordance with the present invention. In particular, STING inhibitors that may be utilized in the methods of the present invention include, but are not limited to, H-151, CAS No. 941987-60-6, or C-178, CAS No. 329198-87-0, or C-176, CAS No. 314054-00-7 (for H-151, C-178, and C-176, see Haag SM et al., 2018. Nature 559:269-73). Another STING antagonist is compound 18 or C18 (see Siu, et al. ACS Med. Chem. Lett., 10(1)(2019), pp. 92-97).

[0071] cGAS or STING Biological Assays: Exemplary assays used to test the inhibitory potential of candidate agents for CGAS and / or STING are as follows: cGAS inhibitors can be measured for cGAS activity using mass spectrometry assays by reaction with ATP, GTP, double-stranded DNA, and CGAS. Other cGAS activity assays include the Cy5-cGAMP fluorescence polarization assay, in which candidate cGAS inhibitors react with ATP or GTP, or double-stranded DNA and cGAS. Cy5-labeled cGAMP and cGAMP antibodies are added, the reaction is read at 620 nm on an Envision plate reader, and Ki is determined (see Hall et al., PLOS ONE 2017, 12, e0184843). Another assay that can be utilized is the soluble adenylyl cyclase (sAC) inhibition assay, in which candidate cGAS inhibitors are incubated with human sACt protein, and then ATP is added. The formation of cAMP and consumption of ATP are compared to standards using RF-MS, and IC50 is calculated.

[0072] mTOR Pathway: The mechanistic target of rapamycin (mTOR) is a highly conserved serine / threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress.

[0073] Exemplary mTOR inhibitors: Any mTOR inhibitor currently known or developed in the art can be tested and developed for the treatment of retinal vasculopathy disclosed in accordance with the present invention.In particular, STING inhibitors that can be utilized in the methods of the present invention include, but are not limited to, rapamycin, Palomid529, sirolimus, everolimus, temsirolimus, ridaforolimus, umirolimus, zotarolimus, and ATP-competitive mTOR kinase inhibitors.

[0074] General Control Non-Repressible 2 (GCN2) Pathway: To thrive, cells need to be able to sense and adapt to their surroundings. The key to adapting to low-nutrient environments is the integrated stress response (ISR), a pathway that allows cells to restore cellular homeostasis or promote apoptosis during periods of stress. Central to the ISR is the serine / threonine protein kinase GCN2, which is involved in sensing starvation. Upon amino acid deprivation, GCN2 is activated and initiates the ISR by phosphorylating the translation initiation factor eIF2α, halting protein translation and activating the transcription factor ATF4, which then upregulates autophagy and biosynthetic pathways.

[0075] Exemplary GCN2 Activators: Any GCN2 activator currently known in the art or developed can be tested and developed for the treatment of retinal vasculopathy as disclosed in accordance with the present invention. In particular, agonists of GCN2 that may be utilized in the methods of the present invention include, but are not limited to, 4-(2-amino-4-methyl-3-(2-methylquinolin-6-yl)benzoyl)-1-methyl-2,5-diphenyl-1H-pyrazol-3(2H)-one hydrochloride or compound 44, 1-(5-(4-amino-2,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)indolin-1-yl)-2-(3-fluoro-5-(trifluoromethyl)phenyl)-ethanone or compound 52, 4-(2-amino-4-methyl-3-(2-(methylamino)benzo[d]thiazol-6-yl)benzoyl)-1-methyl-2,5-diphenyl-1H-pyrazol-3(2H)-one or compound 39 (see Smith, Adrian L., et al., J of Med, 2002, for compounds 39, 44, and 52). Chem. 2015 Feb 12:58(3):1426-41), leucinol, histidinol, threoninol, SB-203207, SB-219383 (see Cavener, D., et al., WO 2008 / 085921), dovitinib, neratinib, sunitinib, and erotinib.

[0076] GCN2 Biological Assays: Exemplary assays used to test the activating potential of candidate agents for GCN2 may include, but are not limited to, the following. One such assay is the eIF2a phosphorylation assay, in which a reaction combining GCN2, eIF2a, and a candidate GCN2 activator is performed upon the addition of ATP. The reaction is quenched, then denatured by boiling, then run on a protein gel, transferred to a nitrocellulose or PVDF membrane, blocked, incubated with an antibody against fluorophore-eIF2a, and then visualized after incubation with a chemiluminescent substrate. A cellular assay that can be used to test candidate GCN2 activators is to produce recombinant retroviruses using a stable cell line, such as HEK293T cells transfected with an ATF4 reporter fused to firefly luciferase (FLuc). Such cells are seeded and then treated with an appropriate amount of candidate GCN2 activator for a period of time. Luminescence is measured and expressed as EC50.

[0077] Methods for monitoring and diagnosing retinal vasculopathy: Standardized ophthalmic examination techniques known in the art include detailed slit lamp biomicroscopic evaluation, which allows for evaluation of the central retinal anatomical structures, including, for example, the eyelids, ocular adnexa, eyelashes, corneal surface, anterior chamber, pupil, lens, vitreous cavity, and optic nerve and macula. Another method is gonioscopy, which allows for detailed examination of the anterior chamber angle. Indirect ophthalmic examination allows for evaluation of the retinal periphery, which is important in monitoring vitreous and peripheral retinal disorders.

[0078] Functional tests of vision are known in the art and include, for example, best corrected visual acuity, contrast visual acuity, and low light visual acuity, color vision (including Ishihara test and Farnsworth-Munsell test) and visual field assessment (including Humphrey automated perimetry and microperimetry), tear production (Schirmer test), and tonometry to measure intraocular pressure (IOP). These are used in conjunction with structural tests including, for example, anterior and posterior segment photography, corneal thickness measurements, ultrasound, ultrasound biomicroscopy, optical coherence tomography (OCT), optical coherence tomography angiography (OCTA), fluorescein angiography (FA), intravenous fluorescein angiography (IVFA), and fundus autofluorescence (FAF). Imaging, such as computed tomography (CT) or magnetic resonance imaging (MRI) scans, are utilized to evaluate the eye, peribulbar and orbital structures, as well as the intracranial portions of the optic nerve, visual pathways, and visual cortex in the brain. These tests allow visualization of the structural integrity and thickness of layers of the eye and surrounding structures, as well as assessment of blood flow and circulation. Advanced functional testing of the retina, optic nerve and visual pathway / cortex, including full-field and multifocal retinal examinations, electrophysiological tests such as visual evoked potentials and microperimetry, are also used to diagnose and monitor disease progression and the effects of treatment. Those skilled in the art will be able to deploy appropriate methodologies known in the art to diagnose, measure and monitor the retinal vasculopathy described herein.

[0079] Patients: In some embodiments of the claimed invention, the candidate inhibitors or agonists of the invention used to treat patients suffering from DME are administered to patients suffering from not only DME but also diabetic retinopathy (DR). In other embodiments, the DME patient suffers from not only DME but also non-proliferative DR. In other embodiments, the DME patient also suffers from proliferative DR. In other embodiments of the invention, the patient suffers from age-related macular degeneration (AMD). In other embodiments, the patient suffering from AMD suffers from neovascular AMD.

[0080] Administration: Intravitreal (IVT) is one exemplary route of administration of the agents described and claimed herein in a procedure in which the drug is placed directly into the space at the back of the eye called the vitreous cavity, which is filled with a jelly-like fluid called vitreous humor gel. This procedure is usually performed by a trained retina specialist in an office setting. Another route of administration of the agents described and claimed herein is intracameral (IC). Intracameral injections are usually injections into the anterior chamber of the eye. Other routes of administration of the agents described and claimed herein can be intravenous, intramuscular, oral, parenteral, topical, and subcutaneous. EXAMPLES

[0081] The following are examples of methods and compositions of the present invention: Given the general description above, it will be understood that various other embodiments may be practiced.

[0082] Example 1: Testing of candidate agents in aged retinal vascular or epithelial or endothelial cells The ability of a candidate agent to eliminate senescent retinal vascular endothelial cells or retinal pigment epithelial cells can be directly measured in the following assay: Human retinal microvascular endothelial cells (HRMEC) are available from Neuromics® under the designation HEC09 or Cell Systems ACBRI181. Human umbilical vein endothelial cells (HUVEC) are available from Lonza® under the designation CC-2519. Cells are cultured in 3% O2 ​​at 4°C for 1 h. 2 , 10% CO 2 Human retinal pigment epithelial cells (RPE) can be obtained from Lonza® under the designation 194987. RPE cells can be maintained and grown in ENDO-Growth® medium (Neuromics®, Edina MN) at less than 75% confluency at 3% O, 5% CO, and approximately 95% humidity. 2 , 10% CO 2 They can be maintained and grown at less than 75% confluency in RPE medium as defined by Sonoda et al. in 2009, containing 5% FBS and Pen / Strep, in an atmosphere of approximately 95% humidity.

[0083] The cells can be divided into three groups: irradiated cells (cultured for 7 days after irradiation before use), proliferating normal cells (cultured at low density for 1 day before use), and quiescent cells (cultured to confluency over 4 days).

[0084] On day 0, irradiated HRMEC or RPE or HUVEC cells can be incubated with TrypLE trypsin-containing reagent (Thermofisher® Scientific, Waltham, Massachusetts) until the cells congregate and begin to detach from the plate. The cells can then be dispersed, counted, and prepared in medium at a concentration of 100,000 cells per mL. This cell suspension can be placed in a T175 flask at a density of 100,000 cells per mL and irradiated at 10-15 Gy. After irradiation, the cells can be seeded in 100 μL in a 96-well plate. On days 1, 3, 4, or 6, the medium in each well can be aspirated and replaced with fresh medium. On day 3, quiescent healthy non-senescent HRMEC or RPE cells can be trypsinized from the culture flask as described above, and the cells can be dispersed, counted, and prepared in medium at a concentration of 80,000 cells per mL. Each well of a 96-well plate can be seeded with 100 μL of cells and medium can be changed on days 1, 3, 6 and 10.

[0085] On day 10, the candidate agent can be appropriately diluted to the desired concentration and then combined with the cells after aspirating from the medium. The candidate agent can be cultured with the cells for an appropriate number of days, such as, for example, 3-7 days. The assay system can use the properties of thermostable luciferase to allow reaction conditions that generate a stable luminescent signal while simultaneously inhibiting endogenous ATPase that is released during cell lysis. At the end of the culture period, the plate can be removed from the incubator and allowed to equilibrate at room temperature for 20 minutes, and then 100 μL of CellTiter-Glo® reagent (Promega® Corp., Madison, Wisconsin) can be added to each well. The cell plate can be placed on an orbital shaker for 30 seconds and then left at room temperature for 10 minutes before luminescence can be measured, for example, using an EnVision® plate reader (Perkin Elmer). Luminescence readings can be normalized to determine % cell viability / growth and plotted against candidate agent concentrations and controls, and potency (IC50 values) can be determined by non-linear curve fitting in Graphpad® Prism.

[0086] This experiment was performed by testing the GPX4 inhibitor RSL3 ((1S,3R)-methyl 2-(2-chloroacetyl)-1-(4-(methoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate; CAS No. 1219810-16-8), which was prepared at 200x in a 1-3 dilution series in DMSO and then diluted to 1.5x the final concentration in culture medium and applied to irradiated senescent HRMEC cells and non-senescent HRMEC cells as a control, see Figure 3A. The data show that RSL3 is selectively senolytic for senescent HRMEC cells compared to non-senescent HRMEC. Similarly, RSL3 was applied to irradiated senescent HUVEC cells and non-senescent HUVEC cells as a control, see Figure 3B. The data indicate that RSL3 is selectively senolytic for senescent HUVEC cells compared to non-senescent HUVEC.

[0087] The experiment was also performed testing a different GPX4 inhibitor, JKE-1674 ((E)-1-(4-(bis(4-chlorophenyl)methyl)piperazin-1-yl)-2-(hydroxyimino)-3-nitropropan-1-one; CAS No. 2421119-60-8), which was prepared at 200x in a 1-3 dilution series in DMSO, then diluted to 1.5x the final concentration in culture medium and applied to irradiated senescent HUVEC cells and non-senescent HUVEC cells as control, see Figure 4. The data show that JKE-1674 is selectively senolytic for senescent HUVEC cells compared to non-senescent HUVEC.

[0088] This experiment was also performed testing yet another GPX4 inhibitor, ML210 (see Eaton et al., BioRxiv, September 5, 2018; CAS No. 1360705-96-9), which was prepared at 200x in a 1-3 dilution series in DMSO, then diluted to 1.5x the final concentration in medium and applied to irradiated senescent HRMEC cells and non-senescent HRMEC cells as a control, see Figure 5. The data show that ML210 is selectively senolytic for senescent HRMEC cells compared to non-senescent HRMEC.

[0089] Similar experiments were performed testing two other candidate GPX4 inhibitors, compounds #19 (see compound 28 described in US2019 / 0263802) and #25 (see compound 156 described in US2019 / 0263802), which were prepared at 200x in a 1-3 dilution series in DMSO, then diluted to 1.5x the final concentration in medium and applied to irradiated senescent HUVEC cells and non-senescent HUVEC cells as a control, see Figures 6A and 6B. The data show that both compounds #19 (Figure 6A) and #25 (Figure 6B) are selectively senolytic for senescent HUVEC cells compared to non-senescent HUVEC.

[0090] Example 2: Evidence of senescent cells in AMD, DR / DME and PDR tissues To obtain evidence of the presence of senescent cells in AMD, DR / DME, and PDR tissue samples from affected patients, we stained for p16.

[0091] Human post-mortem whole donor eyes were prospectively procured from various ocular tissue banks. Patient diagnoses were confirmed using patient history. After enucleation, eyes were immediately placed in Davidson's reagent and fixed for at least 48 hours. After fixation, the solution was replaced with 70% ethanol. Tissues were then placed in histological cassettes and processed for paraffin infiltration and embedding. Paraffin blocks were mounted on a microtome and sectioned to a thickness of 5-9 μm. Tissue sections cut at 4 mm sagittal sections were placed in a 45°C water bath and sections were picked up using Superfrost®Plus microscope slides and excess water was removed with Kimwipe®. Slides were then dried upright at room temperature for 30 minutes, followed by overnight incubation in a 37°C incubator. Slides were baked at 60°C for 15-20 minutes and then cooled to room temperature. Slides were dewaxed using xylene or a xylene substitute (e.g., Histoclear®) for 4 min, and the incubation was repeated a total of three times. Slides were then rehydrated by successive incubations in decreasing concentrations of ethanol as follows: 2 times for 5 min each in 100% ethanol, 2 times for 2 min each in 90% ethanol, 2 times for 2 min each in 75% ethanol, 2 times for 2 min each in 50% ethanol, then washed in water for 4 min.

[0092] Antigen retrieval was performed by incubation in acidic sodium citrate buffer at 120°C for 3 min (or 95°C for 20 min) using a steamer or pressure heater. Slides were cooled to room temperature for 15 min and then washed twice for 2 min each in Tris-buffered saline supplemented with 0.1% Triton-X100 (TBST). Slides were washed again twice for 5 min each in TBST and then blocked for 1 h at room temperature in TBST containing 5% normal serum from the species of origin of the secondary antibody used. After blocking, slides were incubated with p16 primary mouse anti-human p16 diluted 1:2 in TBST. INK4a Incubation with antibody (CINtec clone E6H4, Roche, Cat. No. 705-4793) was overnight at 4°C (or 2 hours at room temperature). P16 was visualized using a diethylaminocoumarin DCC fluorescent staining assay on a Roche Ventana Discovery Platform. After full-slide scanning using a Zeiss Axioscan® microscope (Zeiss®, Oberkochen, Germany), the number of p16INK4a positive cells was quantified using Visiopharm image analysis software (Visiopharm®, Hoersholm, Denmark), and total cells were quantified from total DAPI stained nuclei. Here, p<0.0001 vs. normal by Kruskal-Wallis with Dunn's multiple comparison test.

[0093] Figures 1 and 2 are p16 INK4a Quantification of positive cells is shown, demonstrating an increase in retinas of AMD, DR / DME and PDR patients compared to control retinas.

[0094] Example 3: Efficacy of candidate agents in a bleomycin-induced glaucoma model This example illustrates the testing of candidate agents of the invention in a mouse model of primary open angle glaucoma (POAG). Male C57Bl6 / J mice, 8-10 weeks old, can be sedated in an isofluorane chamber for 3 minutes and then placed on the operating table in a nose cone to maintain constant isofluorane anesthesia. One drop of 2.5% phenylephrine-tropicamide is instilled into the eye for dilation. Baseline intraocular pressure (IOP) measurements can be made in both eyes using a Tonolab™ prior to surgery. IOP values ​​are reported as the average of six measurements. To induce a glaucoma-like phenotype, 2 μL of bleomycin (0.25 U / kg) or PBS (control) can be injected intracamerally into the right eye.

[0095] IOP measurements can be performed on days 7 (pre-treatment), 14, and 21 after injury. Treatment can be performed 7 days after bleomycin injury. Mice can be sedated in an isofluorane chamber for 3 minutes and then placed on a surgical table in a nose cone to maintain constant isofluorane anesthesia. A drop of 2.5% phenylephrine-tropicamide can be instilled into the eye for dilation. Microliter volumes of the candidate agents in combination or in vehicle at the appropriate concentration can be injected intracamerally into one eye.

[0096] Ocular samples can be taken 14 and 21 days after bleomycin injury. Trabecular meshwork can be collected and quickly frozen in liquid nitrogen. Sample storage can be at -80°C until RNA extraction. RNA extraction can be performed using chloroform extraction followed by Direct-Zol Microprep™ RNA Extraction Kit (VWR®). cDNA can be prepared using 500 nanograms of RNA using the High Capacity Reverse Transcriptase™ Kit (ThermoFisher®). One-tenth of the cDNA can be used for levels of RNA expression measurement using PerfeCTa qPCR ToughMix Low Rox™ and Taqman™ primers / probes (QuantaBio™).

[0097] Example 4: Efficacy of candidate agents in a diabetic-induced retinopathy model The streptozotocin (STZ) rodent model (Feit-Leichman et al, IOVS46:4281-87, 2005) recapitulates features of diabetic retinopathy and diabetic macular edema through induction of hyperglycemia via the direct cytotoxic effect of STZ on pancreatic β-cells. Hyperglycemia occurs within days of STZ administration, and phenotypic aspects of diabetic retinopathy occur within weeks, with vascular leakage and reduced visual acuity and contrast sensitivity exhibited in these rodents. Therefore, this model is widely used to evaluate therapeutic agents in diabetic eye disease.

[0098] Six to seven week old C57BL / 6J mice are weighed and their baseline blood glucose is measured (Accu-Chek®, Roche). Mice can be injected intraperitoneally with STZ (Sigma-Alderich®, St. Louis, MO) at 55 mg / Kg for five consecutive days. Age-matched controls can be injected with buffer only. Blood glucose can be measured again one week after the last STZ injection, and mice are considered diabetic if non-fasting blood glucose is higher than 17 mM (300 mg / dL). STZ-treated diabetic C57BL / 6J mice can be intravitreally injected with microliter volumes of candidate agents at 8 and 9 weeks after STZ administration. Retinal vascular permeability can be measured by Evans Blue permeability assay 10 weeks after STZ treatment as follows:

[0099] Mice are anesthetized and Evans Blue dye dissolved in saline is injected via the tail vein. Two hours after tail vein injection, mice can be anesthetized with ketamine and xylazine and perfused through the left ventricle using saline. After perfusion, retinas are dissected, weighed, and placed in formamide at 70°C for 18 hours to extract the Evans Blue dye. The next day, retinas are centrifuged for 45 minutes and removed from the formamide. Evans Blue extravasation is measured using a plate reader at A620. A standard curve is used to convert to units of ng Evans Blue / wet tissue weight.

[0100] Example 5: Efficacy of candidate agents in a laser-induced choroidal neovascularization (CNV) mouse model The laser-induced CNV model involves rupture of Bruch's membrane, resulting in an inflammatory / wound healing response and associated CNV, thereby mimicking wet or neovascular AMD. The choriocapillaris is explicitly involved in the neovascular response, and this model produces an angiographic appearance similar to wet or neovascular AMD.

[0101] Male C57BL / 6J mice (6-8 weeks) can be anesthetized with a ketamine / xylazine cocktail prior to laser procedure. CNV lesions are induced by laser photocoagulation using a diode laser (IRIDEX®, Oculight® GL) and slit lamp (Zeiss®) with a spot size of 50 um, power of 180 mW, and exposure time of 100 ms. Typically, four laser burns are induced at the 3, 6, 9, and 12 o'clock positions around the optic disc of each eye. Candidate agents and appropriate controls can be injected intraperitoneally one day prior to laser induction, with a total of three injections every three days. Nine days after laser induction, mice can be perfused with FITC-lectin or TRITC-dextran via the tail vein. After perfusion, eyes are enucleated and fixed in 4% paraformaldehyde (PFA) for 15 minutes.

[0102] The choroid-sclera complex and retina can be separated, and anti-CD31 immunofluorescence (IF) can be performed to demonstrate the vasculature by whole mount staining of both retina and choroid tissue. For CD31 IF, rat anti-mouse antibody BD550274 was diluted 1:100 and incubated overnight at 4°C. After 4 hours of incubation with secondary anti-rat antibody (Life Technologies®, A11006), whole mounts can be imaged at 488 nm. Quantification of neovascularization in the lesions and blood vessel density in the retina can be performed by ImageJ, an open source software developed by the National Institutes of Health. p-values ​​can be evaluated by Student's t-test (significant change, p<0.05).

[0103] Example 6: Efficacy of candidate agents in a mouse model of oxygen-induced retinopathy (OIR) The OIR model is based on exposure of mouse pups to hyperoxia at a stage when their retinal vasculature is still developing, which leads to capillary depletion and, upon return to room air, to retinal ischemia and proliferative vascular disease in the retinal vasculature, or oxygen-induced retinopathy.

[0104] C57BL / 6J pups are housed (n=10 per cage) on postnatal day 7 (P7) for 5 days in a hyperoxic chamber (75% O2) resulting in vascular regression in the central part of the retina. CD-1 foster mothers are rotated before entering the chamber and 2-3 days after. At P12, pups are returned to room air, where relative hypoxia induces aberrant angiogenesis, and then appropriate amounts of control and candidate agents are administered intraperitoneally. At P17, all groups, including naïve OIR mice, can be euthanized. Eyes are enucleated and fixed in 4% paraformaldehyde for 1 h.

[0105] Retinas were dissected and stained with rhodamine-labeled lectin from Bandeiraea simplicifolia (Griffonia simplicifolia) (1:100) in 1 mM CaCl in PBS. 2The stained retinas are flat mounted on slides and imaged on a Zeiss® AxioScan. Images can be analyzed on a Visiopharm® to determine %VO or %NV of the entire retina.

[0106] Example 7: GPX4 competitive target-engaged gel shift assay A gel shift assay was developed for detection of either in vitro cell line or in vivo tissue target engagement (TE) of GPX4 inhibitors. See Figure 7 for a schematic of the assay.

[0107] In vitro labeling protocol: Two frozen mouse retinas were added directly to a 0.5 mL Precellys® tube with ceramic beads and 200 μL of cold mPER with 1× Roche EDTA-free protease inhibitors and 5 mM TCEP. Retinal tissue was homogenized using a single 15 s burst at 4500 RPM at 4° C. To remove foam, the tube was spun at 5 k×g for 5 min at 4° C., then all liquid was transferred to 1.5 mL Eppendorfs®. Samples were spun at 20 k×g for 10 min at 4° C. and the clarified lysate was transferred to a normal tube. 27.5 μL of each vehicle control was combined into one tube to provide the labeled standard sample.

[0108] For labeling controls, 50 μL of sample was added to 1 μL of 5 mM RSL3 and another 50 μL of sample was added to DMSO as a control. Samples were mixed and incubated at room temperature (RT) for 1 hour. This 1 μL of 5 mM GPX4 biotin probe was added to each sample, which was then vortexed briefly and incubated at room temperature for an additional 1 hour and 20 minutes. At the end of the incubation, 300 μL of -20°C acetone was added and the samples were stored at -20°C. For all other samples, 50 μL of lysate was added to 1 μL of 5 mM GPX4 biotin probe and mixed. The reaction was allowed to proceed for 2 hours at room temperature before acetone precipitation as above.

[0109] Samples were thawed and spun at 20,000xg for 10 minutes at 4°C. The supernatant was aspirated and discarded. 500μL of ice-cold acetone was added to each tube and briefly sonicated in a bath sonicator to break up the pellets. The tubes were then spun at 20,000xg and 4°C for 5 minutes, rotated 180 degrees, and spun for an additional 5 minutes. The supernatant was aspirated and the pellets were allowed to air dry for approximately 10 minutes.

[0110] 10 μL of SDS solution (0.5% SDS, 150 mM NaCl, 50 mM Tris 7.4, 5 mM TCEP, 1x Roche protease inhibitor) was added to each tube and left at room temperature for 1 hour. The tubes were then individually sonicated at the focus of a bath sonicator, then vortexed and spun briefly to consolidate the samples at the bottom of the tube. 40 μL of NP40 solution (1.25% NP-40, 150 mM NaCl, 50 mM Tris 7.4, 5 mM TCEP, 1x Roche protease inhibitor) was added and the samples were sonicated again briefly, vortexed, and collected at the bottom of the tube.

[0111] For the streptavidin binding reaction, 20 μL of sample was added to 5 μL of 5 mg / mL streptavidin (Promega®). The second 20 μL of each reaction was added to a "mock" tube containing 5 μL of 150 mM NaCl. These two 25 μL samples were shaken overnight at 4° C. at 550 RPM.

[0112] Western blot analysis: Samples were diluted with 25 μL of 2× NuPAGE® sample buffer (4× dHO) 2 The gels were diluted with 100 mM NaCl (diluted from 0.01%) and loaded onto 4-12% NuPAGE Midi, 1 mm, 12+2 well Bis / Tris gels (Invitrogen). The gels were eluted at 150 V for 1 h 15 min and then transferred to nitrocellulose membranes using iBlot® (ThermoFisher) using program 3 setting for 7 min.

[0113] Blots were blocked with 5% milk in TBST for 1 hour. After washing with TBST, anti-GPX4 antibody (Abcam#ab125066) diluted 1:2000 in 5% BSA in TBST was added and blots were incubated overnight at 4°C with shaking. Anti-rabbit HRP was used at 1:5000 in 5% milk in TBST for approximately 1 hour at room temperature and signals were detected using Western Pico® ECL reagents. All analyses were performed using a 5 minute exposure from an Azure Biosystems® c500. Integrated density of + / - streptavidin bands was quantified using ImageJ® (National Institutes of Health, USA).

[0114] This experiment demonstrated dose-dependent GPX4 TE of RSL3 in the adult mouse retina. See Figures 8A-8C.

[0115] This experiment was also performed using GPX4 inhibitors compounds #19 and #25 (see Figures 9A and 9B, respectively) by incubating with senescent HUVEC cells for 6 hours. The data showed potent sub-micromolar in vitro TE.

[0116] This gel shift assay can be used in parallel with the OIR model described above to test the TE of candidate GPX4 inhibitors in an in vivo situation. Specifically, once abnormal neovascularization is induced in mouse pups, an appropriate amount of vehicle control or candidate GPX4 inhibitor can be administered, for example, intravitreally (IVT). After a period of time, the mice can be euthanized and the eyes collected and processed in the gel shift assay described herein.

[0117] Example 8: Determination of GCN2 in vitro activity A specific compound, Compound 39, originally described as a PERK inhibitor with off-target GCN2 inhibitor activity (Smith, Adrian L., et al., J of Med Chem. 2015 Feb 12:58(3):1426-41), was tested to determine whether it had GCN2 activation activity using various in vitro assays. Without any limitations, such assays can be used to test other compounds for GCN2 pathway activation. Activating transcription factor 4 (ATF4) was C-terminally tagged in H2122 cells (CRL-5985™) with high affinity luciferase complementary fragment (HiBiT) through a knock-in approach using CRISPR-Cas12a technology and a single-stranded oligonucleotide DNA (ssODN) template carrying a HiBiT tag. After reporter cell line generation (H2122 ATF4-HiBiT), cells were seeded at near confluency in 96-well plates and treated with Compound 39 for 5 hours the following day. Using 10 μM as the starting concentration, dose responses were generated using quarter-log dilutions. Nano-Glo® HiBiT lysis detection (Promega®) reagent was added 5 hours later to allow plate-based bioluminescence quantification of cellular ATF4 protein using an add-mix-read assay format. Peak activation of ATF4 expression was detected at approximately 250 nM of compound 39. See Figure 10A.

[0118] Mouse embryonic fibroblast (MEF) cells were then cultured to near confluency and treated with a fixed concentration (250 nM) of compound 39 for 5 hours. Cells were harvested in LSD lysis buffer and homogenates were separated by SDS-PAGE. Immunoblotting was performed overnight using antibodies targeting P-eIF2a (clone D9G8), ATF4 (clone D4B8), and DNA damage-induced transcript 3 (DDIT3) (clone 9C8). Compound 39 treatment increases the levels of phospho-eIF2a (p-eIF2a), as well as ATF4 and DDIT3 (CHOP). See Figure 10B.

[0119] Primary human retinal microvascular endothelial cells (HRMEC-Cell Systems™ Corp., ACBRI181, Lot #181.04.02.02.02) were cultured in Vascular Cell Basal Medium (ATCC® Lot 90909277) using Endothelial Cell Growth Kit-VEGF (ATCC Lot 80720201) and additional FBS was added to a final concentration of 5% FBS. Cells were seeded at near confluency in 96-well plates and treated the next day with Compound 39 for 5 hours. A dose response was generated using quarter-log dilutions using 10 μM as the starting concentration. After treatment, RNA was extracted using RNeasy® 96-Kit (Qiagen®) and then cDNA was generated using Superscript™ IV VILO™ Master Mix (Thermofisher®). qPCR was performed using standard TaqMan® reagents and conditions. Primer Hs00358796_g1 was used to detect DDTI3 gene expression, and primer (Hs99999910_m1) was used to detect Tbp as a control gene. Peak activation of DDIT3 expression was detected at approximately 250 nM of compound 39. See Figure 10C.

[0120] To determine whether DDIT3 activation by compound 39 is dependent on GCN2 activation rather than PERK, the following experiment was performed. GCN2 knockout cells (CRL-2978™), PERK knockout cells (CRL-2976™), or WT control cells (ATCC CRL-2977™) were cultured to near confluency and treated with a fixed concentration (250 nM) of compound 39 for 5 hours. After treatment, RNA was extracted using RNeasy™ 96-Kit (Qiagen), and then cDNA was generated using Superscript™ IV VILO™ Master Mix (Thermofisher™). qPCR was performed using standard TaqMan reagents and conditions. DDIT3 gene expression was detected using primer Hs00358796_g1, and Tbp was used as a control gene using primer (Hs99999910_m1). Knockout cell lines demonstrated that DDIT3 induction by compound 39 was dependent on GCN2 but not on PERK. See Figure 10D.

[0121] Example 9: Determination of GCN2 in vivo activity Compound 39 was tested in an animal model that shares many characteristics with human ischemic retinopathy, oxygen-induced retinopathy (OIR) neonatal mice. To investigate the pharmacodynamic effects of compound 39, it was administered intraperitoneally (IP) at either 10, 30, or 90 mpk. Dosing formulations were based on a vehicle containing 1% Pluronic F-68 / 1% HPMC, 15% Captisol. Both normoxic and OIR neonates were treated at P12 (end of hyperoxia) and removed 4 hours later or 24 hours later at P13. Retinas were dissected and RNA was extracted using a fully automated spin-column-based nucleic acid extraction instrument (Qiacube Connect™). qPCR was performed using standard TaqMan® reagents and conditions. Atf4, Ddit3, and Tbp (control) gene expression was determined using primers: Mm00515324_m1, Mm00492097_m1, and Mm01277042_m1, respectively. Compound 39 was shown to have a dose-dependent induction of gene expression for both ATF4 (FIG. 11A) and DDIT3 (FIG. 11B) after IP administration.

[0122] Compound 39 administered intraperitoneally activated ATF4 protein expression at 30 and 60 mpk. Adult ocular retinas were dissected 4 hours after administration of compound 39, and the collected samples were kept on ice and homogenized in 300 uL MSD Tris lysis buffer containing Pierce Protease Inhibitor Complete Mini tablets (ThermoScientific™ A32963) using a Precellys™ (500 μL tube) tissue homogenizer. The homogenized samples were then centrifuged to separate the supernatant from the debris. The lysates were separated by SDS-PAGE and immunoblotted for ATF4 (D4B8) and the control protein actinin (Cell Signaling, cat.3134S). See Figure 11C. The results demonstrate that compound 39 increased the levels of ATF4 but not the control protein actinin.

[0123] Example 10: Modulation of stress response genes with relevance to retinal disease using GCN2 agonists To investigate the pharmacodynamic effects of compound 39, it was administered intraperitoneally at either 10, 30, or 90 mpk in both normoxic and OIR neonatal mice. The dosing formulation was based on a vehicle containing 1% Pluronic F-68 / 1% HPMC, 15% Captisol. Both normoxic and OIR neonatal mice were treated at P12 (end of hyperoxia) and removed 4 hours later or 24 hours later at P13. Retinas were dissected and RNA was extracted using a fully automated spin-column-based nucleic acid extraction instrument (Qiacube Connect™). qPCR was performed using standard TaqMan reagents and conditions. Slc7a11, Serpinf1, and Tbp (control) gene expression were determined using primers Mm00442530_m1, Mm00441270_m1, and Mm01277042_m1, respectively. Dose-dependent induction of gene expression by compound 39 was detected for both retinal Slc7a11 (FIG. 12A) and retinal Serpinf1 (FIG. 12B).

[0124] Normoxic and OIR ocular retinas were dissected 4 hours after Compound 39 administration, and the collected samples were kept on ice and homogenized in 300 μL of LSD sample / lysis buffer using a Precellys (500 μL tube) tissue homogenizer. The homogenized samples were then centrifuged to separate the supernatant from the debris. The lysates were separated by SDS-PAGE and immunoblotted for ATF4 (D4B8), p-eIF2a (D9G8), and HIF1α (Novus™, cat.NB100-449) and the control protein actinin (Cell Signaling™, cat.3134S). See Figure 13. Compound 39 treatment resulted in a pharmacodynamic response characterized by increased levels of ATF4 and phospho-eIF2α, as well as downregulation of HIF1α protein levels, indicative of reduced hypoxic stress signaling.

[0125] Example 11: Improvement of retinal vasculature using GCN2 agonists The efficacy of compound 39 was studied in a mouse OIR model. Exposure of young mice to a hyperoxic environment leads to occlusion of the retinal vasculature, followed by pathological neovascularization (angiogenesis) upon return to ambient air. Compound 39 was administered either intraperitoneally (90 mpk) twice at the indicated time points (P12 and P14 in the model) or once only (at P12 followed by bt vehicle control at P14).

[0126] Avascular and neovascular areas were quantified after harvesting and staining of retinal flat specimens at P17 of the model. OIR pups receiving two 90 mpk intraperitoneal doses of Compound 39 showed significant antagonism of neovascularization (FIG. 14A) but no vascular blockade (FIG. 14B) at P17. These results indicate that systemically administered Compound 39 can functionally inhibit pathogenic angiogenesis in the OIR disease model.

[0127] OIR pups were treated with an intraperitoneal injection of 90 mpk Compound 39 on P12, then these pups were transferred to room air with their nursing mothers, dosed repeatedly on P14, and sacrificed on P17. Eyes were enucleated on P17 and retinas were dissected for vascular staining. To determine avascular or neovascular areas, retinas were flat mounted and stained with isolectin B4 (IB4) for fluorescence microscopy. Isolectin stained flat preparations show preservation of retinal vasculature in Compound 39 treated pups (Figure 15B) compared to vehicle treated pups (Figure 15A). Vehicle treated pups had ischemic posterior retinas compared to pups repeatedly treated with Compound 39, indicating that two intraperitoneal doses of 90 mpk Compound 39 significantly inhibited neovascularization.

[0128] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

**Claim 1**: A composition for inhibiting pathogenic angiogenesis in an affected eye, comprising an agent selected from the group consisting of an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2. **Claim 2**: A composition for inhibiting vascular leakage in an affected eye, comprising an agent selected from the group consisting of an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2. **Claim 3** The composition according to claim 1 or 2, wherein the affected eye is a result of aging endothelial cells. **Claim 4** The composition according to claim 1 or 2, wherein the affected eye is a result of aging pericytes. **Claim 5** The composition according to claim 1 or 2, wherein the affected eye is a result of diabetic macular edema (DME), diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR), dry age-related macular degeneration (dAMD), and neovascular age-related macular degeneration (nvAMD). **Claim 6**: A pharmaceutical composition for use in a method of removing aging endothelial cells, comprising an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2, the method comprising contacting the cells with an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2. **Claim 7**: A pharmaceutical composition for use in a method of removing aging pericytes, comprising an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2, the method comprising contacting the cells with an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2. **Claim 8**: A composition for treating a patient suffering from retinovascular disease, comprising an agent selected from the group consisting of an inhibitor of GPX4, an inhibitor of GLS1, an inhibitor of PAPP-A, an inhibitor of cGAS, an inhibitor of STING, an inhibitor of mTOR, or an agonist of GCN2. **Claim 9** The composition according to claim 8, wherein the retinopathy is diabetic macular edema (DME), diabetic retinopathy (DR), proliferative diabetic retinopathy (PDR), dry age-related macular degeneration (dAMD), and neovascular age-related macular degeneration (nvAMD).

10. The composition according to claim 1 or 2, wherein the agent is a small molecule, an antibody, a polypeptide, an antisense oligonucleotide, or a small interfering ribonucleic acid.

11. The composition according to claim 6 or 7, wherein the inhibitor or agonist is a small molecule, an antibody, a polypeptide, an antisense oligonucleotide, or a small interfering ribonucleic acid.

12. The composition according to claim 1 or 2, wherein the inhibitor of GPX4 is selected from the group consisting of RSL3, ML210, ML162, JKE-1674, DPI-7, buthionine sulfoximine (BSO), FIN56, auranofin, elastin, artemisinin, sulfasalazine, artesunate, dihydroartemisinin, compound #19, compound #25, sorafenib, altretamine, almitrine, artemether, artemisone, and lampelizone.

13. The composition according to claim 1 or 2, wherein the inhibitor of GLS1 is 6-diazo-5-oxo-L-norleucine, GK921, UPGLO00004, telaglenastat, JHU395, ethyl 2-(2-amino-4-methylpentanamide)-DON, IPN60090, and BPTES.

14. The composition according to claim 1 or 2, wherein the inhibitor of PAPP-A is an antibody.

15. The composition according to claim 14, wherein the antibody is PAC-1scFv, PAC-1-D8scFv, PAC-2scFv, PAC-5scFv, and mAb-PA1 / 41.

16. The composition according to claim 1 or 2, wherein the inhibitor of PAPP-A is a polypeptide.

17. The composition according to claim 16, wherein the polypeptide is pro-MBP, stanniocalcin-1 (STC1), stanniocalcin-2 (STC2), and bikunin.

18. The composition according to claim 1 or 2, wherein the inhibitor of PAPP-A is an antisense oligonucleotide.

19. The inhibitor of cGAS is 3-(1-(6,7-dichloro-1H-benzo[d]imidazol-2-yl)-5-hydroxy-3-methyl-1H-pyrazol-4-yl)isobenzofuran-1(3H)-one, 1-(6,7-dichloro-9-(1-methyl-1H-pyrazol-3-yl)-1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl)-2-hydroxyethan-1-one, 1-[9-(6-amino-3-pyridinyl)-6,7-dichloro-1,3,4,5-tetrahydro-2H-pyrido[4,3b]indol-2-yl]-2-hydroxy-ethanone, or (1R,2S)-2-(7-oxo-5-phenyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carboxamide)cyclohexane-1-carboxylic acid, and the composition according to claim 1 or 2.

20. The inhibitor of STING is H-151, C-178, C-176, and Compound 18, and the composition according to claim 1 or 2.

21. The inhibitor of mTOR is rapamycin, Palomid 529, sirolimus, everolimus, temsirolimus, ridafolimus, umirolimus, zotarolimus, and an ATP-competitive mTOR kinase inhibitor, and the composition according to claim 1 or 2.

22. The agonist of GCN2 is 4-(2-amino-4-methyl-3-(2-methylquinolin-6-yl)benzoyl)-1-methyl-2,5-diphenyl-1H-pyrazol-3(2H)-one, 1-(5-(4-amino-2,7-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)indolin-1-yl)-2-(3-fluoro-5-(trifluoromethyl)phenyl)-ethanone, 4-(2-amino-4-methyl-3-(2-(methylamino)benzo[d]thiazol-6-yl)benzoyl)-1-methyl-2,5-diphenyl-1H-pyrazol-3(2H)-one, leucenol, histidinol, threoninol, SB-203207, SB-219383, dovitinib, neratinib, sunitinib, and erotinib, and the composition according to claim 1 or 2.

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