Activators of the xc- system and / or gpx-4 for treating conditions associated with cone photoreceptor degeneration

Abstract

The present invention provides methods of treatment of conditions associated with cone photoreceptor degeneration, wherein the treatment comprises activating the Xc- transporter or the GPX4 enzyme in cone photoreceptors. In particular, the present invention provides compounds, preferably ferroptosis inhibitors, for use in such methods.

Description

[0001] ACTIVATORS OF THE Xc- SYSTEM AND / OR GPX-4 FOR TREATING CONDITIONS ASSOCIATED WITH CONE PHOTORECEPTOR DEGENERATION.

[0002] INTRODUCTION

[0003] The retina is a light-sensitive neuronal tissue located at the posterior part of the eyeball. Retinas of humans have two distinct regions. The retinal periphery has low spatial acuity and is responsible for night-vision and different aspects of motion vision. The fovea (or macula) is at the retinal centre and drives high spatial acuity vision that is essential for reading and face recognition. Primates are the only mammals with a fovea. Retina is arranged in three layers of cells, namely the outer nuclear layer (ONL), the inner nuclear layer (INL), and the retinal ganglion cell (RGC) layer. The ONL harbours photoreceptors, which are unique neurons dedicated to converting light into electrochemical signals and as such essential for vision. Two different types of photoreceptors are distinguished: rod photoreceptors respond to dim light and enable vision at night, whereas cone photoreceptors respond to bright daylight and mediate high-resolution and colour vision.

[0004] Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Photoreceptor death is the common cause in 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 linked primarily to a loss of cones. It is thus particularly important to be able to prevent cone photoreceptors degeneration. It would enable therapeutic intervention at the earliest stages to stop the disease developing, thereby ultimately preventing loss of vision.

[0005] To this day, there is no effective therapy for retinal degenerative diseases which remain poorly treatable, presenting an urgent need for developing novel treatment strategies. There are few approved therapies for these diseases, but they only address the eye neovascularisation subsequent to cone photoreceptor degeneration. On the other hand, no molecules capable of preventing or treating the earliest stages of the pathologies, i.e., the death of the cone photoreceptors, have yet been identified.

[0006] Further understanding of the mechanisms leading to cone photoreceptor death is essential for the development of these treatments. Most of the earliest stages of retinal diseases are still not understood, notably the molecular and cellular mechanisms which result in cone photoreceptor death. Several mechanisms of cell death have been suggested to be implicated 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 been shown to specifically drive death of the cone photoreceptors.

[0007] There is thus still an urgent need for molecules that can effectively treat conditions associated with cone photoreceptor degeneration.

[0008] SUMMARY OF THE DISCLOSURE

[0009] In a first aspect, the present disclosure relates to a method of treating a condition associated with cone photoreceptor degeneration. The present disclosure shows that modulation of the Xc- transporter and / or the GPX4 enzyme in cone photoreceptors prevents degeneration of these cells.

[0010] Hence, a first aspect of the disclosure relates to a compound, e.g., a ferroptosis inhibitor, for use in treating a condition associated with cone photoreceptor degeneration, wherein the treatment comprises modulating, in particular specifically modulating, the Xc- transporter or the GPX4 enzyme in cone photoreceptors. More particularly, said ferroptosis inhibitor specifically modulates the Xc- transporter or the GPX4 enzyme in cone photoreceptors, i.e., the Xc- transporter or the GPX4 enzyme are modulated by said ferroptosis inhibitor in cone photoreceptors but are not substantially affected in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, at identical concentrations of ferroptosis inhibitor. Preferably, the activity of the Xc- transporter and / or of the GPX4 enzyme is increased, in particular is specifically increased, in cone photoreceptors. More particularly, said ferroptosis inhibitor specifically increases the activity of the Xc- transporter and / or of the GPX4 enzyme in cone photoreceptors, i.e., the activity of the Xc- transporter or the GPX4 enzyme is increased by said ferroptosis inhibitor in cone photoreceptors but is not substantially affected in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, at identical concentrations of ferroptosis inhibitor. In another preferred instance, the expression of the Xc- transporter and / or the GPX4 enzyme is increased, in particular is specifically increased, in cone photoreceptors. In an instance, the treatment comprises maintaining or increasing cone photoreceptor viability. In an instance, the treatment does not affect rod photoreceptor viability and / or retinal pigment epithelial cell viability.

[0011] In an instance, the treatment comprises preventing an increase in the cell membrane permeability to dyes in cone photoreceptors. More particularly, the treatment comprises specifically preventing an increase in the cell membrane permeability to dyes in cone photoreceptors, i.e. the treatment does not substantially prevent an increase in the cell membrane permeability to dyes in other retinal cell types, such as rod photoreceptors.

[0012] In an instance, the treatment comprises maintaining or increasing intracellular glutathione level in cone photoreceptors. More particularly, the treatment comprises specifically maintaining or increasing intracellular glutathione level in cone photoreceptors.

[0013] In an instance, the treatment comprises maintaining or decreasing, in particular specifically maintaining or decreasing, intracellular NADP / NADPH ratio in cone photoreceptors. More particularly, the treatment comprises specifically maintaining or decreasing intracellular NADP / NADPH ratio in cone photoreceptors.

[0014] In an instance, the treatment comprises preventing or reducing microglial migration.

[0015] In an instance, the treatment comprises preventing or reducing subretinal deposit formation.

[0016] In an instance, the treatment comprises maintaining or increasing cone photoreceptor activity. More particularly, the treatment comprises specifically maintaining or increasing cone photoreceptor activity.

[0017] The compound used in the methods disclosed herein is preferably a ferroptosis inhibitor. More preferably, the compound is selected in the group consisting of DKK1 , OTUB1 , cystine, B-mercaptoethanol, selenium, bardoxolone, carvacrol (CAR), rehmanniosideA, bioflavonoids including galangin, xanthohumol, naringenin, britanin, entacapone, capsiate, resveratrol, dexmedetomidine, irisin, 2-cyano-3,12-dioxoolean- 1 ,9-dien-28-oic acid (CDDO), GW 5074, kaempferol, 1 -O-hexyl-2,3,5- trimethylhydroquinone (HTHQ), 1 ,3-dicaffeoylquinic acid, rosmarinic acid, nobergenin and tinoridine hydrochloride. Most preferably, the compound is selected in the group consisting of of DKK1 , OTUB1 , cystine, B-mercaptoethanol, selenium, bardoxolone, carvacrol (CAR), rehmanniosideA, bioflavonoids including galangin, xanthohumol, naringenin, britanin, entacapone, capsiate, resveratrol, dexmedetomidine, irisin, 2- cyano-3,12-dioxoolean-1 ,9-dien-28-oic acid (CDDO), GW 5074 and kaempferol.

[0018] Preferably the condition associated with cone photoreceptor degeneration of the methods disclosed herein is a condition in which cones are specifically affected. In particular, the condition is a condition in which cone photoreceptor degeneration is not a secondary consequence of the degeneration of the retinal pigment epithelium. More preferably, the condition is selected in the group consisting of age-related macular degeneration, cone dystrophies, cone-rod dystrophies, rod-cone dystrophies, macular dystrophy (e.g. Stargardt disease), central serous chorioretinopathy, Best disease and bestrophinopathies, retinal detachment including rhegmatogenous, serous and fractional causes, solar retinopathy, laser-induced retinopathy, achromatopsia, Usher syndrome, Leber congenital amaurosis, Alstrom syndrome, and Refsum disease.

[0019] In an instance, the compound for the use in the methods disclosed herein, is administered in the subretinal space, the suprachoroidal space, the anterior chamber, the vitreous humour, the subconjunctival space, on the corneal surface. For example, the compound can be administered through systemic administration (inhalator, intravenous, intramuscular or intraperitoneal) or under solution, gel or implant.

[0020] In an instance, the present treatment comprises 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, AKST4290 and Bl 836880. More preferably, administration of the second ferroptosis inhibitor results in reduction or inhibition of lipid peroxidation in cone photoreceptors and / or reduction of intracellular iron levels in cone photoreceptors.

[0021] In an instance, the present treatment comprises administering the compound and the second therapeutic compound simultaneously, sequentially, or separately.

[0022] LEGENDS OF THE FIGURES FIGURE 1. Evidence of ferroptosis occurring in pure cone photoreceptors.

[0023] Mean values and standard errors of the mean are shown. A-B. Brightfield image of cone photoreceptors after 3 days of incubation with control (Ctrl = 0.2% DMSO, A) and treated solutions (3 M imidazole ketone erastin, IKE, B). C- D. Cone photoreceptor staining with calcein labelling viable cells in control (C) and treated (D) conditions. E. Glutamate concentration-dependent cell viability of pure cone photoreceptors with or without adding 1 mM L-Cystine (n=7, p<0.0001 , IC50 = 4.15 M). F. Levels of glutathione in pure cone photoreceptors in control, 3 M IKE and 500|JM glutamate mediums over 3 days (D1 , D2, D3 n=18, p<0.0001 ). G. NADP / NADPH percentages in pure cone photoreceptors in control and 3 M IKE conditions at 3 days (n=4, p=0.0159). H. Concentration-dependent plot of pure cone photoreceptors cell viability after treatment with RSL3, from 0.01 nM to 25nM (n=3, IC50 =8.67nM). I. Western Blot revealing 5 -lipoxygenase (5LOX) and 5-lipoxygenase-activating protein (FLAP) bands in pure cone photoreceptors, absent in rod photoreceptors. J. Lipidomic analysis showing the amount 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 in control and 3 M IKE conditions (n= 6, p=0.0079 (“), p=0.0159 (*)). K. Pure cone photoreceptors viability in control and 3 M IKE conditions when adding various treatments (with antiferroptotic or iron chelating effects): L-Cystine, Zileuton (ZEN), Ferrostatin-1 (FST1 ) and deferiprone (DF).

[0024] FIGURE 2. Alteration of mRNA expression levels for proteins involved in ferroptosis in control versus treated (3pM IKE) pure porcine cone photoreceptors (n=6). Solute Carrier Family 11 Member 2 (SLC11 A2) is involved in the releasing of Fe2+from the endosome into the cell cytoplasm; its decline suggests an iron metabolism dysregulation (p=0.0260). Solute Carrier Family 3 Member 2 (SLC3A2) corresponds to the heavy chain of the System Xc- (p=0.0022). Expression of transferrin receptor (TFRC) modestly increased under treatment condition (p=0.0286). Ferroptosis suppressor protein 1 (AIFM2) is involved to reduce ferroptosis mechanism (p=0.0022). Heat shock protein family B small member 1 (HSPB1 ) carries a role in the iron uptake and may promote ferroptosis22(p=0.0022).

[0025] FIGURE 3. Immunofluorescence localisation of Flap on retinal sections from 3 different species (pig, rat and non-human primate) with respect to the DAPI nuclear staining. In all species, Flap is localised in cone photoreceptors. FIGURE 4. Immunofluorescence of Ferritin heavy chain (B, G) and NCOA4 (C, H) in control (A-E) and treated (F-J) isolated porcine cone photoreceptors. The IKE treatment increased the fluorescence intensities (G, H) as compared to control cone photoreceptors. IKE condition (G, H) showing more immunolabelling than controls (B, C). E. Zoom of the merged labelling showing control cells with a more intense ferritin heavy chain immunolabelling than NCOA4. J. Zoom on treated cells showing a more intense NCOA4 immunolabelling than heavy chain.

[0026] FIGURE 5. Cone photoreceptors degeneration and microglial migration in ex vivo retinal explants. A - H. Histology of retinal sections showing an increase in permeability to ethidium in the first nuclear retinal row representing cone photoreceptors in the treated explant (20pM IKE, F) versus the control tissue (0.2% DMSO, B). Note also the disorganisation of Flap-immunolabeled cone photoreceptors in the treated retina (G) as compared to the control condition (C) as well as the presence of large amoeboid microglial cells in the treated retina (H) up to the outer nuclear layer (ONL) not present in the control retina (D). The nuclear layers are underlined in the control (A) and treated retina (E) by DAPI. I-M. Cone photoreceptor destruction indicated by the loss of their Flap-immunopositive outer segment on a treated flat mounted retinal explant (20pM IKE) and their protection by antiferroptotic agents FST-1 (L) and DF (M) as compared to the control condition (J). The quantification of cone photoreceptor outer segments demonstrates a significant decrease in treated explant and a rescue by the antiferroptotic agents (I, n=3). N-O. Microglial cell distributions in the retinal explant thickness showing their migration toward the outer retina in treated retina (IKE, RSL3) and a prevention of this effect by DF or FST-1 (n=3). Scale bars = 20pm.

[0027] ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, Iba1 : ionised calcium-binding adapter molecule 1 , Ctrl: control condition, IKE: imidazole ketone erastin, OS: outer segment, FST-1 : ferrostatin-1 , DF: deferiprone

[0028] FIGURE 6. Maintenance of different retinal cell types on sections from treated porcine retinal explants (20pM IKE) as compared to the control condition (Ctrl). Rod photoreceptors were immunolabelled against rhodopsin, bipolar cells against PKC alpha and Muller cells against vimentin. Note that retinal structure underlined by the DAPI stained nuclei, and the morphologies of the labelled cells appear preserved in the treated condition, without evident cell loss. FIGURE 7. Effects of RSL3 on retinal explants. RSL3 permeability to ethidium revealed dying cells in control (0.2% DMSO, B) and treated (200nM RSL3, F) retinae. C, G. Morphological changes of Flap-immunolabelled cone photoreceptors in a treated retinal explant (G) as compared to a control explant (C) with the migration of iba1 - immunopositive microglial cells toward the outer nuclear layer (ONL) in treated explant (H) with respect to the control condition (D). I-L. Loss of Flap-immunopositive cone outer segments (OS) in retinal explants treated by RSL3 (J) and their preservation by FST-1 (K) or DF (L) as related to the control condition (I). M. Quantification of the RSL3 toxicity on cone photoreceptor outer segments and their preservation by DF in retinal explants (n=3, p=0.0286). N. Distribution of microglial cells showing their migration towards the outer nuclear layer (ONL) in the presence of RSL3 as compared to the control condition (see Fig. 5N) with the effects of DF or FST-1 (n=3) .

[0029] Scale bars = 20pm. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, Iba1 : ionised calcium-binding adapter molecule 1 , Ctrl: control condition, IKE: imidazole ketone erastin, OS: outer segment, FST-1 : ferrostatin-1 , DF: deferi prone.

[0030] FIGURE 8. Cone photoreceptor degeneration and microglial migration in rats following in vivo subretinal delivery of ferroptosis inducers. A. Schematic representation of the experimental protocol and timeline. Rats were delivered 20 M IKE or 200nM RSL3 (treated) or 0.2% DMSO (control, Ctrl) in the subretinal space. Retinas were retrieved at day 6 (D6) to assess microglial migration (K-O, n=4) or at day 30 (D30) to assess cone photoreceptor degeneration (H-J, n=4). B-G. Sample of in vivo retinal imaging of rats: fundus photography (B, C) and optical coherence tomography (OCT) (D-G) showing the induced retinal detachment (DO, B,D) beside the optic nerve (ON), with fully reattached retina at D6 (C, E); the injection site (arrowheads) and the OCT slice (dashed lines) are shown on fundus photography; (F, G) OCT of the injected area at D6 showing hyperreflective punctuations (arrows) spotted in the inner nuclear layer (INL) and at the outer plexiform layer (OPL) / outer nuclear layer (ONL) junction, more abundantly in the treated condition (G). H, I. Retinal sections (same rats as in B-G) within the injected area, collected at D30 showing a decrease in cone-arrestin immunolabelled cone photoreceptors (CAR) in treated animals (I) as compared to control (H) with the corresponding DAPI nuclear stainings. The eccentricity relative to the optic nerve is the same in both cases. J. Quantification of cone densities (in elements / mm) measured on retinal sections in treated and control rats at D30 (n=4). Outer segments (OS) and perikarya of cones were quantified separately. K. Quantification of microglial density on whole-mounted retinae of treated and control rats at D6. Three retinal plans were evaluated separately: the level of the outer segments (OS), the ONL plan and the pedicles (Ped) plan, which corresponds to the OPL / ONL junction. Each field corresponds to a 500x500pm window, repeated over the entire injected area. L-O. Distributions of cone-arrestin positive cells and Iba1 -positive microglial cells in the whole-mounted retinae of treated and control rats collected at D6, showing two different plans on the z axis (illustrated in H-l): OS (L, M) and Ped (N, O). The samples were picked in the detached area but away from the injection site to illustrate the change in cone OS morphology and the invading microglial cells for treated animals. The eccentricity relative to the optic nerve is the same in both cases.

[0031] Scale bars: 50pm. INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, EZ: ellipsoid zone, RPE: retinal pigment epithelium, DAPI: 4', 6- diamidino-2-phenylindole, CAR: cone arrestin, Iba1 : ionised calcium binding adaptor molecule 1 , Ctrl: control.

[0032] FIGURE 9. Macular changes in non-human primates (NHP) following submacular delivery of 20pM IKE. A. Schematic representation of the experiment protocol. The subretinal delivery of 20pM IKE in the right eye and DMSO diluted in PBS at 1 / 500 in the left eye allowed to detach the superior hemifovea in both eyes. B-C. Fundus photography at baseline (B), 1 (C) and 3 months (D) following the subretinal injection of 20pM IKE. Demarcation line at 1 month (arrowheads in C) and macular pigmentation surrounding subretinal deposits occurring at 3 months (highlighted in D) are shown. E-F. OCT vertical b-scans through the foveal spot of NHP three months after a subretinal delivery of 1 / 500 DMSO (ctrl, E) and 20pM IKE (F). Note the eroded ellipsoid zone (EZ, black asterisk), punctuate hyperreflectivities (arrows) and subretinal deposits (arrowhead). G. OCT horizontal b-scan through the upper parafoveal area within the subretinal delivery of 20pM IKE. The EZ is eroded (black asterisks) within the detached area borderlines (dashed lines). Note also bunches of hyperreflective foci mainly located in the ONL and below the inner limiting membrane (arrows). The uninjected area (outside of the dashed lines) remains perfectly normal. H-J. OCT horizontal b-scans through a macular subretinal deposit (arrowheads) that appeared at M2 (I) and grew until M3 (J). K-L. En face adaptive optics imaging of the foveal photoreceptor layer at 2 degrees of eccentricity, within the detached area, 3 months following the subretinal delivery. Cones are less reflective and clusters with vanishing cones appeared following 20pM IKE delivery (K), whereas the cone mosaic of the control side remains regular (L). M-N. Illustration of the ONL thickness changes in the macular area of NHP 3 months after the subretinal delivery. The grey scale shows ONL thickness changes (in pm). O. Cone density changes (in cones / deg2) measured at 2 degrees within and without the injected area using adaptive optics, 3 months after the subretinal delivery (n=2). P. Changes in N1 - and P1 -wave amplitudes as a percentage of baseline measured on multifocal electroreti nogram (n=2). Only hexagons within the injected area were analysed.

[0033] FIGURE 10. Ferroptosis pathways elucidated in mammalian cone photoreceptors. Ferroptosis ultimately drives ROS formation that leads to cone photoreceptor degeneration. Three subcellular pathways must be considered in ferroptosis in cones. Firstly, the System Xc-, that can be inhibited by high extracellular concentration of glutamate of by ferroptosis inducers (e.g. , IKE), stimulates the entry of L-cystine to produce glutathione. Inhibition of the glutathione redox cycle results in glutathione peroxidase 4 (GPX4) inactivation and accumulation of NADPH. Additionally, the reduced activity of GPX4 enzyme leads to the production of peroxidised lipids such as 5-oxo-ETE via the lipoxygenase pathway. Finally, a high ferritinophagy activity of NCOA4 leads to a dysregulated iron metabolism, resulting in an excessive amount of iron release. Radical species are formed by Fenton’s reaction, and allow for reactive oxygen species production via interaction with peroxidised lipids.

[0034] GPX4: Glutathione peroxide 4, 12-LOX: 12 lipoxygenase, 15-LOX: lipoxygenase, 5-LOX: 5 lipoxygenase, FLAP: 5-lipoxygenase-activating protein, 12-HPETE: 12 hydroxyperoxyeicosatetraenoic, 15-HPETE: 15 hydroxyperoxyeicosatetraenoic, 5- HPETE: 5 hydroxyperoxyeicosatetraenoic, 5-oxo-ETE: 5-0xo-eicosatetraenoic, 12- HETE: 12-hydroxyeicosatetraenoic, 15-HETE: 15-hydroxyeicosatetraenoic, 5-HETE: 5- hydroxyeicosatetraenoic, STEAP 3: STEAP family member 3, DMT1 : Dimetal transporter 1 , NCOA4: nuclear receptor coactivator 4, Keapl : Kelch-like ECH- associated protein 1 , NFE2L2: Nuclear factor (erythroid-derived 2)-like 2. ROS: reactive oxygen species.

[0035] FIGURE 11. Cell viability after pro-ferroptotic treatment on different cell type in the retina. Cone photoreceptors (Cone PRs) are represented by a light grey line and a round shape, ARPE-19s by a dark grey line and a triangular shape, and RPE primary cells by a black line and a square shape. A. Cells were treated with IKE (Imidazole Ketone Erastin) from 0.20 pM to 25 pM. All cone PRs died at 25 pM while 40% of ARPE-19 (n=3, p=0.0001 ) and 80% of RPE primary cells (n=3, p=0.0019) remained alive. B. Cells were treated with RSL3 (Ras Selective Ligand 3) from 0.01 pM to 10 pM. All PR cone cells died at all concentrations, whereas at 5 pM 50% of ARPE-19 (n=3, p=0.0008) and primary RPE cells remained alive. C. Cells were treated with glutamate from 0.001 mM to 1 mM showing no effect on ARPE-19 and primary RPE cells. In contrast, cone PRs all died at 1 mM compared to ARPE-19 cells (n=3, p=0.0050) and primary RPE cells.

[0036] FIGURE 12. Ferroptosis inhibitors reverse cone degeneration in rd1 mice between P15 and P45 ex vivo and in vivo. A-D. PNA-immunolabeled retinal explants of rd1 mice exposed to a control solution (A) and to several ferroptosis inhibitors in culture for 30 days (from P15 to P45) (B-D). E. Automated cone counting on retinal explants of rd1 mice exposed in culture with various ferroptosis inhibitors (Ctrl, HTHQ, 1 ,3-Dicaffeoylquinic acid, SRS16-86, Astilbin, caffeic acid, Tinoridin Hydrochloride n=3; GW5074 n=6; Nobergenin n=2; Mangiferin n=5). White bars represent antioxidants or 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. F-l. PNA- immunolabeled whole-mounted retinae of rd1 mice daily injected intraperitoneally with the vehicle (Ctrl, F and H) or a 2 mg / kg GW 5074 solution for 30 days (from P15 to P45) (G and I). The asterisks indicate the position of H and I. J. Automated cone counting on whole-mounted retinae after daily intraperitoneal injections (n=5) . Scale bars = 50 m in A-D, H, I; 300 m in F, G. PNA: peanut agglutinin, Ctrl: control, HTHQ: 1-0-hexyl-2,3,5-trimethylhydroquinone, 1-3-DCFA: 1 ,3- Dicaff eoylquinic acid, RA: Rosmarinic acid, SMNL: Sonlicromanol, CA: Caffeic acid, Tinoridin HCL: Tinoridin hydrochloride, OH puerarin: Hydroxy purrarain, 5-ACQ: Chlorogenic acid.

[0037] FIGURE 13. Ferroptosis inhibitors do not reverse rod degeneration in rd1 mice between P15 and P45 ex vivo and in vivo. A. Automated cone counting on retinal explants of rd1 mice exposed in culture with various ferroptosis inhibitors that rescued cones (Ctrl, HTHQ, 1 ,3-Dicaffeoylquinic acid, SRS16-86, Astilbin, caffeic acid, Tinoridin Hydrochloride n=3; GW5074 n=6; Nobergenin n=2; Mangiferin n=5). B. Automated cone counting on whole-mounted retinae after daily intraperitoneal injections (n=5).

[0038] FIGURE 14. Rods immunolabeled with rhodopsin (Rho) and DAPI in the nonhuman primate retina injected with the control solution and with 20 pM IKE.

[0039] DESCRIPTION

[0040] Definitions

[0041] The term “about” or “approximately” refers to the normal range of error for a given value or range known to the person of skills in the art. It usually means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0042] As used herein, the terms “activate”, “stimulate, or “induce”, and any grammatical derivative thereof, refer to a comparative increase in a specified response of a designated material (e.g., expression, enzymatic activity) in the presence of a specified reagent. The reagent is described herein as an “activator”.

[0043] As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In particular, “administering” refers to a method of giving a dosage of the compound-disclosed herein to a subject having a condition associated with cone photoreceptor degeneration. The compositions utilised in the methods described herein can be administered, for example, intravitreally (e.g., by intravitreal injection), ocularly (e.g., by ocular injection), or intraocularly (e.g., by intraocular injection). The method of administration can vary depending on various factors (e.g., the compound or composition being administered, and the severity of the condition, disease, or disorder being treated).

[0044] Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; or nanoparticles.

[0045] The term “decreased” or “reduced”, as used herein, refers to the activity of a protein at least 1 -fold (e.g. , 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) lower than its reference value. “Decreased” or “reduced”, as it refers to the activity of a protein of a subject, signifies also at least 5% lower (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%) than the activity of the protein in the reference sample or with respect to the reference value for said protein. The term “decreased” or “reduced”, as used herein, also refers to the level of a biomarker of a subject (such as e.g., the iron concentration in a specific cell type, e.g., cone photoreceptors) at least 1 -fold (e.g. , 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) lower than its reference value. “Decreased” or “reduced”, as it refers to the level of a biomarker of a subject (such as e.g., the iron concentration in a specific cell type, e.g., cone photoreceptors), signifies also at least 5% lower (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%) than the level in the reference sample or with respect to the reference value for said marker.

[0046] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In one instance, the disease is a condition affecting the eye, notably a condition associated with cone photoreceptor degeneration.

[0047] As used herein, “electroretinogram (ERG)” refers to a diagnostic test which measures electrical activity of the retina in response to a light stimulus. ERG can for example record a massed potential from the whole retina; see e.g., full-field ERG. Alternatively, multifocal ERG (mfERG) assesses ERG activity in small areas of retina, whilst pattern 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 a combination 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.

[0048] As used herein, “ferroptosis” refers to a form of cell death understood in the art as involving generation of reactive oxygen species mediated by iron, and characterised by, in part, lipid peroxidation. In ferroptosis, lethality occurs as a result of peroxidation of polyunsaturated fatty acids (PUFAs)that self-propagate unless halted by the lipid peroxidase, glutathione peroxidase 4 (GPX4).

[0049] As used herein, “GPX4” refers to glutathione peroxidase 4, a glutathione metabolism enzyme.

[0050] The term “increased”, as used herein, refers to the activity of a protein, at least 1 -fold (e.g., 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) greater than its reference value. “Increased”, as it refers to the activity of a protein of a subject, signifies also at least 5% greater (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%) than the activity of the protein in the reference sample or with respect to the reference value for said protein. The term “increased”, as used herein, also refers to the level of a biomarker of a subject at least 1 -fold (e.g. , 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) greater than its reference value. “Increased”, as it refers to the level of a biomarker of a subject, signifies also at least 5% greater (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%) than the level in the reference sample or with respect to the reference value for said marker.

[0051] As used herein, the terms “inhibit” or “inhibition” refer to a comparative decrease in a specified response of a designated material (e.g., expression, enzymatic activity) in the presence of a specified reagent. The reagent is described herein as an “inhibitor”.

[0052] As used herein, “measure” or “determine” refers to any qualitative or quantitative determinations.

[0053] As used herein, “operably linked” means that the elements being linked are arranged so that they function in concert for their intended purposes. For example, a promoter is operably linked to a nucleic acid molecule if the promoter effects transcription from the transcription initiation to the terminator of said nucleic acid molecule in a permissive host cell.

[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 a retina. OCT can be used to distinguish layers of the retina, map and measure thickness, inform treatment decisions, provide diagnostic information, monitor disease progression, or a combination thereof. Notably, OCT can be used to quantify cone degeneration. In embodiments, subjects who have not been diagnosed with a retinal degenerative disease have a peak cone density in the fovea of about 200,000 cells / mm2as measured by OCT, and / or a foveal outer nuclear layer thickness of about 100 pm as measured by OCT. Preferably, patients diagnosed with retinal degenerative disease have a peak cone density in the fovea of less than about 200,000 cells / mm2as measured by OCT. Patients diagnosed with a retinal degenerative disease can have a foveal outer nuclear layer thickness of less than about 100 pm as measured by OCT. Additional examples of OCT values from healthy subjects and patients diagnosed with a retinal degenerative disease can be found in Lorenz et al., 2008.

[0055] As used herein, the terms “prevent”, “prevention” and “preventing” refer to the reduction in the risk for a subject of acquiring or developing a given disease, disorder, or condition. The terms "prevent", "prevention" and "preventing" also comprise delaying the onset and / or reducing the frequency and / or intensity of clinical, histological and / or biochemical symptoms or parameters associated with this given disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis such that a therapy is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the risk of acquiring or developing the disease, disorder, or condition, and / or a statistically significant delay of the onset and / or a statistically significant decrease of the frequency and / or intensity of clinical, histological and / or biochemical symptoms or parameters associated with the disease, disorder or condition, is observed in a population susceptible to the disease, disorder, or condition.

[0056] As used herein, the expression “regulatory elements” or “regulatory sequence” refers to any element that allows, contributes or modulates the expression of the encoded nucleic acid(s) in a given host cell or subject, including replication, duplication, transcription, splicing, translation, stability and / or transport of the nucleic acid(s) or its derivative (i.e., m RNA).

[0057] As used herein, the term “specifically” means that the compound, at a given concentration, affects one cell type (namely cone photoreceptors), while not substantially affecting other retinal cell types such as rod photoreceptors or retinal pigment epithelial cells, at the same given / identical concentration. In particular, when referring to an in vivo use, the term “specifically” means that the compound affects one cell type (namely cone photoreceptors) before substantially affecting or without substantially affecting other retinal cell types such as rod photoreceptors of retinal pigments epithelial cells.

[0058] As used herein, a “subject” includes all mammals, including without limitation humans, but also non-human primates such as cynomolgus monkeys. It also includes dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents (such as mice and rats). It will be appreciated that a particularly preferred subject according to the disclosure is a human subject, such as a human suffering from (or at risk of suffering from) a disorder, disease or condition, notably a condition associated with cone photoreceptor degeneration, for example a human patient.

[0059] The term “system Xc-” refers to an amino acid antiporter that mediates the exchange of extracellular L-cystine and intracellular L-glutamate across the cellular plasma membrane. The system Xc- exchanges intracellular glutamate for extracellular cystine, thereby supporting intracellular glutathione (GSH) synthesis as well as non- vesicular glutamate release thus leading to glutathione production and oxidative protection. As used herein, inhibition of system Xc- refers to the prevention of glutamate exit and / or cystine entry by inhibitors. As used herein, activation of the system Xc-refers to the stimulation of glutamate exit and / or cystine entry by activators. System Xc- is a heterodimeric amino acid transporter which consists of xCT, a light-chain subunit that confers cystine transport function and is encoded by the SLC7A11 gene, and the SLC3A2 heavy-chain subunit, which localises system Xc- to the plasma membrane.

[0060] As used herein, the term “therapy” refers to any protocol, method and / or agent that can be used in the prevention, management, treatment and / or amelioration of a disease, disorder, or condition in a patient. The patient may be at risk of contracting the disease, disorder, or condition, or suspected to have contracted the disease, disorder, or condition. Alternatively, the patient may have been diagnosed as suffering from the disease, disorder or condition. Non-limiting examples of therapy comprise administration of a composition (e.g., a pharmaceutical or a vaccinal composition), physical therapy (e.g., radiotherapy, ultrasound therapy, electrotherapy, phototherapy, cryotherapy, etc.), physiotherapy, psychological therapy, etc. Therapy also includes the possibility to use combination therapy. “Combination therapy” and any variation thereof such as “combined use” refers to the action of delivering to the same subject several distinct therapies. Such a combination encompasses the cases where the distinct therapies are delivered to the subject as a single composition (together in the same unit dosage) or separately (i.e. , dissociate arrangement), in which case the distinct therapies may be delivered simultaneously or sequentially. “Simultaneously”, as used herein, refers to the action of delivering at the same time several therapies mixed in the same composition. “Separately”, as used herein, refers to the action of delivering essentially at the same time or over the same period of time (e.g., within one hour or less) several therapies in separate presentations (e.g., separate compositions). “Sequentially” refers to “one after the other”, meaning one therapy being delivered first followed, immediately or after a suitable period of time (e.g., more than one hour), by the delivery of another therapy.

[0061] When therapy comprises administering or the administration of a composition, the composition is administered in an amount, manner, and / or mode effective to treat or prevent the patient’s disease, disorder, or condition. Therapy may require administration of the composition more than once. In such case, the intervals between two successive deliveries of the therapy may be identical or different. In case of combination therapy, the intervals between two successive deliveries of the several distinct therapies may be identical or different. As used herein, the term “treating” or “treatment” means an improvement of the patient’s disease, disorder, or condition, which may be observed at the clinical, histological, and / or biochemical level. The terms “treating” or “treatment” notably include improving a clinical, histological, and / or biochemical symptom or parameter associated with the patient’s disease, disorder, or condition or inhibiting, reducing, or delaying progression or exacerbation of the patient’s disease, disorder, or condition (including secondary damage caused by the disease, disorder, or condition) to either a statistically significant degree or to a degree detectable to one skilled in the art. In some embodiments, treatment is assessed on a population basis such that a therapy is considered to “treat” a particular disease, disorder or condition if a statistically significant improvement of the patient’s disease, disorder, or condition is observed in a population suffering from the disease, disorder, or condition.

[0062] The term “vector” as used herein refers to a vehicle, preferably a nucleic acid molecule or a viral particle that contains the elements necessary to allow delivery, propagation and / or expression of any of the nucleic acid molecule(s) 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 produce additional copies of the nucleic acid molecules when the host cell replicates) as well as shuttle vectors (e.g. functioning in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g. for transferring nucleic acid molecule(s) in a viral genome). For the purpose of the disclosure, the vectors may be of naturally occurring genetic sources, synthetic or artificial, or some combination of natural and artificial genetic elements.

[0063] In the context of the disclosure, the term “vector” has to be understood broadly as including mRNA, plasmid and viral vectors. Vectors which are appropriate in the context of the present disclosure, include, without limitation, bacteriophage, plasmid or cosmid vectors for expression in prokaryotic host cells such as bacteria (e.g. E. coli, BCG or Listeria); vectors for expression in yeast (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g. Sf 9 cells); as well as plasmid and viral vectors for expression in higher eukaryotic cells or subjects. Typically, such vectors are commercially available (e.g., in Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositary institutions such as the American Type Culture Collection (ATCC, Rockville, Md.) or have been the subject of numerous publications describing their sequence, organization and methods of producing, allowing the artisan to apply them. The present disclosure also encompasses vectors (e.g., plasmid DNA and mRNA) complexed to lipids or polymers to form particulate structures such as liposomes, lipoplexes or nanoparticles.

[0064] Methods of treatment

[0065] The present disclosure provides a method for treating conditions associated with cone photoreceptor degeneration.

[0066] The present inventors have surprisingly found that degeneration of cone photoreceptors is caused by activation of specific cell death pathways, specifically ferroptosis, in these cells.

[0067] The present inventors have shown that inhibitors of the Xc- transporter and / or inhibitors of the GPX4 enzyme such as glutamate, erastin, imidazole ketone erastin (IKE), and RSL3 cause a specific drop in cone photoreceptors viability. This loss of viability of cone photoreceptors is accompanied by a decrease in glutathione (GSH) levels, an increase in cell membrane permeability to dyes, an increase in peroxided lipids, and a dysregulation of iron metabolism, such as, notably, an increase in intracellular iron levels. Not only is this effect specifically observed in pure cone photoreceptors in culture, it is also apparent in an ex vivo model of retina, wherein in addition microglia cells migrated in the outer nuclear layer (ONL) as they do in vivo. The significance to in vivo pathological processes was further emphasised by the finding that administration of inducers of ferroptosis into the eyes of non-human primates leads not only to specific cone loss, but also to formation of subretinal deposits and decrease in electroreti nogram amplitude, features highly similar to those exhibited by AMD patients. Importantly, administration of such inducers of ferroptosis into the eyes does not affect the viability of rod photoreceptors or retinal pigment epithelial cells. Thus, surprisingly, even though the Xc- system was described in the prior art as being present in both cone and rod photoreceptors (Hu et al. Characterization of the cystine / glutamate transporter in the outer plexiform layer of the vertebrate retina. Eur J Neurosci. 2008 28(8):1491 -502), only the cone photoreceptors are affected by ferroptosis. Therefore, induction of ferroptosis by inhibitors of the Xc- system or inhibitors of the GPX4 enzyme in cones triggers specific cone photoreceptor degeneration, thereby resulting in phenotypes highly similar to the in vivo pathological situation. Significantly, cone photoreceptors degeneration can be rescued by antiferroptotic treatments. For example, addition of cystine, which activates the Xc- system, to cone photoreceptors specifically restores cell viability in presence of inducers of ferroptosis.

[0068] This demonstrates that proper and specific activation of the Xc- system and / or of the GPX-4 enzyme in cone photoreceptors can block the cell death observed in the present conditions.

[0069] This was further confirmed by the demonstration of the present inventors that administration of ferroptosis inhibitors, in particular ferroptosis inhibitors involved in the Xc system pathway, specifically reversed cone degeneration in rd1 mice, a well- known model of retinal degeneration, while not rescuing rods in these mice. These results thus confirm that, surprisingly, the treatment of the condition associated with cone photoreceptor degeneration disclosed herein, is due to the specific and direct activation of the Xc- transporter or the GPX4 enzyme in cone photoreceptors, while not involving or affecting rod photoreceptors or retinal pigment epithelial cells.

[0070] In a first aspect, the disclosure relates to a compound for use in treating conditions associated with cone photoreceptor degeneration, wherein the treatment comprises activating the Xc- transporter and / or the GPX4 enzyme in cone photoreceptors, more particularly wherein the treatment comprises specifically activating the Xc- transporter and / or the GPX4 enzyme in cone photoreceptors, i.e. the treatment does not substantially activates the Xc- transporter and / or the GPX4 enzyme in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, at identical concentrations of ferroptosis inhibitor.

[0071] The disclosure also related to the use of a compound for making a medicament for treating conditions associated with cone photoreceptor degeneration, wherein the treatment comprises activating the Xc- transporter and / or the GPX4 enzyme in cone photoreceptors, more particularly wherein the treatment comprises specifically activating the Xc- transporter and / or the GPX4 enzyme in cone photoreceptors, i.e. the treatment does not substantially activates the Xc- transporter and / or the GPX4 enzyme in other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells, at identical concentrations of ferroptosis inhibitor.

[0072] The disclosure also relates to a method of treatment of a condition associated with cone photoreceptor degeneration, the method comprising administering the compound to a subject in need thereof, thereby activating the Xc- transporter and / or the GPX4 enzyme, in particular specifically activating the Xc- transporter and / or the GPX4 enzyme in cone photoreceptors, i.e. the treatment does not substantially activates the Xc- transporter and / or the GPX4 enzyme in other retinal cells types, such as rod photoreceptors or retinal pigment epithelial cells, at identical concentrations of ferroptosis inhibitor.

[0073] Ferroptosis is an iron-dependent, lipid peroxidation-dependent form of regulated cell death pathway. Ferroptosis can be initiated by the inhibition of the cystine / glutamate transporter, system xc-, by glutamate leading to a drop in glutathione (GSH) levels. The ferroptosis inducer erastin also inhibits system xc -and cystine deprivation has an analogous effect. Once the GSH levels fall significantly, reactive oxygen species (ROS) levels start to increase exponentially, leading to lipid peroxidation. GSH depletion also results in the inhibition of glutathione peroxidase 4 (GPX4) activity since this enzyme depends on an adequate supply of GSH for its function. Among antioxidant enzymes, GPX4 has the ability to directly reduce complex lipid hydroperoxides that are incorporated in cellular membranes or lipoproteins. Inhibition of GPX-4 activity thus also contributes to increased lipid peroxidation.

[0074] In an instance, the activity of the Xc- transporter and / or of the GPX4 enzyme is increased, in particular specifically increased, in cone photoreceptors. In another preferred instance, the expression of the Xc- transporter and / or the GPX4 enzyme is increased, in particular specifically increased, in cone photoreceptors.

[0075] The treatment according to the present disclosure is particularly advantageous in that it inhibits specifically the death of cone photoreceptors. In particular, the viability of cone photoreceptors is maintained by the treatment. More particularly, the viability of cone photoreceptors is specifically maintained by the treatment, i.e. the viability of other retinal cell types such as rod photoreceptors or retinal pigments epithelial cells is not substantially affected by the treatment. Alternatively, the treatment results in an increased viability of cone photoreceptor cells. More particularly, the treatment results in a specific increased viability of cone photoreceptor cells, i.e. the treatment does not substantially affect viability of other retinal cell types, such as rod photoreceptors or retinal pigment epithelial cells. Viability of the cone photoreceptor cells can be measured by any means known to the skilled person. For example, the viability of these cells can be assayed in vitro or ex vivo with vital dyes e.g., calcein AM, MTT, alamarBlue, ethidium, CellTiterGlo. Alternatively, cone viability can be assayed in an ex vivo model of retina, by e.g., measuring the thickness of the retina, measuring the number of nuclei in the ONL (e.g. after DAPI staining), measuring their permeability to dyes and / or determining the number of cone outer segments or cones after labelling the retinal tissue with a reagent specific of cones (e.g., an anti-FLAP antibody, anti-cone opsin antibody, the peanut agglutinin lectin, or an anti -cone-arrestin antibody). In yet another instance, cone viability can be assayed in vivo by optical coherence tomography (OCT) or adaptative optics retinal imaging. For example, OCT is widely employed clinically to characterise potential retinal degeneration in patients, because it permits to define the location and nature of the changes in the retina and adjacent structures and objectively evaluates the thickness of the retina and surrounding structures. The prevention of decrease of cone degeneration can thus be measured via OCT. All these techniques are well known to the skilled person. The cone cell survival can also be demonstrated on histological samples by measuring the thickness of the retina, measuring the number of nuclei in the ONL (e.g. after DAPI staining), or determining the number of cones after labelling the retinal tissue with a reagent specific of cone outer segments or cones (e.g., an anti-FLAP antibody, anti-cone opsin antibody, the peanut agglutinin lectin or an anti -cone-arrestin antibody). It is understood that the skilled person will be capable of adapting each of these methods as required by the circumstances to effectively measure the cone photoreceptor viability.

[0076] In another instance, the treatment disclosed herein is particularly advantageous in that it blocks, in particular specifically blocks, the decrease of intracellular glutathione levels in cone photoreceptors. In particular, the intracellular glutathione levels in cone photoreceptors are maintained, in particular specifically maintained, by the treatment. Alternatively, the treatment results in increased, in particular specific increased, intracellular glutathione levels in cone photoreceptors. Many commercial assays for measuring glutathione levels are available to the skilled person, including the one used in the examples. In another instance, the treatment disclosed herein is particularly advantageous in that it blocks, in particular specifically blocks, the intracellular NADP / NADPH ratio from increasing in cone photoreceptors. In particular, the intracellular NADP / NADPH ratio in cone photoreceptors is maintained, in particular specifically maintained, by the treatment. Alternatively, the treatment results in a decreased, in particular specific decreased, intracellular NADP / NADPH ratio in cone photoreceptors. Many commercial assays for measuring NADP / NADPH ratio are available to the skilled person, including the one used in the examples.

[0077] In another instance, the treatment disclosed herein is particularly advantageous in that it inhibits microglia from migrating in the ONL. In particular, the treatment results in a decreased migration of the microglia cells. Alternatively, microglia migration is prevented by the treatment. Microglia migration is well known to the skilled person and can be determined by any means known in the art (Rashid et al. Front Immunol. 10:1975, 2019).

[0078] In another instance, the treatment disclosed herein is particularly advantageous in that it blocks the formation of subretinal deposits. Such deposits are observed in a number of conditions associated with cone degeneration, e.g., AMD. In particular, the treatment results in a decreased formation of subretinal deposits. Alternatively, formation of subretinal deposits is prevented by the treatment. Subretinal deposits are well known to the skilled person and can be determined by any means known in the art (Monge et al. Taiwan J Ophthalmol. 12(2): 138-146, 2022).

[0079] In another instance, the treatment disclosed herein is particularly advantageous in that it blocks, in particular specifically blocks, the decrease of the cone photoreceptor activity. In particular, the cone photoreceptor activity is maintained, in particular specifically maintained, by the treatment. Alternatively, the treatment results in increased, in particular specific increased, cone photoreceptor activity. Cone receptor activity may be assayed by electroreti nogram (ERG) or multifocal electroreti nogram (mfERG); see e.g. Asanad S, Karanjia R. Multifocal Electroreti nogram. [Updated 2022 Oct 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan. Preferably, the treatment disclosed herein comprises obtaining at least 2, at least 3, at least 4, at least 5 of the above features. More preferably, the treatment disclosed herein comprises obtaining all 6 above features.

[0080] The present therapy for conditions associated with cone photoreceptor degeneration can be combined with, or adjunctive to, one or more other treatments.

[0081] Combination therapy as provided herein involves the administration of at least two agents to a patient, the first of which is a therapeutic compound as disclosed herein, and the second of which is another therapeutic compound. Accordingly, the disclosure relates to the therapeutic compound described herein, for the treatment of conditions associated with cone photoreceptor degeneration, wherein the therapeutic compound is administered with the second therapeutic compound.

[0082] Combination therapies of the present disclosure can result in a greater than additive, or a synergistic, effect, providing therapeutic benefits where neither the therapeutic compound disclosed herein nor the second therapeutic compound is administered in an amount that is, alone, therapeutically effective. Thus, such agents can be administered in lower amounts, reducing the possibility and / or severity of adverse effects.

[0083] For example, the second therapeutic compound may be a second ferroptosis inhibitor. In particular, administration of the second ferroptosis inhibitor may results in reduction or inhibition, in particular specific reduction or inhibition, of lipid peroxidation in cone photoreceptors and / or reduction, in particular specific reduction, of intracellular iron levels in cone photoreceptors. Suitable second ferroptosis inhibitors include notably SCD1 , Prominin , miR-522, and / or RP11 -89; an RNA inhibiting any one of ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5, ALOX5AP, FTL, FTH1 , and / or NCO4; zileuton; ferrostatin-1 ; liproxstatin-1 ; PDA NPs; 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; o-tocopherol; tolmetin; caffeic acid; diallyl trisulfide; trelagliptin (succinate); ellagic acid; norbergenin; chlorogenic acid; 3'-hydroxypuerarin; 1 ;3-dicaffeoylquinic acid; rosmarinic acid; deferiprone; ferrostatin-1 ; deferoxamine (DFO); gossypol acetic acid; histochrome; ciclopirox olamine (CPX); bafilomycin A1 ; quercitrin; baicalein; dexrazoxane; 2;2'- bipyridine; 1 ;10-phenanthroline; mangiferin; astilbin; YL-939; nuclear enriched transcript 1 (NEAT1 ); and deferasirox (DFX).

[0084] Alternatively, the second therapeutic compound can treat or alleviate another aspect of the present conditions. In particular, the present therapeutic compound is advantageously administered with a second therapeutic compound which prevents neoangiogenesis in the eye. These compounds include e.g., aflibercept, ranibizumab, pegaptanib, bevacizumab, brolucizumab, faricimab, AKB-9778, nesvacumab, and Bl 836880.

[0085] The therapeutic compound and the second therapeutic compound can be administered simultaneously, successively, or separately.

[0086] Therapeutic compounds for treating conditions associated with cone degeneration.

[0087] The present disclosure provides therapeutic compounds that are effective against conditions associated with cone photoreceptor degeneration. The compounds disclosed herein are notably capable of activating, in particular specifically activating, the Xc- system and / or the GPX4 enzyme in cone photoreceptors. Indeed, the present disclosure demonstrates that inhibition of the Xc- system and / or the GPX4 enzyme (in cone photoreceptors) induces ferroptosis in cone photoreceptors, eventually resulting in a phenotype similar to the in vivo pathological situation. Moreover, this can be reversed by activators of Xc- and / or GPX4 such as cystine.

[0088] Accordingly, the therapeutic compounds for use in the methods disclosed herein are preferably inhibitors of ferroptosis.

[0089] Therapeutic compounds disclosed herein include notably chemical and biological agents (e.g., an agent, including a small molecule drug or biologic, such as an antibody or cell) useful in the treatment of conditions associated with cone photoreceptor degeneration, wherein administration of this compound results in activation of the Xc- system and / or the GPX4 enzyme.

[0090] Examples of biological agents comprise the SLC7A11, SLC3A2, GPX4, DKK1 and / or OTUB1 gene products. DKK1 stimulates SLC7A11 expression, whilst OTUB1 stabilises SLC7A11 (Liu et al. Cancer Res. 79(8):1913-1924, 2019; Wu et al. Wat Commun. 13(1 ) : 1371 , 2022). DKK1 (Genbank ID No.: NMJ312242) encodes a 266-amino acid protein (Genbank ID No.: NPJ336374) which is eventually matured and secreted. 0TUB1 (Genbank ID No.: NMJ317670) encodes a 271 -residue amino acid (Genbank ID No.: NP_060140). Other examples of biological agents include DKK1 and / or OTUB1 polynucleotides. Preferably, the polynucleotide used herein is inserted within a vector, e.g., a viral vector such as e.g., an adenovirus-associated (AAV) vector, including AAV8, AAV2, AAV2tYF, and AAV5. Preferably, the polynucleotide is operably linked to regulatory sequences. The regulatory sequences are advantageously suitable for expression in the retinal or the subretinal tissue. More preferably, the polynucleotide disclosed herein is operably linked to adequate regulatory sequences in a vector.

[0091] Delivery may be accomplished via gene therapy, e.g., by administering vector as disclosed herein encoding an therapeutic product to the suprachoroidal space, subretinal space (with vitrectomy, or without vitrectomy (e.g., with a catheter through the suprachoroidal space, or via peripheral injection), intraretinal space, and / or outer surface of the sclera (i.e., juxtascleral administration) in the eye(s) of a human patient, to create a permanent depot in the eye that continuously supplies the therapeutic product ( e.g., a post-translationally modified therapeutic product)

[0092] Alternatively, the compound of the disclosure is a small molecule. Example of small molecules which activate the Xc- system and / or the GPX-4 enzyme include notably cystine, B-mercaptoethanol, selenium, bardoxolone, carvacrol (CAR), rehmanniosideA, bioflavonoids including galangin, xanthohumol, naringenin, britanin, entacapone, capsiate, resveratrol, dexmedetomidine, irisin, 2-cyano-3,12-dioxoolean- 1 ,9-dien-28-oic acid (CDDO), GW 5074, kaempferol, 1 -O-hexyl-2,3,5- tri methyl hydroquinone, 1 ,3-dicaffeoylquinic acid, rosmarinic acid, nobergenin and tinoridine hydrochloride.

[0093] Preferably, the compound disclosed herein is selected in the group consisting of DKK1 , OTUB1 , cystine, B-mercaptoethanol, selenium, bardoxolone, carvacrol (CAR), rehmanniosideA, bioflavonoids including galangin, xanthohumol, naringenin, britanin, entacapone, capsiate, resveratrol, dexmedetomidine, irisin, 2-cyano-3,12-dioxoolean- 1 ,9-dien-28-oic acid (CDDO), GW 5074, kaempferol, 1 -O-hexyl-2,3,5- tri methyl hydroquinone, 1 ,3-dicaffeoylquinic acid, rosmarinic acid, nobergenin and tinoridine hydrochloride. More preferably, the compound disclosed herein is selected in the group consisting of DKK1 , OTUB1, cystine, B-mercaptoethanol, selenium, bardoxolone, carvacrol (CAR), rehmanniosideA, bioflavonoids including galangin, xanthohumol, naringenin, britanin, entacapone, capsiate, resveratrol, dexmedetomidine, irisin, 2-cyano-3,12-dioxoolean-1 ,9-dien-28-oic acid (CDDO), GW 5074 and kaempferol.

[0094] Conditions associated with cone photoreceptor degeneration

[0095] The present disclosure provides a method for treating conditions associated with cone photoreceptor degeneration. A condition associated with cone photoreceptor degeneration as disclosed herein is condition in which specific cone degeneration is observed, i.e., a condition in which cone photoreceptor degeneration is not a secondary consequence of the degeneration of the retinal pigment epithelium.

[0096] In some instances, cone photoreceptors will be the only cells affected by degeneration. In some instances, other cell types may also be subject to degeneration. For example, in some instances, the conditions associated with cone photoreceptor degeneration may also present degeneration of adjacent supporting tissue, such as e.g., the retinal pigment epithelium. It must be understood that whenever the retinal pigment epithelium is also subject to degeneration, cone photoreceptors are nonetheless specifically affected by degeneration, i.e., cone photoreceptor degeneration is not a secondary consequence of the degeneration of the retinal pigment epithelium. In preferred instances, the other cell types affected by degeneration in the conditions associated with cone photoreceptor degeneration described herein include not only the adjacent supporting tissue, such as e.g., the retinal pigment epithelium, but also the retinal neural cells, including e.g., rod photoreceptors, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. In this very specific instance, whenever any of these other cell types are also subject to degeneration, cone photoreceptors are nonetheless specifically affected by degeneration, i.e., cone photoreceptor degeneration is not a secondary consequence of the degeneration of one or more of the other cell types.

[0097] In particular, conditions associated with cone photoreceptor degeneration include age-related macular degeneration (AMD), cone dystrophies, cone-rod dystrophies, rod-cone dystrophies, macular dystrophy (e.g. Stargardt disease), central serous chorioretinopathy, Best disease and bestrophinopathies, retinal detachment including rhegmatogenous, serous and fractional causes, solar retinopathy, laser- induced retinopathy, achromatopsia, Usher syndrome, Leber congenital amaurosis, Alstrom syndrome, Refsum disease, etc.

[0098] Preferably, the condition associated with cone photoreceptor degeneration of the methods disclosed herein are selected in the group consisting of age-related macular degeneration (AMD), cone dystrophies, cone-rod dystrophies, rod-cone dystrophies, macular dystrophy (e.g. Stargardt disease), central serous chorioretinopathy, Best disease and bestrophinopathies, retinal detachment including rhegmatogenous, serous and fractional causes, solar retinopathy, laser-induced retinopathy, achromatopsia, Usher syndrome, Leber congenital amaurosis, Alstrom syndrome, and Refsum disease. More preferably, the condition associated with cone photoreceptor degeneration of the methods disclosed herein is age-related macular degeneration (AMD). In another more preferred instance, the condition associated with cone photoreceptor degeneration of the methods disclosed herein is a cone dystrophy. In another more preferred instance, the condition associated with cone photoreceptor degeneration of the methods disclosed herein is retinal detachment.

[0099] Conditions associated with cone photoreceptor can be diagnosed by any technique or method known to the skilled person.

[0100] Standardised ophthalmic examination techniques known in the art include, for example, a detailed slit lamp biomicroscopic evaluation which allows evaluation of the lids, ocular adnexa, lashes, corneal surface, anterior chamber, pupils, lens, vitreous cavity and central retinal anatomy including the optic nerve and macula. Another method is gonioscopy which allows detailed examination of the anterior chamber angle. Indirect ophthalmoscopy allows for evaluation of the retinal periphery, important in the monitoring of vitreous and peripheral retinal disorders.

[0101] Functional tests of visual acuity are known in the art and include, for example, best corrected acuity, contrast acuity, and low luminance acuity, colour vision (including Ishihara and Farnsworth Munsell tests) and visual field evaluation (including Flumphrey automated perimetry and microperimetry), tear production (Schirmer test), and tonometry to measure intraocular pressure (IOP). These are used in conjunction with structural tests, which include, for example, anterior and posterior segment photographs, corneal pachymetry, ultrasound, ultrasound biomicroscopy, optical coherence tomography (OCT), optical coherence tomography angiography (OCTA), fluorescence angiography (FA), intravenous fluorescein angiography (IVFA), and fundus autofluorescence (FAF). Imaging such as computerised tomography (CT) or magnetic resonance imaging (MRI) scans are utilised to evaluate ocular, periocular and orbital structures, and the intracranial portion of the optic nerve, visual pathway and visual cortex in the brain. These tests allow visualisation of structural integrity and thickness of the layers of the eye and surrounding structures, and assessment of blood flow and circulation. Advanced functional testing of the retina, optic nerve and visual pathway / cortex is also used, including electrophysiologic tests such as full field and multifocal electroretinography, visual evoked potentials and microperimetry to diagnose and monitor disease progression and impact of therapy. The person of skill in the art will be able to deploy the appropriate methodologies known in the art to diagnose, measure and monitor the conditions associated with cone photoreceptor degeneration described herein.

[0102] Pharmaceutical compositions

[0103] The present compounds for treating conditions associated with cone photoreceptor degeneration can be formulated in compositions. Optionally, the compositions can comprise one or more additional therapeutic agents, such as the second therapeutic agents described below. The compositions will usually be supplied as part of a sterile, pharmaceutical composition that will normally include a pharmaceutically acceptable carrier and / or excipient. In another aspect, the disclosure thus provides a pharmaceutical composition comprising the compound for treating conditions associated with cone photoreceptor degeneration and a pharmaceutical acceptable vehicle and / or an excipient.

[0104] Accordingly, provided herein is a pharmaceutical composition comprising one or more compound for use in treating conditions associated with cone photoreceptor degeneration, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic and / or prophylactic ingredients. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition. A pharmaceutically acceptable or suitable composition includes an ophthalmologically suitable or acceptable composition. This composition can be in any suitable form (depending upon the desired method of administering it to a patient). The compositions utilised in the methods described herein can be administered, for example, intravitreally (e.g., in the subretinal space, the suprachoroidal space, the anterior chamber, the vitreous humour, the subconjunctival space, or on the corneal surface), by eye drop, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intra tracheally, intra thecally, intranasally, intravaginally, intrarectally, topically, intra tumourally, peritoneally, subcutaneously, subconjunctivally, intra vesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localised perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions.

[0105] The compositions utilised in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). The most suitable route for administration in any given case will depend on the particular therapeutic compound, the subject, and the nature and severity of the disease and the physical condition of the subject. The compound for treating conditions associated with cone photoreceptor degeneration can be administered by systemic administration (inhalator, intravenous, intramuscular or intraperitoneal) or under solution, gel or implant.

[0106] The compositions described herein may be typically formulated as liquid or fluid compositions, semisolids (e.g., gels or hydrogels), foams, or porous solids (e.g., polymeric matrices, composites, calcium phosphate derivatives, and the like, as appropriate for ophthalmic tissue engineering). Pharmaceutical compositions can be conveniently presented in unit dose forms containing a predetermined amount of the therapeutic compound disclosed herein per dose. Such a unit can contain for example but without limitation 5 mg to 5 g, for example 10 mg to 1 g, or 20 to 50 mg. Pharmaceutically acceptable carriers for use in the disclosure can take a wide variety of forms depending, e.g., on the condition to be treated or route of administration. The dosage forms can notably be 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, ophthalmic bioerodible implant, and non- bioerodible ophthalmic inserts or depots, nasal sprays and ointment, various rectal or vaginal preparations.

[0107] Pharmaceutical compositions of the disclosure can be prepared for storage as lyophilised formulations or aqueous solutions by mixing the compound having the desired degree of purity with optional pharmaceutically-acceptable carriers, excipients or stabilisers typically employed in the art (all of which are referred to herein as “carriers”), i.e., buffering agents, stabilising agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, Remington’s Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipients at the dosages and concentrations employed.

[0108] Buffering agents help to maintain the pH in the range which approximates physiological conditions. They can be present at concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid- monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid- disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid- potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used. Preservatives can be added to retard microbial growth, and can be added in amounts ranging from 0.2%-1% (w / v). Suitable preservatives for use with the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides (e.g. , chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonicifiers sometimes known as “stabilisers” can be added to ensure isotonicity of liquid compositions of the present disclosure and include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilisers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilises the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilisers can be polyhydric sugar alcohols (enumerated 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 and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, o- monothioglycerol and sodium thio sulfate; low molecular weight polypeptides (e.g. , peptides of 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccacharides such as raffinose; and polysaccharides such as dextran. Stabilisers can be present in the range from 0.1 to 10,000 weights per part of weight active protein.

[0109] Non-ionic surfactants or detergents (also known as “wetting agents”) can be added to help solubilise the therapeutic compound as well as to protect the therapeutic compound against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc. ), polyoxamers (184, 188, etc. ), pluronic polyols, polyoxyethylene sorbitan monoethers (TWEENO-20, TWEENO-80, etc. ). Non-ionic surfactants can be present in a 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.

[0110] Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0111] The present disclosure is further directed to a pharmaceutical composition comprising at least: i) a therapeutic compound as described herein, i.e., a compound for treating conditions associated with cone photoreceptor degeneration, and ii) a second therapeutic agent, for example as described below, as combination products for simultaneous, separate or sequential use.

[0112] Compositions of therapeutic compound and a second therapeutic agents can be administered singly, as mixtures of one or more therapeutic compounds and / or one or more a second therapeutic agent, in mixture or combination with other agents useful for treating conditions associated with cone photoreceptor degeneration. Examples of suitable combinations are provided below.

[0113] Encompassed by the present disclosure are pharmaceutical kits containing therapeutic compounds described herein. The pharmaceutical kit is a package comprising a compound for treating conditions associated with cone photoreceptor degeneration (e.g., either in lyophilised form or as an aqueous solution) and one or more of the following:

[0114] • A second therapeutic agent, for example as described below;

[0115] • A device for administering the immune checkpoint inhibitor, for example a pen, needle and / or syringe; and

[0116] • Pharmaceutical grade water or buffer to resuspend the inhibitor if the inhibitor is in lyophilised form.

[0117] Each unit dose of the therapeutic compound can be packaged separately, and a kit can 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 a specific embodiment, the one or more unit doses are each housed in a syringe or pen. Effective dosages

[0118] The therapeutic compounds as described herein, i.e., compounds for treating conditions associated with cone photoreceptor degeneration, will generally be used in an amount effective to achieve the intended result, for example an amount effective for preventing or decreasing cone photoreceptor degeneration by using any of the methods described above. Pharmaceutical compositions comprising the therapeutic compounds can be administered to such patients (e.g., human subjects) at therapeutically effective dosages.

[0119] The term “therapeutically effective dosage” means an amount of active compound or conjugate that elicits the desired biological response in a subject. Such response includes alleviation of the symptoms of the disease or disorder being treated, prevention, inhibition or a delay in the recurrence of symptom of the disease or of the disease itself, or prevention, inhibition or delay in the progression of symptom of the disease or of the disease itself. More specifically, a “therapeutically effective” dosage as used herein is an amount that confers a therapeutic benefit. A therapeutically effective dosage is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.

[0120] In the context of the present disclosure, a therapeutically effective amount refers to an amount of the therapeutic compound disclosed herein that is sufficient to prevent or decrease cone photoreceptor degeneration.

[0121] Preferably, a therapeutically effective amount is an amount of the therapeutic compound which is sufficient to obtain at least one of the followings:

[0122] • maintaining or increasing cone photoreceptor viability;

[0123] • preventing an increase in cell membrane permeability to dyes in cone photoreceptors;

[0124] • maintaining or increasing intracellular glutathione level in cone photoreceptors;

[0125] • maintaining or decreasing intracellular NADP / NADPH ratio in cone photoreceptors;

[0126] • preventing or reducing glial migration;

[0127] • preventing or reducing subretinal deposit formation; and maintaining or increasing cone photoreceptor activity.

[0128] Preferably, a therapeutically effective amount as disclosed herein is sufficient to obtain at least 2, at least 3, at least 4, at least 5, at least 6 of the above features. More preferably, a therapeutically effective amount as disclosed herein is sufficient to obtain all 7 above features.

[0129] Effective dosages can be estimated initially from in vitro assays. For example, an initial dose may be formulated to achieve a concentration of the present therapeutic compound that is at least the concentration of the compound capable of inhibiting cone photoreceptor degeneration in an in vitro or ex vivo assay, such as those described in the present examples. Calculating dosages to achieve such concentrations in the eye taking into account the bioavailability of the particular compound is well within the capabilities of skilled artisans. For guidance, the reader is referred to Fingl & Woodbury, “General Principles” in Goodman and Gilman’s The Pharmaceutical Basis of Therapeutics, Chapter 1 , latest edition, Pagamonon Press, and the references cited therein.

[0130] Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Toxicity and therapeutic efficacy of a compound or a conjugate can be determined by standard pharmaceutical procedures in cell cultures and in experimental animals. The effective amount of present therapeutic compound or second therapeutic compound to be administered to a subject will depend on the stage, category and status of the multiple myeloma and characteristics of the subject, such as general health, age, sex, body weight and drug tolerance. The effective amount of the present therapeutic compounds or second therapeutic compound to be administered will also depend on administration route and dosage form. Dosage amount and interval can be adjusted individually to provide levels in the eye of the active compound that are sufficient to maintain desired therapeutic effects.

[0131] The amount of the 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 therapeutic regimen (e.g., whether another therapeutic compound is used), the age and condition of the particular subject being treated, the sensitivity of the patient being treated to the present therapeutic compounds disclosed. The appropriate dosage can be readily determined by a person skilled in the art. Ultimately, a physician will determine appropriate dosages to be used. This dosage can be repeated as often as appropriate. If side effects develop the amount and / or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice. The proper dosage and treatment regimen can be established by monitoring the progress of therapy using conventional techniques known to the people skilled of the art.

[0132] The effective dose of a therapeutic compound as described herein can range from about 0.001 to about 75 mg / kg per single (e.g., bolus) administration, multiple administrations or continuous administration, or to achieve a serum concentration of 0.01 -5000 pg / ml serum concentration per single (e.g., bolus) administration, multiple administrations or continuous administration, or any effective range or value therein depending on the condition being treated, the route of administration and the age, weight and condition of the subject. In a certain instance, each dose can range from about 0.5 pg to about 50 pg per kilogram of body weight, for example from about 3 pg to about 30 pg per kilogram body weight.

[0133] Amount, frequency, and duration of administration will depend on a variety of factors, such as the patient’s age, weight, and disease condition.

[0134] In various instances, the period of treatment is at least one day. Preferably, the period of treatment 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, is at least one week. Preferably, the period of treatment 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 up to about 20 years. In embodiment, the treatment period comprises 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 about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, or about 24 months. In certain instances, the treatment can continue for the lifetime of the patient following diagnosis of a condition associated with cone photoreceptor degeneration. Other characteristics and advantages of the disclosure appear in the continuation of the description with the examples and the figures whose legends are represented below.

[0135] EXAMPLES

[0136] EXAMPLE 1

[0137] METHODS

[0138] Data availability

[0139] Animal model: rats

[0140] Eight-weeks-old Long Evans wild-type rats were obtained from Janvier Labs (ISO 9001 certification). Controls and treated were administered a subretinal injection at day 0 (DO) of either 0.2% DMSO (D2650, Millipore Sigma, Burlington, MA) in PBS (14190- 094 Life Technologies Europe B.V., Bleiswijk, The Netherlands) or 0.2% of a 20pM IKE in DMSO solution diluted in PBS, respectively.

[0141] Surgical and dissection procedures in rats

[0142] Rats were anesthetized by an intraperitoneal (IP) injection of 40mg / kg ketamine (Ketamidor 100mg / ml, Axience SAS, Pantin, France) and 0.14mg / kg medetomidine (Domitor 0.85mg / ml, Vetoquinol S.A., Paris, France). Local anesthesia was achieved with oxybuprocaine eyedrops (1.6mg / 0.4ml, Thea, Clermont-Ferrand, France). Mydriasis in the left eye was obtained with 0.5% tropicamide eyedrops (Mydriaticum, Thea, Clermont-Ferrand, France). Tear gel (Lubrithal, Dechron, Shrewsbury, UK) was used as a lens-eye interface during the whole procedure. Using an ophthalmic microscope (Lumera 700, Carl Zeiss, Oberkochen, Germany), a conjunctivoscleral channel was created with a 30 Gauge (G) needle (BD Microlance 3, Becton, Dickinson S.A., Fraga, Spain). A flat contact lens (Cover slips, mini, 8mm, World Precision Instruments, Sarasota, FL) allowed for visualisation of the retinal plane. Nasal subretinal delivery of 9pL of control or IKE solution was performed using a 30G cannula with a nonbeveled metal tip on a 10pL syringe (Hamilton, Reno, NV) mounted on a microinjector (Micro 4, World Precision Instruments, Sarasota, FL). After surgery, chloramphenicol-retinol eye ointment (Ophtalon 10mg / g, TVM, Lempdes, France), IP injection of 1 mL of 5% glucose monohydrate (Osalia, Paris, France) and subcutaneous injection of 0.9mg / kg atipamezole (Antidorm 4.27mg / ml, Axience SAS, Pantin, France) were administered. In vivo anatomical investigations, i.e., optical coherence tomography (OCT, Bioptigen, Durham, NC) and funuds photography (Micron IV, Phoenix-Micron, Inc., Bend, OR), were performed at DO to ensure the subretinal delivery and the day of the sacrifice to confirm retinal reattachment. Sacrifice, using intracardiac injection of 1 ml / kg pentobarbital (Exagon, Axience SAS, Patin, France), was performed at day 6 (D6) for one group of animals (n=4) to assess microglial migration, and at D30 (D30) for one group (n=8) to assess 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. Globes were dissected to obtain whole retinae, immersed in a 24-well plate (#351147 Corning Inc., Corning, NY) for immunohistochemistry. In addition, labelled eyecup slices (with CAR and Iba1 ) were performed to assess another quantitative analysis. To do so, eyes were collected from rats and the cornea was removed. Then, eyes were immerged in 4% PFA for 1 h before successive sucrose baths from 10% to 30%. Lastly, lens was gently removed and the eye cup placed into a freezing medium in liquid nitrogen. They were stocked at -20° C until to be cut into slices of 12pm with a cryostat (CM3050 S, Leica, Wetzlar, Germany).

[0143] Mammalian eyes

[0144] Porcine eyes were obtained from a local slaughterhouse in agreement with the local regulatory department and the veterinarians from the French Ministry of Agriculture (agreement FR75105131 ).

[0145] Pure cone photoreceptors

[0146] Pig eyes were dissected to obtain retinae that were cut into pieces and then digested by papain at 4U / mL (LSO 3124, Worthington,) and L-Cysteine (5.5 mM, Sigma- Aldrich) for 20 minutes at 37° C. The enzyme activity was stopped by Neurobasal-A (NBA) medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) which contained 5% Fetal Bovine Serum (FBS) and DNase I at 15pg / ml (D4263, Millipore Sigma, Burlington, MA). A series of short centrifugations (30sec, 5 to 6 time) was performed in order to collect supernatant excluding the first one which contains mainly rod photoreceptors. Pellets were resuspended with a P1000 to dissociate the remaining cells between each centrifugation. Afterwards, the cell suspension was centrifuged for 10 minutes at 800 rpm and pellets were resuspended in NBA medium containing 1% of L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, ThermoFisher Scientific, Waltham, MA, USA).

[0147] Cone PR were purified by lectin-panning selection as previously described (Balse et al. (2005) IOVS 46:367), with slight modifications to obtain millions of cones in a suspension. The cells were then seeded in 384 well plates (781091 , Greiner, Ulis, France) to get 3000cells / well using a robot (Viaflo Assist, Integra-Biosciences, Saint- Ouen, France) and incubated at 37° C and 5% of CO2 for 3 days.

[0148] Retinal explants

[0149] Porcine and non-human primate retinae were cultivated as previously described (Greferath et al. (2016) Ophthalmology 123:1320). After isolation of retinae in CO?- independent medium, pieces from the region behind the optic nerve were obtained using biopsy punches of 2mm, were placed on polycarbonate membrane (140652, Thermo, Waltham, Massachusetts) and photoreceptors were turned upwards. The explants were kept in culture in a CO? incubator at 37° C for 3 days.

[0150] Treatments

[0151] To induce ferroptosis, the following components were used: Glutamate (49621 , Sigma Aldrich, Missouri, USA) between 1 pM to 500pM in vitro, Imidazole Ketone Erastine (IKE) (HY-114481 , MedChemExpress, Monmouth, USA) either at 3pM on purified cones photoreceptors or at 20pM for ex vivo and in vivo experiments, and RSL3 (HY- 114481 , MedChemExpress, Monmouth, USA) either at 20nM, 200nM and 10pM respectively for cells, ex vivo and in vivo experiments. To inhibit ferroptosis, Ferrostatin-1 (FST-1 , HY-100579, MedChemExpress, Monmouth, USA) was used at 50nM on purified cones or at 400nM on retinal explants. Also, Deferiprone (HY-B0568, MedChemExpress, Monmouth, USA) was efficient at 50pM on pure cones and Zileuton (HY-14164, MedChemExpress, Monmouth, USA) at 20pM on purified cones.

[0152] Cell viability measurement

[0153] After 3 days of incubation, cell viability was measured with Calcein (C1430, Thermo, Waltham, Massachusetts) at 1 / 4000 which is incubated for 1 hour at 37° C. The number of viable cells was calculated with an automated fluorescence microscope, ArrayScan (Cellomics ArrayScan VTI HCS reader, Thermo, Waltham, AAA).

[0154] Screening on primary cells

[0155] Purified cone photoreceptors were seeded into 384-well plates with clear flat bottom (781091 , Greiner, Ulis, France) by a robot having a 384-well head (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) in order to have 4*103 cells / well. In the first two columns, cells were treated with only DMSO (D2650, Millipore Sigma, Burlington, MA) and in the rest of the plate cells were treated with, either RSL3 (HY- 114481 , MedChemExpress, Monmouth, USA) at 20nM final or IKE (HY-114481 , MedChemExpress, Monmouth, USA) at 3pM final. 17 hours after the induction of cell degeneration, a ferroptosis library compounds (HY-L051 , MedChemExpress, Monmouth, USA) was added to cells by a robot having a 384-well head (BRAVO, Assist, Integra- Biosciences, Saint-Ouen, France) in order to obtain a final concentration at 10pM of compounds. Each treatment was diluted into neurobasal medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) and 1 / 100 of L-glutamine (G3126, Millipore Sigma, Burlington, MA). The cells were then incubated for another 2 days at 37° C and 5% CO?. Then on the 3rdday after seeding, cell viability was measured.

[0156] Results were analysed with TIBCO Spotfire® software (California, USA). The robust Z’ factor was calculated for each plate, and those above 0.45 were selected. Then, for each plate, the percentage of live cells was calculated from the positive controls. The average number of cells counted is calculated and reported at 100%. This ratio of positive controls was reported to all the molecules in the plate. Next, all the plates from the same treatment (IKE or RSL3) were displayed in order to select the compounds having a cell survival higher than 50% and we extract these compounds on Excel.

[0157] Li pi domic analysis

[0158] Purified cone photoreceptors either treated or not with 3pM IKE were washed 3 times with PBS, scrapped in 100pL and snap-frozen with liquid nitrogen immediately after collection and stored at -80° C until extraction. Extraction and analysis of eicosanoids was performed by the lipidomic facility core: MetaToul-Lipidomique (I2MC, Inserm, Toulouse, France), MetaboHUB-ANR-1 1— INBS-0010. Western blot

[0159] Cells were collected, separated from medium by centrifugation and whole cellular proteins were extracted in lysis buffer (10mM HEPES [pH 7], 100mM NaCl, 2mM EDTA, 0.5% NP-40 and protease inhibitor cocktails (Millipore Sigma, Burlington, MA). After a 13000rpm centrifugation, the supernatant was kept for further WB analysis. Samples were then loaded in 4% to 15% Mini-PROTEAN TGX Tris-glycine buffer SDS- PAGE and transferred onto a 0.2pm Trans-Blot Turbo nitrocellulose membrane (BioRad, California, USA). Membranes were blocked for 1 hour at room temperature in 1 x Tris-Buffer Saline (TBS, 10mM Tris-HCl [pH 8], 150 mM NaCl) supplemented with 5% (w / v) dried skim milk powder. Primary antibody incubation was carried out overnight at 4°C. The presence of 5LOX and FLAP in total lysates at the expected molecular weight was confirmed by Western blotting with anti-5LOX (1 :200; Abeam AB169755) and anti-FLAP (1 :500; Abeam AB85227). The secondary HRP-coupled antibody used was goat anti-rabbit (1 :20,000; 111 -035-003, Jackson ImmunoResearch Laboratories, Pennsylvania) for detection of both 5LOX and FLAP. In between and after antibodies incubations, membranes were extensively washed in TBS containing 2.5% Tween-20 (TBS-T). Western blots were visualised using the enhanced chemiluminescence method (ECL Prime detection reagent, Amersham, UK).

[0160] NADPH assay

[0161] The NADP / NADPH assay kit (ab176724, Abeam, Cambridge, UK) was used to quantify the level of NADP and NADPH respectively according to the manufacturer’s instructions.

[0162] Glutathione (GSH) assay

[0163] We quantified the level of GSH according to the manufacturer’s instruction for GSH assay kit (V6912, Promega, Madison, Wl).

[0164] Cryostat sections

[0165] After 3 days of incubation, retinal explants were fixed with 4% paraformaldehyde (15714, Electron Microscopy Sciences, Hatfield, Pennsylvania) for 1 h at room temperature and washed 3 times with PBS. Then, retinal explants were conserved at +4°C or transversally sectioned. The last were cryopreserved in successive sucrose baths of 10%, 20% and 30%. Then, samples were frozen in a tissue freezing medium (72592, Electron Microscopy Sciences, Hatfield, Pennsylvania) by immersion in liquid nitrogen until the tissue freezing medium appeared stiff. Samples were conserved at -20° C until cutting in transversal slices of 10pm with a cryostat (CM3050 S, Leica, Wetzlar, Germany). Retinal cross sections were conserved at -20° C. Immunolabelling

[0166] Samples (fixed retinal explants and retinal cross-sections) were firstly permeabilised with 0.5% Triton 100X (T8787, Sigma-Aldrich, Missouri, USA) diluted in PBS 1 x. Then, unspecific areas were blocked with a saturated buffer composed of 10% of Normal Donkey Serum (S30-100ML, Millipore Sigma, Burlington, MA) diluted in PBS 1 x. The primary and secondary antibodies used in this study are listed in table 1 .

[0167] Table 1. List of antibodies.

[0168] The PNA lectin diluted at 1 / 50 (L21409, Thermo, Waltham, Massachusetts) and rabbit FLAP antibody with a working concentration at 5pg / mL (ab85227, Abeam, Cambridge, UK) are targeting antigenic sites that are at the extracellular side of the cell membrane. For those labelling, no permeabilization step was done.

[0169] Finally, retinal explants were washed 3 times in PBS and flat-mounted with Permafluor (TA-030-FM, Thermo, Waltham, Massachusetts) in Cellvis plates (P12-1.5H- N, IBL, Gerasdorf, Austria) for fluorescence microscopy. Retinal transversal slices and whole-mounted retinae were also mounted with Permafluor on coverslips or in a 6-well plate (P06-1.5H-N, Cellvis, Mountain View, CA), respectively.

[0170] Confocal microscopy

[0171] Retinae were imaged using a laser-scanning confocal microscope (Fluoview V- 1000, Olympus, Tokyo, Japan). In rats’ retinae, cone counting (outer segments and perikaryon per mm) was performed manually on retinal slices.

[0172] CQ1 imaging

[0173] The CQ1 confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan) was used to image whole-mounted retinae of rats and flat mounted porcine explants. For injected rats’ retinae, fields of the inferonasal and infero temporal petals were manually reviewed in 4 z-axis planes defined by the cone labelling: the outer segments (OS) plane, the cone nucleus (CN) plane, the inner fibre (IF) plane and the cone pedicle (CP) plane. Emphasis was given to microglial migration. Representative samples were extracted from the CQ1 software. Z stack images were analysed using Cell pathfinder software (Yokogawa, Tokyo, Japan) which enables 2D, 3D or slice quantification. Algorithms were generated (one for the porcine explants and one for the whole- mounted retinae of rats) to quantify cones perikarya or outer segments and microglial cells according to the z axis. Extracted quantification data were analysed using TIBCO Spotfire® (California, USA).

[0174] Non-human primates (NHP)

[0175] Two NHP were used in this study: a 5-year-old male and a 15-year-old female. Animals were born in captivity and originated from approved suppliers with AAALAC certification (SARL Bioprim, Bazieges, France; Cynologics-Silabe, Niederhausbergen, France). Ethical approvals were obtained from the local ethical committee CETEA n°44 of MIRCen and then the French Ministry of Education and Research. A full description of housing and perioperative care is available in Dentel et al. (2023) Ophthalmol. Sci. 3:100316.

[0176] IKE toxicity in NHP was assessed by a subretinal delivery of a 20pM IKE solution in one eye and a control solution (0.2% DMSO diluted in PBS) in the fellow eye. A comprehensive description of anaesthesia, surgery and data acquisition is available in Dentel et al. Briefly, the subretinal injection was performed in order to detach the superior hemifovea in each eye. Anatomical (slit-lamp exam, optical coherence tomography -OCT-, adaptive optics -AO-) and functional (full-field and multifocal electroretinogram, ffERG and mfERG, respectively) investigations were performed at baseline (within a week prior to surgery), at day 3 (to confirm that retinal reattachment was completed), at one month, and then monthly until month 4. The embedded software of the rtx1 (AOdetect, Imagine Eyes, Orsay France) was used to quantify adaptive optics cone mosaic parameters. Also, the additionally used power spectrum spacing method to quantify cone mosaic in NHP is detailed in Dentel et al.

[0177] Analyses and statistics

[0178] Analyses, graphs and charts were achieved using GraphPad Prism 8.4.0 Software (GraphPad Software Inc., San Diego, CA). Treatments and controls were statistically compared with the Mann-Whitney t-test and were considered to be significant when p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) or p < 0.0001 (****).

[0179] In charts, data were normalised to controls when possible and represented as mean + / - standard error of the mean. The Mann-Whitney t-test was used to compare controls with treatments in the in vitro and ex vivo analyses. In rats, differences in cone density and microglial density were compared using a multiple t-test and statistical significance determined using the Holm-Sidak method. In NHPs, differences in cone density, regularity, spacing values and ERG amplitude waves were compared using a two-tailed t test. For mfERG in NHP, each hexagon was compared with the contralateral matching hexagon, considering the symmetry between the two eyes.

[0180] RESULTS

[0181] Glutamate-elicited ferroptosis in purified cone photoreceptors

[0182] Photoreceptors release their neurotransmitter, glutamate, at their synaptic terminals but they only express glutamate transporters (not classic ionotropic receptors). When applying glutamate on purified porcine cone photoreceptors (Fig. 1 A- D), their viability decreased by 20% already with 10pM; and up to 82% at the highest tested concentration of 500pM (Fig. 1 E). This range of glutamate is encountered physiologically as indicated by the glutamate affinity of receptors and transporters (Gielen (2010) medecine / sciences 26:65). To define if this toxicity can be related to the expression of the system Xc anti porter, which has been reported in cone photoreceptor by immunocytochemistry (Dun et al. (2006) Cell Tissue Res 324:189; Bridges et al. (2004) Ophthalmol. Vis. Sci. 45:2906-2914), we applied glutamate in the presence of 1 mM L-cystine. At every glutamate concentration, L-cystine prevented the toxicity of glutamate (n=7, p<0.0001 , IC50 = 4.15pM) (Fig. 1 E). Because the system Xc transporter has a very distinct pharmacology from other glutamate transporters or receptors, we examined how the different agonist and antagonist of this transporter affected cone survival. Erastin and imidazole ketone erastin (IKE), two functional inhibitors of the System Xc, produced toxicity on isolated cone photoreceptors, similarly than with glutamate but in a lower dose range in accordance with their greater affinity (fig. 1A-D, F). L-Cystine also rescued cones exposed to IKE (n=6, p=0.0022) (Fig. 1 K).

[0183] To assess the molecular mechanisms that underlie this glutamate-elicited toxicity in cone photoreceptors, we measured GSH levels in cones exposed to 500pM glutamate and imidazole ketone erastin (IKE 3pM). A 50% decrease in GSH at day 1 and later was observed under both conditions when compared to a control condition (Fig. 1 F, n=18, p<0.0001 ) . Cone photoreceptor cell death is therefore due to a dysregulation of the cellular levels of GSH, induced by a System Xc dysfunction. Knowing that GSH is involved in a redox cycle where its oxidized form is reduced with a hydrogen atom carried by NADPH (Dixon et al. (2019) Annu. Rev. Cancer Biol. 3:35), we measured the NADPH / NADP ratio in cones treated with 3pM IKE. This condition increased the amount of NADPH from 25% in controls to 85% in treated cultures (n=4, p=0.0159) (Fig. 1G). The intracellular NADPH / NADP ratio is indirectly related to the activity of glutathione peroxidase 4 (GPX4) during the oxidation reduction of glutathione. When GPX4 becomes inactive, this cycle also becomes inactive and can induce this accumulation of NADPH suggesting that the redox cycle is downregulated when the System Xc is inhibited (Azuma et al. (2022) J. Biol. Chem. 298:101824). To investigate if GSH is crucial for cones survival, a specific inhibitor of GPX4, Ras-selective lethal 3 (RSL3), was used. This inhibitor showed a toxic effect on cone photoreceptor at very low concentrations (n=3, IC50 =8.67nM) (Fig. 1 H), highlighting the key role of GPX4 in cone survival. RNA extraction from treated and control pure cone photoreceptors was then performed, and we showed that expressions of proteins involved in regulating the glutathione redox cycle and downstream mechanisms were altered (Fig. 2).

[0184] GPX4 is also known to prevent peroxided lipids formation that react with free radical species (Xie et al. (2016) Cell Death Differ. 23:369; Dixon et al. 2019). Those peroxided lipids are formed by lipoxygenases such as 5 -lipoxygenase (5LOX), which is activated by 5-lipoxygenase-activating protein (FLAP). To examine presence of these mechanisms in cone photoreceptors, western blots were performed, and we discovered the presence of both 5LOX and FLAP in cones but not in rod photoreceptors (Fig. 11). Specific immunostaining on retinal tissues from different species revealed the specific FLAP localisation in cone photoreceptors (Fig. 3). To assess the role of this peroxided lipid pathway in glutamate-elicited cell death, cone cell extracts were analysed by lipidomic after 2 days incubation with 3pM IKE. Analyses revealed enhanced amounts of peroxided lipids such as 5-Oxo-6,8,11 ,14-eicosatetraenoic acid (5-oxo-ETE) (n=6, p= 0.0079 (**), p= 0.0159 (*)) (Fig. 1 J), a metabolic product of 5LOX. To demonstrate the contribution of this lipid peroxidation in the observed glutamate-elicited toxicity, we tested the effects of Zileuton (ZEN), an enzymatic antagonist of 5LOX, in the presence of IKE. ZEN rescues 80% of cone photoreceptors (Fig. 1 K, n=3, p=0.0238), suggesting that lipid peroxidation plays a key role in glutamate-elicited cone degeneration.

[0185] Lipid peroxidation induces iron metabolism changes during ferroptotic processes. Ferritinophagy processes can be highlighted by immunolabelling ferritin and nuclear receptor coactivator 4 (NCOA4), a ferritinophagy inducer. Cones treated with IKE showed an increase in immunolabelling intensity of both ferritin heavy chain and NCOA4. Also, NCOA4 seemed to be slightly more intense than ferritin heavy chain, suggesting an increase of the activity of iron metabolism in treated cones (Fig. 4). Ferritinophagy processes can be inhibited by Ferrostatin-1 (FST1 , a specific ferroptosis inhibitor) and deferiprone (DF, an iron chelator preventing Fenton’s reaction by limiting intracellular accumulation of free iron). Both rescued cones from IKE-induced toxicity, by 68.5% for FST1 (n=8, p=0.0047) and by 77.2% for DF (n=5, p=0.0079) (Fig. 1K). In addition, other antiferroptotic molecules were tested after treatment with RSL3 at 20nM and IKE at 3pM, targeting different components involved in ferroptosis activation (Table 2). Thus, it seems that pure cones photoreceptors exposed to proferroptotic agents (e.g., IKE) can be rescued by antiferroptotic treatments, enhancing the role of ferroptosis in cone survival.

[0186] TABLE 2. Anti ferroptotic molecules after treatment by either 20nM RSL3 or 3 M IKE

[0187] A preliminary screening was performed on cone photoreceptors treated either by 20nM RSL3 or 3pM IKE. A day after inducing ferroptosis in cells, bank compounds were added at 10pM. Best hits were recorded in the table above, according to each treatment. Some compounds did not increase cell survival over 50% depending on treatment and were not reported in the table ( / ). Cells treated with only 20nM RSL3 or 3pM IKE showed only 30% and 25% of cell survival respectively, 3 days after treatments (n=3) .

[0188] Molecules involved in lipoxygenases pathway

[0189] Molecules involved in iron pathway

[0190] Ferroptosis of cone cells in an ex vivo retinal model

[0191] To show that cone photoreceptors can degenerate by ferroptosis in an integrated retinal model without retinal pigment epithelium, we applied IKE on freshly prepared porcine retinal explants. Specificity and selectivity of ferroptosis in retina was assessed by examining damaged cells (cell membrane permeability to ethidium dye) after incubation for 3 days. Whereas ethidium labelled few scattered cells in the control condition, the first row of photoreceptor nuclei was labelled by ethidium in the IKE-treated explants (Fig.5B,F). This outstanding layer corresponds to cone photoreceptors’ nuclei in the porcine retina (Pattnaik et al. (2000) J. Neurosci. 29:6789), as indicated by the Flap immunolabelling (Fig.5C,G). Figure 5G illustrates the morphological alterations of cone photoreceptors with 20pM IKE. Other types of retinal cells were also labelled, showing no major qualitative changes (Fig. 6). In situ cellular toxicity was then assessed in cone photoreceptors by counting (automatically with the CellPath software of the CQ1©) at the level of outer segments (OS) immunolabelled by Flap (Fig. 5J-M). 20pM IKE induced a 50% decrease of the amount of cone OS (n=3, p=0.0286) (Fig. 5I). 1pM FST-1 and 100pM DF rescued 84% and 78.4% of OS, respectively (n=3, p=0.1000 and p=0.0286, respectively) (Fig. 5I). Similarly, when cone degeneration was triggered by 200nM RSL3 in retinal explants (Fig.7), 100pM DF rescued 78.5% of OS (n=3, p=0.0286) (Fig. 7M). These data are suggestive for ferroptosis being an effective and selective pathway of cone photoreceptor degeneration in an integrated retinal tissue. Additionally, microglial migration from the inner retina (normal localisation) toward the outer retina (normally free of microglia) seemed to be triggered in retinal tissues exposed to ferroptosis inducers. Activation of microglial cells is suggested by their increased volume and modified shape, with cellular extensions embracing cones (Fig. 5H). Automated quantitative analyses revealed significant intraretinal microglial displacement toward the outer nuclear layer (ONL) triggered by both 20pM IKE and 200nM RSL3 (n=3) (Fig. 5N). The migration was partially prevented by ferroptosis inhibitors (FST-1 and DF) (n=3) (Fig. 50; Fig. 7N). This microglial cell morphological change and their migration toward the photoreceptor layers provide another evidence of cone degeneration by ferroptosis.

[0192] Cone degeneration and microglial migration in rats

[0193] To assess whether proferroptotic agents enable for initiation of degenerative and inflammatory processes in living retinae, ferroptosis inducers were injected in the subretinal space of Long Evans rats (20pM IKE, see Fig. 8A). At day 6, optical coherence tomography (OCT) showed numerous punctuate hyperreflectivities suggestive of microglial cells in rats injected with ferroptosis inducers (arrows in Fig 8G). The same retinae were then examined with cone and microglial immunostaining. Cone photoreceptors density was evaluated at day 30 on retinal slices through the injected area. OS and perikarya were substantially reduced in animals treated by ferroptosis inducers through subretinal delivery (Fig 8H-I). The quantification on retinal sections showed a reduction of perikarya approximately by one third, and of OS by almost a half with ferroptosis inducers (Fig. 8J). Microglial migration within the injected area was quantified at D6 on whole mounted retinae, showing an increase of microglial cells at the level of the OS and at the cone synaptic terminals (Fig. 8K). Illustration of microglial cells spotted with high density in the photoreceptor layers day 6 is provided in Fig. 8L-0. These data suggest that triggering ferroptosis in living rat retinae leads to cone degeneration and microglial migration toward the photoreceptor layers.

[0194] Macular degeneration in non-human primates

[0195] A ferroptosis inducer was similarly applied in vivo on retinae of living non- human primates. A subretinal delivery of 20pM IKE was performed in one eye and the control solution (1 / 500 DMSO) in the fellow eye with a subsequent follow-up of 4 months (see Fig 9A). A previous study showed that injection of the vehicle solution in the subretinal space had no functional or anatomical ocular or systemic adverse effect in NHP (6 months of follow-up to date in 2 NHPs), but resulted in a transient change of photoreceptors identified with adaptive optics ophthalmoscopy, which underwent a full recovery after 4 months (Dentel et al. 2023).

[0196] In eyes injected with 20pM IKE, changes in retinal structure and function were observed in vivo. Firstly, macular pigmentation surrounding foveal subretinal deposits occurred within 3 months (Fig. 9B-D). OCT also showed eroded ellipsoid zone (i.e., zone of inner and outer segments of photoreceptors) and punctuate hyperreflectivities spotted in the ONL only within the detached area with 20pM IKE (Fig 9E-G). Several subretinal deposits appeared at the foveal level at 1 month (Fig 9F) and at the macular level at 2 months (Fig 9H-J). Adaptive optics imaging (a technology that allows for in vivo cone photoreceptor visualisation) of several subretinal deposits appeared identical to those seen in patients affected by age-related macular degeneration (AMD) and showed substantial vanishing of cones within the detached area with 20pM IKE (Fig. 9K-L). Embedded OCT software allowing for ONL segmentation through the injected area showed significant ONL thickness changes within the injected area with 20pM IKE (Fig. 9M, N).

[0197] Adaptive optics was also used to quantify cone density in the injected area. The effect of a standard subretinal delivery of DMSO 0.2% on cone reflectivity in adaptive optics imaging has already been reported (Dentel et al. 2023). Cone loss was observed in eyes injected with 20pM IKE: cone density decreased of 661.00 cones / deg2versus 37.50 cones / mm2at 4 months in a 2-degree superior eccentricity area (n=2, p<0.001 ). No changes were observed in a 2-degree inferior eccentricity (i.e., outside of the injected area).

[0198] Functional alterations of cone photoreceptors were assessed by multifocal electroreti nogram (mfERG): changes from baseline in the N1 -wave amplitudes (an indicator of photoreceptor’s hyperpolarized response to light) and P1 -wave amplitudes (indicating inner retinal layers’ functionality) were analysed. N1 -wave amplitudes were reduced significantly in eyes injected with 20pM IKE (-52.19%) versus eyes injected with control solution (-2.95%) (n=2, p=0.0396). A non-significant decreasing trend of P1 -wave amplitudes was also noted (-33.03% versus -3.83%, n=2, p=0.0874). The subretinal delivery of a ferroptosis inducer at the macular level in non-human primates resulted in alterations highly reminiscent of clinical features that can be seen in macular degeneration.

[0199] EXAMPLE 2: Ferroptosis does not affect primary retinal pigment epithelium cells

[0200] MATERIALS AND METHODS

[0201] Purification of porcine cone photoreceptors

[0202] Pig eyes were dissected to obtain retinae, which were cut into pieces and digested with 4 U / ml papain (LSO 3124, Worthington,) and L-cysteine (5.5 mM, Sigma-Aldrich) for 20 minutes at 37° C. The enzymatic reaction was stopped by adding Neurobasal-A (NBA) medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) and 15 pg / ml DNase I (D4263, Millipore Sigma, Burlington, MA). Several brief centrifugations (30 s at 110 x g, 5 to 6 times) were performed and the supernatants were collected, excluding that from the first centrifugation, which contained mostly rod photoreceptors. Pellets were resuspended by using a P1000 pipette to dissociate the remaining cells between centrifugations. The cell suspension was then centrifuged for 10 minutes at 110 x g and pellets were resuspended in NBA medium containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA).

[0203] Cone photoreceptors were purified by lectin panning, with a slightly modified version of a previously described method (Balse et al, IOVS 2005) to obtain millions of cones in suspension. The cells were then used to seed in a transparent 384-well plates (781091 , Greiner, Ulis, France) or in a white 384-well plates (781080, Greiner, Ulis, France) at a density of either 3,000 or 4,000 cells / well with a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated for three days at 37° C under an atmosphere containing 5% CO?.

[0204] Purification of porcine retinal pigmented epithelial (RPE) cells

[0205] Porcine eyes were dissected and the retinas were removed to obtain an eyecup with RPE cells at the fundus. Trypsin 0.25% (25200-056, Thermo Fisher Scientific, Waltham, MA, USA), previously heated, was added to the eyecup and incubated for one hour at 37° C. The cells were then removed from the eyecup by flushing with a pipette and were transferred to a tube containing DMEM medium (41966-029, Thermo Fisher Scientific, Waltham, AAA, USA) and 20% FBS (A31605-01 , Thermo Fisher Scientific, Waltham, Massachusetts) (=D20). After centrifugation at 110 x g for 5 min, the pellet was resuspended in D20 and the cells were seeded in 60 mm Petri dishes and incubated at 37 °C and 5% CO2. The following day, the medium was changed.

[0206] The cells were then used to seed in a white 384-well plates (781080, Greiner, Ulis, France) at 4,000 cells / well with a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated for three days at 37° C under an atmosphere containing 5% CO?.

[0207] ARPE- 19 cell line

[0208] The cell line was obtained from ATCC, distributed by LGC. The cells were used between P15 and P30 and seeded in D20 medium.

[0209] The cells were then used to seed in a white 384-well plates (781080, Greiner, Ulis, France) at 4,000 cells / well with a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated for three days at 37° C under an atmosphere containing 5% CO?.

[0210] Treatments

[0211] The following compounds were used to induce ferroptosis: glutamate (49621 , Millipore Sigma, Burlington, MA) at a concentration of 1 pM to 1 mM in vitro, IKE (HY-114481 , MedChemExpress, Monmouth, USA) at a concentration of 200 nM to 25 pM, including 3 pM for purified cone photoreceptors and 20 pM for ex vivo and in vivo experiments and RSL3 (HY-100218A, MedChemExpress, Monmouth, USA) at concentrations of 10 nM to 10 pM, including 20 nM, 200 nM and 10 pM for cells, ex vivo and in vivo experiments, respectively. For the inhibition of ferroptosis, FST-1 (HY-100579, MedChemExpress, Monmouth, USA) was used at a concentration of 50 nM on purified cones or at 400 nM on retinal explants. DF (HY-B0568, MedChemExpress, Monmouth, USA) was effective at a concentration of 50 pM on pure cones and ZEN (HY-14164, MedChemExpress, Monmouth, USA) was effective at 20 pM on purified cones (see Figures 1 K, 5I and 7M).

[0212] CellTiter Gio (CTG)

[0213] A cell viability assay was performed using CellTiter Gio (G7573, Promega, Madison, Wl) on RPE cells, ARPE-19 cells and cone photoreceptors. Viability was measured according to the manufacturer's instructions. RESULTS

[0214] IKE and RSL3 do not alter primary RPE cells in vitro

[0215] The inventors further investigated the sensitivity threshold of retinal pigment epithelium cells (RPE) following administration of a pro-ferroptotic agent. 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 is to determine the most sensitive cell type between RPE cells and cone PRs. The inventors therefore used the ARPE-19 cell line, which has been used in all research to characterize the RPE cells, and performed a range of concentrations of IKE, RSL3 and glutamate. When the cells were treated with IKE, a weaker effect was observed on ARPE-19 showing 34.3% living cells at 25 pM compared to 0% in cones (n=3, p = 0.0001 ) (Fig. 11A). Similarly, treatment of ARPE-19 with RSL3 resulted in a higher mortality rate, however, at concentrations considerably higher than those used to kill the cones. At 10 pM RSL3, only 5.3% of ARPE-19 cells were still alive, but at 5 pM, only 50% of cells remained alive (n=3, p = 0.0008) (Fig. 11 B), whereas the EC50 of RSL3 on cones is 1 .58 nM, demonstrating a much greater sensitivity to GPX4 inhibition in cones. Finally, treatment with glutamate had no effect on ARPE-19, unlike the cone PRs where at 1 mM all died (n=3, p= 0.0050) (Fig. 11C).

[0216] However, the inventors were also interested in comparing the effect of pro-ferroptotic molecules between the ARPE-19 cell line and primary porcine RPE cells. There was a major difference with IKE treatment, which did not affect primary cells at all, even at 25 pM (n=3, p = 0.0019) (Fig. 11A). But survival trends were similar between RSL3 and glutamate treatments (Fig. 11 B, C).

[0217] Conclusions

[0218] These results show that cone photoreceptors are more sensitive to pro-ferroptotic agents than RPE cells. Thus, a lower level of oxidative stress can trigger the death of cone photoreceptors without triggering the death of the RPE cells. Furthermore, it should be noted that the articles talking about ferroptosis in RPE cells use a cell line that divides easily and can therefore become immortal (Kozlowski et al. (2015) Curr. Eye Res. 40:501 ). However, ferroptosis is very well described in RPE cells. EXAMPLE 3: Ferroptosis rescued cones from degeneration in RD1 mice ex vivo and in vivo

[0219] MATERIALS AND METHODS

[0220] Animal model: mice

[0221] Mice experiments and procedures were approved by the Local Animal Ethics Committee Charles Darwin CEEACD #5 and performed in the approved facilities associated to the Institut de la Vision (Paris, France) in accordance with European Directive 2010 / 63 / UE. All experimental work were performed according to the institutional policies on biosecurity and safety procedures. Mice were enclosed in a controlled environment and maintained under a reverse half-day dark / light cycle, with ad libitum access to food and water except during surgery.

[0222] The C3H / HeNRj line (rd1 obtained by crossing a Bagg Albino female and a DBA male) was purchased from Janvier Laboratories (Le Genest Saint-Isle, France, ISO 9001 certification), for production and breeding.

[0223] Retinal explants of rd1 mice

[0224] Eyes were collected at P15, cleaned and incubated for 20 min at 37 °C and 5% CO? in a solution of 1 / 10 L-Cysteine (Millipore Sigma, Burlington, MA) at 3.5 mg / 10 mL in a CO? independent medium containing glucose (6.5 g / L) in which papain (LSO 3124, Worthington) was diluted at 1 / 50. Eyes were then immersed for 5 min in neurobasal medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) with 1% of L- glutamine (G3126, Millipore Sigma, Burlington, MA) = NBAg, and 10% SVF (A31605-01 , Thermo Fisher Scientific, Waltham, Massachusetts) on ice. Dissection was performed at +4°C to obtain the whole retina with the RPE. Whole-mounted retinae were flattened onto polycarbonate membranes (140652, Thermo Fisher Scientific, Waltham, Massachusetts). Tested components were diluted to 10 pM in NBAg and 1 / 50 B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA) and changed twice a week for 30 days (until P45 is reached). Explants were then fixed with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA) for 1 h. Finally, PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA) was performed and explants were mounted in Cellvis plates (P12-1.5H-N, IBL, Gerasdorf, Austria) with mounting medium for imaging. Intraperitoneal injections in rd1 mice

[0225] Rd1 mice were daily injected intraperitoneally from P15 to P45 with 2 mg / kg GW 5074 (HY-10542, MedChemExpress, Monmouth, USA) diluted in 40% PEG300 (HY-Y0873, MedChemExpress, Monmouth, USA) and 5% Tween80 (HY-Y1891 , MedChemExpress, Monmouth, USA) in PBS (14190144, Thermo Fisher Scientific, Waltham, MA, USA) or the vehicle only (control group). Eyes were recovered at P45 and fixed for 1 h with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA). Retinae were retrieved for PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA). The CQ1© confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan) was used to image rd1 mice retinae at X10.

[0226] Imaging and statistics

[0227] The Cell PathFinder analysis software was used to quantify the number of PNA-labeled objects in the hand-drawn center of retinal explants for ex vivo experiments, and in whole-mounted retinae for in vivo experiments. The number of detected objects (= cones) was quantified over the entire area and then reported per mm2.

[0228] A Mann-Whitney U two-sided test was used to assess differences of cone density in retinal explants and in injected mice.

[0229] RESULTS

[0230] Various ferroptosis inhibitors were screened on isolated porcine cones after initial exposure to 20 nM RSL3 and 3 pM IKE to seek for protective components to prevent cone loss in retinal degeneration.

[0231] The inventors then tested the neuroprotective effect of several of these components on a well-studied model of retinal degeneration, the rd1 mouse (Leveillard et al. (2004) Nat. Genet. 36:755). In vivo in rd1 mice, cone degeneration is early, starting at P15 with subsequent OS loss at P15-P20; only half of the cell bodies remain at P45. Retinae were retrieved from rd1 mice at P15 and the retinal explants were exposed to ferroptosis inhibitors in culture until P45. Several treatments have provided cone protection (Fig. 12 A-E). However, while this model leads to a rapid degeneration, the inventors may have shown molecules that can rescue cone photoreceptor degeneration in a slower degenerating disease such as AMD. Thanks to automatic cone counting, the inventors have identified five antioxidant molecules with a significant neuroprotective effect (1 ,3-Dicaffeoylquinic acid (n=3, p=O.OO55), HTHQ (1 -0-hexyl-2,3,5-trimethylhydroquinone, n=3, p=0.0176), rosmarinic acid (n=4, p=0.0205), nobergenin (n=2, p=0.0256), tinoridine hydrochloride (n=3, p=0.0055)). On the other hand, the inventors have identified five molecules which inhibits ferroptosis process, with a significant neuroprotective effect: two of them acts directly on 5-LOX (SRS16-86 (n=3, p=0.0001 ) and caffeic acid (n=3, p=0.0055)), two of them play a role in iron metabolism (Mangiferin (n=5, p=0.0005) and astilbin (n=3, p=O.OO55). The last molecule involved in specific ferroptosis inhibition (through the system Xc pathway) demonstrated a 138.92% neuroprotective effect on pure cone photoreceptors and also proved effective on retinal explants, which led to in vivo injections with this molecule GW 5074 (n=6, p=0.0001). It was injected intraperitoneally and daily at 2mg / kg from P15 to P45. Immunolabeling on retinae retrieved at P45 showed a rescue of +81.48% of cones in rd1 mice injected with GW 5074 in comparison with the controls daily injected with the vehicle only (1597 vs 880 cones per mm2, n=5 p=0.0079) (Fig. 12J).

[0232] The findings demonstrate that a ferroptosis inhibitor can reverse cone degeneration in an animal model of retinal degeneration ex vivo and in vivo.

[0233] EXAMPLE 4: Ferroptosis inhibitors do not rescue rods in RD1 mice

[0234] MATERIALS AND METHODS

[0235] Similarly to cones, rods were assessed on retinal explants of rd1 mice following exposure to ferroptosis inhibitors in culture from P15 to P45, as described in Example 3 above. The inventors also conducted the same assessment following intraperitoneal injections of GW 5074 that was described in Example 3 above.

[0236] Retinae were labeled with Rho, were imaged with the CQ1 and Cell Pathfinder was used to count rods.

[0237] Whereas cones were plotted in “fold change”, results dealing with rods were plotted in density (rods per mm2) because their amounts were not enough to consider the changes from baseline in each retina (changes sometimes exceeded the total amount of rods). RESULTS

[0238] There were no significant differences between controls and ferroptosis inhibitors, either in retinal explants or in vivo (Fig. 13). EXAMPLE 5: Ferroptosis inducers do not alter rods in non-human primates

[0239] MATERIALS AND METHODS

[0240] Similarly to cone labeling in a NHP injected with 20 pM IKE in the subretinal space, rods were labeled with a rhodopsin antibody.

[0241] RESULTS No significant changes were observed at the level of the outer segments of rods in the retina injected with 20 pM IKE with respect to the control (Fig. 14).

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Claims

CLAIMS1 . A compound, e.g., a ferroptosis inhibitor, for use in treating a condition associated with cone photoreceptor degeneration, wherein the treatment comprises activating the Xc- transporter or the GPX4 enzyme in cone photoreceptors.

2. The compound for the use of claim 1 , wherein the activity of the Xc- transporter or the GPX4 enzyme is increased or decreased.

3. The compound for the use of any one claims 1 or 2, wherein the expression of the Xc- transporter or the GPX4 enzyme is increased.

4. The compound for the use of any one of claims 1 to 3, wherein the treatment comprises maintaining or increasing cone photoreceptor viability.

5. The compound for the use of any one of claims 1 to 4, wherein the treatment comprises maintaining or increasing intracellular glutathione level in cone photoreceptors.

6. The compound for the use of any one of claims 1 to 5, wherein the treatment comprises maintaining or decreasing intracellular NADP / NADPH ratio in cone photoreceptors.

7. The compound for the use of any one of claims 1 to 6, wherein the treatment comprises preventing or reducing glial migration.

8. The compound for the use of any one of claims 1 to 7, wherein the treatment comprises preventing or reducing subretinal deposit formation.

9. The compound for the use of any one of claims 1 to 8, wherein the treatment comprises maintaining or increasing cone photoreceptor activity.

10. The compound for the use of any one of claims 1 to 9, wherein the ferroptosis inhibitor is selected in the group consisting of cystine, B-mercaptoethanol, selenium, bardoxolone, carvacrol (CAR), rehmanniosideA, bioflavonoids including galangin, xanthohumol, naringenin, britanin, entacapone, capsiate, resveratrol, dexmedetomidine, irisin, kaempferol, fluoflavine, GKT136901 hydrochloride, and ML171.11 . The compound for the use of any one of claims 1 to 10, wherein the condition is selected in the group consisting of age-related macular degeneration (AMD), cone dystrophies, cone-rod dystrophies, rod-cone dystrophies, macular dystrophy (e.g. Stargardt disease), central serous chorioretinopathy, Best disease and bestrophinopathies, retinal detachment including rhegmatogenous, serous and fractional causes, solar retinopathy, laser-induced retinopathy, achromatopsia, Stargardt disease, Usher syndrome, Leber congenital amaurosis (LCA), Alstrom disease, and Refsum disease, preferably AMD or retinitis pigmentosa, more preferably AMD.

12. The compound for the use of any one of claims 1 to 11 , wherein the use comprises the administration of the component in the subretinal space, the suprachoroidal space, the anterior chamber, the vitreous humor, the subconjunctival space, on the corneal surface, through systemic administration (inhalator, intravenous, intramuscular or intraperitoneal) or under solution, gel or implant.

13. The compound for the use of any one of claims 1 to 12, wherein the use comprises the administration of a further therapeutic agent.

14. The compound for the use of claim 13, wherein the further therapeutic agent is a second ferroptosis inhibitor, pegaptanib, ranibizumab, bevacizumab, brolucizumab, faricimab, AKB-9778, nesvacumab, Bl 836880, or aflibercept.

15. The compound for the use of claim 14, wherein administration of the second ferroptosis inhibitor results in reduction or inhibition of lipid peroxidation in cone photoreceptors and / or reduction of intracellular iron levels in cone photoreceptors.

16. The compound for the use of any one of claims 13 to 15, wherein the component and the further therapeutic agent are administered simultaneously, sequentially, or separately.

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