Lipoxygenase modulators for treating conditions associated with cone photoreceptor degeneration

Abstract

The present invention provides methods of treating conditions associated with cone photoreceptor degeneration, wherein the treatment comprises reducing or inhibiting lipid peroxidation in a cone photoreceptor. In particular, the present invention provides compounds for use in such methods, preferably metro death inhibitors.

Description

[0001] introduction

[0002] The retina is a light-sensitive neuronal structure located at the back of the eyeball. The human retina has two distinct regions. The peripheral retina has low spatial acuity and is responsible for different aspects of night vision and motion vision. The fovea (or macula), located in the center of the retina, drives high spatial acuity vision, crucial for reading and face recognition. Primates are the only mammals with a fovea. The retina is arranged in three layers of cells: the outer nuclear layer (ONL), the inner nuclear layer (INL), and the retinal ganglion cell layer (RGC). The ONL contains photoreceptors, unique neurons dedicated to converting light into electrochemical signals, and are therefore essential for vision. Two different types of photoreceptors can be distinguished: rod photoreceptors respond to dim light and enable night vision, while cone photoreceptors respond to bright sunlight and mediate high-resolution and color vision.

[0003] Retinal neurodegeneration associated with dysfunction or death of photoreceptors is a leading cause of incurable vision loss. Photoreceptor death is a common cause of many retinal disorders, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). In most of these diseases (e.g., AMD), the pathology appears to be primarily related to the loss of cone cells. Therefore, the ability to prevent cone photoreceptor degeneration is particularly important. This would enable therapeutic interventions at the earliest stages to halt disease progression and ultimately prevent vision loss.

[0004] To date, there are no effective treatments for retinal degenerative diseases, which remain difficult to treat, thus necessitating the development of new treatment strategies. Approved therapies for these diseases are few, and they only address neovascularization in the eye following cone photoreceptor degeneration. Furthermore, molecules capable of preventing or treating the earliest stage of the pathology (i.e., cone photoreceptor death) have not yet been identified.

[0005] Further understanding of the mechanisms leading to cone photoreceptor death is crucial for developing these therapies. Most of the earliest stages of retinal diseases remain unknown, particularly the molecular and cellular mechanisms leading to cone photoreceptor death. Several cell death mechanisms have been shown to be involved in ocular diseases, including apoptosis, pyroptosis, necrosis, and ferroptosis (Yang et al., Int J Mol Sci. 21(19):7279, 2020; Lin et al., J Ophthalmol. 2022:2695212, 2022; Zhang et al., Front Nutr. 9:844757, 2022). However, none of these mechanisms has shown a specific driver for cone photoreceptor death.

[0006] Therefore, there remains an urgent need for molecules that can effectively treat conditions associated with cone photoreceptor degeneration. Summary of the Invention

[0007] In a first aspect, this disclosure relates to methods for treating conditions associated with cone photoreceptor degeneration. This disclosure shows that regulation of lipid peroxidation in cone photoreceptors prevents degeneration of these cells. In particular, inhibition of lipid peroxidation in cone photoreceptors prevents degeneration of these cells.

[0008] Therefore, a first aspect of this disclosure relates to compounds, such as ferroptosis inhibitors, for use in the treatment of conditions associated with cone photoreceptor degeneration, wherein said treatment includes regulating, particularly specifically regulating, lipid peroxidation in cone photoreceptors. More specifically, the ferroptosis inhibitor specifically regulates lipid peroxidation in cone photoreceptors, i.e., lipid peroxidation in cone photoreceptors is regulated by the ferroptosis inhibitor, but lipid peroxidation in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, is substantially unaffected by the ferroptosis inhibitor. Preferably, the regulation of lipid peroxidation is due to a reduction or inhibition, particularly a specific reduction or inhibition, of the activity of at least one lipoxygenase and / or phospholipase A2 in cone photoreceptors. More specifically, the ferroptosis inhibitor specifically reduces or inhibits the activity of at least one lipoxygenase and / or phospholipase A2 in cone photoreceptors; that is, the activity of at least one lipoxygenase and / or phospholipase A2 in cone photoreceptors is reduced or inhibited by the ferroptosis inhibitor, but the activity of at least one lipoxygenase and / or phospholipase A2 in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, is substantially unaffected by the ferroptosis inhibitor. In another preferred embodiment, the activity of 5-lipoxygenase (5-LOX) and / or 5-lipoxygenase activator protein (FLAP) in cone photoreceptors is reduced or inhibited, particularly specifically reduced or inhibited. More specifically, the ferroptosis inhibitor specifically reduces or inhibits the activity of 5-lipoxygenase (5-LOX) and / or 5-lipoxygenase-activating protein (FLAP) in cone photoreceptors; that is, the activity of 5-lipoxygenase (5-LOX) and / or 5-lipoxygenase-activating protein (FLAP) in cone photoreceptors is reduced or inhibited by the ferroptosis inhibitor, but is substantially unmodified in other retinal cell types such as rod photoreceptors or retinal pigment epithelial cells.

[0009] In one instance, the treatment involved maintaining or increasing cone photoreceptor activity. In another instance, the treatment did not affect rod photoreceptor activity and / or retinal pigment epithelial cell activity.

[0010] In one instance, the treatment includes preventing increased permeability of the cell membrane to dyes in cone photoreceptors. More specifically, the treatment includes specifically preventing increased permeability of the cell membrane to dyes in cone photoreceptors, i.e., the treatment substantially does not prevent increased permeability of the cell membrane to dyes in other retinal cell types, such as rod photoreceptors.

[0011] In one instance, the treatment comprises maintaining or reducing the intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE), in at least one cone photoreceptor. More specifically, the treatment comprises specifically maintaining or reducing the intracellular formation of lipid peroxides, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid, in at least one cone photoreceptor.

[0012] In one instance, treatment included preventing or reducing microglial migration.

[0013] In one instance, treatment included preventing or reducing the formation of subretinal deposits.

[0014] In one instance, the treatment involves maintaining or increasing cone photoreceptor activity. More specifically, the treatment involves specifically maintaining or increasing cone photoreceptor activity, meaning that the treatment does not substantially maintain or increase the activity of other retinal cell types such as rod photoreceptors or retinal pigment epithelial cells.

[0015] The compounds used in the methods disclosed herein are preferably ferroptosis inhibitors. More preferably, the compounds are selected from the group consisting of: SCD1; RNA inhibiting any one of PLA2, ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5 and ALOX5AP; ziluton; ferrostatin-1; liproxstatin-1; PDANP; 16-86; XJB-5-131; UAMC-3203; SRS15-72B; SRS15-72A; SRS16-80; SRS16-86; GSK2190915; MK-886; ML355; BLX-3887; VKH2; ferrostatin-1, liproxstatin-1; PDA NP, 16-86, XJB-5-131, UAMC-3203, SRS15-72B, SRS15-72A, SRS16-80, SRS16-86, VKH2, nuclear enriched transcript 1 (NEAT1), α-tocopherol, BAY x 1005; tolmetin; caffeic acid; diallyl trisulfide; trelagliptin (succinate); tannic acid; norbergenin; chlorogenic acid; 3'-hydroxypuerarin; 1,3-dicaffeoylquinic acid; rosmarinic acid; (-)-epicatechin, CP-24879 (hydrochloride); FPL 62064; hexahydrocurcumin; Mito-TEMPO; MEL351; Nelonemdaz (potassium); Reynoutrin; Sonlicromanol (hydrochloride); SP-141; tenoridin hydrochloride; Trolox; and 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ).Most preferably, the compound is selected from the group consisting of: SCD1; inhibiting RNA of any one of PLA2, ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5 and ALOX5AP; ziluton; ferrostatin-1; liproxstatin-1; PDA NP; 16-86; XJB-5-131; UAMC-3203; SRS15-72B; SRS15-72A; SRS16-80; SRS16-86; GSK2190915; MK-886; ML355; BLX-3887; VKH2; ferrostatin-1, liproxstatin-1; PDA NP, 16-86, XJB-5-131, UAMC-3203, SRS15-72B, SRS15-72A, SRS16-80, SRS16-86, VKH2, nuclear enriched transcript 1 (NEAT1), α-tocopherol, BAYx 1005; tometetin; caffeic acid; diallyl trisulfide; treglitazone (succinate); tannic acid; norbergenin; chlorogenic acid; 3'-hydroxypuerarin; 1,3-dicaffeoylquinic acid; rosmarinic acid; (-)-epicatechin, CP-24879 (hydrochloride); FPL 62064; hexahydrocurcumin; Mito-TEMPO; MEL351; Nelonemdaz (potassium); renoside; Sonlicromanol (hydrochloride); SP-141, tenoridin hydrochloride; and Trolox.

[0016] Preferably, the conditions associated with cone photoreceptor degeneration in the methods disclosed herein are conditions in which cones are specifically affected. In particular, the conditions are those in which cone photoreceptor degeneration is not a secondary consequence of retinal pigment epithelium degeneration. More preferably, the conditions are selected from the group consisting of: age-related macular degeneration, cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt's disease), central serous chorioretinopathy, Best's disease and bestrophinopathies, retinal detachment (including rhegmatogenous, serous, and tractional causes), solar retinopathy, laser-induced retinopathy, achromatopsia, Usher syndrome, Leber congenital amaurosis, Alström syndrome, and Refsum disease.

[0017] In one instance, the compound used in the methods disclosed herein is administered in the subretinal space, suprachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or on the corneal surface. For example, the compound can be administered systemically (inhaler, intravenously, intramuscularly, or intraperitoneally) or as a solution, gel, or implant.

[0018] In one example, the treatment of the present invention includes the administration of a second therapeutic agent. The second therapeutic agent is preferably a second ferroptosis inhibitor, aflibercept, ranibizumab, pegaptanib, bevacizumab, brolucizumab, faricimab, AKB-9778, nesvacumab, and BI 836880. More preferably, the administration of the second ferroptosis inhibitor results in a reduction or inhibition of lipid peroxidation in cone photoreceptors and / or a decrease in intracellular iron levels in cone photoreceptors.

[0019] In one instance, the treatment of the present invention includes simultaneous, sequential, or separate administration of the compound and a second therapeutic compound. Attached Figure Description

[0020] Figure 1Evidence of ferroptosis occurring in pure cone photoreceptors. Mean values ​​and standard errors of the mean are shown. AB. Bright-field images of cone photoreceptors after 3 days of incubation with control (Ctrl = 0.2% DMSO, A) and treatment solution (3 µM imidazole ketone erastin, IKE, B). CD. Cone photoreceptor staining with calcein-labeled live cells under control (C) and treatment (D) conditions. E. Glutamate concentration-dependent cell viability of pure cone photoreceptors with or without 1 mM L-cysteine ​​(n=7, p<0.0001, IC50=4.15 µM). F. Glutathione levels in pure cone photoreceptors at 3 days in control, 3 µM IKE, and 500 µM glutamate medium (D1, D2, D3 n=18, p<0.0001). G. Percentage of NADP / NADPH in pure cone photoreceptors on day 3, compared with control and 3 µM IKE conditions (n=4, p=0.0159). H. Concentration-dependent plot of cell viability of pure cone photoreceptors after treatment with 0.01 nM to 25 nM RSL3 (n=3, IC50=8.67 nM). I. Western blot analysis showed bands of 5-lipoxygenase (5LOX) and 5-lipoxygenase activator protein (FLAP) in pure cone photoreceptors, which were absent in rod photoreceptors. J. Lipomics analysis showed the amounts of 15- and 5-hydroxyeicosatetraenoic acid (15-HETE and 5-HETE, respectively) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5oxoETE) detected in pure cone photoreceptors under control and 3 μM IKE conditions (n=6, p=0.0079(**), p=0.0159(*)). K. Activity of pure cone photoreceptors under control and 3 µM IKE conditions when various treatments (with antiferroptosis or iron chelation effects) were added: L-cysteine, zileutone (ZEN), ferrostatin-1 (FST1), and deferoxone (DF).

[0021] Figure 2 Changes in mRNA expression levels of proteins involved in ferroptosis in pure porcine cone photoreceptors of control and treatment (3 µM IKE) (n=6). Solute carrier family 11 member 2 (SLC11A2) is involved in ferroptosis. 2+Released from endosomes into the cytoplasm; its decrease suggests iron metabolism disorder (p=0.0260). Solute carrier family 3 member 2 (SLC3A2) corresponds to the heavy chain of systemic Xc- (p=0.0022). Transferrin receptor (TFRC) expression moderately increased under treatment conditions (p=0.0286). Ferroplasm inhibitor 1 (AIFM2) is involved in mechanisms reducing ferroptosis (p=0.0022). Heat shock protein family B small member 1 (HSPB1) plays a role in iron uptake and may promote ferroptosis. 22 (p=0.0022).

[0022] Figure 3 Regarding DAPI nuclear staining, immunofluorescence localization of flap was observed on retinal sections from three different species (pig, rat, and non-human primates). In all species, the flap was located within the cone photoreceptor.

[0023] Figure 4 Immunofluorescence of ferritin heavy chain (B, G) and NCOA4 (C, H) in isolated porcine cone photoreceptors from control (AE) and treatment (FJ) cells. IKE treatment increased fluorescence intensity (G, H) compared to control cone photoreceptors. IKE conditions (G, H) showed more immunomarking than controls (B, C). E. Magnified view of combined markings, showing stronger ferritin heavy chain immunomarking than NCOA4 in control cells. J. Magnified view of treated cells, showing stronger NCOA4 immunomarking than heavy chain.

[0024] Figure 5Degeneration of cone photoreceptors and microglial migration in ex vivo retinal explants. Histological examination of retinal sections A–H showed increased permeability of the first row of nuclear retina, representing cone photoreceptors, to etidon in treated explants (20 μM IKE, F) compared to control tissue (0.2% DMSO, B). Also noted was the disorganization of Flap-immunolabeled cone photoreceptors in the treated retina (G) compared to control (C), and the presence of large amoeba-like microglia up to the outer nuclear layer (ONL) in the treated retina (H), which were absent in the control retina (D). In both control (A) and treated retina (E), the nuclear layer was highlighted by DAPI. IM. Loss of Flap-immunopositive outer segments in treated flat-mounted retinal explants (20 μM IKE) compared to control (J) indicated destruction of cone photoreceptors and their protection by the antiferroptosis agents FST-1 (L) and DF (M). Quantification of the outer segments of cone photoreceptors showed a significant reduction in treated explants and rescue by the antiferroptosis agents (I, n=3). NO. Distribution of microglia in the thickness of retinal explants, showing their migration toward the outer retina in treated retina (IKE, RSL3), and prevention of this effect by DF or FST-1 (n=3). Scale bar = 20 µm.

[0025] ONL: Outer nuclear layer, INL: Inner nuclear layer, GCL: Ganglion cell layer, Iba1: Ionized calcium-binding adaptor molecule 1, Ctrl: Control condition, IKE: Imidazolone ferroptosis inducer, OS: Outer segment, FST-1: Ferrostatin-1, DF: Deferoxone

[0026] Figure 6 Compared to the control condition (Ctrl), different retinal cell types were maintained on sections from treated porcine retinal explants (20 μM IKE). Rod photoreceptors were immunolabeled for rhodopsin, bipolar cells for PKCα, and Müller cells for vimentin. It was noted that retinal structures were highlighted by DAPI-stained cell nuclei, and the morphology of the labeled cells appeared to be preserved under the treatment conditions, with no significant cell loss.

[0027] Figure 7Effects of RSL3 on retinal explants. RSL3 permeability to etidon revealed dead cells in the retinas of control (0.2% DMSO, B) and treated (200 nM RSL3, F). C, G. Morphological changes of Flap-labeled cone photoreceptors in treated retinal explants (G) compared to control explants (C), and migration of iba1-immunopositive microglia toward the outer nuclear layer (ONL) in treated explants (H) relative to control condition (D). IL. Loss of Flap-immunopositive outer cone segments (OS) in RSL3-treated retinal explants (J) relative to control condition (I) and their preservation via FST-1 (K) or DF (L). M. Quantification of RSL3 toxicity to outer cone photoreceptor segments and their preservation in retinal explants via DF (n=3, p=0.0286). N. Distribution of microglia, showing their distribution in the presence of RSL3 compared to control conditions (see...). Figure 5 The migration of N towards the outer nuclear layer (ONL), and the effects of DF or FST-1 (n=3).

[0028] Scale bar = 20 µm. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, Iba1: ionized calcium-binding adaptor molecule 1, Ctrl: control condition, IKE: imidazolidin ferroptosis inducer, OS: outer segment, FST-1: ferrostatin-1, DF: deferoxone.

[0029] Figure 8In vivo subretinal delivery of ferroptosis inducers to induce cone photoreceptor degeneration and microglial migration in rats. A. Schematic representation of experimental protocol and timeline. Rats were delivered subretinally with 20 μM IKE or 200 nM RSL3 (treatment) or 0.2% DMSO (control, Ctrl). Retinas were removed on day 6 (D6) to assess microglial migration (KO, n=4) or on day 30 (D30) to assess cone photoreceptor degeneration (HJ, n=4). BG. In vivo retinal imaging samples from rats: fundus photography (B, C) and optical coherence tomography (OCT) (DG) show retinal detachment induced near the optic nerve (ON) (D0, B, D), with complete reattachment of the retina at D6 (C, E); injection sites (arrows) and OCT sections (dashed lines) are shown on the fundus photographs; (F, G) OCT of the injection area at D6 shows highly reflective markings (arrows) found at the junction of the inner nuclear layer (INL) and outer plexiform layer (OPL) / outer nuclear layer (ONL), which are more abundant under treatment conditions (G). H, I. Retinal sections of the injection area collected at D30 (same rats as in BG) show a reduction in cone photoreceptors (CARs) immunolabeled with cone inhibitory protein in the treated animal (I) compared to the control (H) with corresponding DAPI nuclear staining. The eccentricity relative to the optic nerve is the same in both cases. J. Quantification of cone density (in elements / mm²) measured on retinal sections of treated and control rats at D30 (n=4). The outer segment (OS) and perinuclear body of the cones were quantified separately. K. Quantification of microglial density on whole-mounted retina of treated and control rats at D6. Three retinal planes were assessed: the outer segment (OS) level, the ONL plane, and the pedicle (Ped) plane corresponding to the OPL / ONL junction. Each field corresponded to a 500 × 500 μm window and was repeated throughout the injection area. LO. Distribution of cone suppressor protein-positive cells and Iba1-positive microglia in whole-mounted retina of treated and control rats collected at D6, showing two distinct planes on the z-axis (as shown in HI): OS (L, M) and Ped (N, O). Samples were taken from the detachment area but away from the injection site to illustrate changes in cone OS morphology and invading microglia in the treated animals. The degree of eccentricity relative to the optic nerve is the same in both cases.

[0030] Scale bar: 50 μm. INL: Inner nuclear layer, OPL: Outer plexiform layer, ONL: Outer nuclear layer, EZ: Ellipsosome zone, RPE: Retinal pigment epithelium, DAPI: 4',6-diamidinyl-2-phenylindole, CAR: Cone repressor protein, Iba1: Ionized calcium-binding adaptor molecule 1, ctrl: Control.

[0031] Figure 9 Macular changes in non-human primates (NHPs) following subretinal delivery of 20 μM IKE. A. Schematic representation of the experimental protocol. Subretinal delivery of 20 μM IKE in the right eye and DMSO diluted 1 / 500 in PBS in the left eye allowed for superior foveal detachment in both eyes. BC. Fundus photographs at baseline (B), 1 month (C), and 3 months (D) after subretinal injection of 20 μM IKE. The demarcation line at 1 month (arrow in C) and macular pigmentation around the subretinal deposits at 3 months are shown (highlighted in D). EF. OCT vertical b-scan of NHPs across the fovea three months after subretinal delivery of 1 / 500 DMSO (ctrl, E) and 20 μM IKE (F). Note the eroded ellipsoidal band (EZ, black asterisk), punctate hyperreflectance (arrow), and subretinal deposits (arrow). G. Horizontal b-scan OCT scan through the superior parafoveal region after 20 μM IKE subretinal delivery. EZ is eroded within the boundary of the detachment region (dashed line) (black asterisk). High-reflectivity foci (arrows) also noted, primarily located in the ONL and below the internal limiting membrane. The uninjected area (outside the dashed line) remains completely normal. HJ. Horizontal b-scan OCT scan through the subretinal deposits in the macular region (arrows), which appear in M2 (I) and grow to M3 (J). KL. Frontal adaptive optics imaging of the foveal photoreceptor layer at an eccentricity of 2 degrees within the detachment region 3 months after subretinal delivery. Cones show reduced reflectivity and clusters of cone disappearance (K) after 20 μM IKE delivery, while cone mosaicism remains regular on the control side (L). MN. Illustration of ONL thickness changes in the NHP macular region 3 months after subretinal delivery. Grayscale shows ONL thickness changes (in μm). O. Changes in cone density (in cones / deg²) measured using adaptive optics at 2 degrees outside and within the injection area 3 months after subretinal delivery (n=2). P. Percentage changes in N1 and P1 wave amplitudes measured on multifocal electroretinography relative to baseline (n=2). Only hexagons within the injection area were analyzed.

[0032] Figure 10Ferraphobia pathways elucidated in mammalian cone photoreceptors. Ferraphobia ultimately drives ROS formation, leading to cone photoreceptor degeneration. Ferraphobia in cones must be considered through three subcellular pathways. First, systemic Xc- can be inhibited by high extracellular concentrations of glutamate or ferrophobia inducers (e.g., IKE), stimulating L-cysteine ​​influx to produce glutathione. Inhibition of the glutathione redox cycle leads to inactivation of glutathione peroxidase 4 (GPX4) and NADPH accumulation. Additionally, reduced GPX4 enzyme activity results in the production of lipid peroxides, such as 5-oxo-ETE via the lipoxygenase pathway. Finally, high ferritinophagy activity of NCOA4 leads to iron metabolism dysregulation, resulting in excessive iron release. Free radicals are formed via the Fenton reaction and allow the production of reactive oxygen species through interaction with lipid peroxides.

[0033] GPX4: Glutathione peroxide 4, 12-LOX: 12-lipoxygenase, 15-LOX: lipoxygenase, 5-LOX: 5-lipoxygenase, FLAP: 5-lipoxygenase activator, 12-HPETE: 12-hydroxyperoxyeicosatetraenoic acid, 15-HPETE: 15-hydroxyperoxyeicosatetraenoic acid, 5-HPETE: 5-hydroxyperoxyeicosatetraenoic acid, 5-oxo-ETE: 5-oxo-eicosatetraenoic acid, 12-HETE: 12-hydroxyeicosatetraenoic acid, 15-HETE: 15-hydroxyeicosatetraenoic acid, 5-HETE: 5-hydroxyeicosatetraenoic acid, STEAP 3: STEAP family member 3, DMT1: Divalent metallotransferase 1, NCOA4: Nuclear receptor coactivator 4, Keap1: Kelch-like ECH-associated protein 1, NFE2L2: Nuclear factor (erythroid-derived 2)-like 2. ROS: Reactive oxygen species.

[0034] Figure 11Cell viability after ferroptosis-inducing treatments of different cell types in the retina. Cone photoreceptors (PRs) are represented by light gray lines and circles, ARPE-19 by dark gray lines and triangles, and primary RPE cells by black lines and squares. A. Cells were treated with IKE (imidazolidinone ferroptosis inducer) from 0.20 μM to 25 μM. All PRs died at 25 µM, while 40% of ARPE-19 (n=3, p=0.0001) and 80% of primary RPE cells (n=3, p=0.0019) remained viable. B. Cells were treated with RSL3 (Ras selective ligand 3) from 0.01 μM to 10 μM. At all concentrations, all PR cone cells died, while at 5 µM, 50% of ARPE-19 (n=3, p=0.0008) and primary RPE cells remained viable. C. Treatment of cells with 0.001 mM to 1 mM glutamate showed no effect on ARPE-19 and primary RPE cells. In contrast, all cone PR cells died at 1 mM compared to ARPE-19 cells (n=3, p=0.0050) and primary RPE cells.

[0035] Figure 12Ferraphobia inhibitors reversed cone degeneration in rd1 mice between P15 and P45 in vitro and in vivo. AD. PNA-immunolabeled rd1 mouse retinal explants were cultured for 30 days in control solution (A) and several feraphobia inhibitors (from P15 to P45) (BD). E. Automated cone counting was performed on retinal explants from rd1 mice exposed in culture to various feraphobia inhibitors (Ctrl, HTHQ, 1,3-dicaffeoylquinic acid, SRS16-86, astilbin, caffeic acid, tenoridin hydrochloride n=3; GW5074 n=6; nobergenin n=2; mangiferin n=5). White bars represent antioxidants or free radical scavengers, gray bars represent molecules involved in the Xc system pathway, checkerboard bars represent molecules acting on the lipoxygenase pathway, and vertical bars represent molecules involved in the iron pathway. FI. PNA-labeled full-thickness retinal slices (G and I) of rd1 mice after daily intraperitoneal injection of the mediator (Ctrl, F, and H) or 2 mg / kg GW 5074 solution for 30 days (from P15 to P45). Asterisks indicate the locations of H and I. J. Automated cone counting (n=5) of full-thickness retinal slices following daily intraperitoneal injection. Scale bar = 50 µm for AD, H, and I; 300 µm for F and G. PNA: peanut lectin, Ctrl: control, HTHQ: 1-O-hexyl-2,3,5-trimethylhydroquinone, 1-3-DCFA: 1,3-dicaffeoylquinic acid, RA: rosmarinic acid, SMNL: Sonlicromanol, CA: caffeic acid, Tinoridin HCl: tenoridin hydrochloride, OH puerarin: hydroxypuerarin, 5-ACQ: chlorogenic acid.

[0036] Figure 13 Ferroplasmosis inhibitors cannot reverse rod degeneration between P15 and P45 in rd1 mice, either in vitro or in vivo. A. Automated cone counting was performed on retinal explants from rd1 mice exposed in culture to various ferroplasmosis inhibitors for rescuing cones (Ctrl, HTHQ, 1,3-dicaffeoylquinic acid, SRS16-86, astilbin, caffeic acid, tenoridin hydrochloride n=3; GW5074 n=6; nobergenin n=2; mangiferin n=5). B. Automated cone counting was performed on full-thickness retinal slices following daily intraperitoneal injection (n=5).

[0037] Figure 14Rods were immunolabeled with rhodopsin (Rho) and DAPI in the retinas of nonhuman primates injected with control solution and 20 µM IKE. Detailed Implementation

[0038] definition

[0039] The terms “about” or “approximately” refer to a normal range of error for a given value or range, as known to those skilled in the art. It typically means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range. References to “about” in this document include (and describe) embodiments for that value or parameter itself.

[0040] As used herein, the terms “activate,” “stimulate,” or “induce,” and any of their grammatical derivatives, refer to a comparative increase in a specific response (e.g., expression, enzyme activity) of a specified substance in the presence of a particular reagent. This reagent is described herein as an “activator.”

[0041] As used herein, “administer” or “administration” means the act of injecting or otherwise physically delivering a substance, which is present outside the body, into a patient’s body, such as via mucous membranes, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, administration of a substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of a substance typically occurs before the onset of the disease or its symptoms. In particular, “administering” refers to the method of administering a dose of the compound disclosed herein to a subject suffering from a condition associated with degeneration of cone photoreceptors. The compositions used in the methods described herein may be administered, for example, intravitreal (e.g., by intravitreal injection), ocular (e.g., by ocular injection), or intraocular (e.g., by intraocular injection). The method of administration can vary depending on various factors, such as the compound or composition administered and the severity of the condition, disease, or disorder being treated.

[0042] Application can be made via a carrier or medium, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pills; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; or nanoparticles.

[0043] As used herein, the terms “decreased” or “reduced” mean that the activity of a protein is at least one-fold lower than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000, or more times lower). When referring to the activity of a subject’s protein, “decreased” or “reduced” also means that the activity of the protein is at least 5% lower than that in a reference sample or relative to the reference value of said protein (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). As used herein, the term “reduced” or “lower” also refers to a subject’s level of a biomarker (e.g., lipid peroxidation in a specific cell type, such as cone photoreceptors) being at least 1-fold lower than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000, or more times). When referring to a subject’s level of a biomarker (e.g., lipid peroxidation in a specific cell type, such as cone photoreceptors), “reduced” or “lower” also means at least 5% lower than the level in a reference sample or relative to a reference value for said biomarker (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

[0044] The term "disease" refers to any condition or disorder that impairs or interferes with the normal function of cells, tissues, or organs. In one instance, the disease is a condition affecting the eye, particularly one related to degeneration of the cone photoreceptors.

[0045] As used herein, “electroretinogram (ERG)” refers to a diagnostic test that measures the electrical activity of the retina in response to light stimulation. For example, ERG can record convergent potentials from the entire retina; see, for example, full-field ERG. Alternatively, multifocal ERG (mfERG) assesses ERG activity in small regions of the retina, while graphic ERG (pERG) assesses macular retinal ganglion cell (RGC) activity. ERG can be an objective measure of retinal function that can be recorded under physiological conditions. For example, ERG can be used to provide diagnostic information, monitor the progression of retinal diseases and disorders, or combinations thereof. ERG can be used to determine the effectiveness of any of the various therapeutic compounds disclosed herein. Examples of ERG values ​​from healthy subjects and patients can be found in Lorenz et al., Invest. Ophthalmol. Vis. Sci. 49:5235-5242, 2008.

[0046] As used herein, “ferroptosis” refers to a form of cell death as understood in the art, involving the generation of iron-mediated reactive oxygen species and characterized in part by lipid peroxidation. In ferroptosis, lethality occurs due to the peroxidation of self-replicating polyunsaturated fatty acids (PUFAs), unless prevented by the lipid peroxidase glutathione peroxidase 4 (GPX4).

[0047] As used herein, the term “increased” means that the activity of a protein is at least 1 time greater than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more). When referring to the activity of a subject’s protein, “increased” also means that the activity of the protein is at least 5% higher than that in a reference sample or relative to the reference value of said protein (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). As used herein, the term “increased” also refers to a subject’s biomarker level being at least 1 times higher than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000, or more times). When referring to a subject’s biomarker level, “increased” also means at least 5% higher than the level in the reference sample or relative to the reference value of said biomarker (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%).

[0048] As used herein, the term "inhibit" or "inhibition" refers to a relative reduction in a specific response (e.g., expression, enzyme activity) of a specified substance in the presence of a particular reagent. This reagent is described herein as an "inhibitor".

[0049] As used herein, the term "lipid peroxidation" refers to the result of free radical damage to lipids. Lipid peroxidation forms a number of oxidation products, including lipid peroxides and aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).

[0050] As used herein, the terms “lipid peroxides” or “peroxidized lipids” refer to the oxidation products of phospholipids and polyunsaturated fatty acids (PUFAs).

[0051] As used herein, “lipoxygenase” is an enzyme that oxidizes polyunsaturated fatty acids or alkenes with two oxygen atoms into arachidic acid-like substances. It catalyzes the first reaction in the so-called lipoxygenase pathway, participating in responses to external trauma and stress. Examples of lipoxygenases in humans include 15-LOX, 15-LOX-2, 12-LOX, 12R-LOX, eLOX-3, and 5-LOX. In some instances, lipoxygenase activity requires the presence of an activator. For example, the accessory protein 5-LOX activator protein (FLAP) is essential for the activation of 5-LOX enzymes. As used herein, the term “lipoxygenase” also includes those coactivators, particularly FLAP.

[0052] As used in this article, “measure” or “determine” refers to any qualitative or quantitative determination.

[0053] As used herein, “operably linked” means that the linked elements are arranged such that they work together for their intended purpose. For example, if a promoter affects transcription initiation from the start of transcription to the terminator of a nucleic acid molecule in a permitted host cell, then the promoter is operably linked to the nucleic acid molecule.

[0054] As used herein, "optical coherence tomography (OCT)" refers to a non-invasive imaging test that uses light waves to capture cross-sectional images of the retina. OCT can be used to differentiate retinal layers, map and measure thickness, inform treatment decisions, provide diagnostic information, monitor disease progression, or a combination thereof. Notably, OCT can be used to quantify retinal cone degeneration. In this implementation, subjects diagnosed without retinal degenerative disease have approximately 200,000 cells / mm² in the fovea, as measured by OCT. 2 The peak cone density, and / or the outer nuclear layer thickness of the fovea, as measured by OCT, is approximately 100 μm. Preferably, the fovea of ​​a patient diagnosed with retinal degenerative disease has less than approximately 200,000 cells / mm², as measured by OCT. 2 Peak cone density. Patients diagnosed with retinal degenerative diseases may have an extrafocal nuclear layer thickness of less than about 100 μm, as measured by OCT. Other examples of OCT values ​​from healthy subjects and patients diagnosed with retinal degenerative diseases can be found in Lorenz et al., 2008.

[0055] As used herein, the term "phospholipase A2" or "PLA2" refers to the PLA2 superfamily of lipases. PLA2 activity refers to the ability of PLA2 enzymes to hydrolyze fatty acids from the sn-2 position of membrane phospholipids (e.g., phosphatidylcholine). The PLA2 superfamily comprises four main types of enzymes, including secretory PLA2 (sPLA2), cytoplasmic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2), and platelet-activating factor (PAF) acetylhydrolase / oxidized lipid lipoprotein-associated PLA2 ((Lp)PLA2). In particular, PLA2, especially sPLA2 and cPLA2, is known to hydrolyze phosphatidylcholine, producing lysophosphatidylcholine and simultaneously releasing arachidonic acid.

[0056] As used in this article, “polyunsaturated fatty acid (PUFA)” refers to fatty acids containing at least two -CH=CH- groups, such as linoleic acid, linolenic acid, and arachidonic acid (AA).

[0057] As used herein, the terms “prevent,” “prevention,” and “preventing” refer to reducing the risk of a subject acquiring or developing a given disease, disorder, or condition. The terms “prevent,” “prevention,” and “preventing” also include delaying the onset and / or reducing the frequency and / or intensity of clinical, histological, and / or biochemical symptoms or parameters associated with the given disease, disorder, or condition. In some implementations, prevention is assessed on a population basis such that the therapy is considered to “prevent” the specific disease, disorder, or condition if a statistically significant reduction in the risk of developing or progressing to the disease, disorder, or condition is observed in a susceptible population, and / or a statistically significant delay in the onset of clinical, histological, and / or biochemical symptoms or parameters associated with the disease, disorder, or condition, and / or a statistically significant reduction in their frequency and / or intensity, is observed.

[0058] As used herein, the terms "regulatory elements" or "regulatory sequence" refer to any element that allows, facilitates, or regulates the expression of the encoded nucleic acid in a given host cell or subject, including the replication, duplication, transcription, splicing, translation, stabilization, and / or transport of the nucleic acid or its derivatives (i.e., mRNA).

[0059] As used herein, the term "specifically" means that a compound affects one cell type (i.e., cone photoreceptors) at a given concentration, while having substantially no effect on other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, at the same given / same concentration. In particular, when referring to in vivo use, the term "specifically" means that a compound affects one cell type (i.e., cone photoreceptors) while having substantially no effect on other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells.

[0060] As used herein, “subject” includes all mammals, including but not limited to humans, and also includes non-human primates such as cynomolgus monkeys. It also includes dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents (such as mice and rats). It should be understood that a particularly preferred subject according to this disclosure is a human subject, such as a person suffering from a disorder, disease, or condition (or at risk of suffering from a disorder, disease, or condition) (particularly conditions related to cone photoreceptor degeneration), such as a human patient.

[0061] As used herein, the term "therapy" means any program, method, and / or agent that can be used to prevent, manage, treat, and / or improve a patient's disease, disorder, or condition. A patient may be at risk of having a disease, disorder, or condition, or is suspected of having one. Alternatively, a patient may have been diagnosed with a disease, disorder, or condition. Non-limiting examples of therapy include the administration of a composition (e.g., a drug or vaccine composition), physical therapy (e.g., radiation therapy, ultrasound therapy, electrotherapy, phototherapy, cryotherapy, etc.), physiotherapy, psychotherapy, etc. Therapy also includes the possibility of using combination therapy. "Combination therapy" and any variations thereof, such as "combined use," refers to the act of delivering several different therapies to the same subject. This combination covers situations where different therapies are delivered to the subject as a single composition (together at the same unit dose) or separately (i.e., arranged separately), in which case the different therapies may be delivered simultaneously or sequentially. As used herein, "simultaneously" means the act of simultaneously delivering several therapies mixed in the same composition. As used herein, “separately” refers to the act of delivering several therapies in a separate manner (e.g., separate compositions) substantially simultaneously or within the same time period (e.g., within an hour or less). “Sequentially” means “one after the other,” implying that one therapy is delivered first, followed immediately by or after a suitable time period (e.g., more than an hour).

[0062] When a therapy involves the administration of a composition or the application of a composition, the composition is administered in a quantity, manner, and / or pattern that is effective in treating or preventing a patient's disease, disorder, or ailment. The therapy may require more than one administration of the composition. In this case, the interval between two consecutive deliveries of the therapy may be the same or different. In the case of combination therapy, the interval between two consecutive deliveries of several different therapies may be the same or different.

[0063] As used herein, the term "treating" or "treatment" means an improvement in a patient's disease, disorder, or condition that can be observed at the clinical, histological, and / or biochemical levels. The term "treating" or "treatment" specifically includes improving clinical, histological, and / or biochemical symptoms or parameters associated with a patient's disease, disorder, or condition, or suppressing, reducing, or delaying the progression or worsening of a patient's disease, disorder, or condition (including secondary damage resulting from the disease, disorder, or condition) to a statistically significant degree or to a degree detectable by a person skilled in the art. In some embodiments, treatment is evaluated on a population basis such that if a statistically significant improvement in a patient's disease, disorder, or condition is observed in a population of patients with the disease, disorder, or condition, the treatment is considered to "treat" the specific disease, disorder, or condition.

[0064] As used herein, the term "vector" refers to a medium, preferably a nucleic acid molecule or viral particle, containing elements necessary to allow the delivery, proliferation, and / or expression of any nucleic acid molecule described herein within a host cell or subject. This term encompasses vectors for maintenance (cloning vectors) or vectors for expression in various host cells or subjects (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids) or integration vectors (e.g., designed to integrate into the host cell genome and generate additional copies of the nucleic acid molecule during host cell replication), as well as shuttle vectors (e.g., functioning in prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., for transferring nucleic acid molecules from a viral genome). For the purposes of this disclosure, vectors may be of a naturally occurring genetic source (synthetic or artificial) or a combination of natural and artificial genetic elements.

[0065] In the context of this disclosure, the term "vector" must be understood broadly to include mRNA, plasmids, and viral vectors. Suitable vectors in the context of this disclosure include, but are not limited to, phage, plasmid, or granular vectors for expression in prokaryotic host cells such as bacteria (e.g., *Escherichia coli*, *BCG*, or *Listeria*); vectors for expression in yeast (e.g., *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, *Pichia pastoris*); baculovirus vectors for expression in insect cell systems (e.g., *Sf9* cells); and plasmids and viral vectors for expression in higher eukaryotic cells or subjects. Typically, such vectors are commercially available (e.g., at Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or from depository institutions such as the American Type Culture Collection (ATCC), Rockville, Md., or have become the subject of numerous publications describing their sequences, tissues, and methods of production, thus allowing those skilled in the art to use them. This disclosure also covers carriers (e.g., plasmid DNA and mRNA) that are combined with lipids or polymers to form particulate structures such as liposomes, lipid complexes, or nanoparticles.

[0066] Treatment

[0067] This disclosure provides a method for treating conditions associated with degeneration of cone photoreceptors.

[0068] The inventors of this disclosure have surprisingly discovered that cone photoreceptor degeneration is caused by the activation of specific cell death pathways in these cells, particularly ferroptosis.

[0069] The inventors of this disclosure have demonstrated that ferroptosis inducers such as glutamate, ferroptosis inducers, imidazole ketone erastin (IKE), and RSL3 induce a specific decrease in cone photoreceptor activity. This loss of cone photoreceptor activity is accompanied by decreased glutathione (GSH) levels, increased cell membrane permeability to dyes, increased lipid peroxidation, and dysregulation of iron metabolism. This effect was observed specifically not only in pure cone photoreceptors in cultures but also evident in an in vitro model of the retina, where additional microglia migrated in the outer nuclear layer (ONL), just as they do in vivo. The importance of this in vivo pathological process is further highlighted by the following finding: administration of ferroptosis inducers to the eyes of non-human primates not only leads to specific cone loss but also to the formation of subretinal deposits and a decrease in electroretinogram amplitude, features highly similar to those exhibited in patients with AMD. Importantly, administration of such ferroptosis inducers to the eye does not affect the activity of rod photoreceptors or retinal pigment epithelial cells.

[0070] Therefore, ferroptosis induced in cones triggers specific cone photoreceptor degeneration via lipid peroxidation, resulting in a phenotype highly similar to the in vivo pathological condition. Notably, cone photoreceptor degeneration can be rescued by antiferroptosis therapy. In particular, lipoxygenase inhibitors, especially zileutone (a 5-LOX inhibitor) and ferrostatin-1, which inhibit lipid peroxidation in cone photoreceptors, specifically reverse the effects induced by ferroptosis.

[0071] This indicates that specific inhibition of lipid peroxidation in cone photoreceptors, for example by inhibiting lipoxygenase activity, can block cell death observed under the conditions disclosed herein.

[0072] This is further confirmed by the inventors of this disclosure, namely that administration of ferroptosis inhibitors, particularly ferroptosis inhibitors of lipid peroxidation, specifically reversed cone degeneration in rd1 mice (a known model of retinal degeneration) without rescuing rods in these mice. Therefore, these results surprisingly confirm that the treatment of the condition associated with cone photoreceptor degeneration disclosed herein is attributable to a specific and direct inhibition of lipid peroxidation in cone photoreceptors, without involving or affecting rod photoreceptors or retinal pigment epithelial cells.

[0073] In a first aspect, this disclosure relates to compounds for use in treating conditions associated with cone photoreceptor degeneration, wherein said treatment includes inhibiting lipid peroxidation in cone photoreceptors, and more particularly, wherein said treatment includes specifically inhibiting lipid peroxidation in cone photoreceptors, i.e., said treatment substantially does not inhibit lipid peroxidation in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells.

[0074] This disclosure also relates to the use of the compounds in the preparation of medicaments for treating conditions associated with cone photoreceptor degeneration, wherein said treatment includes inhibiting lipid peroxidation in cone photoreceptors, and more particularly, wherein said treatment includes specifically inhibiting lipid peroxidation in cone photoreceptors, i.e., said treatment substantially does not inhibit lipid peroxidation in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells.

[0075] This disclosure also relates to a method of treating a condition associated with degeneration of cone photoreceptors, the method comprising administering the compound to a subject in need to inhibit lipid peroxidation in cone photoreceptors, particularly specifically inhibiting lipid peroxidation in cone photoreceptors, i.e., the treatment substantially does not inhibit lipid peroxidation in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells.

[0076] Ferroptosis is a regulatory cell death pathway that is iron-dependent and lipid peroxidation-dependent. Decreased glutathione (GSH) levels can trigger ferroptosis. Once GSH levels decrease significantly, reactive oxygen species (ROS) levels begin to increase exponentially. Polyunsaturated fatty acids (PUFAs), particularly arachidonic acid (AA), are sensitive to lipid peroxidation and are essential for ferroptosis. Acyl-CoA synthetase long chain family member 4 (ACSL4), voltage-dependent anion channels (VDACs), and the enzyme phospholipase A2 are involved in the generation and regulation of ROS in cells. Furthermore, ACSL4, along with lysophosphatidylcholine acyltransferase 3 (LPCAT3), participates in the synthesis of endogenous PUFAs. Lipooxygenases, which catalyze the formation of lipid peroxides from PUFAs, represent other key players in this pathway.

[0077] Therefore, the inhibition of lipid peroxidation in the methods described herein preferably includes inhibiting the activity of one or more of ACSL4, VDAC, phospholipase A2, and / or lipoxygenase in the cone photoreceptors. Specifically, the inhibition of lipid peroxidation in the methods described herein preferably includes specifically inhibiting the activity of one or more of ACSL4, VDAC, phospholipase A2, and / or lipoxygenase in the cone photoreceptors, i.e., in other retinal cell types (such as rod photoreceptors or retinal pigment epithelial cells), the activity of one or more of ACSL4, VDAC, phospholipase A2, and / or lipoxygenase is substantially unaffected by the treatment. More preferably, in the methods disclosed herein, the activity of one or more lipoxygenases is reduced or inhibited, thereby resulting in the inhibition of lipid peroxidation in the cone photoreceptors, particularly specifically. Even more preferably, in the cone photoreceptors, the activity of 5-lipoxygenase (5-LOX) and / or 5-lipoxygenase activator protein (FLAP) is reduced or inhibited, particularly specifically.

[0078] According to this disclosure, 5-LOX is an enzyme that catalyzes the oxidation of arachidonic acid (AA) to 5(S)-peroxyhydro-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) and further dehydration to allyl epoxide leukotrienes A4. FLAP is a protein required for the biosynthesis of leukotrienes via 5-LOX. FLAP binds to arachidonic acid and may play an important role in the transfer of arachidonic acid to 5-LOX.

[0079] The treatment according to this disclosure is particularly advantageous because it specifically inhibits the death of cone photoreceptors. Specifically, the treatment maintains the vitality of cone photoreceptors. More specifically, the treatment specifically maintains the vitality of cone photoreceptors, meaning that the vitality of other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, is substantially unaffected by the treatment. Alternatively, the treatment leads to an increase in the vitality of cone photoreceptor cells. More specifically, the treatment leads to a specific increase in the vitality of cone photoreceptor cells, meaning that the treatment substantially does not increase the vitality of other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells. The vitality of cone photoreceptor cells can be measured by any method known to those skilled in the art. For example, the vitality of these cells can be determined in vitro using, for example, calcein AM, MTT, alamarBlue, ethidium, or CellTiterGlo. Alternatively, cone vitality can be determined in an ex vivo model of the retina by, for example, measuring retinal thickness, measuring the number of cell nuclei in the ONL (e.g., after DAPI staining), measuring their permeability to the dye, and / or determining the number of cones after labeling retinal tissue with reagents specific to the outer segment of the cone or the cone itself (e.g., anti-FLAP antibody, anti-opsin antibody, peanut agglutinin (PNA) or anti-cone inhibitory protein antibody). In another instance, cone vitality can be determined in vivo by optical coherence tomography (OCT) or adaptive optics retinal imaging. For example, OCT is widely used clinically to characterize potential retinal degeneration in patients because it allows for defining the location and nature of changes in the retina and adjacent structures, and objectively assessing the thickness of the retina and surrounding structures. Therefore, a reduction or prevention of cone degeneration can be measured by OCT. All of these techniques are well known to those skilled in the art. Cone cell viability can also be demonstrated on histological samples by measuring retinal thickness, measuring the number of cell nuclei in the ONL (e.g., after DAPI staining), or determining the number of cones after labeling retinal tissue with a cone-specific reagent (e.g., anti-FLAP antibody, anti-opsin antibody, peanut agglutinin lectin, or anti-cone inhibitory protein antibody). It should be understood that those skilled in the art will be able to adapt each of these methods to the needs of the environment to effectively measure cone photoreceptor activity.

[0080] In another instance, the treatment disclosed herein is particularly advantageous because it blocks, in particular specifically, the formation of lipid peroxides, especially 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), in the cone photoreceptors. Specifically, by said treatment, the intracellular formation of lipid peroxides (especially 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)) is maintained, particularly specifically maintained. Alternatively, said treatment results in a reduction, particularly a specific reduction, of the intracellular formation of lipid peroxides (especially 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)) in the cone photoreceptors. Lipid peroxidation can be measured quantitatively or qualitatively by various methods. It can be measured by the loss of fatty acids; by the amount of primary peroxidation products; by the amount of secondary products such as carbonyl groups and hydrocarbon gases; and by a decrease in antioxidant activity. Many commercial assays for measuring lipid peroxides are available to those skilled in the art, including the assay used in the examples.

[0081] In another instance, the treatment disclosed herein is particularly advantageous because it inhibits microglial migration in the ONL. Specifically, the treatment results in reduced microglial cell migration. Alternatively, the treatment can prevent microglial migration. Microglial migration is well known to those skilled in the art and can be measured by any method known in the art (Rashid et al., Front Immunol. 10:1975, 2019).

[0082] In another instance, the treatment disclosed herein is particularly advantageous because it blocks the formation of subretinal deposits. Such deposits are observed in many conditions associated with cone degeneration, such as AMD. In particular, the treatment results in a reduction in the formation of subretinal deposits. Alternatively, the treatment prevents the formation of subretinal deposits. Subretinal deposits are well known to those skilled in the art and can be determined by any method known in the art (Monge et al., China Taiwan J Ophthalmol. 12(2):138-146, 2022).

[0083] In another instance, the treatment disclosed herein is particularly advantageous because its blocking, especially specific blocking, of the reduction in cone photoreceptor activity. Specifically, cone photoreceptor activity is maintained by the treatment, particularly specifically. Alternatively, the treatment results in an increase in cone photoreceptor activity, particularly a specific increase. Cone receptor activity can be measured by electroretinography (ERG) or multifocal electroretinography (mfERG); see, for example, Asanad S, Karanjia R. Multifocal Electroretinogram. [Updated 9 October 2022]. See: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; January 2023.

[0084] Preferably, the treatment disclosed herein includes obtaining at least two, at least three, or at least four of the above-described features. More preferably, the treatment disclosed herein includes obtaining all five of the above-described features.

[0085] The treatment of the present invention for conditions related to cone photoreceptor degeneration can be combined with one or more other treatments or used as an adjunct therapy.

[0086] The combination therapy provided herein involves administering at least two agents to a patient, wherein the first agent is a therapeutic compound disclosed herein, and the second agent is another therapeutic compound. Therefore, this disclosure relates to the therapeutic compound described herein for treating conditions associated with cone photoreceptor degeneration, wherein the therapeutic compound is administered together with the second therapeutic compound.

[0087] When neither the therapeutic compound nor the second therapeutic compound disclosed herein is administered at a level that is individually therapeutically effective, the combination therapy of this disclosure may result in a greater than additive effect or synergistic effect, thereby providing therapeutic benefit. Therefore, such agents can be administered at lower doses, thereby reducing the likelihood and / or severity of adverse reactions.

[0088] For example, the second therapeutic compound can be a second ferroptosis inhibitor. Specifically, administration of a second ferroptosis inhibitor can lead to a reduction or inhibition of lipid peroxidation in cone photoreceptors, particularly a specific reduction or inhibition, and / or a decrease in intracellular iron levels in cone photoreceptors, particularly a specific decrease. Suitable second ferroptosis inhibitors specifically include Prominin2; miR-522 and / or RP11-89 gene inhibitors; FTL; FTH1; and / or NCO4 RNA inhibitors; deferoxone; ferrostatin-1; deferoxamine (DFO); raffinol acetate; histochrome; ciclopirox olamine (CPX); bafloxacin A1; quercetin; baicalein; dexrazoxane; 2,2'-bipyridine; 1,10-phenanthroline. Mangiferin; Astilbene; YL-939; and deferasirox (DFX); DKK1; OTUB1; Cystine; β-mercaptoethanol; Selenium; Bardosorone; Carvacrol (CAR); Rehmannia glutinosa; Bioflavonoids, including galangin; xanthohumol; naringenin; Inula japonica; entacapone; capsaicin ester, resveratrol, dexmedetomidine, irisin, 2-cyano-3,12-dioxoolean-1,9-diene-28-acid (CDDO) and kaempferol.

[0089] Alternatively, a second therapeutic compound may treat or alleviate another aspect of the condition described herein. In particular, the therapeutic compounds of this disclosure are advantageously administered in combination with a second therapeutic compound that prevents intraocular neovascularization. These compounds include, for example, aflibercept, ranibizumab, pegaptanib, bevacizumab, brolucizumab, faricimab, AKB-9778, nesvacumab, and BI 836880.

[0090] Therapeutic compounds and second therapeutic compounds may be administered simultaneously, sequentially, or separately.

[0091] Therapeutic compounds used to treat conditions related to cone degeneration.

[0092] This disclosure provides therapeutic compounds that are effective against conditions associated with cone photoreceptor degeneration. The compounds disclosed herein are particularly effective at inhibiting, especially specifically inhibiting, lipid peroxidation in cone photoreceptors. In fact, this disclosure demonstrates that ferroptosis induced in cones triggers cone cell photoreceptor degeneration via lipid peroxidation, ultimately leading to a phenotype similar to the in vivo pathological condition. Furthermore, this phenotype can be reversed by inhibitors of lipoxygenases such as zileutone.

[0093] Therefore, the therapeutic compound used in the methods disclosed herein is preferably a ferroptosis inhibitor.

[0094] The therapeutic compounds disclosed herein include, in particular, chemical and biological agents (e.g., pharmaceuticals, including small molecule drugs or biological products such as antibodies or cells) that can be used to treat conditions associated with degeneration of the cone photoreceptor, wherein administration of the compound results in inhibition of lipid peroxidation.

[0095] One example of a biopharmaceutical is the product of the SCD1 gene. Stearoyl-CoA desaturase 1 (SCD1) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids (MUFA). Overexpression of SCD1 is known to protect cells from ferroptosis (Tesfay et al., Cancer Res. 79(20):5355-5366, 2019). SCD1 (Genbank ID No.: NM_005063) encodes a 359-amino acid protein (Genbank ID No.: NP_005054). Other examples of biopharmaceuticals used to inhibit any gene involved in lipid peroxidation and known to promote ferroptosis include RNAs, including antisense RNA, siRNA, shRNA, etc. This type of gene specifically includes ACSL4 (Genbank ID No.:NM_004458), LPCAT3 (Genbank ID No.:NM_005768), PLA2 (Huang et al., Int J Mol Med. 2015 35(3):587-596), and the genes ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5, and ALOX5AP (Genbank ID No.:NM_004458), which encode lipoxygenases 15-LOX, 15-LOX-2, 12-LOX, 12R-LOX, eLOX-3, 5-LOX, and FLAP, respectively. The numbers are: NM_001140, NM_001141, NM_000697, NM_001139, NM_021628, NM_000698.5, and NM_001204406. Preferably, the polynucleotides used herein are inserted into a vector, such as a viral vector, like an adeno-associated virus (AAV) vector, including AAV8, AAV2, AAV2tYF, and AAV5. Preferably, the polynucleotide is operatively linked to a regulatory sequence. The regulatory sequence is advantageously adapted for expression in the retina or subretinal tissue. More preferably, the polynucleotides disclosed herein are operatively linked to a suitable regulatory sequence in the vector.

[0096] Delivery can be accomplished via gene therapy, for example, by administering a carrier of the therapeutic product disclosed herein into the suprachoroidal space, subretinal space (with or without vitrectomy, e.g., via a catheter through the suprachoroidal space or via peripheral injection), intraretinal space, and / or outer surface of the sclera (i.e., parascleral administration) of the eyeball of a human patient to create a permanent reservoir in the eye that continuously supplies the therapeutic product (e.g., a post-translational modified therapeutic product).

[0097] Alternatively, the compounds disclosed herein are small molecules. Examples of small molecules that inhibit lipid peroxidation include, in particular, ziruton, ferrostatin-1, liproxstatin-1, PDA NP, 16-86, XJB-5-131, UAMC-3203, SRS15-72B, SRS15-72A, SRS16-80, SRS16-86, GSK2190915, MK-886, ML355, BLX-3887, VKH2, BAY x 1005, α-tocopherol, tometetin, caffeic acid, diallyl trisulfide, treglitazone (succinate), tannic acid, norbergenin, chlorogenic acid, 3'-hydroxypuerarin, 1,3-dicaffeoylquinic acid, rosmarinic acid, 1-O-hexyl-2,3,5-trimethylhydroquinone, and tenoridin hydrochloride.

[0098] Preferably, the compounds disclosed herein are selected from the group consisting of: SCD1; RNA that inhibits any one of PLA2, ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5 and ALOX5AP; ziluton; ferrostatin-1; liproxstatin-1; PDA NP; 16-86; XJB-5-131; UAMC-3203; SRS15-72B; SRS15-72A; SRS16-80; SRS16-86; GSK2190915; MK-886; ML355; BLX-3887; VKH2; BAY x 1005; α-Tocopherol; Tometrine; Caffeic acid; Diallyl trisulfide; Treglitazone (succinate); Tannic acid; Glechoderma lucidum; Chlorogenic acid; 3'-Hydroxypusrin; 1,3-Dicaffeoylquinic acid; Rosmarinic acid; 1-O-Hexyl-2,3,5-Trimethylhydroquinone and Tenoridine hydrochloride. More preferably, the compounds disclosed herein are selected from the group consisting of: SCD1; RNA that inhibits any one of PLA2, ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5 and ALOX5AP; ziluton; ferrostatin-1; liproxstatin-1; PDA NP; 16-86; XJB-5-131; UAMC-3203; SRS15-72B; SRS15-72A; SRS16-80; SRS16-86; GSK2190915; MK-886; ML355; BLX-3887; VKH2; BAY x 1005; α-Tocopherol; Tometrine; Caffeic acid; Diallyl trisulfide; Treglitazone (succinate); Tannic acid; Norbergenin; Chlorogenic acid; 3'-Hydroxypusrin; 1,3-Dicaffeoylquinic acid and Rosmarinic acid.

[0099] Diseases related to cone photoreceptor degeneration

[0100] This disclosure provides a method for treating conditions associated with cone photoreceptor degeneration. As disclosed herein, conditions associated with cone photoreceptor degeneration are those in which specific cone degeneration is observed, i.e., conditions in which the cone photoreceptor degeneration is not a secondary consequence of retinal pigment epithelium degeneration.

[0101] In some instances, the cone photoreceptor may be the only cell affected by degeneration. In some instances, other cell types may also undergo degeneration. For example, in some instances, conditions associated with cone photoreceptor degeneration may also present with degeneration of the adjacent supporting tissue (retinal pigment epithelium). It must be understood that regardless of when the retinal pigment epithelium also undergoes degeneration, the cone photoreceptor remains specifically affected by degeneration; that is, cone photoreceptor degeneration is not a secondary consequence of retinal pigment epithelium degeneration. In a preferred embodiment, the other cell types affected by degeneration in conditions associated with cone photoreceptor degeneration described herein do not solely include adjacent supporting tissue, such as the retinal pigment epithelium. It must be understood that regardless of when the retinal pigment epithelium also undergoes degeneration, the cone photoreceptor remains specifically affected by degeneration; that is, cone photoreceptor degeneration is not a secondary consequence of retinal pigment epithelium degeneration. In a preferred embodiment, other cell types affected by degeneration in conditions associated with cone photoreceptor degeneration described herein include not only adjacent supporting tissues (e.g., retinal pigment epithelium) but also retinal neurons (including, for example, rod photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells). In this very specific embodiment, the cone photoreceptor remains specifically affected by degeneration regardless of whether any of these other cell types also undergo degeneration at any given time; that is, cone photoreceptor degeneration is not a secondary consequence of degeneration of one or more other cell types.

[0102] Specifically, conditions associated with degeneration of cone photoreceptors include age-related macular degeneration (AMD), cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt's disease), central serous chorioretinopathy, Best's disease and bestrophinopathies, retinal detachment (including rhegmatogenous, serous, and tractional causes), solar retinopathy, laser-induced retinopathy, achromatopsia, Usher syndrome, Leber congenital amaurosis, Alström syndrome, Refsum disease, etc.

[0103] Preferably, the conditions associated with cone photoreceptor degeneration in the methods disclosed herein are selected from the group consisting of: age-related macular degeneration (AMD), cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt's disease), central serous chorioretinopathy, Best's disease and bestrophinopathies, retinal detachment (including rhegmatogenous, serous, and tractional causes), solar retinopathy, laser-induced retinopathy, achromatopsia, Usher syndrome, Leber congenital amaurosis, Alström syndrome, and Refsum disease. More preferably, the condition associated with cone photoreceptor degeneration in the methods disclosed herein is age-related macular degeneration (AMD). In another more preferred example, the condition associated with cone photoreceptor degeneration in the methods disclosed herein is cone dystrophy. In yet another more preferred example, the condition associated with cone photoreceptor degeneration in the methods disclosed herein is retinal detachment.

[0104] Conditions related to cone photoreceptors can be diagnosed using any technique or method known to those skilled in the art.

[0105] Standardized ophthalmic examination techniques known in the art include, for example, detailed slit-lamp biomicroscopy, which allows assessment of the eyelids, ocular appendages, eyelashes, corneal surface, anterior chamber, pupil, lens, vitreous cavity, and central retinal anatomy including the optic nerve and macula. Another approach is gonioscopy, which allows for detailed examination of the anterior chamber angle. Indirect ophthalmoscopy allows for assessment of the peripheral retina, which is important in monitoring vitreous and peripheral retinal disorders.

[0106] Functional tests of visual acuity are known in the art and include, for example, best-corrected visual acuity, contrast visual acuity and low-light visual acuity, color vision (including the Ishihara and Farnsworth Munsell tests) and visual field assessments (including the Flumphrey automated visual field test and micro-visual field test), tear production (the Schirmer test), and intraocular pressure (IOP) measurements. These are used in conjunction with structural tests, including, for example, anterior and posterior segment photography, corneal thickness measurement, 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 is used to assess the ocular, periocular, and orbital structures and the intracranial portions of the optic nerve, visual pathways, and visual cortex in the brain. These tests visualize the structural integrity and thickness of the ocular and surrounding structural layers and assess blood flow and circulation. Advanced functional tests of the retina, optic nerve, and visual pathway / cortex, including electrophysiological tests such as full-field and multifocal electroretinography, visual evoked potentials, and micro-field examinations, were also used to diagnose and monitor disease progression and the effects of treatments. Those skilled in the art will be able to deploy appropriate methods known in the art to diagnose, measure, and monitor the conditions described herein related to cone photoreceptor degeneration.

[0107] Pharmaceutical Composition

[0108] The compounds of the present invention for treating conditions associated with cone photoreceptor degeneration can be formulated in compositions. Optionally, the composition may contain one or more additional therapeutic agents, such as the second therapeutic agent described below. The compositions are generally provided as part of a sterile pharmaceutical composition that typically includes a pharmaceutically acceptable carrier and / or excipient. In another aspect, this disclosure therefore provides pharmaceutical compositions comprising compounds for treating conditions associated with cone photoreceptor degeneration and pharmaceutically acceptable carriers and / or excipients.

[0109] Therefore, this document provides pharmaceutical compositions comprising one or more compounds for treating conditions associated with cone photoreceptor degeneration, and one or more pharmaceutically acceptable carriers and optional other therapeutic and / or prophylactic ingredients. A carrier (or excipient) is acceptable or suitable if it is compatible with the other components of the composition and is harmless to the recipient of the composition (i.e., the subject). Pharmaceutically acceptable or suitable compositions include ophthalmologically suitable or acceptable compositions.

[0110] The composition can be in any suitable form (depending on the method of administration to the patient). The compositions used in the methods described herein can be applied, for example, intravitreal (e.g., in the subretinal space, suprachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or on the corneal surface), via eye drops, intramuscular, intravenous, intradermal, percutaneous, intraarterial, intraperitoneal, intralesional, intra-articular, intraprostatic, intrapleural, intratracheal, intrasheath, intranasal, intravaginal, intrarectal, surface, intratumoral, intraperitoneal, subcutaneous, subconjunctival, intracystic, mucosal, intraperitoneal, intraumbilical, intraocular, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraoral, intraperitoneal, intradermal, via inhalation, via injection, via implantation, via infusion, via continuous infusion, via direct local perfusion of target cells, via catheter, via irrigation, in cream form, or as a lipid composition. The compositions used in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., depending on the compound or composition administered and the severity of the condition, disease, or disorder being treated). In any given situation, the most suitable route of administration will depend on the specific compound, the subject, the nature and severity of the disease, and the subject's physical condition. Therefore, various delivery systems can be used to administer the compounds disclosed herein. Preferably, the compositions of this disclosure are administered in the subretinal space, suprachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or on the corneal surface. In some preferred embodiments of the invention, the compounds are formulated as an aqueous solution and administered via intravenous infusion. In some embodiments, administration is performed via intravenous infusion over one hour.

[0111] The compositions described herein can generally be formulated as liquid or fluid compositions, semi-solids (e.g., gels or hydrogels), foams, or porous solids (e.g., polymer matrices, complexes, calcium phosphate derivatives, etc., as long as they are suitable for ophthalmic tissue engineering). The compositions used in the methods described herein can also be administered systemically or topically. The method of administration can vary depending on various factors (e.g., depending on the compound or composition administered and the severity of the condition, disease, or disorder being treated). In any given situation, the most appropriate route of administration will depend on the specific therapeutic compound, the subject, the nature and severity of the disease, and the subject's physical condition. Compounds used to treat conditions associated with cone photoreceptor degeneration can be administered systemically (inhaler, intravenously, intramuscularly, or intraperitoneally) or in solutions, gels, or implants. Specifically, dosage forms may include tablets, capsules, intravenous injections, intramuscular injections, local injections, topical creams, gels and ointments, eye drops, ophthalmic solutions, ophthalmic suspensions, ophthalmic emulsions, intravitreal injections, subtenon injections, bioerodible ophthalmic implants and non-bioerodible ophthalmic inserts or reservoirs, nasal sprays and ointments, and various rectal or vaginal preparations.

[0112] The pharmaceutical composition can be conveniently present in unit dose form, with each dose containing a predetermined amount of the therapeutic compound disclosed herein. Such a unit may contain, for example, but not limited to, 5 mg to 5 g, such as 10 mg to 1 g, or 20 mg to 50 mg. The pharmaceutically acceptable carrier used in this disclosure may take various forms, depending on, for example, the condition to be treated or the route of administration.

[0113] The pharmaceutical compositions disclosed herein can be prepared by mixing an antibody of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”) commonly used in the art, for storage in lyophilized or aqueous forms, i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (edited by Osol, 1980). Such additives must be non-toxic to the recipient at the dosage and concentration used.

[0114] Buffers help maintain pH within a range close to physiological conditions. They can be present at concentrations from about 2 mM to about 50 mM. Buffers suitable for use in this disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., mixtures of monosodium citrate and disodium citrate, mixtures of citrate and trisodium citrate, mixtures of citrate and monosodium citrate, etc.), succinate buffers (e.g., mixtures of succinate and monosodium succinate, mixtures of succinate and sodium hydroxide, mixtures of succinate and disodium succinate, etc.), tartrate buffers (e.g., mixtures of tartaric acid and sodium tartrate, mixtures of tartaric acid and potassium tartrate, mixtures of tartaric acid and sodium hydroxide, etc.), and fumarate buffers (e.g., mixtures of fumaric acid and monosodium fumarate). This includes buffers such as fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Alternatively, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0115] Preservatives may be added to delay microbial growth, and may be added in amounts of 0.2%-1% (w / v). Preservatives suitable for use in this disclosure include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride halides (e.g., chlorides, bromides, and iodides), hexamethyl chloride, and alkyl esters of parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes referred to as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this disclosure, and include polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol. Stabilizers are a broad class of excipients whose functions range from fillers to additives used to solubilize therapeutic agents or help prevent their denaturation or adhesion to container walls. Typical stabilizers can be polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; amino acid polymers; and those containing... Sulfur reducing agents, such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 residues or fewer); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides, such as lactose, maltose, and sucrose; and trisaccharides, such as raffinose; and polysaccharides, such as dextran. Stabilizers may be present in the range of 0.1 to 10,000 parts by weight of active protein.

[0116] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve therapeutic compounds and protect them from agitation-induced aggregation. This also allows formulations to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamer (184, 188, etc.), pranic polyols, and polyoxyethylene sorbitol monoethers (TWEEN®-20, TWEEN®-80, etc.). Nonionic surfactants can be present in the range of about 0.05 mg / ml to about 1.0 mg / ml, for example, about 0.07 mg / ml to about 0.2 mg / ml.

[0117] Other excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0118] This disclosure also relates to pharmaceutical compositions comprising at least:

[0119] i) Therapeutic compounds as described herein, namely compounds for treating conditions associated with cone photoreceptor degeneration, and

[0120] ii) A second therapeutic agent, such as those described below,

[0121] As a combination product used simultaneously, separately, or sequentially.

[0122] Combinations of therapeutic compounds and second therapeutic agents can be administered alone, as a mixture of one or more therapeutic compounds and / or one or more second therapeutic agents, or in combination with other agents that can be used to treat conditions associated with cone photoreceptor degeneration. Examples of suitable combinations are provided below.

[0123] This disclosure covers pharmaceutical kits containing the therapeutic compounds described herein. A pharmaceutical kit is a package containing a compound (e.g., in lyophilized form or as an aqueous solution) for treating conditions associated with cone photoreceptor degeneration and one or more of the following:

[0124] • A second therapeutic agent, such as those described below;

[0125] • Devices for administering immune checkpoint inhibitors, such as pens, needles, and / or syringes; and

[0126] • If the inhibitor is in lyophilized form, use pharmaceutical-grade water or buffer to resuspend the inhibitor.

[0127] Each unit dose of the therapeutic compound may be packaged separately, and the kit may contain one or more unit doses (e.g., two unit doses, three unit doses, four unit doses, five unit doses, eight unit doses, ten unit doses, or more). In some embodiments, one or more unit doses are each contained in a syringe or pen.

[0128] Effective dose

[0129] Therapeutic compounds, as described herein, which are compounds used to treat conditions associated with cone photoreceptor degeneration, are typically used in amounts that effectively achieve the desired outcome, such as amounts that effectively prevent or reduce cone photoreceptor degeneration by any of the methods described above. Pharmaceutical compositions containing therapeutic compounds may be administered to such patients (e.g., human subjects) at therapeutically effective doses.

[0130] The term "therapeuticly effective dose" refers to the amount of an active compound or conjugate that elicits a desired biological response in a subject. Such responses include relief of symptoms of the treated disease or disorder, prevention, suppression, or delay of the recurrence of symptoms of the disease or the disease itself, or prevention, suppression, or delay of the progression of symptoms of the disease or the disease itself. More specifically, as used herein, a "therapeuticly effective" dose is the amount that imparts a therapeutic benefit. A therapeutically effective dose is also the dose in which the therapeutically beneficial effect of a pharmaceutical agent outweighs any of its toxic or harmful effects.

[0131] In the context of this disclosure, a therapeutically effective amount refers to an amount of the therapeutic compound disclosed herein that is sufficient to prevent or reduce degeneration of the cone photoreceptor.

[0132] Preferably, the therapeutically effective amount is an amount sufficient to obtain a therapeutic compound of at least one of the following:

[0133] • Maintain or increase the activity of cone photoreceptors;

[0134] • To prevent increased permeability of the cell membrane to dyes in the cone photoreceptors;

[0135] • Maintain or reduce the intracellular formation of lipid peroxides in at least one cone photoreceptor, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE).

[0136] • Prevent or reduce the migration of small gelatinous substances;

[0137] • To prevent or reduce the formation of subretinal deposits; and

[0138] • Maintain or increase the activity of cone photoreceptors.

[0139] Preferably, the therapeutically effective amount disclosed herein is sufficient to obtain at least two, at least three, at least four, at least five, or at least six of the above-described features. More preferably, the therapeutically effective amount disclosed herein is sufficient to obtain all of the above-described features.

[0140] The effective dose can initially be estimated from in vitro assays. For example, the initial dose can be formulated to a concentration of the therapeutic compound disclosed herein that is capable of inhibiting cone photoreceptor degeneration in at least in vitro or ex vivo assays (such as those described in the embodiments of this disclosure). Calculating the dose to achieve such a concentration in the eye, taking into account the bioavailability of a particular compound, is entirely within the capabilities of someone skilled in the art. For reference, the reader is advised to refer to Goodman and Gilman's *The Pharmaceutical Basis of Therapeutics*, Chapter 1, latest edition, Fingl & Woodbury, “General Principles,” Pagamonon Press, and the references cited therein.

[0141] Determining the effective amount is entirely within the competence of those skilled in the art, particularly based on the detailed disclosure provided herein. The toxicity and therapeutic efficacy of the compound or conjugate can be determined in cell cultures and laboratory animals using standard pharmaceutical procedures. The effective amount of the therapeutic compound or second therapeutic compound of this disclosure administered to a subject will depend on the stage, class, and state of multiple myeloma, as well as the characteristics of the subject, such as general health status, age, sex, weight, and drug tolerance. The effective amount of the therapeutic compound or second therapeutic compound of this disclosure to be administered will also depend on the route of administration and dosage form. The amount and interval of dose adjustment can be individualized to provide an effective level of the active compound sufficient to maintain the desired therapeutic effect.

[0142] The amount of therapeutic compound administered will depend on a variety of factors, including the nature and stage of the condition being treated, the form, route, and site of administration, the treatment regimen (e.g., whether another therapeutic compound is used), the age and condition of the specific subject being treated, and the patient's sensitivity to the disclosed therapeutic compound. An appropriate dosage can be readily determined by those skilled in the art. Ultimately, the physician will determine the appropriate dosage to be used. This dosage may be repeated frequently as needed. If side effects occur, the amount and / or frequency of the dosage may be modified or reduced according to general clinical practice. Appropriate dosage and treatment regimens can be established by monitoring the progress of the therapy using conventional techniques known to those skilled in the art.

[0143] Effective doses of the therapeutic compounds described herein can be in the range of about 0.001 to about 75 mg / kg for a single, multiple, or continuous administration, or to achieve serum concentrations of 0.01-5000 μg / ml in a single, multiple, or continuous administration, or any effective range or value thereof, depending on the condition being treated, the route of administration, and the age, weight, and physical condition of the subject. In some instances, each dose can range from about 0.5 μg to about 50 μg / kg body weight, for example, from about 3 μg to about 30 μg / kg body weight.

[0144] The amount, frequency, and duration of application will depend on a variety of factors, such as the patient's age, weight, and disease condition.

[0145] In various instances, the treatment period is at least 1 day. Preferably, the treatment period is at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days. In other instances, it is at least 1 week. Preferably, the treatment period is at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, or at least 6 weeks. The treatment period can be as long as approximately 20 years. In embodiments, the treatment period includes 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. The treatment period can be approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 13 months, approximately 14 months, approximately 15 months, approximately 16 months, approximately 17 months, approximately 18 months, approximately 19 months, approximately 20 months, approximately 21 months, approximately 22 months, or approximately 24 months. In some cases, treatment can continue for a patient's entire life after a diagnosis of a condition related to cone photoreceptor degeneration.

[0146] Other features and advantages of this disclosure are presented in the continuation of the description and in the embodiments and accompanying drawings (legends of which are shown below).

[0147] Example

[0148] Example 1

[0149] method

[0150] Data availability

[0151] Animal model: rat

[0152] Eight-week-old Long Evans wild-type rats were obtained from Janvier Labs (ISO 9001 certified). On day 0 (D0), the control and treatment groups were administered 0.2% DMSO (D2650, Millipore Sigma, Burlington, MA) in PBS (14190-094 Life Technologies Europe BV, Bleiswijk, The Netherlands) or 0.2% 20 μM IKE in a DMSO solution diluted in PBS via subretinal injection.

[0153] Surgical and anatomical procedures for rats

[0154] Rats were anesthetized by intraperitoneal (IP) injection of 40 mg / kg ketamine (Ketamidor 100 mg / ml, Axience SAS, Pantin, France) and 0.14 mg / kg metomidine (Domitor 0.85 mg / ml, Vetoquinol SA, Paris, France). Local anesthesia was achieved using oxybuprocaine eye drops (1.6 mg / 0.4 ml, Théa, Clermont-Ferrand, France). Mydriasis of the left eye was achieved using 0.5% tropicamide eye drops (Mydriaticum, Théa, Clermont-Ferrand, France). Tear gel (Lubrithal, Dechron, Shrewsbury, UK) was used as the lens-ocular interface throughout the procedure. A conjunctival-scleral canal was created using an ophthalmic microscope (Lumera 700, Carl Zeiss, Oberkochen, Germany) with a 30 Gauge (G) needle (BD Microlance 3, Becton, Dickinson SA, Fraga, Spain). A flat contact lens (Coverslips, mini, 8mm, World Precision Instruments, Sarasota, FL) enabled visualization of the retinal plane. 9 μL of control or IKE solution was delivered subretinally using a 10 μL syringe (Hamilton, Reno, NV) with a 30G cannula fitted to a microsyringe (Micro 4, World Precision Instruments, Sarasota, FL) and a non-beveled metal tip. Postoperatively, chloramphenicol-retinol eye ointment (Ophtalon 10 mg / g, TVM, Lempdes, France) was applied, followed by an intraperitoneal injection of 1 mL of 5% glucose monohydrate (Osalia, Paris, France) and a subcutaneous injection of 0.9 mg / kg atemetazol (Antidorm 4.27 mg / ml, Axience SAS, Pantin, France). Vivisection studies, including optical coherence tomography (OCT, Bioptigen, Durham, NC) and fundus photography (Micron IV, Phoenix-Micron, Inc., Bend, OR), were performed on day 0 to ensure subretinal delivery and on the day of euthanasia to confirm retinal reattachment.Animals were sacrificed on day 6 (D6) for microglial migration using 1 ml / kg pentobarbital (Exagon, Axience SAS, Patin, France) and on day 30 (D30) for cone degeneration. Left eyes were collected and immediately immersed in 4% paraformaldehyde (PFA, J61899.AP ThermoFisher Scientific, Waltham, MA) for 2 hours, then stored in PBS at +4 °C. The eyeballs were dissected to obtain the intact retina, which was then immersed in 24-well plates (#351147 Corning Inc., Corning, NY) for immunohistochemistry. Additionally, labeled eyecup sections (using CAR and Iba1) were performed for another quantitative analysis. For this, eyes were collected from rats and the cornea was removed. The eyes were then immersed in 4% PFA for 1 hour, followed by sequential immersion in 10% to 30% sucrose baths. Finally, the lens is gently removed and the eye cup is placed in liquid nitrogen in a freezing medium. They are stored at -20 °C until they are cut into 12 µm sections using a cryostat (CM3050 S, Leica, Wetzlar, Germany).

[0155] mammalian eyes

[0156] Pig eyes were obtained from a local slaughterhouse in accordance with an agreement between the local regulatory authorities and the French Ministry of Agriculture Veterinarian (Agreement FR75105131).

[0157] Pure cone photoreceptors

[0158] Pig eyes were dissected to obtain the retina, which was then cut into small pieces and digested at 37 °C for 20 minutes with 4 U / mL papain (LSO 3124, Worthington) and L-cysteine ​​(5.5 mM, Sigma-Aldrich). Enzyme activity was terminated by Neurobasal-A (NBA) medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) and 15 µg / mL DNase I (D4263, Millipore Sigma, Burlington, MA). A series of short centrifugations (30 seconds, 5 to 6 times) were performed to collect the supernatant, discarding the first supernatant, which mainly contained rod photoreceptors. Between centrifugations, the pellet was resuspended in P1000 to dissociate the remaining cells. The cell suspension was then centrifuged at 800 rpm for 10 minutes, and the pellet was resuspended in NBA medium containing 1% L-glutamine (G3126, MilliporeSigma, Burlington, MA) and 1% B27 supplement (17504044, ThermoFisher Scientific, Waltham, MA, USA).

[0159] As previously described (Balse et al., (2005) IOVS 46:3672), cone PR was purified by lectin panning, with slight modifications to obtain millions of cones in suspension. Cells were then seeded in 384-well plates (781091, Greiner, Ulis, France) using a robotic system (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) to obtain 3000 cells / well, and incubated at 37 °C and 5% CO2 for 3 days.

[0160] retinal explants

[0161] Retinas from pigs and non-human primates were cultured as previously described (Greferath et al., (2016) Ophthalmology 123:1320). After isolating the retina in a CO2-independent medium, fragments from the region behind the optic nerve were obtained using a 2 mm biopsy puncturist and placed on a polycarbonate membrane (140652, Thermo, Waltham, Massachusetts), with the photoreceptors flipped upwards. The explants were cultured in a CO2 incubator at 37 °C for 3 days.

[0162] deal with

[0163] To induce ferroptosis, the following components were used: glutamate (49621, Sigma Aldrich, Missouri, USA) at in vitro levels ranging from 1 µM to 500 µM; imidazolidinone ferroptosis inducer (IKE) (HY-114481, MedChemExpress, Monmouth, USA) at 3 µM for purified cone photoreceptors and 20 µM for in vitro and in vivo experiments; and RSL3 (HY-114481, MedChemExpress, Monmouth, USA) at 20 nM, 200 nM, and 10 µM for cellular, in vitro, and in vivo experiments, respectively. To inhibit ferroptosis, ferrostatin-1 (FST-1, HY-100579, MedChemExpress, Monmouth, USA) was used at 50 nM for purified cones or 400 nM for retinal explants. In addition, deferiphenone (HY-B0568, MedChemExpress, Monmouth, USA) was highly effective against pure cones at 50 µM, and ziluton (HY-14164, MedChemExpress, Monmouth, USA) was highly effective against purified cones at 20 µM.

[0164] Cell viability measurement

[0165] After 3 days of incubation, cell viability was measured using calcein (C1430, Thermo, Waltham, Massachusetts) at 1 / 4000 concentration at 37 °C for 1 hour. The number of viable cells was counted using an automated fluorescence microscope, ArrayScan (Cellomics ArrayScan VTI HCS reader, Thermo, Waltham, MA).

[0166] Screening of primary cells

[0167] Purified cone photoreceptors were seeded into 384-well plates with transparent flat bottoms (781091, Greiner, Ulis, France) using a robot with a 384-well head (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) until they reached a depth of 4*10. 3Cells / well. In the first two columns, cells were treated with DMSO only (D2650, MilliporeSigma, Burlington, MA), while in the remaining plates, cells were treated with either RSL3 (HY-114481, MedChemExpress, Monmouth, USA) at a final concentration of 20 nM or IKE (HY-114481, MedChemExpress, Monmouth, USA) at a final concentration of 3 µM. Seventeen hours after inducing cell denaturation, a ferroptosis library compound (HY-L051, MedChemExpress, Monmouth, USA) was added to the cells using a robotic system with a 384-well head (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) to obtain a final concentration of 10 μM. Each treatment was diluted in neurobasal medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) and 1 / 100 L-glutamine (G3126, Millipore Sigma, Burlington, MA). Cells were then incubated at 37 °C and 5% CO2 for 2 days. Cell viability was measured on day 3 post-inoculation.

[0168] Results were analyzed using TIBCO Spotfire® software (California, USA). Robust Z' factors were calculated for each plate, and those higher than 0.45 were selected. Then, for each plate, the percentage of viable cells was calculated relative to the positive control. The mean cell count was calculated and reported as 100%. The ratio of the positive control to all molecules in the plate was reported. Next, all plates from the same treatment (IKE or RSL3) are shown to select compounds with cell viability higher than 50%, and these compounds were extracted in Excel.

[0169] Lipidomics analysis

[0170] Purified cone photoreceptors, whether treated with 3 μM IKE or not, were washed three times with PBS, scraped into 100 μL, and immediately flash-frozen in liquid nitrogen and stored at -80 °C until extraction. Echienoic acid extraction and analysis were performed at the core of the lipidomics facility: MetaToul-Lipidomique (I2MC, Inserm, Toulouse, France), MetaboHUB-ANR-1 1—INBS-0010.

[0171] Western blot

[0172] Cells were collected, separated from the culture medium by centrifugation, and whole-cell proteins were extracted in lysis buffer (10 mM HEPES [pH 7], 100 mM NaCl, 2 mM EDTA, 0.5% NP-40, and a mixture of protease inhibitors) (Millipore Sigma, Burlington, MA). After centrifugation at 13,000 rpm, the supernatant was retained for further Western blotting analysis. The samples were then loaded into 4%–15% Mini-PROTEAN TGX Tris-glycine buffered SDS-PAGE and transferred to a 0.2 μm Trans-Blot Turbo nitrocellulose membrane (Bio-Rad, California, USA). The membrane was blocked at room temperature for 1 hour in 1x Tris-buffered saline (TBS, 10 mM Tris-HCl [pH 8], 150 mM NaCl) supplemented with 5% (w / v) dried skim milk powder. The membrane was incubated overnight with primary antibody at ℃. Western blotting with anti-5LOX (1:200; Abcam AB169755) and anti-FLAP (1:500; Abcam AB85227) confirmed the presence of 5LOX and FLAP in the total lysate at the expected molecular weight. A second HRP-conjugated antibody, goat anti-rabbit antibody (1:20,000; 111-035-003, Jackson ImmunoResearch Laboratories, Pennsylvania), was used to detect 5LOX and FLAP. The membrane was thoroughly washed with TBS (TBS-T) containing 2.5% Tween-20 between and after antibody incubation. Western blotting was visualized using enhanced chemiluminescence (ECL Prime detection reagent, Amersham, UK).

[0173] NADPH Measurement

[0174] According to the manufacturer's instructions, use the NADP / NADPH assay kit (ab176724, Abcam, Cambridge, UK) to quantify the levels of NADP and NADPH, respectively.

[0175] Glutathione (GSH) assay

[0176] GSH levels were quantified according to the manufacturer's instructions for the GSH assay kit (V6912, Promega, Madison, WI).

[0177] Cryostat section

[0178] After 3 days of incubation, retinal explants were fixed for 1 hour at room temperature with 4% paraformaldehyde (15714, Electron Microscopy Sciences, Hatfield, Pennsylvania) and washed three times with PBS. The retinal explants were then stored at +4 °C or as transverse sections. Finally, they were cryopreserved in continuous 10%, 20%, and 30% sucrose baths. The samples were then frozen in tissue cryopreservation medium (72592, Electron Microscopy Sciences, Hatfield, Pennsylvania) by immersion in liquid nitrogen until the medium hardened. The samples were stored at -20 °C until they were cut into 10 µm transverse sections using a cryostat (CM3050 S, Leica, Wetzlar, Germany). The transverse sections of the retina were stored at -20 °C.

[0179] Immunomarkers

[0180] Samples (fixed retinal explants and retinal cross sections) were first permeabilized with 0.5% Triton 100X (T8787, Sigma-Aldrich, Missouri, USA) diluted in 1X PBS. Then, nonspecific regions were blocked with a saturated buffer consisting of 10% normal donkey serum (S30-100ML, Millipore Sigma, Burlington, MA) diluted in 1X PBS. The primary and secondary antibodies used in this study are listed in Table 1.

[0181] Table 1. Antibody List.

[0182]

[0183] PNA lectin diluted 1 / 50 (L21409, Thermo, Waltham, Massachusetts) and rabbit FLAP antibody at a working concentration of 5 µg / mL (ab85227, Abcam, Cambridge, UK) were targeted at antigen sites on the extracellular side of the cell membrane. For those labeled, no permeabilization step was performed.

[0184] Finally, the retinal explants were washed three times in PBS and mounted in Cellvis plates (P12-1.5HN, IBL, Gerasdorf, Austria) with Permafluor (TA-030-FM, Thermo, Waltham, Massachusetts) for fluorescence microscopy. Transverse sections and full-thickness retinal slices were also mounted with Permafluor on coverslips or in 6-well plates (P06-1.5HN, Cellvis, Mountain View, CA).

[0185] Confocal microscopy

[0186] Retinal imaging was performed using a laser scanning confocal microscope (Fluoview V-1000, Olympus, Tokyo, Japan). Cone counting (outer segments and perinuclear bodies per millimeter) was performed manually on retinal sections in rat retina.

[0187] CQ1 imaging

[0188] Full-thickness spread retina of rats and porcine explants were imaged using a CQ1 confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan). For injected rat retinas, the visual fields of the infranasal and infratemporal flaps were manually examined in four z-axis planes defined by cone markers: the outer segment (OS) plane, the cone nucleus (CN) plane, the inner fiber (IF) plane, and the cone endfoot (CP) plane. Microglial migration was a key focus. Representative samples were extracted from the CQ1 software. Z-stacked images were analyzed using Cell pathfinder software (Yokogawa, Tokyo, Japan), which is capable of 2D, 3D, or slice quantification. Peribody or outer segment cone nuclei and microglia were quantified using z-axis generation algorithms (one for porcine explants and one for rat full-thickness spread retina). The extracted quantitative data were analyzed using a TIBCO Spotfire® (California, USA).

[0189] Non-human primates (NHP)

[0190] Two NHPs were used in this study: a 5-year-old male and a 15-year-old female. The animals were born in captivity and from AAALAC-approved suppliers (SARL Bioprim, Bazièges, France; Cynologics-Silabe, Niederhausbergen, France). Ethical approvals were obtained from the local ethics committee CETEA n°44 in MIRCen and the French Ministry of Education and Research. A complete description of housing and perioperative care is available in Dentel et al., (2023) Ophthalmol.Sci. 3:100316.

[0191] IKE toxicity in NHP was assessed by subretinal delivery of 20 µM IKE solution in one eye and a control solution (0.2% DMSO diluted in PBS) in the other eye. Dentel et al. provide a comprehensive description of anesthesia, surgery, and data acquisition. In short, subretinal injection was performed to detach the superior fovea in each eye. Anatomical (slit-lamp examination, optical coherence tomography-OCT-, adaptive optics-AO-) and functional (full-field and multifocal electroretinography, ffERG, and mfERG, respectively) studies were performed at baseline (within one week preoperatively), day 3 (to confirm completion of retinal reattachment), and month 1, and then monthly until month 4. Built-in software in the RTX1 (AOdetect, Imagine Eyes, Orsay France) was used to quantify the adaptive optics cone mosaic parameters. Furthermore, Dentel et al. detail the additional use of the power spectrum spacing method to quantify cone mosaic in NHP.

[0192] Analysis and Statistics

[0193] GraphPad Prism 8.4.0 software (GraphPad Software Inc., San Diego, CA) was used for analysis, plotting, and graphing. The Mann-Whitney t-test was used to statistically compare the treatments and controls, and p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****) was considered significant.

[0194] In charts, normalize the data relative to the control as much as possible and express it as the mean plus or minus the standard error of the mean.

[0195] The Mann-Whitney t-test was used to compare the control and treatment groups in in vitro and ex vivo analyses. In rats, multiple t-tests were used to compare differences in cone density and microglial density, and the Holm-Sidak method was used to determine statistical significance. In NHP, two-tailed t-tests were used to compare differences in cone density, regularity, interocular spacing, and ERG amplitude. For mfERG in NHP, each hexagon was compared to the contralateral matching hexagon, taking into account the symmetry between the two eyes.

[0196] result

[0197] Glutamate-induced ferroptosis of purified cone photoreceptors

[0198] Photoreceptors release their neurotransmitter glutamate at their synaptic terminals, but they express only the glutamate transporter (rather than the classic ionotropic receptor). When glutamate is applied to purified porcine cone photoreceptors ( Figure 1 AD), at 10 µM, the activity of pure porcine cone photoreceptors decreased by 20%; and at the highest tested concentration of 500 µM, it reached 82% ( Figure 1 E). This range of glutamate is physiologically possible, as evidenced by the glutamate affinity of receptors and transporters (Gielen (2010) médecine / sciences 26:65). To determine whether this toxicity is related to systemic Xc - The expression of the antitransporter (already reported in cone photoreceptors by immunocytochemistry) (Dun et al., (2006) Cell Tissue Res 324:189; Bridges et al., (2004) Ophthalmol.Vis.Sci 45:2906-2914) was involved in the administration of glutamate in the presence of 1 mM L-cysteine. At each glutamate concentration, L-cysteine ​​prevented glutamate toxicity (n=7, p<0.0001, IC50). 50 =4.15 µM)( Figure 1 E). Due to system Xc - This transporter possesses pharmacology quite different from other glutamate transporters or receptors, and we investigated how different agonists and antagonists of this transporter affect cone survival. Ferraphobia inducers and imidazolid ferrophobia inducers (IKE) are systemic Xc - Two functional inhibitors of glutamate are toxic to isolated cone photoreceptors, similar to glutamate, but with a lower dose range due to their greater affinity. Figure 1 AD, F). L-cysteine ​​also rescued cones exposed to IKE (n=6, p=0.0022) Figure 1 K).

[0199] To assess the molecular mechanisms underlying this glutamate-induced toxicity in cone photoreceptors, we measured GSH levels in cones exposed to 500 µM glutamate and an imidazolidinedione ferroptosis inducer (IKE 3 µM). A 50% reduction in GSH was observed under both conditions on day 1 and thereafter compared to the control condition. Figure 1 F, n=18, p<0.0001). Therefore, the death of cone photoreceptor cells is attributed to system Xc. - Dysfunction-induced dysregulation of GSH levels in cells. GSH is known to participate in redox cycles, in which its oxidized form is reduced by hydrogen atoms carried by NADPH (Dixon et al., (2019) Annu. Rev. CancerBiol. 3:35). We measured the NADPH / NADP ratio in cones treated with 3 µM IKE. This condition increased the amount of NADPH from 25% in the control to 85% in the treated culture (n=4, p=0.0159). Figure 1 G). The intracellular NADPH / NADP ratio is indirectly related to the activity of glutathione peroxidase 4 (GPX4) during glutathione redox. When GPX4 becomes inactive, the cycle also becomes inactive and can induce this accumulation of NADPH, suggesting that when the system Xc - When inhibited, the redox cycle is downregulated (Azuma et al., (2022) J. Biol. Chem. 298:101824). To investigate whether GSH is essential for cone survival, a specific inhibitor of GPX4, Ras selective lethal molecule 3 (RSL3), was used. This inhibitor showed toxic effects on cone photoreceptors at very low concentrations (n=3, IC50). 50 =8.67 nM)( Figure 1 H), highlighting the crucial role of GPX4 in cone survival. RNA was then extracted from treated and control pure cone photoreceptors, and we found altered expression of proteins involved in regulating the glutathione redox cycle and downstream mechanisms. Figure 2 ).

[0200] GPX4 is also known to prevent the formation of lipid peroxides that react with free radicals (Xie et al., (2016) Cell Death Differ. 23:369; Dixon et al., 2019). These lipid peroxides are formed by lipoxygenases such as 5-lipoxygenase (5LOX) (which is activated by 5-lipoxygenase-activated protein (FLAP)). To examine the presence of these mechanisms in cone photoreceptors, Western blotting was performed, and we found both 5LOX and FLAP in cones, but neither in rod photoreceptors. Figure 1 I). Specific immunostaining on retinal tissues from different species revealed specific FLAP localization in cone photoreceptors. Figure 3 To assess the role of this lipid peroxidation pathway in glutamate-induced cell death, cone cell extracts were analyzed by lipidomics after incubation with 3 μM IKE for 2 days. The analysis revealed an increase in the amount of lipid peroxidation, such as 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) (a metabolite of 5LOX) (n=6, p= 0.0079 (**), p= 0.0159 (*)). Figure 1 J). To demonstrate the contribution of this lipid peroxidation to the observed glutamate-induced toxicity, we tested the effect of zileutone (ZEN), a 5-LOX enzyme antagonist, in the presence of IKE. ZEN rescued 80% of the cone photoreceptors (J). Figure 1 K (n=3, p=0.0238) suggests that lipid peroxidation plays a key role in glutamate-induced cone degeneration.

[0201] Changes in iron metabolism during lipid peroxidation-induced ferroptosis. Ferritin autophagy can be highlighted by immunomarking of ferritin and nuclear receptor coactivator 4 (NCOA4) (a ferritin autophagy inducer). Cones treated with IKE showed increased immunomarking intensity of ferritin heavy chain and NCOA4. Furthermore, NCOA4 appeared to be slightly stronger than ferritin heavy chain, suggesting increased iron metabolism activity in the treated cones. Figure 4 Ferritin autophagy can be inhibited by ferrostatin-1 (FST1, a specific inhibitor of ferroptosis) and deferoxone (DF, an iron chelator that prevents the Fenton response by limiting the accumulation of intracellular free iron). Both of these rescued cones from IKE-induced toxicity, with FST1 (n=8, p=0.0047) rescuing 68.5% and DF (n=5, p=0.0079) rescuing 77.2%. Figure 1K). In addition, other anti-ferroptosis molecules were tested after treatment with 20 nM RSL3 and 3 μM IKE, targeting different components involved in ferroptosis activation (Table 2). Therefore, it appears that pure cone photoreceptors exposed to ferroptosis promoters (such as IKE) can be rescued by anti-ferroptosis treatment, which highlights the role of ferroptosis in cone survival.

[0202] Table 2. Antiferroptosis molecules after treatment with 20 nM RSL3 or 3 µM IKE

[0203] Preliminary screening of cone photoreceptors was performed using 20 nM RSL3 or 3 μM IKE. Library compounds were added at a concentration of 10 μM one day after induced ferroptosis in the cells. Best hits are recorded in the table above for each treatment. Some compounds did not increase cell viability by more than 50% depending on the treatment and are not reported in the table ( / ). Cells treated with only 20 nM RSL3 or 3 µM IKE showed only 30% and 25% cell viability, respectively, three days after treatment (n=3).

[0204]

[0205]

[0206] Ferrocyte retinal death in isolated retinal models

[0207] To demonstrate that cone photoreceptors can undergo ferroptosis degeneration in an integrated retinal model lacking retinal pigment epithelium, we applied IKE to freshly prepared porcine retinal explants. The specificity and selectivity of ferroptosis in the retina were assessed by examining damaged cells (cell membrane permeability to ethidium dye) after 3 days of incubation. In control conditions, ethidium labeled a few scattered cells, while in IKE-treated explants, the first row of photoreceptor nuclei were ethidium-labeled (…). Figure 5 B, F). This prominent layer corresponds to the nucleus of the cone photoreceptor in the porcine retina (Pattnaik et al., (2000) J. Neurosci. 29:6789), as shown by the Flap immunomarker. Figure 5 C, G). Figure 5 G shows morphological changes in cone photoreceptors using 20 μM IKE. Other types of retinal cells were also labeled, showing no severe quality changes. Figure 6 In situ cytotoxicity in cone photoreceptors was then assessed by counting outer segment (OS) layers immunolabeled by Flap (automatically using CellPath software of CQ1©). Figure 5 JM). 20 μM IKE induced a 50% reduction in the amount of visual cone OS (n=3, p=0.0286) ( Figure 5 I). 1 µM FST-1 and 100 µM DF salvaged 84% and 78.4% of the overall survival (OS) respectively (n=3, p=0.1000 and p=0.0286 respectively). Figure 5 I). Similarly, when 200 nM RSL3 in the retinal explant triggers cone degeneration ( Figure 7 ), 100 μM DF saved 78.5% of OS (n=3, p=0.0286) Figure 7 These data suggest that ferroptosis is an effective and selective pathway for integrating cone photoreceptor degeneration in retinal tissue.

[0208] Furthermore, microglial migration from the inner retina (normally located) towards the outer retina (typically lacking microglia) appears to be triggered in retinal tissue exposed to ferroptosis inducers. Microglial activation, indicated by increased volume and shape changes, suggests that the cells extend to surround the cones (…). Figure 5 H). Automated quantitative analysis revealed significant intraretinal microglial shifts toward the outer nuclear layer (ONL) triggered by 20 μM IKE and 200 nMRSL3 (n=3). Figure 5 N). Ferraphobia inhibitors (FST-1 and DF) (n=3) partially blocked migration (Figure 50; Figure 7 N). These microglial morphological changes and their migration toward the photoreceptor layer provide further evidence of cone degeneration via ferroptosis.

[0209] Cone degeneration and microglial migration in rats

[0210] To assess whether ferroptosis inducers could initiate degenerative and inflammatory processes in the living retina, ferroptosis inducers were injected into the subretinal space of Long Evans rats (20 μM IKE, see below). Figure 8 A). On day 6, optical coherence tomography (OCT) showed numerous punctate hyperreflectances, suggesting microglia in rats injected with ferroptosis inducers. Figure 8 (Arrow in G). The same retina was then examined with cone and microglial immunostaining. Cone photoreceptor density was assessed on retinal sections passing through the injection area on day 30. In animals treated with ferroptosis inducers via subretinal delivery, OS and perinuclear bodies were significantly reduced ( Figure 8 Quantitative analysis on retinal sections showed that, using ferroptosis inducers, the perinuclear corpuscle was reduced by about one-third, and the overall survival (OS) was reduced by nearly half. Figure 8 J). At D6, microglial migration within the injection area was quantified on the full-thickness retinal lamina, showing an increase in microglial cells in the OS layer and at the cone synapse terminals (J). Figure 8 K). Figure 8The Locator Image (LO) provides an illustration of the high density of microglia found in the photoreceptor layer on day 6. These data suggest that triggered ferroptosis in the live rat retina leads to cone degeneration and microglia migration toward the photoreceptor layer.

[0211] Macular degeneration in non-human primates

[0212] Similarly, ferroptosis inducers were administered subretinally to the retina of live nonhuman primates. A 20 μM IKE solution was administered subretinally in one eye, and a control solution (1 / 500 DMSO) was administered subretinally in the contralateral eye, followed by a 4-month follow-up (see [link to original text]). Figure 9 A). A previous study showed that in NHP, subretinal injection of the mediator solution did not cause any functional or anatomical ocular or systemic adverse effects (6-month follow-up in 2 NHPs to date), but resulted in transient changes in photoreceptors identified by adaptive optic ophthalmoscopy, which fully recovered after 4 months (Dentel et al., 2023).

[0213] In eyes injected with 20 µM IKE, changes in retinal structure and function were observed in vivo. Firstly, macular pigmentation occurred within 3 months, surrounding subretinal deposits at the fovea. Figure 9 BD). OCT also showed eroded ellipsoidal bands (i.e., regions of the inner and outer segments of the photoreceptor) and punctate high reflectivity found only in the ONL within the 20 μM IKE detachment region. Figure 9 EG). Several subretinal deposits appeared in the fovea at 1 month ( Figure 9 F), and appeared in the macular layer at 2 months (F), and appeared in the macula ( Figure 9 HJ). Adaptive optics imaging of several subretinal deposits (a technique that allows visualization of cone photoreceptors in vivo) appears identical to that seen in patients affected by age-related macular degeneration (AMD), showing that cones in the detached areas are essentially absent under 20 μM IKE. Figure 9 KL). Built-in OCT software, which allows for ONL segmentation across the injection area, shows a significant change in ONL thickness within the injection area at 20 µM IKE. Figure 9 M, N).

[0214] Adaptive optics has also been used to quantify cone density in the injection region. The effect of standard subretinal delivery of 0.2% DMSO on cone reflectance in adaptive optic imaging has been reported (Dentel et al., 2023). Cone loss was observed in eyes injected with 20 µMIKE: at 4 months, there was a decrease in cone cell density of 661.00 cones / deg² in the region with an eccentricity of 2 degrees or higher, compared to 37.50 cones / mm² (n=2, p<0.001). No change was observed in the region with an eccentricity of 2 degrees or lower (i.e., outside the injection region).

[0215] Functional alterations in cone photoreceptors were assessed using multifocal electroretinography (mfERG): changes in N1 wave amplitude (an indicator of the hyperpolarization response of photoreceptors to light) and P1 wave amplitude (an indicator of the function of the inner retinal layer) relative to baseline were analyzed. Eyes injected with 20 µM IKE showed a significant decrease in N1 wave amplitude (-52.19%) compared to eyes injected with the control solution (-2.95%) (n=2, p=0.0396). A non-significant decreasing trend in P1 wave amplitude was also observed (-33.03% vs. -3.83%, n=2, p=0.0874). In non-human primates, subretinal delivery of ferroptosis inducers at the macular level resulted in alterations highly similar to clinical features seen in macular degeneration.

[0216] Example 2 Ferrocytes do not affect primary retinal pigment epithelial cells.

[0217] Materials and methods

[0218] Purification of pig cone photoreceptors

[0219] Pig eyes were dissected to obtain the retina, which was then cut into pieces and digested at 37 °C for 20 minutes with 4 U / ml papain (LSO3124, Worthington) and L-cysteine ​​(5.5 mM, Sigma-Aldrich). Enzyme activity was terminated by adding Neurobasal-A (NBA) medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) and 15 µg / ml DNase I (D4263, Millipore Sigma, Burlington, MA). Several short centrifugations were performed (5 to 6 times at 110 × g for 30 s) and the supernatant was collected, discarding the supernatant containing the main rod photoreceptors from the first centrifugation. The pellet was resuspended between centrifugations using a P1000 pipette to dissociate the remaining cells. The cell suspension was then centrifuged at 110 xg for 10 minutes, and the pellet was resuspended in NBA medium containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA).

[0220] Cone photoreceptors were purified by lectin panning using a slightly modified version of the previously described method (Balse et al., IOVS 2005) to obtain millions of suspended cones. Cells were then seeded at a density of 3,000 or 4,000 cells / well in clear 384-well plates (781091, Greiner, Ulis, France) or white 384-well plates (781080, Greiner, Ulis, France) using a robotic system (ViafloAssist, Integra-Biosciences, Saint-Ouen, France) and incubated for three days at 37 °C in an atmosphere containing 5% CO2.

[0221] Purification of porcine retinal pigment epithelial (RPE) cells

[0222] Pig eyes were dissected and the retina removed to obtain eyecups containing fundus RPE cells. Preheated 0.25% trypsin (25200-056, Thermo Fisher Scientific, Waltham, MA, USA) was added to the eyecups and incubated at 37 °C for 1 hour. Cells were then removed from the eyecups by pipetting and transferred to tubes containing DMEM (41966-029, Thermo Fisher Scientific, Waltham, MA, USA) and 20% FBS (A31605-01, Thermo Fisher Scientific, Waltham, Massachusetts) (=D20). After centrifugation at 110 × g for 5 min, the pellet was resuspended in D20, and cells were seeded in 60 mm Petri dishes and incubated at 37 °C and 5% CO2. The medium was changed the following day.

[0223] The cells were then seeded at 4,000 cells / well in white 384-well plates (781080, Greiner, Ulis, France) using a robotic system (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated at 37 °C in an atmosphere containing 5% CO2 for three days.

[0224] ARPE-19 cell line

[0225] Cell lines were obtained from ATCC and distributed by LGC. Cells were used between P15 and P30 and seeded in D20 medium.

[0226] The cells were then seeded at 4,000 cells / well in white 384-well plates (781080, Greiner, Ulis, France) using a robotic system (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated at 37 °C in an atmosphere containing 5% CO2 for three days.

[0227] deal with

[0228] The following compounds were used to induce ferroptosis: glutamate at in vitro concentrations of 1 μM to 1 mM (49621, Millipore Sigma, Burlington, MA); IKE at concentrations of 200 nM to 25 μM (HY-114481, MedChemExpress, Monmouth, USA) (including 3 μM for purifying cone photoreceptors and 20 μM for in vitro and in vivo experiments); and RSL3 at concentrations of 10 nM to 10 μM (HY-100218A, MedChemExpress, Monmouth, USA) (including 20 nM, 200 nM, and 10 µM for in vitro and in vivo cell experiments). To inhibit ferroptosis, FST-1 (HY-100579, MedChemExpress, Monmouth, USA) was used at a concentration of 50 nM on pure cones or at a concentration of 400 nM on retinal explants. DF (HY-B0568, MedChemExpress, Monmouth, USA) is effective against pure cones at a concentration of 50 µM, and ZEN (HY-14164, MedChemExpress, Monmouth, USA) is effective against pure cones at a concentration of 20 µM (see [link to product]). Figure 1 K, 5I and 7M).

[0229] CellTiter Glo (CTG)

[0230] Cell viability was measured using a CellTiter Glo (G7573, Promega, Madison, WI) instrument for RPE cells, ARPE-19 cells, and cone photoreceptors. Viability was measured according to the manufacturer's instructions.

[0231] result

[0232] In vitro, IKE and RSL3 do not alter primary RPE cells.

[0233] The inventors further investigated the sensitivity threshold of retinal pigment epithelial (RPE) cells after administration of ferroptosis-promoting agents. It is well known in the literature that RPE cells can undergo ferroptosis-mediated death (Lee et al., (2022) Oxid. Med. Cell. Longev. 2022:1792894). However, the question was how to determine the most sensitive cell type between RPE cells and cone PR cells. Therefore, the inventors used the ARPE-19 cell line, which has been used in all studies to characterize RPE cells, and investigated a range of concentrations of IKE, RSL3, and glutamate. When cells were treated with IKE, a weaker effect on ARPE-19 was observed, showing 34.3% viable cells at 25 μM, compared to 0% in cones (n=3, p=0.0001). Figure 11 A). Similarly, treatment of ARPE-19 with RSL3 resulted in a higher mortality rate; however, its concentration was significantly higher than that used to kill cones. At 10 μM RSL3, only 5.3% of ARPE-19 cells remained viable, but at 5 μM, only 50% of cells remained viable (n=3, p=0.0008). Figure 11 B), while the EC50 of RSL3 for cones was 1.58 nM, demonstrating that cones are much more sensitive to GPX4 inhibition. Finally, glutamate treatment had no effect on ARPE-19, unlike cone PR, which was completely killed at 1 mM glutamate (n=3, p=0.0050). Figure 11 C).

[0234] However, the inventors were also interested in comparing the effects of ferroptosis-promoting molecules between the ARPE-19 cell line and primary porcine RPE cells. Significant differences were observed with IKE treatment, which had no effect on primary cells, even at 25 μM (n=3, p=0.0019). Figure 11 A). However, the survival trends between RSL3 and glutamate treatments were similar ( Figure 11 B, C).

[0235] in conclusion

[0236] These results indicate that cone photoreceptors are more sensitive to ferroptosis-inducing agents than RPE cells. Therefore, lower levels of oxidative stress can trigger cone photoreceptor death without triggering RPE cell death. Furthermore, it should be noted that articles discussing ferroptosis in RPE cells use cell lines that are prone to division and thus can become immortal (Kozlowski et al., (2015) Curr. Eye Res. 40:501). However, ferroptosis is well-described in RPE cells.

[0237] Example 3Ferroresorption in RD1 mice, both in vitro and in vivo, rescues cones from degeneration.

[0238] Materials and methods

[0239] Animal model: Mouse

[0240] Mouse experiments and procedures were approved by the Local Animal Ethics Committee Charles Darwin CEEACD #5 and conducted in an approved facility associated with the Institut de la Vision (Paris, France) in accordance with European Directive 2010 / 63 / UE. All experimental work was performed in accordance with the institution's biosafety and safety procedures. Mice were kept in a controlled environment under a reverse half-day dark / light cycle and had free access to food and water except during surgery.

[0241] The C3H / HeNRj strain (rd1 obtained by crossing female Bagg Albino and male DBA) was purchased from Janvier Laboratories (Le Genest Saint-Isle, France, ISO 9001 certified) for production and breeding.

[0242] retinal explants of rd1 mice

[0243] Eyes were collected at P15, washed, and placed in CO2-independent medium containing glucose (6.5 g / L) with 1 / 10 L-cysteine ​​(Millipore Sigma, Burlington, MA) at 3.5 mg / 10 mL, with papain (LSO 3124, Worthington) diluted 1 / 50, and incubated at 37 °C with 5% CO2 for 20 min. The eyes were then immersed on ice in neurobasal medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) = NBAg and 10% SVF (A31605-01, Thermo Fisher Scientific, Waltham, Massachusetts). Dissection was performed at +4 °C to obtain the entire retina with RPE. Full-thickness retinal lamina were flattened onto a polycarbonate film (140652, Thermo Fisher Scientific, Waltham, Massachusetts). The test component was diluted to 10 µM in NBAg and 1 / 50 B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA) and changed twice weekly for 30 days (until P45 was reached). The explants were then fixed for 1 hour with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA). Finally, PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA) was performed, and the explants were mounted on Cellvis plates (P12-1.5HN, IBL, Gerasdorf, Austria) for imaging.

[0244] rd1 mice intraperitoneal injection

[0245] From P15 to P45, Rd1 mice were intraperitoneally injected daily with 2 mg / kg GW 5074 (HY-10542, MedChemExpress, Monmouth, USA) diluted in PBS (14190144, Thermo Fisher Scientific, Waltham, MA, USA) containing 40% PEG300 (HY-Y0873, MedChemExpress, Monmouth, USA) and 5% Tween80 (HY-Y1891, MedChemExpress, Monmouth, USA), or the mediator alone (control group). At P45, the eyes were retrieved and fixed for 1 hour with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA). The retinas were retrieved for PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA). The retina of rd1 mice was imaged at X10 using a CQ1© confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan).

[0246] Imaging and Statistics

[0247] For this purpose, Cell PathFinder analysis software was used to quantify the number of PNA-labeled objects in the hand-drawn center of retinal explants in ex vivo experiments and in full-thickness retinal patches in in vivo experiments. The number of objects (= cones) detected throughout the entire area was quantified and then reported per mm².

[0248] The Mann-Whitney U two-sided test was used to assess the difference in cone density between retinal explants and injected mice.

[0249] result

[0250] Following initial exposure to 20 nM RSL3 and 3 µM IKE, various ferroptosis inhibitors were screened on isolated porcine cones to identify protective components that prevent cone cell loss in retinal degeneration.

[0251] The inventors then tested the neuroprotective effects of several of these components on the mature retinal degeneration model rd1 mice (Léveillard et al., (2004) Nat. Genet. 36:755). In rd1 mice, cone degeneration is early, starting at P15, followed by loss of optical survival (OS) at P15–P20; at P45, only half of the cell bodies are retained. Retinas were retrieved from rd1 mice at P15, and the retinal explants were exposed to ferroptosis inhibitors in culture until P45. Several treatments provided cone protection (…). Figure 12 (AE). However, while this model leads to rapid degeneration, the inventors may have demonstrated molecules that can rescue cone photoreceptor degeneration in slower degenerative diseases such as AMD.

[0252] Using automated cone counting, the inventors have identified five antioxidant molecules with significant neuroprotective effects (1,3-dicaffeoylquinic acid (n=3, p=0.0055), HTHQ (1-O-hexyl-2,3,5-trimethylhydroquinone, n=3, p=0.0176), rosmarinic acid (n=4, p=0.0205), nobergenin (n=2, p=0.0256), and tenoridone hydrochloride (n=3, p=0.0055)). On the other hand, the inventors have identified five molecules that inhibit ferroptosis and have significant neuroprotective effects: two of them act directly on 5-LOX (SRS16-86 (n=3, p=0.0001) and caffeic acid (n=3, p=0.0055)), and two of them play a role in iron metabolism (mangiferin (n=5, p=0.0005) and astilbin (n=3, p=0.0055). The last molecule involved in specific ferroptosis inhibition (via the systemic Xc pathway) demonstrated a 138.92% neuroprotective effect on pure cone photoreceptors and was also shown to be effective in retinal explants, which led to in vivo injection of this molecule GW 5074 (n=6, p=0.0001). Intraperitoneal injections of 2 mg / kg were administered daily from P15 to P45. Immunolabeling on the retina retrieved at P45 showed that, compared with the control group which received only the carrier daily, injection of GW... Cone cells in 5074 rd1 mice were rescued by +81.48% (1597 vs 880 cones / mm², n=5, p=0.0079). Figure 12 J).

[0253] These findings suggest that ferroptosis inhibitors can reverse cone degeneration in both in vitro and in vivo animal models of retinal degeneration.

[0254] Example 4 Ferraphobia inhibitors do not rescue rods in RD1 mice

[0255] Materials and methods

[0256] Similar to cones, as described in Example 3 above, rods were evaluated on retinal explants in rd1 mice after exposure to ferroptosis inhibitors in the culture from P15 to P45. The inventors also performed the same evaluation as described in Example 3 above after intraperitoneal injection of GW 5074.

[0257] The retina was marked with Rho, imaged with CQ1, and the bar counted using Cell Pathfinder.

[0258] Since the cones are plotted as "multiple changes", the processed rods are plotted as density (rods / mm²) because the number of rods is insufficient to account for the variation in each retina relative to the baseline (the variation sometimes exceeds the total number of rods).

[0259] result

[0260] There was no significant difference between the control and ferroptosis inhibitors, either in retinal explants or in vivo. Figure 13 ).

[0261] Example 5 Ferrocyte death inducers do not alter the visual rod in non-human primates.

[0262] Materials and methods

[0263] Similar to cone labeling in NHP with 20 μM IKE injected into the subretinal space, rods were labeled with rhodopsin antibodies.

[0264] result

[0265] No significant changes were observed in the outer segment level of the rod in the retina injected with 20 μM IKE compared to the control. Figure 14 ).

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Claims

1. A compound, such as an inhibitor of ferroptosis, for use in the treatment of conditions associated with degeneration of cone photoreceptors, wherein said treatment comprises reducing or inhibiting lipid peroxidation in cone photoreceptors.

2. The compound for use according to claim 1, wherein the activity of phospholipase A2 and / or the activity of 5-lipoxygenase (5-LOX) and / or the activity of 5-lipoxygenase activator protein (FLAP) are reduced or inhibited.

3. The compound for use according to any one of claims 1 or 2, wherein the treatment comprises maintaining or increasing the activity of cone photoreceptors.

4. The compound for use according to any one of claims 1 to 3, wherein the treatment comprises maintaining or reducing the intracellular formation of lipid peroxides in at least one cone photoreceptor, particularly 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE).

5. The compound for use according to any one of claims 1 to 4, wherein the treatment comprises preventing or reducing colloidal migration.

6. The compound for use according to any one of claims 1 to 5, wherein the treatment comprises preventing or reducing the formation of subretinal deposits.

7. The compound for use according to any one of claims 1 to 6, wherein the treatment comprises maintaining or increasing cone photoreceptor activity.

8. The compound according to any one of claims 1 to 7, wherein the compound is selected from the group consisting of: SCD1; RNA inhibiting any one of PLA2, ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5 and ALOX5AP; ziluton; ferrostatin-1; liproxstatin-1; PDA NP; 16-86; XJB-5-131; UAMC-3203; SRS15-72B; SRS15-72A; SRS16-80; SRS16-86; GSK2190915; MK-886; ML355; BLX-3887; VKH2; ferrostatin-1, liproxstatin-1, PDA NP, 16-86, XJB-5-131, UAMC-3203, SRS15-72B, SRS15-72A, SRS16-80, SRS16-86, VKH2, nuclear enriched transcript 1 (NEAT1), α-tocopherol, BAY x 1005; tometetin; caffeic acid; diallyl trisulfide; treglitazone (succinate); tannic acid; norbergenin; chlorogenic acid; 3'-hydroxypuerarin; 1,3-dicaffeoylquinic acid; rosmarinic acid; (-)-epicatechin, CP-24879 (hydrochloride); FPL62064; hexahydrocurcumin; Mito-TEMPO; MEL351; Nelonemdaz (potassium); renoside; Sonlicromanol (hydrochloride); SP-141, tenoridin hydrochloride; and Trolox.

9. The compound for use according to any one of claims 1 to 8, wherein the condition is selected from the group consisting of: age-related macular degeneration (AMD), cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt's disease), central serous chorioretinopathy, Best disease and bestrophinopathies, retinal detachment including rhegmatogenous, serous and tractional causes, solar retinopathy, laser-induced retinopathy, achromatopsia, Stargardt's disease, Usher syndrome, Leber congenital amaurosis (LCA), Alström's disease and Refsum's disease, preferably AMD or retinitis pigmentosa, more preferably AMD.

10. The compound for use according to any one of claims 1 to 9, wherein the use comprises applying the compound to the subretinal space, suprachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, corneal surface, by systemic administration (inhaler, intravenous, intramuscular, or intraperitoneal) or as a solution, gel, or implant.

11. The compound for use according to any one of claims 1 to 10, wherein the use comprises the administration of an additional therapeutic agent.

12. The compound for use according to claim 11, wherein the additional therapeutic agent is a second ferroptosis inhibitor, pilgatanib, ranibizumab, bevacizumab, buxizumab, farexizumab, AKB-9778, nevasumab, BI 836880, or aflibercept.

13. The compound for use according to claim 12, wherein the administration of the second ferroptosis inhibitor results in the activation of the Xc-transporter or GPX4 enzyme in the cone photoreceptor and / or a decrease in the intracellular iron level in the cone photoreceptor.

14. The compound for use according to any one of claims 11 to 13, wherein the compound and the additional therapeutic agent are administered simultaneously, sequentially, or separately.

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