Iron metabolism modulators for treating conditions associated with cone cell degeneration.

Abstract

The present invention provides a method for treating a condition associated with cone cell degeneration, wherein the treatment comprises reducing intracellular iron levels in cone cells. In particular, the present invention provides compounds for use in such a method, preferably ferroptosis inhibitors. [Solution] This disclosure relates to a method for treating conditions associated with cone photoreceptor degeneration. This disclosure shows that regulating iron metabolism in cone photoreceptor cells prevents degeneration of these cells. In particular, reducing intracellular iron levels in cone photoreceptor cells prevents degeneration of these cells.

Description

[Background technology]

[0001] The retina is the light-sensing nerve tissue located at the back of the eyeball. The human retina has two distinct regions. The peripheral retina has lower spatial vision and is responsible for various aspects of night vision and motion vision. The fovea (or macula) is located in the center of the retina and is responsible for high spatial vision essential for reading and face recognition. Among mammals, only primates have a fovea. The retina is composed of three cell layers: the outer granular layer (ONL), the inner granular layer (INL), and the retinal ganglion cell (RGC) layer. The ONL contains photoreceptor cells, which are the only neurons specialized in converting light into electrochemical signals and are therefore essential for vision. Two different types of photoreceptor cells are distinguished: rod photoreceptor cells respond to faint light and enable night vision, while cone photoreceptor cells respond to bright daylight and mediate high resolution and color vision.

[0002] Retinal neurodegeneration associated with dysfunction or death of photoreceptor cells is a leading cause of incurable vision loss. Photoreceptor cell death is a common cause of many retinal disorders, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). In many of these diseases (e.g., AMD), the pathology appears to be primarily related to the loss of cones. Therefore, preventing cone photoreceptor degeneration is of particular importance. This allows for the earliest possible therapeutic intervention to stop the onset of the disease, thereby ultimately preventing vision loss.

[0003] To date, there are no effective treatments for retinal degenerative diseases, and the situation remains difficult to manage, making the development of new treatment strategies urgently needed. While there are a few approved treatments for these diseases, they only address neovascularization of the eye following cone cell degeneration. Meanwhile, molecules capable of preventing or treating the earliest stages of the disease—namely, the death of cone cells—have yet to be identified.

[0004] A deeper understanding of the mechanisms causing cone photoreceptor cell death is essential for the development of these treatments. The molecular and cellular mechanisms leading to cone photoreceptor cell death, particularly in the earliest stages of retinal disease, remain largely unknown. Several mechanisms of cell death, including apoptosis, pyroptosis, necrosis, and ferroptosis, have been suggested to be involved in ocular diseases (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 have been shown to specifically drive cone photoreceptor cell death. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, there is still an urgent need for molecules that can effectively treat conditions associated with cone cell degeneration. [Means for solving the problem]

[0006] In a first aspect, the disclosure relates to a method for treating conditions associated with cone photoreceptor degeneration. The disclosure shows that regulating iron metabolism in cone photoreceptor cells prevents degeneration of these cells. In particular, reducing intracellular iron levels in cone photoreceptor cells prevents degeneration of these cells.

[0007] Accordingly, a first aspect of the present disclosure relates to compounds for use in the treatment of conditions associated with cone photoreceptor degeneration, for example, ferroptosis inhibitors, wherein the treatment comprises reducing, in particular specifically reducing, intracellular iron levels in cone photoreceptor cells, or sequestering, in particular specifically sequestering, intracellular iron molecules. More specifically, the ferroptosis inhibitors specifically reduce intracellular iron levels in cone photoreceptor cells or specifically sequester intracellular iron molecules.

[0008] In the example, the treatment involves maintaining or increasing cone photoreceptor cell viability. In the example, with the same dose of treatment, and in particular with the same concentration of ferroptosis inhibitor, the treatment does not affect rod photoreceptor cell viability and / or the treatment does not affect retinal pigment epithelial cell viability.

[0009] In this example, the treatment involves blocking an increase in the pigment permeability of the cell membrane of cone photoreceptor cells. More specifically, the treatment involves specifically blocking an increase in the pigment permeability of the cell membrane of cone photoreceptor cells, i.e., the treatment does not substantially block an increase in the pigment permeability of the cell membranes of other retinal cell types, such as rod photoreceptor cells.

[0010] In this example, the treatment includes maintaining or reducing the activity of ferritin and / or nuclear receptor coactivator 4 (NCOA4), particularly specifically maintaining or reducing the activity of ferritin and / or nuclear receptor coactivator 4 (NCOA4) in cone photoreceptor cells.

[0011] In this example, the treatment involves inhibiting or reducing the migration of microglia.

[0012] In this example, the treatment includes preventing or reducing the formation of subretinal deposits.

[0013] In this example, the treatment involves maintaining or increasing cone cell activity. More specifically, the treatment involves specifically maintaining or increasing cone cell activity, i.e., the treatment does not substantially affect the activity of other retinal cell types such as rod cells or retinal pigment epithelial cells.

[0014] The compounds used in the methods disclosed herein are preferably ferroptosis inhibitors. More preferably, the compounds are selected from the group consisting of prominin 2, miR-522, and / or RP11-89 genes; RNA that inhibits FTL, FTH1, and / or NCO4; deferipron, ferrostatin-1, deferoxamine (DFO), gossypol acetate, histochrome, cyclopiroxolamine (CPX), bafilomycin A1, quercitrin, baicalein, dexrazoxane, 2,2'-bipyridine, 1,10-phenanthroline, mangiferin, astilbin, YL-939, deferasirox (DFX), 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), 1,3-dicaffeoylquinic acid, rosmarinic acid, norbergenin, and tinoridine hydrochloride. Most preferably, the compounds are from the group consisting of prominin 2, miR-522, and / or RP11-89 genes; RNA that inhibits FTL, FTH1, and / or NCO4; deferipron, ferrostatin-1, deferoxamine (DFO), gossypol acetate, histochrome, cyclopiroxolamine (CPX), bafilomycin A1, quercitrin, baicalein, dexrazoxane, 2,2'-bipyridine, 1,10-phenanthroline, mangiferin, astilbin, YL-939, and deferasirox (DFX).

[0015] Preferably, the conditions associated with cone photoreceptor degeneration of the methods disclosed herein are conditions in which cones are specifically affected. In particular, this condition is one in which cone photoreceptor degeneration is not associated with a specific degenerative mechanism affecting retinal cell types other than photoreceptor cells. More preferably, this condition is selected from the group consisting of age-related macular degeneration, cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt disease), central serous chorioretinopathy, Best's disease and bestrofinopathy, retinal detachment including rhegmatogenous retinal detachment, serous retinal detachment and tractional retinal detachment, photoretinopathy, laser-induced retinopathy, monochromacy, Usher syndrome, Leber congenital amaurosis, Alström syndrome, and Refsum disease.

[0016] For example, the compounds for use in the methods disclosed herein are administered intravitreously (e.g., into the subretinal space, extrachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or corneal surface) by instillation, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intratracheally, intrathecally, intranasally, vaginally, intrarectally, locally, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosa, intrapericardially, intracardiacally, intraumbilically, intraocularly, intraorbitally, orally, transdermally, by inhalation, by injection, implantation, infusion, continuous infusion, directly to target cells by local perfusion bath, by catheter, by lavage, as a cream, or as a lipid composition.

[0017] In this example, the treatment includes the administration of a second therapeutic agent. The second therapeutic agent is preferably a second ferroptosis inhibitor, aflibercept, ranibizumab, pegaptanib, bevacizumab, brolucizumab, falisimab, AKB-9778, nesbakumab, AKST4290, and BI 836880. More preferably, the administration of the second ferroptosis inhibitor results in a decrease or inhibition of lipid peroxidation in cone photoreceptor cells, and / or an increase in the activity of the Xc- system and / or GPX-4 enzyme in cone photoreceptor cells.

[0018] In this example, the procedure involves administering the compound and the second therapeutic compound simultaneously, sequentially, or separately. [Brief explanation of the drawing]

[0019] [Figure 1]This figure shows evidence of ferroptosis occurring in pure cone photoreceptor cells. The mean and standard error of the mean are shown. A-B. Bright-field images of cone photoreceptor cells incubated for 3 days in control (Ctrl=0.2% DMSO, A) and treatment solution (3 μM imidazole ketone elastin, IKE, B). C-D. Staining of cone photoreceptor cells using calcein-labeled viable cells under control (C) and treatment (D) conditions. E. Glutamate concentration-dependent cell viability of pure cone photoreceptor cells with or without the addition of 1 mM L-cysteine ​​(n=7, p<0.0001, IC50=4.15 μM). F. Glutathione levels of pure cone photoreceptor cells over 3 days in control, 3 μM IKE, and 500 μM glutamate medium (D1, D2, D3 n=18, p<0.0001). G. H. NADP / NADPH percentage of pure cone photoreceptor cells on day 3 under control and 3 μM IKE conditions (n=4, p=0.0159). I. Concentration-dependent plot of pure cone photoreceptor cell viability after treatment with RSL3, 0.01 nM to 25 nM (n=3, IC50=8.67 nM). I. Western blot showing 5-lipoxygenase (5LOX) and 5-lipoxygenase-activated protein (FLAP) bands in pure cone photoreceptor cells, which are not present in rod photoreceptor cells. J. Lipidomic analysis (n=6, p=0.0079(**), p=0.0159(*)) showing the amounts of 15- and 5-hydroxyeicosatetraenoic acid (15-HETE and 5-HETE, respectively) and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) detected in pure cone photoreceptor cells under control and 3 μM IKE conditions. K. Various treatments (with anti-ferroptotic or iron chelating effects): Survival rates of pure cone photoreceptor cells under control and 3 μM IKE conditions when L-cysteine, diloton (ZEN), ferrostatin-1 (FST1), and deferipron (DF) were added. [Figure 2]This figure shows the changes in mRNA expression levels of proteins involved in ferroptosis in control-treated (3 μM IKE) pure porcine cone photoreceptor cells (n=6). Solute Carrier Family 11 Member 2 (SLC11A2) is involved in the release of Fe2+ from endosomes to the cytoplasm, and its decrease suggests dysregulation of iron metabolism (p=0.0260). Solute Carrier Family 3 Member 2 (SLC3A2) corresponds to the heavy chain of the Xc- system (p=0.0022). Transferrin receptor (TFRC) expression was moderately increased under the treatment conditions (P=0.0286). Ferroptosis suppressor protein 1 (AIFM2) is involved in the suppression of the ferroptosis mechanism (p=0.0022). Heat shock protein family B minor member 1 (HSPB1) plays a role in iron uptake and may promote ferroptosis.22 (p=0.0022) [Figure 3] This figure shows the immunofluorescence localization of flap in retinal sections of three species (pig, rat, and non-human primate) with respect to DAPI nuclear staining. In all species, flap is localized to cone photoreceptor cells. [Figure 4] This figure shows immunofluorescence assays of isolated porcine cone photoreceptor cells with control ferritin heavy chain (B, G) and NCOA4 (C, H), as well as treated cells (F-J). IKE treatment increased fluorescence intensity (G, H) compared to control cone photoreceptor cells. IKE conditions (G, H) show more immunolabeling than control (B, C). E. Magnification of merged labels showing control cells with stronger ferritin heavy chain immunolabeling than NCOA4. J. Magnification of treated cells showing stronger NCOA4 immunolabeling than heavy chain immunolabeling. [Figure 5]These figures show cone cell degeneration and microglia migration in ex vivo retinal explants. A-H. Histological structure of retinal sections showing increased ethidium permeability in the first nucleus of cone cells in the treated explant (20 μM IKE, F) compared to control tissue (0.2% DMSO, B). Note that compared to the control condition (C), the order of cone cells immunolabeled with Flap is disrupted in the treated retina (G), and that in the treated retina (H), large amoeboid microglia cells, which are not present in the control retina (D), are present even in the outer granular layer (ONL). The nuclear layer is highlighted in the control retina (A) and treated retina (E) with DAPI. I-M. Cone cell destruction, indicated by loss of its flap-immunopositive outer segments in treated, flat-mounted retinal explants (20 μM IKE), and its protection by the anti-ferroptotic agents FST-1 (L) and DF (M) compared to control condition (J). Quantification of cone cell outer segments demonstrates a significant reduction in treated explants and rescue by the anti-ferroptotic agents (I, n=3). N~O. Microglia cell migration toward the retinal lateral side (IKE, RSL3) and microglia cell distribution at retinal explant thickness (n=3) demonstrating inhibition of this effect by DF or FST-1 in treated retina. Scale bar = 20 μm. ONL: Outer granular layer, INL: Inner granular layer, GCL: Ganglion cell layer, Iba1: Ionized calcium-binding adapter molecule 1, Ctrl: Control condition, IKE: Imidazole ketone elastin, OS: Outer segment, FST-1: Ferrostatin-1, DF: Deferipron [Figure 6] This figure shows the maintenance of various retinal cell types in sections of treated porcine retinal explants (20 μM IKE) compared to the control condition (Ctrl). Rod photoreceptor cells were immunolabeled with rhodopsin, bipolar cells with PKC-alpha, and Müller cells with vimentin. Note that the retinal structure is enhanced by DAPI-stained nuclei, and under the treated condition, there is no evidence of cell loss, and the morphology of the labeled cells appears to be preserved. [Figure 7]This is a figure showing the effect of RSL3 on retinal explants. The ethidium permeability of RSL3 showed dying cells in the control (0.2% DMSO, B) retina and the treated (200 nM RSL3, F) retina. C, G. Morphological changes of Flap-immunolabeled cone photoreceptor cells in the treated retinal explants (G) compared to the control explants (C), and migration of iba1-immunopositive microglial cells towards the outer granular layer in the treated explants (H) compared to the control condition (D). I - L. Loss of Flap-immunopositive outer segments (OS) in the retinal explants treated with RSL3 (J) and its retention by FST-1 (K) or DF (L) in relation to the control condition (I). M. Quantification of RSL3 toxicity to cone photoreceptor outer segments in retinal explants and its retention by DF (n = 3, p = 0.0286). N. Distribution of microglial cells showing migration of microglial cells towards the outer nuclear layer (ONL) in the presence of RSL3 compared to the control condition (see Fig. 5N) with the effect of DF or FST-1 (n = 3). Scale bar = 20 μm. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, Iba1: ionized calcium-binding adapter molecule 1, Ctrl: control condition, IKE: imidazole ketone elastin, OS: outer segment, FST-1: ferrostatin-1, DF: deferiprone. [Figure 8]This figure shows cone photoreceptor degeneration and microglia migration in rats after in vivo subretinal delivery of a ferroptosis-inducing substance. A. Schematic diagram of the experimental protocol and schedule. 20 μM IKE, 200 nM RSL3 (treatment), or 0.2% DMSO (control, Ctrl) was delivered into the subretinal space of rats. The retinas were collected on day 6 (D6) and microglia migration was evaluated (K~O, n=4), or cone photoreceptor degeneration was evaluated on day 30 (D30) (H~J, n=4). B~G. In vivo retinal imaging samples from rats: Fundus photography (B, C) and optical coherence tomography (OCT) (D-G) showing induced retinal detachment near the optic nerve (ON) (D0, B, D) and fully reattached retina at D6 (C, E); injection site (arrowhead) and OCT thin section (dotted line) are shown in the fundus photography; (F, G) OCT of the injection area at D6 showing punctate, highly reflective small spots (arrows) (more abundant in treatment condition (G)) at the junction of the inner granular layer (INL) and outer plexiform layer (OPL) / outer granular layer (ONL). H, I. Retinal section within the injection area recovered at D30 (same rats as B-G) showing a decrease (I) in cone arrestin-labeled cone photoreceptor cells (CARs) in treated animals compared to control (H), with corresponding DAPI nucleus staining. The degree of eccentricity relative to the optic nerve is the same in both cases. J. Quantification of cone density (units, cells / mm) measured in retinal sections of treated and control rats at D30 (n=4). The outer segment (OS) and cell body of cones were quantified separately. K. Quantification of microglia density in whole-mounted retinas of treated and control rats at D6. Three retinal planes, namely the outer segment (OS) level, the ONL plane, and the pedicle (Ped) plane corresponding to the OPL / ONL junction, were evaluated separately. Each field of view corresponded to a 500 × 500 μm window and was repeated throughout the entire injection area. L~O. Distribution of cone arrestin-positive cells and Iba1-positive microglia cells in whole-mounted retinas of treated and control rats recovered at D6, showing two different planes in the z-axis direction (illustrated in H~I), OS (L, M), and Ped (N, O). For the treated animals, samples were collected from the detached area to illustrate changes in cone OS morphology and invading microglia cells, but away from the injection site. The degree of eccentricity relative to the optic nerve was the same in both cases. Scale bar: 50 μm.INL: Inner granular layer, OPL: Outer plexiform layer, ONL: Outer granular layer, EZ: Ellipsoidal zone, RPE: Retinal pigment epithelium, DAPI: 4',6-diamidino-2-phenylindole, CAR: Cone arrestin, Iba1: Ionized calcium-binding adapter molecule 1, ctrl: Control. [Figure 9]This figure shows macular changes in non-human primates (NHPs) after subretinal delivery of 20 μM IKE. A. Schematic diagram of the experimental protocol. Subretinal delivery of 20 μM IKE in the right eye and subretinal delivery of DMSO diluted 1 / 500 with PBS in the left eye caused superior hemifovea detachment in both eyes. B-C. Fundus photographs at baseline (B), 1 month (C), and 3 months (D) after subretinal injection of 20 μM IKE. The boundary line at 1 month (arrowhead in C) and macular pigment deposition around the subretinal deposits that occurred at 3 months (highlighted in D) are shown. E-F. OCT vertical b scan through the foveal spot of an NHP 3 months after subretinal delivery of 1 / 500 DMSO (ctrl, E) and 20 μM IKE (F). Note the eroded ellipsoidal zone (EZ, black asterisk), small, highly reflective areas (arrows), and subretinal deposits (arrowheads). G. OCT horizontal b-scan through the superior parafoveal region within the subretinal delivery of 20 μM IKE. The EZ is eroded within the boundary of the detached area (dotted line) (black asterisk). Note also a cluster of highly reflective lesions located mainly beneath the ONL and internal limiting membrane (arrows). The uninjected area (outside the dotted line) remains completely normal. H-J. OCT horizontal b-scan through macular subretinal deposits (arrowheads) that appeared at M2 (I) and grew to M3 (J). K-L. Frontal adaptive optics imaging of the foveal photoreceptor layer within the detached area at 2 degrees eccentricity, 3 months after subretinal delivery. After delivery of 20 μM IKE, the cones showed reduced reflectivity and clusters of cone disappearance appeared (K), while the cone mosaic on the control side remained regular (L). M~N. Illustration of ONL thickness changes in the macular region of NHP 3 months after subretinal delivery. Grayscale shows ONL thickness changes (unit, μm). O. Changes in cone density (unit, cells / deg2) measured at two degrees, inside and outside the injection area, using adaptive optics, 3 months after subretinal delivery (n=2). P. Changes in N1 and P1 wave amplitudes as a percentage of baseline measured by multifocal electroretinography (n=2). Only hexagons within the injection area were analyzed. [Figure 10]A diagram showing the ferroptosis pathway elucidated in mammalian cone cells. Ferroptosis ultimately drives ROS formation, which causes cone cell degeneration. In cone ferroptosis, three intracellular pathways must be considered. First, the Xc- system, which can be inhibited by high extracellular concentrations of glutamate or ferroptosis inducers (e.g., IKE), stimulates the uptake of L-cysteine to generate glutathione. Inhibition of the glutathione redox cycle leads to the inactivation of glutathione peroxidase 4 (GPX4) and the accumulation of NADPH. Furthermore, the decreased activity of the GPX4 enzyme causes the production of lipid peroxides such as 5-oxo-ETE via the lipoxygenase pathway. Finally, the high ferritinophagy activity of NCOA4 causes iron metabolism dysregulation and results in the release of excessive amounts of iron. Radical species are formed by the Fenton reaction, enabling the production of reactive oxygen species through interaction with lipid peroxides. GPX4: glutathione peroxidase 4, 12-LOX: 12-lipoxygenase, 15-LOX: lipoxygenase, 5-LOX: 5-lipoxygenase, FLAP: 5-lipoxygenase activating protein 12-HPETE: 12-hydroxyperoxyeicosatetraenoic acid, 15-HPETE: 15-hydroxyperoxyeicosatetraenoic acid, 5-HPETE: 5-hydroxyperoxyeicosatetraenoic acid, 5-oxo-ETE: 5-oxo-eicosatetraenoic acid, 12-HETE: 12-hydroxy eicosatetraenoic acid, 15-HETE: 15-hydroxy eicosatetraenoic acid, 5-HETE: 5-hydroxy eicosatetraenoic acid, STEAP 3: STEAP family member 3, DMT1: Dimetal transporter 1, NCOA4: nuclear receptor coactivator 4, Keap1: Kelch-like ECH-associated protein 1, NFE2L2: nuclear factor (erythroid-derived 2)-like 2, ROS: reactive oxygen species [Figure 11]This figure shows the cell viability of different retinal cell types after ferroptosis-promoting treatment. Cone photoreceptor cells (cone PRs) are represented by light gray lines and circles, ARPE-19 cells by dark gray lines and triangles, and primary RPE cells by black lines and squares. A. Cells were treated with 0.20 μM to 25 μM IKE (imidazole ketone elastin). All cone PRs died at 25 μM, but 40% of ARPE-19 cells (n=3, p=0.0001) and 80% of primary RPE cells (n=3, p=0.0019) survived. B. Cells were treated with 0.01 μM to 10 μM RSL3 (Ras Selective Ligand 3). All cone PRs were killed at all concentrations, while at 5 μM, 50% of ARPE-19 cells (n=3, p=0.0008) and primary RPE cells survived. C. Treatment of cells with 0.001 mM to 1 mM glutamate showed no effect on ARPE-19 cells or primary RPE cells. In contrast, cone PRs were killed at 1 mM compared to ARPE-19 cells (n=3, p=0.0050) and primary RPE cells. [Figure 12]This figure shows that ferroptosis inhibitors reverse cone degeneration in rd1 mice from P15 to P45 both ex vivo and in vivo. A-D. PNA immunolabeled retinal explants of rd1 mice cultured for 30 days (P15-P45) exposed to a control solution (A) and several ferroptosis inhibitors (B-D). E. Automated cone counts of retinal explants of rd1 mice cultured exposed to various ferroptosis inhibitors (Ctrl, HTHQ, 1,3-dicaffeoylquinic acid, SRS16-86, astilbin, caffeic acid, tinoridine hydrochloride n=3; GW5074 n=6; norbergenin n=2; mangiferin n=5). White bars represent antioxidants or radical scavengers, gray bars represent molecules involved in the Xc pathway, grid-patterned bars represent molecules acting on the lipoxygenase pathway, and vertical striped bars represent molecules involved in the iron pathway. F-I. Whole-mounted retinas immunolabeled with PNA from rd1 mice that received daily intraperitoneal injections of vehicle (Ctrl, F, and H) or 2 mg / kg of GW 5074 solution (G and I) for 30 days (P15-P45). Asterisks indicate the locations of H and I. J. Automated cone counts of whole-mounted retinas after daily intraperitoneal injections (n=5). Scale bar = 50 μm for A-D, H, and I; 300 μm for F and G. PNA: peanut agglutinin, Ctrl: control, HTHQ: 1-O-hexyl-2,3,5-trimethylhydroquinone, 1-3-DCFA: 1,3-dicaffeoylquinic acid, RA: rosmarinic acid, SMNL: sone chromanol, CA: caffeic acid, Tinoridin HCl: tinoridine hydrochloride, OH-puerarin: hydroxypuerarin, 5-ACQ: chlorogenic acid. [Figure 13] This figure shows that ferroptosis inhibitors do not reverse rod degeneration to P15-P45 in rd1 mice ex vivo and in vivo. A. Automated cone counts of retinal explants of rd1 mice exposed to various ferroptosis inhibitors that rescued cones in culture medium (Ctrl, HTHQ, 1,3-dicaffeoylquinic acid, SRS16-86, astilbin, caffeic acid, tinoridine hydrochloride n=3; GW5074 n=6; norbergenin n=2; mangiferin n=5). B. Automated cone counts of whole-mounted retinas after daily intraperitoneal injections (n=5). [Figure 14] This figure shows rhodopsin (Rho) and DAPI-labeled rods from the retinas of non-human primates injected with a control solution and 20 μM IKE. [Modes for carrying out the invention]

[0020] definition The terms “about” or “approximately” refer to the normal margin of error of a given value or range known to those skilled in the art. It typically means within 20%, for example, 10%, or 5% (or less than or equal to 1%) of the given value or range. In this specification, references to a “about” value or parameter include (and are described) embodiments relating to that value or parameter itself.

[0021] In this specification, the terms “activate,” “stimulate,” or “induce,” and their grammatical derivatives, refer to a relatively increased specific reaction (e.g., expression, enzyme activity) of a specified substance in the presence of a particular reagent. This reagent is referred to herein as an “activator.”

[0022] In this specification, “administer” or “dosage” means the act of injecting a substance that is outside the body into a patient or otherwise physically delivering it, for example, by mucosal delivery, intradermal delivery, intravenous delivery, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. When a disease or its symptoms are treated, administration of the substance is typically performed after the onset of the disease or its symptoms. When a disease or its symptoms are prevented, administration of the substance is typically performed before the onset of the disease or its symptoms. In particular, “administer” means a method of administering a dosage of the compounds disclosed herein to a subject having a condition associated with cone photoreceptor degeneration. Compositions used in the methods described herein can be administered, for example, intravitreously (e.g., by intravitreal injection), into the eye (e.g., by ocular injection), or intraocularly (e.g., by intraocular injection). The method of administration may vary depending on various factors (e.g., the compound or composition being administered, and the severity of the condition, disease, or disorder being treated).

[0023] Administration may be by means of a carrier or vehicle, such as an injectable solution containing a sterile aqueous or non-aqueous solution, or physiological saline; a cream; a lotion; a capsule; a tablet; a granule; a pellet; a powder; a suspension, emulsion, or microemulsion; a patch; a micelle; a liposome; a vesicle; an implant containing a microimplant; eye drops; other proteins and peptides; synthetic polymers; microspheres; or nanoparticles.

[0024] The terms “decreased” or “reduced” as used herein mean that the activity of a protein is at least one-fold (e.g., 1x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 10,000x or more) lower than its reference value. With respect to the activity of the subject protein, “decreased” or “reduced” also means that the activity is at least 5% lower than the activity of the protein in the reference sample or at least 5% lower than the reference value of the protein (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%), 99%), or 100%). The terms “decreased” or “reduced” also, as used herein, mean that the level of the biomarker in question (e.g., iron concentration in a specific cell type, e.g., cone photoreceptor cells) is at least one-fold (e.g., 1x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 10,000x or more) lower than its reference value. With respect to the level of a subject biomarker (e.g., iron concentration in a specific cell type, e.g., cone photoreceptor cells), “decreased” or “reduced” also means that the level is lower than the level in the reference sample or at least 5% lower than the reference value of the marker (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%).

[0025] "Disease" means any condition or disorder that impairs or interferes with the normal function of a cell, tissue, or organ. For example, a disease is a condition affecting the eye, particularly one related to cone cell degeneration.

[0026] In this specification, “electroretinography (ERG)” refers to a diagnostic test that measures the electrical activity of the retina in response to light stimulation. ERG can, for example, record the massed potential from the entire retina; see, for example, whole-field ERG. Alternatively, multifocal ERG (mfERG) assesses ERG activity in small areas of the retina, while patterned ERG (pERG) assesses retinal ganglion cell (RGC) activity in the macula. 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 efficacy of any of the various therapeutic compounds disclosed herein. Examples of ERG values ​​for healthy subjects and patients can be found in Lorenz et al., Invest. Ophthalmol. Vis. Sci. 49:5235-5242, 2008.

[0027] In this specification, “ferroptosis” refers to a form of cell death understood in the art to involve the generation of iron-mediated reactive oxygen species, and is characterized in part by lipid peroxidation. In ferroptosis, lethality occurs as a result of the peroxidation of polyunsaturated fatty acids (PUFAs), which self-replicate unless stopped by the lipid peroxidase, glutathione peroxidase 4 (GPX4).

[0028] The term "increased" as used herein means that the activity of a protein is at least one-fold higher than its baseline value (e.g., 1x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 10,000x or more). With respect to the activity of the subject protein, "increased" also means that the activity of the protein is at least 5% higher than that of the protein in the reference sample or at least 5% higher than the baseline value of the protein (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). The term “increased” also means, as used herein, that the level of the biomarker in question is at least one-fold higher than its reference value (e.g., 1x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 10,000x or more). With respect to the level of the biomarker in question, “increased” also means that the level is at least 5% higher than the level in the reference sample or at least 5% higher than the reference value of the marker (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%).

[0029] In this specification, the terms “inhibit” or “suppress” refer to a comparative reduction in a particular reaction (e.g., expression, enzyme activity) of a specified material in the presence of a particular reagent. This reagent is referred to herein as “inhibitor.”

[0030] In this specification, the term “lipid peroxidation” means the result of free radical damage to lipids. Lipid peroxidation forms lipid peroxides and several oxidation products, including aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).

[0031] In this specification, the terms "lipid peroxide" or "peroxide-treated lipid" refer to oxidation products of phospholipids and polyunsaturated fatty acids (PUFAs).

[0032] In this specification, “lipoxygenase” is an enzyme that oxidizes polyunsaturated fatty acids and alkenes to eicosanoids with two oxygen atoms. It catalyzes the first reaction of the so-called lipoxygenase pathway, which is involved in the response to external trauma and stress. Examples of human lipoxygenases include 15-LOX, 15-LOX-2, 12-LOX, 12R-LOX, eLOX-3, and 5-LOX. In some examples, lipoxygenase activity requires the presence of an activating factor compound ("lipoxygenase activator"). For example, the helper protein 5-LOX activating protein (FLAP) is a 5-LOX activator because it is required for the activation of this enzyme.

[0033] In this specification, “measure” or “determine” refers to any qualitative or quantitative determination.

[0034] In this specification, “operably linked” means that linked elements are arranged to cooperate in order to function for their intended purposes. For example, if a promoter in a permissible host cell brings about transcription of a nucleic acid molecule from transcription initiation to transcription termination, the promoter is operationally linked to the nucleic acid molecule.

[0035] As used herein, “optical coherence tomography (OCT)” refers to a non-invasive imaging technique that uses light waves to capture cross-sectional images of the retina. OCT can be used for distinguishing layers of the retina, illustrating and measuring thickness, notifying treatment decisions, providing diagnostic information, monitoring disease progression, or a combination of these. In particular, OCT can be used to quantify cone degeneration. In embodiments, subjects not diagnosed with retinal degenerative disease have a peak cone density of approximately 200,000 cells / mm³ at the fovea, as measured by OCT. 2The outer granular layer of the fovea has a thickness of approximately 100 μm, as measured by OCT. Preferably, patients diagnosed with retinal degenerative disease have a peak cone density of approximately 200,000 cells / mm² in the fovea, as measured by OCT. 2 Patients diagnosed with retinal degenerative disease may have a thickness of less than approximately 100 μm in the outer granular layer of the fovea, as measured by OCT. Further examples of OCT values ​​in healthy subjects and patients diagnosed with retinal degenerative disease can be found in Lorenz et al., 2008.

[0036] "Polyunsaturated fatty acids (PUFAs)" as used herein refers to fatty acids containing at least two -CH=CH- groups, such as linoleic acid, linolenic acid, and arachidonic acid (AA).

[0037] In this specification, the terms “prevent,” “prevent,” and “prevention” refer to a reduction in the risk that an object will contract or develop a given disease, disorder, or condition. The terms “prevent,” “prevent,” and “prevention” also include delaying the onset of clinical, histological, and / or biochemical symptoms or parameters associated with the given disease, disorder, or condition, and / or reducing the frequency and / or intensity of clinical, histological, and / or biochemical symptoms or parameters associated with the given disease, disorder, or condition. In some embodiments, prevention is evaluated on a population basis, and a therapy is considered to “prevent” a particular disease, disorder, or condition if a statistically significant reduction in the risk of contracting or developing a particular disease, disorder, or condition, and / or a statistically significant delay in the onset of clinical, histological, and / or biochemical symptoms or parameters associated with that disease, disorder, or condition, and / or a statistically significant reduction in the frequency and / or intensity of clinical, histological, and / or biochemical symptoms or parameters associated with that disease, disorder, or condition is observed in a population susceptible to that disease, disorder, or condition.

[0038] In this specification, the terms “regulatory element” or “regulatory sequence” refer to any element that enables, contributes to, or regulates the expression of an encoded nucleic acid in a given host cell or subject, including, but not limited to, the replication, copying, transcription, splicing, translation, stability, and / or transport of nucleic acids or their derivatives (i.e., mRNA).

[0039] In this specification, the term “specifically” means that, at a given concentration, the compound affects one cell type (i.e., cone photoreceptor cells) while substantially not affecting other retinal cell types, such as rod photoreceptor cells or retinal pigment epithelial cells, at the same given / identical concentration. In particular, when referring to in vivo use, the term “specifically” means that the compound affects one cell type (i.e., cone photoreceptor cells) before or without substantially affecting other retinal cell types, such as rod photoreceptor cells or retinal pigment epithelial cells.

[0040] In this specification, “Subjects” includes all mammals, including but not limited to humans, as well as non-human primates such as cynomolgus macaques. This also includes dogs, cats, horses, sheep, goats, cattle, rabbits, pigs, and rodents (such as mice and rats). It will be understood that particularly preferred subjects of this disclosure are human subjects, for example, disorders, diseases, or conditions related to cone photoreceptor degeneration, and especially humans who are suffering from (or at risk of suffering from) the condition, e.g., human patients.

[0041] In this specification, the term “therapy” means any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease, disorder, or condition in a patient. A patient may be at risk of developing a disease, disorder, or condition, or may be suspected of having a disease, disorder, or condition. Alternatively, a patient may have been diagnosed with a disease, disorder, or condition. Non-limiting examples of therapy include the administration of compositions (e.g., pharmaceutical compositions or vaccine compositions), physical therapies (e.g., radiotherapy, ultrasound therapy, electrotherapy, phototherapy, cryotherapy, etc.), physiotherapy, psychotherapy, etc. Therapy also includes the possibility of using combination therapy. “Combination therapy” and any variation thereof, e.g., “combination” means the act of delivering several different therapies to the same subject. Such combinations include cases where different therapies are delivered to the subject as a single composition (together in the same unit dose) or separately (i.e., separated formulations), in the latter case, the different therapies may be delivered simultaneously or sequentially. "Simultaneously" as used herein refers to the act of delivering several therapies mixed in the same composition at the same time. "Separately" as used herein refers to the act of delivering several therapies in separate forms of delivery (e.g., separate compositions) essentially simultaneously or within the same time period (e.g., within one hour or less). "Sequentially" means "in order," meaning that one therapy is delivered first, and immediately thereafter, or after a suitable time (e.g., more than one hour), another therapy is delivered.

[0042] When a therapy involves administering a composition or includes the administration of a composition, the composition is administered in an amount, method, and / or form that is effective in treating or preventing the patient's disease, disorder, or condition. A therapy may require more than one administration of the composition. In such cases, the interval between the delivery of two consecutive therapies may be the same or different. In the case of combination therapy, the interval between the delivery of two consecutive therapies of several different therapies may be the same or different.

[0043] In this specification, the terms “to treat” or “treatment” mean improvement of a patient’s disease, disorder, or condition, which may be observed at clinical, histological, and / or biochemical levels. The terms “to treat” or “treatment” include, among other things, improving clinical, histological, and / or biochemical symptoms or parameters associated with a patient’s disease, disorder, or condition, or inhibiting, suppressing, or delaying the progression or worsening of a patient’s disease, disorder, or condition (including secondary damage caused by the disease, disorder, or condition) to a statistically significant extent or to an extent detectable by a person skilled in the art. In some embodiments, treatment is evaluated on a population basis, and if a statistically significant improvement in a patient’s disease, disorder, or condition is observed in a population suffering from a particular disease, disorder, or condition, the treatment is considered to “treat” that disease, disorder, or condition.

[0044] The term “vector” as used herein refers to a vehicle, preferably a nucleic acid molecule or viral particle, that contains the elements necessary to enable the delivery, proliferation, and / or expression of any of the nucleic acid molecules described herein within a host cell or subject. The term encompasses maintenance vectors (cloning vectors) or expression vectors in various host cells or subjects (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids) or integration vectors (e.g., designed to be integrated into the genome of a host cell and generate further copies of the nucleic acid molecule as the host cell replicates), as well as shuttle vectors (e.g., functioning in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., transporting nucleic acid molecules in a viral genome). For the purposes of this disclosure, vectors may be from naturally occurring gene sources, synthetic or artificial, or any combination of natural and artificial genetic elements.

[0045] In the context of this disclosure, the term “vector” should be broadly understood to include mRNA, plasmids, and viral vectors. Vectors appropriate to the context of this disclosure include, without limitation, bacteriophages, plasmids or cosmid vectors for expression in prokaryotic host cells, e.g., bacteria (e.g., E. coli, BCG, or Listeria); vectors for expression in yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculoviral vectors for expression in insect cell lines (e.g., Sf9 cells); and plasmids and viral vectors for expression in higher eukaryotic cells or subjects. Typically, such vectors are commercially available (e.g., from Invitrogen, Stratagene, Amersham Biosciences, Promega, etc.) or available from depositaries such as the American Type Culture Collection (ATCC, Rockville, Md.), or are the subject of numerous publications describing their sequences, composition, and methods of preparation, making them applicable to those skilled in the art. This disclosure also includes vectors (e.g., plasmid DNA and mRNA) that form complexes with lipids or polymers to form particulate structures such as liposomes, lipoplexes, or nanoparticles.

[0046] Treatment method This disclosure provides a method for treating conditions associated with cone cell degeneration.

[0047] To our surprise, the inventors discovered that the degeneration of cone photoreceptor cells is caused by the activation of a specific cell death pathway in these cells, specifically ferroptosis.

[0048] The inventors have shown that ferroptosis-inducing substances, such as glutamate, elastin, imidazole ketone elastin (IKE), and RSL3, induce a specific decrease in cone cell viability. This decrease in cone cell viability is accompanied by a decrease in glutathione (GSH) levels, increased cell membrane pigment permeability, increased lipid peroxides, and dysregulation of iron metabolism. This effect was not only specifically observed in cultures of pure cone cells but was also evident in an ex vivo model of the retina, where microglia cells migrated to the outer granular layer (ONL) as seen in vivo. The importance of the in vivo pathogenesis is further highlighted by the finding that administration of ferroptosis-inducing substances to the eyes of non-human primates induces not only specific cone loss but also the formation of subretinal deposits and a decrease in electroretinogram amplitude, features very similar to those shown in AMD patients. Importantly, administration of such ferroptosis-inducing substances to the eye does not affect the survival rate of rod photoreceptor cells or retinal pigment epithelial cells.

[0049] Therefore, induction of ferroptosis in cones by activators of iron metabolism dysregulation leads to specific cone photoreceptor degeneration, resulting in a phenotype very similar to the in vivo pathological condition. Importantly, cone photoreceptor degeneration can be rescued by anti-ferroptosis treatment. In particular, there are activators of NRF2, which is responsible for regulating iron metabolism, such as astilbin, ginsenoside Rh3, mangiferin, and torecetin. Other inhibitors of iron metabolism dysregulation are the iron chelators deferlipron and CN128 (hydrochloride), which reverse the effects induced by ferroptosis in cone photoreceptor cells.

[0050] This demonstrates, for example, that specific inhibition of dysregulation of iron metabolism due to a decrease in intracellular iron levels in cone photoreceptor cells can prevent the cell death observed under these conditions.

[0051] This was further confirmed by our demonstration that administration of ferroptosis inhibitors, particularly those involved in iron metabolism, specifically reversed cone degeneration in rd1 mice, a well-known model of retinal degeneration, but did not rescue rod cells in these mice. Therefore, these results surprisingly confirm that the treatment of conditions related to cone cell generation disclosed herein is due to the specific and direct inhibition of dysregulation of iron metabolism in cone cells, while not involving or affecting rod cells or retinal pigment epithelial cells.

[0052] In a first aspect, the present disclosure relates to compounds for use in the treatment of conditions associated with cone photoreceptor degeneration, wherein the treatment comprises reducing intracellular iron levels in cone photoreceptor cells, and more particularly, compounds comprising specifically reducing intracellular iron levels in cone photoreceptor cells.

[0053] The disclosure also relates to the use of compounds for manufacturing pharmaceuticals for treating conditions associated with cone photoreceptor degeneration, wherein the treatment includes reducing intracellular iron levels in cone photoreceptor cells, and more particularly, the treatment includes specifically reducing intracellular iron levels in cone photoreceptor cells.

[0054] The disclosure also relates to a method for treating a condition associated with cone photoreceptor degeneration, comprising administering a compound to a subject in need thereof, thereby reducing intracellular iron levels in cone photoreceptor cells, in particular a method for specifically reducing intracellular iron levels in cone photoreceptor cells.

[0055] Ferroptosis is a controlled cell death pathway characterized by the simultaneous accumulation of large amounts of iron ions and lipid peroxidation. Imbalances in iron uptake, storage, and efflux affect the cell's susceptibility to ferroptosis. Several proteins that regulate iron metabolism have been identified, such as transferrin receptor 1 (TFR1) and divalent metal transporter 1 (DMT1) for iron transport and uptake, ferroportin (FPN) for intracellular iron efflux, and ferritin for iron storage. Dysfunction of these proteins leads to abnormalities in iron metabolism, causing a disruption of iron homeostasis. Iron excess triggers and catalyzes lipid peroxidation via the Fenton reaction, leading to ferroptosis. Furthermore, ferritinophagy, a selective form of autophagy, contributes to the initiation of ferroptosis through the degradation of ferritin, which leads to unstable iron excess. Once autophagy is activated, the NCOA4 protein mediates the binding of ferritin to lysosomes, triggering its degradation and the release of free iron.

[0056] Therefore, reducing intracellular iron levels by the method described herein preferably involves inhibiting ferritinophagy in cone photoreceptor cells. In particular, reducing intracellular iron levels by the method described herein preferably involves specifically inhibiting ferritinophagy in cone photoreceptor cells, i.e., ferritinophagy in other retinal cell types such as rod photoreceptor cells is not substantially inhibited. More preferably, the activity of ferritin or NCO4 is reduced or inhibited in cone photoreceptor cells by the method disclosed herein, particularly specifically, thereby resulting in a reduction, particularly specific reduction, of intracellular iron levels in cone photoreceptor cells.

[0057] The treatment according to this disclosure is particularly advantageous in that it specifically inhibits the death of cone photoreceptor cells. In particular, the viability of cone photoreceptor cells is maintained by the treatment. More specifically, the viability of cone photoreceptor cells is specifically maintained by the treatment, i.e., the viability of other retinal cell types such as rod photoreceptor cells or retinal pigment epithelial cells is substantially unaffected by the treatment. Alternatively, the treatment results in an increase in the viability of cone photoreceptor cells. More specifically, the treatment results in a specific increase in the viability of cone photoreceptor cells, i.e., the treatment does not substantially increase the viability of other retinal cell types such as rod photoreceptor cells or retinal pigment epithelial cells. The viability of cone photoreceptor cells can be measured by any means known to those skilled in the art. For example, the viability of these cells can be evaluated in vitro or ex vivo using, for example, calcein AM, MTT, alamarBlue, ethidium, or CellTiterGlo. Alternatively, cone viability can be assessed in an ex vivo model of the retina by, for example, measuring their pigment permeability, measuring retinal thickness, measuring the number of ONL nuclei (e.g., after DAPI staining), and / or determining the number of cone outer segments or cones after labeling the retinal tissue with cone-specific reagents (e.g., anti-FLAP antibody, anti-cone opsin antibody, peanut agglutinin lectin, or anti-cone arrestin antibody). In yet another example, cone viability can be assessed in vivo by optical coherence tomography (OCT) or adaptive optics retinal imaging. For example, OCT is widely used clinically to characterize the potential for retinal degeneration in patients because it clarifies the location and nature of changes in the retina and surrounding structures and allows for objective assessment of the thickness of the retina and surrounding structures. Prevention of cone degeneration reduction can therefore be measured by OCT. All of these techniques are well known to those skilled in the art. Cone cell viability can also be demonstrated in histological specimens by measuring retinal thickness, measuring the number of nuclei in the ONL (e.g., after DAPI staining), or determining the number of cones after labeling retinal tissue with a reagent specific to the outer segment or cone (e.g., anti-FLAP antibody, anti-cone opsin antibody, peanut agglutinin lectin, or anti-cone arrestin antibody).Those skilled in the art will understand that each of these methods can be adapted as needed depending on the situation to effectively measure the survival rate of cone photoreceptor cells.

[0058] In another example, the treatment disclosed herein is particularly advantageous in that it inhibits the migration of microglia to the ONL. Specifically, the treatment results in a reduction of microglial cell migration. Alternatively, microglial migration is inhibited by the treatment. Microglial migration is well known to those skilled in the art and can be determined by any means known in the art (Rashid et al. Front Immunol. 10:1975, 2019).

[0059] In another example, the treatment disclosed herein is particularly advantageous in that it interferes with the formation of subretinal deposits. Such deposits are observed in several conditions associated with cone degeneration, e.g., AMD. In particular, the treatment results in a reduction of subretinal deposit formation. Alternatively, the formation of subretinal deposits is inhibited by the treatment. Subretinal deposits are well known to those skilled in the art and can be determined by any means known in the art (Monge et al. Taiwan J Ophthalmol. 12(2):138-146, 2022).

[0060] In another example, the treatment disclosed herein is particularly advantageous in that it interferes with, in particular specifically, the reduction of cone photoreceptor activity. In particular, cone photoreceptor activity is maintained by the treatment, in particular specifically. Alternatively, the treatment results in an increase in cone photoreceptor activity, in particular a specific increase. Cone photoreceptor activity may be assessed by electroretinography (ERG) or multifocal electroretinography (mfERG); see, for example, Asanad S, Karanjia R. Multifocal Electroretinogram. [Updated 2022 Oct 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan.

[0061] Preferably, the procedures disclosed herein include obtaining at least two, at least three, or at least four of the above features. More preferably, the procedures disclosed herein include obtaining all five of the above features.

[0062] This therapy for conditions associated with cone cell degeneration can be used in combination with or as an adjunct to one or more other treatments.

[0063] The combination therapy provided herein involves the administration of at least two drugs to a patient, the first of which is a therapeutic compound disclosed herein, and the second of which is another therapeutic compound. Accordingly, this disclosure relates to the therapeutic compounds described herein for the treatment of conditions associated with cone cell degeneration, which are administered together with the second therapeutic compound.

[0064] The combination therapies disclosed herein can produce a greater effect than an additive or synergistic effect, and thus provide therapeutic utility, when neither the therapeutic compound disclosed herein nor the second therapeutic compound is administered in a therapeutically effective amount alone. Therefore, such agents can be administered in lower doses, reducing the potential and / or severity of adverse effects.

[0065] For example, the second therapeutic compound may be a second ferroptosis inhibitor. In particular, administration of a second ferroptosis inhibitor may result in a reduction or inhibition of lipid peroxidation in cone photoreceptor cells, especially a specific reduction or inhibition, and / or a reduction in intracellular iron levels in cone photoreceptor cells, especially a specific reduction. Appropriate secondary ferroptosis inhibitors include, among others, RNAs that inhibit any one of the following: DKK1;OTUB1;SCD1;ACSL4, LPCAT3, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5 and ALOX5AP; cystine; β-mercaptoethanol; selenium; bardoxolone; carvacrol (CAR); rehmannioside A; bioflavonoids, e.g., galangin; xanthohumol; naringenin; britanin; entacapone; capsiate; resveratrol; dexmedetomidine; irisin; 2-cyano-3; 12-dioxolean-1; 9-dien-28-acid (CDDO); and kaempferol; diloton; ferrostatin-1; liproxstatin-1; PDA NP;16-86;XJB-5-131;UAMC-3203;SRS15-72B;SRS15-72A;SRS16-80;SRS16-86;GSK2190915;MK-886;ML355;BLX-3887;VKH2;BAY x 1005;α-tocopherol;tolmetin;caffeic acid;diallyl trisulfide;trelagliptin (succinate), ellagic acid;norbergenin;chlorogenic acid;3'-hydroxypuerarin;1;3-dicaffeoylquinic acid;nuclear enriched transcript 1 (NEAT1);and rosmarinic acid.

[0066] Alternatively, a second therapeutic compound can treat or alleviate another aspect of this condition. In particular, the therapeutic compound is advantageously administered in conjunction with a second therapeutic compound that inhibits ocular angiogenesis. These compounds include, for example, aflibercept, ranibizumab, pegaptanib, bevacizumab, brolucizumab, falisimab, AKB-9778, nesbakumab, and BI 836880.

[0067] The therapeutic compound and the second therapeutic compound can be administered simultaneously, consecutively, or separately.

[0068] Therapeutic compounds for treating conditions associated with pyramidal degeneration This disclosure provides therapeutic compounds effective against conditions associated with cone photoreceptor degeneration. The compounds disclosed herein can, in particular, specifically reduce intracellular iron levels in cone photoreceptor cells. Indeed, this disclosure demonstrates that a decrease in intracellular glutathione levels (in cone photoreceptor cells) induces ferroptosis in the cone photoreceptor cells, ultimately resulting in a phenotype similar to the in vivo pathological condition. Furthermore, this induced ferroptosis of photoreceptor cells can be reversed by iron chelating agents such as deferlipron.

[0069] Therefore, the therapeutic compounds used in the methods disclosed herein are preferably ferroptosis inhibitors.

[0070] The therapeutic compounds disclosed herein include, in particular, chemical and biological agents (e.g., agents including small molecule drugs or biological agents such as antibodies or cells) useful for treating conditions associated with cone cell degeneration, the administration of which results in a decrease in intracellular iron levels.

[0071] Examples of biological agents include prominin 2, miR-522, and / or products of the RP11-89 gene, whose expression is known to protect cells from ferroptosis (Brown et al. Dev Cell. 51(5):575-586.e4, 2019; Zhang et al. Mol Cancer. 19(1):43, 2020; Luo et al. Cell Death Dis. 12(11):1043, 2021). Other examples of biological agents include RNA, including antisense RNA, siRNA, and shRNA, for inhibiting genes known to be involved in lipid peroxidation and promote ferroptosis. Such genes include, among others, FTL (Genbank ID: NM_000146), FTH1 (Genbank ID: NM_002032), which encode the light and heavy chains of ferritin, respectively, and / or NCO4 (Genbank ID: NM_005437). Preferably, the polynucleotides used herein are inserted into a vector, for example, a viral vector, such as an adenovirus-associated (AAV) vector containing AAV8, AAV2, AAV2tYF, and AAV5. Preferably, the polynucleotides are operably ligated to a regulatory sequence, which is advantageously suitable for expression in retinal or subretinal tissue. More preferably, the polynucleotides disclosed herein are operably ligated to a suitable regulatory sequence within a vector.

[0072] Delivery may be achieved via gene therapy, for example, by administering a vector disclosed herein encoding a therapeutic product into the extrachoroidal space, subretinal space (with or without vitrectomy (e.g., by using a catheter through the extrachoroidal space or by injection into the periphery), intraretinal space, and / or the outer surface of the sclera (i.e., scleral-preserving administration) of the eye of a human patient to create a permanent depot in the eye that provides a continuous supply of the therapeutic product (e.g., a post-translationally modified therapeutic product).

[0073] Alternatively, the compounds of this disclosure are small molecules. Examples of small molecules that cause a decrease in intracellular iron levels include, among others, deferipron, ferrostatin-1, deferoxamine (DFO), gossypol acetate, histochrome, cyclopiroxolamine (CPX), bafilomycin A1, quercitrin, baicalein, dexrazoxane, 2,2'-bipyridine, 1,10-phenanthroline, mangiferin, astilbin, YL-939, deferasirox (DFX), 1-O-hexyl-2,3,5-trimethylhydroquinone, 1,3-dicaffeoylquinic acid, rosmarinic acid, norbergenin, and tinoridine hydrochloride.

[0074] Preferably, the compounds disclosed herein are selected from the group consisting of prominin 2, miR-522, and / or RP11-89 genes; RNA that inhibits FTL, FTH1, and / or NCO4; deferipron, ferrostatin-1, deferoxamine (DFO), gossypol acetate, histochrome, cyclopiroxolamine (CPX), bafilomycin A1, quercitrin, baicalein, dexrazoxane, 2,2'-bipyridine, 1,10-phenanthroline, mangiferin, astilbin, YL-939, deferasirox (DFX), 1-O-hexyl-2,3,5-trimethylhydroquinone, 1,3-dicaffeoylquinic acid, rosmarinic acid, norbergenin, and tinoridine hydrochloride. More preferably, the compounds disclosed herein are selected from the group consisting of prominin 2, miR-522, and / or RP11-89 genes; RNA that inhibits FTL, FTH1, and / or NCO4; deferipron, ferrostatin-1, deferoxamine (DFO), gossypol acetate, histochrome, cyclopiroxolamine (CPX), bafilomycin A1, quercitrin, baicalein, dexrazoxane, 2,2'-bipyridine, 1,10-phenanthroline, mangiferin, astilbin, YL-939, and deferasirox (DFX).

[0075] Conditions related to cone cell degeneration This disclosure provides methods for treating conditions associated with cone photoreceptor degeneration. Conditions associated with cone photoreceptor degeneration disclosed herein are conditions in which specific cone degeneration is observed, i.e., conditions in which cone photoreceptor degeneration is not a secondary consequence of degeneration of the retinal pigment epithelium.

[0076] In some cases, cone photoreceptor cells may be the only cells affected by degeneration. In some cases, other cell types may also be affected by degeneration. For example, in some cases, conditions associated with cone photoreceptor degeneration will also show degeneration of adjacent supporting tissues, such as the retinal pigment epithelium. When the retinal pigment epithelium is also affected by degeneration, it must be understood that cone photoreceptor degeneration is not a secondary consequence of the degeneration of the retinal pigment epithelium, but rather specifically affected by degeneration of the cone photoreceptor cells. In preferred cases, other cell types affected by degeneration in conditions associated with cone photoreceptor degeneration described herein include not only adjacent supporting tissues, such as the retinal pigment epithelium, but also retinal neurons, such as rod photoreceptor cells, bipolar cells, ganglion cells, horizontal cells, and amacrine cells. In this very specific case, when any of these other cell types is also affected by degeneration, cone photoreceptor cells are also specifically affected by degeneration, meaning that cone photoreceptor degeneration is not a secondary consequence of the degeneration of one or more other cell types.

[0077] In particular, conditions associated with cone cell degeneration include age-related macular degeneration (AMD), cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt disease), central serous chorioretinopathy, Best's disease and bestrofinopathy, retinal detachment including rhegmatogenous retinal detachment, serous retinal detachment, and tractional retinal detachment, photoretinopathy, laser-induced retinopathy, monochromacy, Usher syndrome, Leber congenital amaurosis, Alström syndrome, and Refsum disease.

[0078] Preferably, the conditions associated with cone photoreceptor degeneration in the methods disclosed herein are selected from the group consisting of age-related macular degeneration (AMD), cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt disease), central serous chorioretinopathy, Best's disease and bestrofinopathy, retinal detachment including rhegmatogenous retinal detachment, serous retinal detachment, and tractional retinal detachment, photoretinopathy, laser-induced retinopathy, monochromacy, Usher syndrome, Leber congenital amaurosis, Alström syndrome, and Refsum disease. More preferably, the condition associated with cone photoreceptor degeneration in the methods disclosed herein is age-related macular degeneration (AMD). In another more preferred example, the condition associated with cone photoreceptor degeneration in the methods disclosed herein is cone dystrophy. In yet another more preferred example, the condition associated with cone photoreceptor degeneration in the methods disclosed herein is retinal detachment.

[0079] Conditions related to cone photoreceptor cells can be diagnosed by any technique or method known to those skilled in the art.

[0080] Standardized ophthalmic examination techniques known in the art include, for example, detailed slit-lamp biomicroscopy evaluation, which allows for the assessment of central retinal anatomical structures, including the eyelids, adnexa, eyelashes, corneal surface, anterior chamber, pupil, lens, vitreous cavity, and the optic nerve and macula. Another method is gonioscopy, which allows for detailed examination of the anterior chamber angle. Indirect ophthalmoscopic examination allows for the assessment of the peripheral retina, which is important for monitoring vitreous and peripheral retinal disorders.

[0081] Functional tests of visual acuity are known in the art and include, for example, best-corrected visual acuity, contrast visual acuity, and low-light visual acuity; color vision (including the Ishihara test and Farnsworth-Munsell test); visual field evaluation (including Humphrey automated perimetry and microperimeter); tear production (Schirmer test); and intraocular pressure (IOP) measurement. These are used in conjunction with structural tests, which include, for example, anterior and posterior segment photography, corneal thickness measurement, 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 computed tomography (CT) or magnetic resonance imaging (MRI) scans, is used to evaluate ocular structures, periorbital structures, and orbital structures, as well as the intracranial portion of the optic nerve, the visual pathway, and the visual cortex of the brain. These tests allow for the visualization of structural integrity, as well as the thickness of layers of the eye and surrounding structures, and the assessment of blood flow and circulation. Advanced functional tests of the retina, optic nerve, and visual pathway / cortex are also used, including whole-field and multifocal electroretinography, electrophysiological tests such as visual evoked potentials, and microperimeter measurements for diagnosis and monitoring disease progression and the effectiveness of therapy. Those skilled in the art will be able to deploy appropriate techniques known in the art to diagnose, measure, and monitor conditions associated with cone photoreceptor degeneration described herein.

[0082] Pharmaceutical composition The compound for treating conditions associated with cone cell degeneration can be formulated into a composition. Optionally, the composition may include one or more additional therapeutic agents, e.g., a second therapeutic agent described below. The composition will typically be supplied as part of a sterile pharmaceutical composition, which will typically contain a pharmaceutically acceptable carrier and / or excipient. In another embodiment, the present disclosure provides a pharmaceutical composition comprising a compound for treating conditions associated with cone cell degeneration and a pharmaceutically acceptable vehicle and / or excipient.

[0083] Accordingly, pharmaceutical compositions are provided herein that include one or more compounds for use in the treatment of conditions associated with cone cell degeneration, together with one or more pharmaceutically acceptable carriers, and optionally with other therapeutic and / or prophylactic components. A carrier (or excipient) is acceptable or suitable if it is compatible with the other components of the composition and is not toxic to the recipient (i.e., subject) of the composition. A pharmaceutically acceptable or suitable composition is an ophthalmologically appropriate or acceptable composition.

[0084] This composition may be in any suitable form (depending on the desired method of administering it to the patient). The composition used in the methods described herein may be administered, for example, intravitreously (e.g., into the subretinal space, extrachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or corneal surface), by eye drops, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intrathecally, intranasally, vaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, orally, topically, transdermally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, directly to target cells by local perfusion bath, by catheter, by lavage, as a cream, or as a lipid composition. The compositions used in the manner described herein may be administered systemically or locally. The method of administration may vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In any given case, the most suitable route of administration will depend on the specific compound, the subject, the nature and severity of the disease, and the subject's health condition. Accordingly, various delivery systems can be used to administer the compounds disclosed herein. Preferably, the compositions are administered to the subretinal space, extrachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or corneal surface. In certain preferred examples of the present invention, the compounds are formulated as aqueous solutions and administered by intravenous infusion. In some examples, administration is by intravenous infusion over one hour.

[0085] The compositions used in the manner described herein may also be administered systemically or locally. The method of administration may vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In any given case, the most suitable route of administration will depend on the specific therapeutic compound, the subject, the nature and severity of the disease, and the subject's health condition. Compounds for treating conditions associated with cone cell degeneration may be administered systemically (by inhaler, intravenously, intramuscularly, or intraperitoneally) or in the form of a solution, gel, or implant.

[0086] The compositions described herein can typically be formulated as liquid or fluid compositions, semi-solids (e.g., gels or hydrogels), foams, or porous solids (e.g., polymer matrices, complexes, calcium phosphate derivatives, etc., suitable for ophthalmic tissue engineering). Pharmaceutical compositions can, for convenience, be provided in unit dose forms, each dose containing a predetermined amount of the therapeutic compound disclosed herein. Such units may include, for example, 5 mg to 5 g, e.g., 10 mg to 1 g, or 20 to 50 mg. Pharmaceutically acceptable carriers for use in this disclosure can take a wide variety of forms, for example, depending on the condition being treated or the route of administration. Dosage forms may include, in particular, tablets, capsules, intravenous injections, intramuscular injections, topical injections, topical creams, gels and ointments, eye drops, ophthalmic solutions, ophthalmic suspensions, ophthalmic emulsions, intravitreal injections, sub-Tenon's injections, biodegradable ophthalmic implants, and non-biodegradable ophthalmic inserts or depots, nasal sprays and ointments, and various rectal or vaginal formulations.

[0087] The pharmaceutical compositions of this disclosure can be prepared for storage as lyophilized formulations or aqueous solutions by mixing compounds of desired purity with any optional, pharmaceutically acceptable carriers, excipients, or stabilizers commonly used in the art (all of which are referred to herein as "carriers"), namely buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipient at the dosage and concentration used.

[0088] Buffers help maintain pH within a range close to physiological conditions. They can be present in concentrations ranging from approximately 2 mM to approximately 50 mM. Suitable buffers for use in this disclosure include both organic and inorganic acids and their salts, e.g., citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartarate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate- The solution includes disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc., gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Furthermore, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0089] Preservatives can be added to slow the growth of microorganisms, and may be added in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives for use in this disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, e.g., methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. To ensure the isotonicity of the liquid compositions of this disclosure, isotonicizers, sometimes known as “stabilizers,” can be added, which include polyhydric sugar alcohols, e.g., trihydric or higher sugar alcohols, e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, and their function can range from volume extenders to additives that help solubilize therapeutic agents or prevent denaturation or adhesion to the container walls. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, etc.; cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, e.g. Examples include urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides with 10 or fewer residues); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides, such as lactose, maltose, sucrose, and trisaccharides, such as raffinose; and polysaccharides, such as dextran.The stabilizer may be present in an amount ranging from 0.1 to 10,000 by weight per 1 part by weight of active protein.

[0090] Nonionic surfactants or detergents ("also known as wetting agents") can be added not only to help solubilize the therapeutic compound but also to protect it from aggregation induced by agitation, which also allows the formulation to be exposed to a pressurized, shear surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), Pluronic® polyols, and polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Nonionic 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.

[0091] Further excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0092] This disclosure further describes combination products for simultaneous, separate, or sequential use, at least i) Therapeutic compounds described herein, i.e., compounds for treating conditions associated with cone cell degeneration and ii) For example, the subject is a pharmaceutical composition containing the second therapeutic agent described below.

[0093] The therapeutic compound and the composition of the second therapeutic agent can be administered alone, as a mixture of one or more therapeutic compounds and / or one or more second therapeutic agents, or in combination with other agents useful for treating conditions associated with cone cell degeneration. Examples of suitable combinations are provided below.

[0094] A pharmaceutical kit containing the therapeutic compounds described herein is encompassed by this disclosure. The pharmaceutical kit is a package comprising a compound (e.g., either in lyophilized form or as an aqueous solution) for treating a condition associated with cone cell degeneration and one or more of the following: • A second therapeutic agent, such as those listed below; • Devices for administering immune checkpoint inhibitors, such as pens, needles, and / or syringes; and • If the inhibitor is in lyophilized form, pharmaceutical-grade water or buffer for resuspending the inhibitor.

[0095] Each unit dose of the therapeutic compound can be packaged separately, and the kit may contain one or more unit doses (e.g., two unit doses, three unit doses, four unit doses, five unit doses, eight unit doses, ten unit doses, or more). In certain embodiments, one or more unit doses are each contained in a syringe or pen.

[0096] Effective dosage The therapeutic compounds 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 by using any of the methods described above, for example, an amount effective to prevent or reduce cone photoreceptor degeneration. Pharmaceutical compositions containing the therapeutic compounds can be administered to such patients (e.g., human subjects) in therapeutically effective doses.

[0097] The term "therapeutic dose" means the amount of an active compound or complex that elicits a desired biological response in a subject. Such responses include the alleviation of symptoms of the disease or disorder being treated, the prevention, inhibition, or delay of recurrence of symptoms of the disease or the disease itself, or the prevention, inhibition, or delay of progression of symptoms of the disease or the disease itself. More specifically, as used herein, a "therapeutic dose" is the amount that provides therapeutic utility. A therapeutic dose is also the amount in which the therapeutically beneficial effects of the drug outweigh the toxic or adverse effects of the drug.

[0098] In the context of this disclosure, the therapeutically effective dose refers to an amount of the therapeutic compound disclosed herein that is sufficient to prevent or reduce cone photoreceptor degeneration.

[0099] Preferably, the therapeutically effective dose is an amount of the therapeutic compound sufficient to obtain at least one of the following: • To maintain or increase the survival rate of cone photoreceptor cells; • To inhibit the increased pigment permeability of the cell membrane in cone photoreceptor cells; • To maintain or decrease the activity of ferritin and / or nuclear receptor coactivator 4 (NCOA4); • To inhibit or reduce the migration of microglia; • To prevent or reduce the formation of subretinal deposits; and • To maintain or increase cone cell activity.

[0100] Preferably, the therapeutically effective dose disclosed herein is sufficient to obtain at least two, at least three, at least four, at least five, or at least six of the above characteristics. More preferably, the therapeutically effective dose disclosed herein is sufficient to obtain all seven of the above characteristics.

[0101] The effective dosage can initially be estimated from an in vitro assay. For example, the initial dose may be formulated to achieve a concentration of the therapeutic compound that is at least the concentration of a compound capable of inhibiting cone photoreceptor degeneration in an in vitro or ex vivo assay, such as those described in this embodiment. Calculating the dosage to achieve such a concentration in the eye, taking into account the bioavailability of a particular compound, is well within the capabilities of those skilled in the art. For guidance, readers should refer 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.

[0102] Determining the effective dose is well within the capabilities of those skilled in the art, especially considering the detailed disclosure provided herein. The toxicity and therapeutic efficacy of the compound or complex can be determined by standard pharmaceutical procedures in cell culture and experimental animals. The effective dose of the therapeutic compound or the second therapeutic compound administered to a subject will depend on the stage, category, and status of multiple myeloma, as well as the subject's characteristics, such as overall health, age, sex, weight, and drug tolerance. The effective dose of the therapeutic compound or the second therapeutic compound administered will also depend on the route of administration and dosage form. Dosage and intervals can be individually adjusted to provide sufficient levels of the active compound in the eye to maintain the desired therapeutic effect.

[0103] The amount of therapeutic compound administered will depend on various factors, such as the nature and stage of the condition being treated, the form, route, and site of administration, the treatment regimen (e.g., whether another therapeutic compound is used), the age and condition of the specific subject being treated, and the patient's sensitivity to the disclosed therapeutic compound. An appropriate dosage can be easily determined by those skilled in the art. Ultimately, the physician will determine the appropriate dosage to be used. This dosage can be repeated at appropriate intervals. If side effects occur, the dosage and / or frequency of administration can be changed or reduced in accordance with standard medical practice. An appropriate dosage and treatment regimen can be established by using conventional techniques known to those skilled in the art and by monitoring the progress of therapy.

[0104] The effective doses of the therapeutic compounds described herein may range from about 0.001 to about 75 mg / kg per single (e.g., bolus), multiple, or consecutive dose, depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject, or may be within a range that achieves a serum concentration of 0.01 to 5000 μg / ml per single (e.g., bolus), multiple, or consecutive dose, or any effective range or value within that range. In specific examples, each dose may range from about 0.5 μg to about 50 μg per kilogram of body weight, for example, from about 3 μg to about 30 μg per kilogram of body weight.

[0105] The dosage, frequency, and duration of administration will depend on various factors such as the patient's age, weight, and disease status.

[0106] In various examples, the treatment period is at least one day. Preferably, the treatment period is at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. In other examples, it is at least one week. Preferably, the treatment period is at least two weeks, at least three weeks, at least four weeks, at least five weeks, or at least six weeks. The treatment period may be up to approximately 20 years. In embodiments, the treatment period includes one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks. The treatment period may be approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, or 24 months. In certain cases, treatment may continue for the patient's lifetime after diagnosis of a condition related to cone cell degeneration.

[0107] Other features and advantages of this disclosure are described in the following description, along with the examples and drawings. [Examples]

[0108] Example 1 method Data usefulness Animal model: rat Eight-week-old Long Evans wild-type rats were obtained from Janvier Labs (ISO 9001 certified). On day 0 (D0), the control and treatment groups were administered either 0.2% DMSO (D2650, Millipore Sigma, Burlington, MA) in PBS (14190-094 Life Technologies Europe BV, Bleiswijk, The Netherlands) or 20 μM IKE in a 0.2% DMSO solution diluted in PBS, respectively, via subretinal injection.

[0109] Surgical and anatomical procedures in rats Rats were anesthetized by intraperitoneal (IP) injection of 40 mg / kg ketamine (Ketamidor 100 mg / ml, Axience SAS, Pantin, France) and 0.14 mg / kg medetomidine (Domitol 0.85 mg / ml, Vetoquinol SA, Paris, France). Local anesthesia was achieved with oxybuprocaine eye drops (1.6 mg / 0.4 ml, Thea, Clermont-Ferrand, France). Left eye pupillary dilation was achieved with 0.5% tropicamide eye drops (Mydriaticum, Thea, Clermont-Ferrand, France). Throughout the procedure, tear gel (Lubrithal, Dechron, Shrewsbury, UK) was used as the lens-eye interface. Using an ophthalmic microscope (Lumera 700, Carl Zeiss, Oberkochen, Germany), a 30-gauge (G) needle (BD Microlance 3, Becton, Dickinson SA, Fraga, Spain) was used to create conjunctivoscleral channels. A flat contact lens (coverslip, small, 8 mm, World Precision Instruments, Sarasota, FL) enabled visualization of the retinal plane. A 30G cannula with a non-bevel metal tip, attached to a 10 μL syringe (Hamilton, Reno, NV) mounted on a microinjector (Micro 4, World Precision Instruments, Sarasota, FL), was used to deliver either a control or IKE solution subretinally through the nasal cavity. Following surgery, the patient received chloramphenicol-retinol ophthalmic ointment (Ophtalon 10 mg / g, TVM, Lempdes, France), 1 mL of 5% glucose monohydrate (Osalia, Paris, France) via intracutaneous injection, and 0.9 mg / kg of atipamezole (Antidorm 4.27 mg / ml, Axience SAS, Pantin, France) via subcutaneous injection.In vivo anatomical investigations, specifically optical coherence tomography (OCT, Bioptigen, Durham, NC) and fundus photography (Micron IV, Phoenix-Micron, Inc., Bend, OR), were performed on Day 0 to ensure subretinal delivery and to confirm retinal reattachment on the day of euthanasia. On Day 6 (D6), one group of animals (n=4) was administered 1 ml / kg pentobarbital (Exagon, Axience SAS, Patin, France) intracardiacally to evaluate microglial migration, and on Day 30 (D30), one group (n=8) was administered 1 ml / kg pentobarbital (Exagon, Axience SAS, Patin, France) intracardiac injection to evaluate cone degeneration, followed by euthanasia. The left eye was retrieved and immediately immersed in 4% paraformaldehyde (PFA, J61899.AP ThermoFisher Scientific, Waltham, MA) for 2 hours, then stored in PBS at 4°C. The eyeballs were dissected to obtain the entire retina, which was immersed in a 24-well plate (#351147 Corning Inc., Corning, NY) for immunohistochemistry. Furthermore, thin sections of the eye cups were labeled (using CAR and Iba1) to evaluate another quantitative analysis. To do this, the eyes were harvested from rats and the corneas were removed. The eyes were then immersed in 4% PFA for 1 hour, followed by immersion in continuous sucrose baths from 10% to 30%. Finally, the lenses were carefully removed and the eye cups were placed in a freezing medium in liquid nitrogen. They were stored at -20°C until they were cut into 12 μm thin sections using a cryostat (CM3050 S, Leica, Wetzlar, Germany).

[0110] Mammalian eyes With the agreement of the local regulatory department and a veterinarian from the French Ministry of Agriculture (Agreement FR75105131), the pig eyes were obtained from a local slaughterhouse.

[0111] Pure cone photoreceptor cells The retina was obtained by dissecting pig eyes and cut into small pieces. These were then digested with 4 U / mL papain (LSO 3124, Worthington) and L-cysteine ​​(5.5 mM, Sigma-Aldrich) at 37°C for 20 minutes. Enzyme activity was stopped with Neurobasal-A (NBA) medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) containing 5% fetal bovine serum (FBS) and 15 μg / mL DNase I (D4263, Millipore Sigma, Burlington, MA). A series of short centrifuges (30 seconds, 5-6 times) were performed to collect the supernatant, except for the first centrifuge which mainly contained rod photoreceptor cells. Between each centrifuge, the pellet was resuspended in P1000 to dissociate any remaining cells. Subsequently, the cell suspension was centrifuged at 800 rpm for 10 minutes, and the pellet was resuspended in NBA medium containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, ThermoFisher Scientific, Waltham, MA, USA).

[0112] Pyramid PR was purified by lectin panning selection as previously described (Balse et al. (2005) IOVS 46:3672) with slight modifications to obtain millions of pylori cells in suspension. Then, cells were seeded into 384-well plates (78109, Greiner, Ulis, France) at 3000 cells / well using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated at 37°C and 5% CO2 for 3 days.

[0113] retinal explant Pig and non-human primate retinas were cultured as previously described (Greferath et al. (2016) Ophthalmology 123:1320). After isolation of the retinas in CO2-independent medium, a 2 mm biopsy punch was used to obtain a small piece from the region near the optic nerve, which was placed on a polycarbonate membrane (140652, Thermo, Waltham, Massachusetts) with the photoreceptor cells facing upward. The explants were cultured in a CO2 incubator at 37°C for 3 days.

[0114] process To induce ferroptosis, the following components were used: glutamate (49621, Sigma Aldrich, Missouri, USA), 1 μM to 500 μM in vitro; imidazole ketone elastin (IKE) (HY-114481, MedChemExpress, Monmouth, USA), 3 μM to purified cone photoreceptor cells or 20 μM in ex vivo and in vitro experiments; and RSL3 (HY-114481, MedChemExpress, Monmouth, USA), 20 nM, 200 nM, and 10 μM in cells, ex vivo experiments, and in vivo experiments, respectively. To inhibit ferroptosis, ferrostatin-1 (FST-1, HY-100579, MedChemExpress, Monmouth, USA) was used at 50 nM to purified cones or 400 nM to retinal explants. Furthermore, deferipron (HY-B0568, MedChemExpress, Monmouth, USA) was effective against pure cones at 50 μM, and Zillowton (HY-14164, MedChemExpress, Monmouth, USA) was effective against purified cones at 20 μM.

[0115] Cell viability measurement After incubation for 3 days, cell viability was measured by incubation at 37°C for 1 hour with 1 / 4000 calcein (C1430, Thermo, Waltham, Massachusetts). The number of viable cells was calculated using an automated fluorescence microscope, ArrayScan (Cellomics ArrayScan VTI HCS reader, Thermo, Waltham, MA).

[0116] Screening in primary cells Robots with 384 well heads (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) can perform 4×10 3 Purified cone photoreceptor cells were seeded into clear, flat-bottomed 384-well plates (781091, Greiner, Ulis, France) to a cell / well ratio. In the first two rows, cells were treated with DMSO (D2650, Millipore Sigma, Burlington, MA) alone, while in the remaining rows, cells were treated with either 20 nM final RSL3 (HY-114481, MedChemExpress, Monmouth, USA) or 3 μM final IKE (HY-114481, MedChemExpress, Monmouth, USA). Seventeen hours after induction of cytopathy, ferroptosis library compounds (HY-L051, MedChemExpress, Monmouth, USA) were added to the cells using a robot with a 384-well head (BRAVO, Assist, Integra-Biosciences, Saint-Ouen, France) to obtain a final concentration of 10 μM of the compound. Each treatment was diluted in neurobasal medium (10888022, ThermoFisher Scientific, Waltham, MA, USA) and 1 / 100 L-glutamine (G3126, Millipore Sigma, Burlington, MA). The cells were then incubated at 37°C and 5% CO2 for a further 2 days. Cell viability was then measured on day 3 after seeding.

[0117] The results were analyzed using TIBCO Spotfire® software (California, USA). For each plate, the robust Z' factor was calculated, and those with a value greater than 0.45 were selected. Then, for each plate, the percentage of viable cells was calculated from the positive control. The average number of cells counted was calculated and reported as 100%. This positive control ratio was reported for all molecules in the plate. Next, compounds with cell viability higher than 50% were selected, and all plates treated with the same process (IKE or RSL3) were displayed so that we could extract these compounds into Excel.

[0118] Lipidomic Analysis Purified cone photoreceptor cells treated with or untreated with 3 μM IKE were washed three times with PBS, collected in 100 μL using a scraper, and immediately flash-frozen in liquid nitrogen. The cells were then stored at -80°C until extraction. Eicosanoid extraction and analysis were performed at the core of the lipidomic facility: MetaToul-Lipidomique (I2MC, Inserm, Toulouse, France), using MetaboHUB-ANR-1 1-INBS-0010.

[0119] Western blot Cells were harvested and separated from the culture medium by centrifugation. Whole cell proteins were extracted in lysis buffer (10 mM HEPES [pH 7], 100 mM NaCl, 2 mM EDTA, 0.5% NP-40, and a protease inhibitor cocktail (Millipore Sigma, Burlington, MA)). After centrifugation at 13000 rpm, the supernatant was set aside for further WB analysis. The samples were then loaded onto 4%-15% Mini-PROTEAN TGX Tris-glycine buffer SDS-PAGE and transferred to a 0.2 μm Trans-Blot Turbo nitrocellulose membrane (Bio-Rad, California, USA). The membrane was lysed in 1× Tris-buffered saline (TBS, 10 mM) with 5% (w / v) dried skim milk powder added. The lysates were blocked in Tris-HCl [pH 8] and 150 mM NaCl at room temperature for 1 hour. Primary antibody incubation was performed overnight at 4°C. The presence of 5LOX and FLAP at their expected molecular weights in the total lysate was confirmed by Western blotting using anti-5LOX (1:200; Abcam AB169755) and anti-FLAP (1:500; Abcam AB85227). For the detection of both 5LOX and FLAP, the HRP-conjugated secondary antibody used was goat anti-rabbit (1:20,000; 111-035-003, Jackson ImmunoResearch Laboratories, Pennsylvania). During and after antibody incubation, the membranes were broadly washed in TBS (TBS-T) containing 2.5% Tween-20. Western blots were visualized using enhanced chemiluminescence (ECL Prime detection reagent, Amersham, UK).

[0120] NADPH assay Using the NADP / NADPH assay kit (ab176724, Abcam, Cambridge, UK), the levels of NADP and NADPH were quantified, respectively, according to the manufacturer's instructions.

[0121] Glutathione (GSH) assay We quantified GSH levels according to the manufacturer's instructions for the GSH assay kit (V6912, Promega, Madison, WI).

[0122] Cryostat section After incubation for 3 days, the retinal explants were fixed in 4% paraformaldehyde (15714, Electron Microscopy Sciences, Hatfield, Pennsylvania) at room temperature for 1 hour and washed three times with PBS. The retinal explants were then kept at +4°C or sectioned transversely. Finally, they were cryopreserved in successive 10%, 20%, and 30% sucrose baths. The samples were then frozen in tissue freezing medium (72592, Electron Microscopy Sciences, Hatfield, Pennsylvania) by immersion in liquid nitrogen until the tissue freezing medium appeared to solidify. The samples were kept at -20°C until cut into 10 μm transverse thin sections using a cryostat (CM3050 S, Leica, Wetzlar, Germany). The retinal cross-sections were kept at -20°C.

[0123] immunolabeling First, the samples (fixed retinal explants and retinal cross-sections) were permeabilized with 0.5% Triton 100X (T8787, Sigma-Aldrich, Missouri, USA) diluted with PBS 1×. Then, nonspecific regions were blocked with a saturated buffer consisting of 10% normal donkey serum (S30-100ML, Millipore Sigma, Burlington, MA) diluted with PBS 1×. The primary and secondary antibodies used in this study are listed in Table 1.

[0124] [Table 1]

[0125] PNA lectin (L21409, Thermo, Waltham, Massachusetts) diluted to 1 / 50 and rabbit FLAP antibody (ab85227, Abcam, Cambridge, UK) at an active concentration of 5 μg / mL target antigenic sites on the extracellular side of the cell membrane. These labels were not subjected to a permeabilization process.

[0126] Finally, the retinal explants were washed three times in PBS and mounted flat in Cellvis plates (P12-1.5HN, IBL, Gerasdorf, Austria) using Permafluor (TA-030-FM, Thermo, Waltham, Massachusetts) for fluorescence microscopy. Transverse retinal thin sections and entire mounted retinas were also mounted on coverslips or in 6-well plates (P06-1.5HN, Cellvis, Mountain View, CA) using Permafluor.

[0127] Confocal microscope The retina was imaged using a laser scanning confocal microscope (Fluoview V-1000, Olympus, Tokyo, Japan). In rat retinas, cone counting (outer segments and cell bodies per 1 mm) was performed manually on retinal thin sections.

[0128] CQ1 Imaging Whole-mounted rat retinas and flat-mounted porcine explants were imaged using a CQ1 confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan). In the injected rat retinas, the inferonasal and inferotemporal petals were manually re-examined in the four z-axis planes defined by cone labeling: the outer segment (OS) plane, the cone nucleus (CN) plane, the inner fiber (IF) plane, and the cone stalk (CP) plane. Emphasis was placed on microglia migration. Representative samples were extracted from the CQ1 software. Z-stack images were analyzed using Cell Pathfinder software (Yokogawa, Tokyo, Japan), which enables 2D, 3D, or slice quantification. Algorithms were developed to quantify cone cell bodies or outer segments and microglia cells along the z-axis (one for porcine explants and one for whole-mounted rat retinas). The extracted quantified data was analyzed using TIBCO Spotfire® (California, USA).

[0129] Non-human primates (NHPs) Two NHPs were used in this study: a 5-year-old male and a 15-year-old female. The animals were born in captivity and acquired from AAALAC-certified approved suppliers (SARL Bioprim, Bazieges, France; Cynologics-Silabe, Niederhausbergen, France). Ethical review approval was obtained from the MIRCen regional ethics committee CETEA n°44 and then from the French Ministry of Education and Research. A complete description of housing and perioperative care is available in Dentel et al. (2023) Ophthalmol. Sci. 3:100316.

[0130] IKE toxicity in NHP was evaluated by subretinal delivery of a 20 μM IKE solution into one eye and a control solution (0.2% DMSO diluted in PBS) into both eyes. A comprehensive description of anesthesia, surgical procedures, and data acquisition is available in Dentel et al. Briefly, subretinal injection was performed in each eye to detach the superior fovea. Anatomical investigations (slit-lamp test, optical coherence tomography (OCT), adaptive optics (AO)) and functional investigations (whole-field electroretinography and multifocal electroretinography, ffERG and mfERG, respectively) were performed at baseline (within one week before surgery), on day 3 (to confirm completion of retinal reattachment), at one month, and then monthly until four months. Adaptive optics cone mosaic parameters were quantified using the integrated software of rtx1 (AOdetect, Imagine Eyes, Orsay France). Furthermore, the power spectral spacing method, which was additionally used to quantify cone mosaicism in NHP, is described in detail by Dentel et al.

[0131] Analysis and Statistics Analysis, graphs, and charts were created using GraphPad Prism 8.4.0 Software (GraphPad Software Inc., San Diego, CA). The treatment and control groups were statistically compared using the Mann-Whitney t-test, and statistical significance was considered to be p<0.05(*), p<0.01(**), p<0.001(***), or p<0.0001(****).

[0132] In the charts, data was normalized relative to the control where possible and expressed as the mean + / - standard error of the mean.

[0133] In in vitro and ex vivo analyses, the Mann-Whitney t-test was used to compare the treated and controlled populations. In rats, differences in cone density and microglia density were compared using multiple t-tests, and statistical significance was determined using the Holm-Sidak method. In NHP, differences in cone density, regularity, interval values, and ERG amplitude waves were compared using two-sided t-tests. In mfERG in NHP, each hexagon was compared to its corresponding contralateral hexagon, taking into account symmetry between the two eyes.

[0134] result Glutamate-induced ferroptosis in purified cone photoreceptor cells Photoreceptor cells release their neurotransmitter, glutamate, at their synaptic terminals, but they express only glutamate transporters (not classical ion channel receptors). When glutamate was applied to purified porcine cone photoreceptor cells (Figure 1A-D), their viability decreased by 20% at 10 μM and by 82% at the highest concentration tested, 500 μM (Figure 1E). This range of glutamate is physiologically observed, as indicated by the glutamate affinity of receptors and transporters (Gielen (2010) medecine / sciences 26:65). To clarify whether this toxicity, which has been reported in cone photoreceptor cells by immunocytochemistry (Dun et al. (2006) Cell Tissue Res 324:189; Bridges et al. (2004) Ophthalmol. Vis. Sci. 45:2906-2914), may be related to the expression of the Xc-system antiporter, we applied glutamate in the presence of 1 mM L-cysteine. At all glutamate concentrations, L-cysteine ​​inhibited the toxicity of glutamate (n=7, p<0.0001, IC50=4.15 μM) (Figure 1E). Because the Xc-system transporter has a pharmacology very different from other glutamate transporters or receptors, we investigated how different agonists and antagonists of this transporter affect cone survival. Two functional inhibitors of the Xc-system, elastin and imidazole ketone elastin (IKE), showed toxicity to isolated cone photoreceptor cells, similar to glutamate, but at lower dose ranges, consistent with their greater affinity (Figures 1A-D, F). L-cysteine ​​also rescued cones exposed to IKE (n=6, p=0.0022) (Figure 1K).

[0135] To evaluate the molecular mechanisms underlying this glutamate-induced toxicity in cone photoreceptor cells, we measured GSH levels in cones exposed to 500 μM glutamate and imidazole ketone elastin (IKE 3 μM). In both conditions, a 50% decrease in GSH was observed from day 1 onward compared to the control condition (Figure 1F, n=18, p<0.0001). The cell death of cone photoreceptor cells is therefore due to dysregulation of cellular GSH levels, induced by Xc-system dysfunction. Knowing that GSH is involved in the redox cycle in which its oxidized form is reduced by hydrogen atoms carried by NADPH (Dixon et al. (2019) Annu. Rev. Cancer Biol. 3:35), we measured the NADPH / NADP ratio in cones treated with 3 μM IKE. This condition increased the amount of NADPH from 25% in the control to 85% in the treated culture (n=4, p=0.0159) (Figure 1G). The intracellular NADPH / NADP ratio is indirectly related to the activity of glutathione peroxidase 4 (GPX4) during the redox of glutathione. When GPX4 is inactive, this cycle is also inactive, which may induce this NADPH accumulation, suggesting that inhibition of the Xc-system also downregulates the redox cycle (Azuma et al. (2022) J. Biol. Chem. 298:101824). To investigate whether GSH is decisive for cone cell survival, we used Ras-selective lethal 3 (RSL3), a specific inhibitor of GPX4. This inhibitor showed toxic effects on cone cell photoreceptor cells at very low concentrations (n=3, IC2). 50 (=8.67nM) (Figure 1H), highlighting the crucial role of GPX4 in cone cell survival. Then, by performing RNA extraction from treated and control pure cone cell photoreceptor cells, we showed altered expression of proteins involved in regulating the glutathione redox cycle and downstream mechanisms (Figure 2).

[0136] GPX4 is also known to inhibit the formation of peroxide lipids that react with free radical species (Xie et al. (2016) Cell Death Differ. 23:369; Dixon et al. 2019). These peroxide lipids are formed by lipoxygenases such as 5-lipoxygenase (5LOX), which is activated by 5-lipoxygenase-activating protein (FLAP). To investigate the presence of these mechanisms in cone photoreceptor cells, we performed Western blotting and found that both 5LOX and FLAP are present in cones but not in rod photoreceptor cells (Figure 1I). Specific immunostaining of retinal tissue from different species revealed specific FLAP localization in cone photoreceptor cells (Figure 3). To evaluate the role of this peroxide lipid pathway in glutamate-induced cell death, cone cell extracts were lipidochemically analyzed after incubation in 3 μM IKE for 2 days. Analysis showed an increase in the amount of peroxides of lipids such as 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) (n=6, p=0.0079(**), p=0.0159(*)) (Figure 1J), a metabolite of 5LOX. To demonstrate the contribution of this lipid peroxidation to the observed glutamate-induced toxicity, we tested the effect of Zillowton (ZEN), an enzymatic antagonist of 5LOX, in the presence of IKE. ZEN rescued 80% of cone photoreceptor cells (Figure 1K, n=3, p=0.0238), suggesting that lipid peroxidation plays a crucial role in glutamate-induced cone degeneration.

[0137] Lipid peroxidation induces changes in iron metabolism during the ferroptosis process. The ferritinophagy process can be enhanced by immunolabeling of ferritin and nuclear receptor coactivator 4 (NCOA4), a ferritinophagy inducer. Pyramid cells treated with IKE showed increased immunolabeling intensity for both ferritin heavy chain and NCOA4. NCOA4 appeared slightly stronger than ferritin heavy chain, suggesting increased iron metabolic activity in treated pyramid cells (Figure 4). The ferritinophagy process can be inhibited by ferrostatin-1 (FST1, a specific ferroptosis inhibitor) and deferlipron (DF, an iron chelator that inhibits the Fenton reaction by limiting the intracellular accumulation of free iron). Both rescued cones from IKE-induced toxicity, with 68.5% (n=8, p=0.0047) rescued by FST1 and 77.2% (n=5, p=0.0079) by DF (Figure 1K). Furthermore, other anti-ferroptosis molecules targeting different components involved in ferroptosis activation were tested after treatment with 20 nM RSL3 and 3 μM IKE (Table 2). Thus, pure cone photoreceptor cells exposed to ferroptosis promoters (e.g., IKE) can be rescued by anti-ferroptosis treatment, suggesting that the role of ferroptosis in cone survival is enhanced.

[0138] [Table 2-1]

[0139] [Table 2-2]

[0140] Ferroptosis of cone cells in an ex vivo retinal model To demonstrate that cone photoreceptor cells can degenerate by ferroptosis in an integrated retinal model lacking retinal pigment epithelium, we applied IKE to newly prepared porcine retinal explants. After incubation for 3 days, the specificity and selectivity of ferroptosis in the retina were evaluated by examining damaged cells (ethidium dye permeability of the cell membrane). Under control conditions, ethidium labeled only a small number of cells, but in IKE-treated explants, the nuclei of the first row of photoreceptor cells were labeled with ethidium (Figure 5B, F). This prominent layer corresponds to the nuclei of cone photoreceptor cells in porcine retina, as shown by flap immunolabeling (Figure 5C, G) (Pattnaik et al. (2000) J. Neurosci. 29:6789). Figure 5G illustrates the morphological changes of cone photoreceptor cells induced by 20 μM IKE. Other types of retinal cells were also labeled and did not show significant qualitative changes (Figure 6). Next, the in-situ cytotoxicity in cone photoreceptor cells was assessed by counting the level of immunolabeled outer segments (OS) with Flap (automatically using CellPath software for CQ1(C)) (Figures 5J-M). 20 μM IKE induced a 50% reduction in cone OS (n=3, p=0.0286) (Figure 5I). 1 μM FST-1 and 100 μM DF rescued 84% and 78.4% of OS, respectively (n=3, p=0.1000 and p=0.0286, respectively) (Figure 5I). Similarly, in retinal explants, 200 nM RSL3 induced cone degeneration (Figure 7), and 100 μM DF rescued 78.5% of OS (n=3, p=0.0286) (Figure 7M). These data suggest that ferroptosis is an effective and selective pathway for cone cell degeneration in integrated retinal tissue.

[0141] Furthermore, the migration of microglia from the inner retina (normal localization) to the outer retina (where microglia are normally absent) appeared to be induced in retinal tissue exposed to ferroptosis-inducing substances. Activation of microglial cells was suggested by an increase in their volume and changes in shape, accompanied by elongation of cells surrounding the cones (Figure 5H). Automated quantitative analysis showed significant intraretinal migration of microglia toward the outer granular layer (ONL) induced by both 20 μM IKE and 200 nM RSL3 (n=3) (Figure 5N). Migration was partially inhibited by ferroptosis inhibitors (FST-1 and DF) (n=3) (Figure 5O; Figure 7N). This morphological change of microglial cells and their migration toward the photoreceptor layer provides further evidence of ferroptosis-induced cone degeneration.

[0142] Pyramidal degeneration and microglial migration in rats To evaluate whether ferroptosis promoters enable the initiation of degenerative and inflammatory processes in living retinas, ferroptosis inducers were injected into the subretinal space of Long Evans rats (20 μM IKE, see Figure 8A). On day 6, optical coherence tomography (OCT) showed numerous small, highly reflected cells suggestive of microglia in rats injected with the ferroptosis inducer (arrows in Figure 8G). Next, the same retinas were examined by immunostaining for cones and microglia. Cone photoreceptor cell density was assessed on day 30 in retinal thin sections passing through the injection area. Overall survival (OS) and cell bodies were substantially reduced in animals treated with the ferroptosis inducer via subretinal delivery (Figures 8H-I). Quantification in retinal sections showed approximately a one-third reduction in cell bodies and nearly a half reduction in OS with the ferroptosis inducer (Figure 8J). Microglia migration within the injection area was quantified using D6 in a fully mounted retina, showing an increase in microglia at OS levels and cone synaptic terminals (Figure 8K). Figures 8L-O illustrate the high density of punctate microglia in the photoreceptor layer on day 6. These data suggest that inducing ferroptosis in living rat retinas leads to cone degeneration and microglia migration toward the photoreceptor layer.

[0143] Macular degeneration in non-human primates The ferroptosis-inducing substance was similarly applied in vivo to the retinas of living non-human primates. Subretinal delivery of 20 μM IKE was performed in one eye, and subretinal delivery of a control solution (1 / 500 DMSO) was performed in both eyes, followed by 4 months of observation (see Figure 9A). In previous studies, injection of vehicle solutions into the subretinal space did not show functional or anatomical ocular or systemic adverse effects in NHPs (two NHPs have been followed for 6 months to date), but resulted in transient changes in photoreceptor cells confirmed by adaptive optics ophthalmoscopic examination, which completely recovered after 4 months (Dentel et al. 2023).

[0144] In eyes injected with 20 μM IKE, structural and functional changes in the retina were observed in vivo. Initially, macular pigment deposition surrounding foveal subretinal deposits occurred within 3 months (Figure 9B-D). OCT showed punctate, small, pinpoint high reflectivity in the eroded ellipsoidal zone (i.e., the zones of the inner and outer segments of photoreceptor cells) and ONL only within the detached area with 20 μM IKE (Figure 9E-G). Some subretinal deposits appeared at the foveal level at 1 month (Figure 9F) and at the macular level at 2 months (Figure 9H-J). Adaptive optics imaging of some subretinal deposits (a technique enabling in vivo visualization of cone photoreceptor cells) appeared similar to that seen in patients with age-related macular degeneration (AMD), showing substantial loss of cones within the detached area with 20 μM IKE (Figure 9K-L). The integrated OCT software, which enables ONL splitting through the injection region, showed a significant change in ONL thickness within the region injected with 20 μM IKE (Figure 9M, N).

[0145] Adaptive optics was also used to quantify cone density in the injection area. The effect of standard subretinal delivery of 0.2% DMSO on cone reflexivity in adaptive optics imaging has already been reported (Dentel et al. 2023). In eyes injected with 20 μM IKE, cone loss was observed: at an eccentric position of 2 degrees superiorly, the cone density was 661.00 cells / deg at 4 months. 2From 37.50 pieces / mm 2 The value decreased to (n=2, p<0.001). No change was observed at an eccentric position 2 degrees downward (i.e., outside the injection area).

[0146] Functional changes in cone photoreceptor cells were evaluated by multifocal electroretinography (mfERG): changes from baseline in N1 wave amplitude (an indicator of the hyperpolarization response of cone photoreceptor cells to light) and P1 wave amplitude (an indicator of the functionality of the inner retina) were analyzed. N1 wave amplitude was significantly reduced in eyes injected with 20 μM IKE (-52.19%) compared to eyes injected with the control solution (-2.95%) (n=2, p=0.0396). A non-significant decreasing trend in P1 wave amplitude was also noted (-33.03% vs. -3.83%, n=2, p=0.0874). Subretinal delivery of ferroptosis-inducing substances at the macular level in non-human primates resulted in changes strongly reminiscent of clinical features that can be seen in macular degeneration.

[0147] Example 2 Ferroptosis does not affect primary retinal pigment epithelial cells. Materials and methods Purification of porcine cone photoreceptor cells The retina was obtained by dissecting a pig eye, cut into small pieces, and digested with 4 U / mL papain (LSO 3124, Worthington) and L-cysteine ​​(5.5 mM, Sigma-Aldrich) at 37°C for 20 minutes. 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 μg / mL DNase I (D4263, Millipore Sigma, Burlington, MA). Several short centrifuges (110 × g for 30 seconds, 5-6 times) were performed, and the supernatant was collected, excluding the first centrifuge which mainly contained rod photoreceptor cells. During centrifugation, the pellet was resuspended using a P1000 pipette to dissociate any remaining cells. The cell suspension was then centrifuged at 110 × g for 10 minutes, and the pellet was resuspended in NBA medium containing 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA) and 1% B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA).

[0148] Cone photoreceptor cells were purified by lectin panning using 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 seeded at a density of either 3,000 or 4,000 cells / well in clear 384-well plates (781091, Greiner, Ulis, France) or white 384-well plates (781080, Greiner, Ulis, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France), and incubated at 37°C for 3 days in an atmosphere containing 5% CO2.

[0149] Purification of porcine retinal pigment epithelial (RPE) cells Pig eyes were dissected, the retina was removed, and eye cups containing RPE cells were obtained from the fundus. Pre-warmed 0.25% trypsin (25200-056, Thermo Fisher Scientific, Waltham, MA, USA) was added to the eye cups and incubated at 37°C for 1 hour. Then, the cells were removed from the eye cups by flushing with a pipette and transferred to tubes containing DMEM medium (41966-029, Thermo Fisher Scientific, Waltham, MA, USA) and 20% FBS (A31605-01, Thermo Fisher Scientific, Waltham, Massachusetts) (=D20). After centrifugation at 110×g for 5 minutes, 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 medium was changed the following day.

[0150] The cells were then seeded at a rate of 4,000 cells / well into white 384-well plates (781080, Greiner, Ulis, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated at 37°C for 3 days in an atmosphere containing 5% CO2.

[0151] ARPE-19 cell line The cell lines were distributed by LGC and obtained from ATCC. The cells were used between P15 and P30 and seeded in D20 medium.

[0152] The cells were then seeded at a rate of 4,000 cells / well into white 384-well plates (781080, Greiner, Ulis, France) using a robot (Viaflo Assist, Integra-Biosciences, Saint-Ouen, France) and incubated at 37°C for 3 days in an atmosphere containing 5% CO2.

[0153] process To induce ferroptosis, the following compounds were used: glutamic acid (49621, Millipore Sigma, Burlington, MA) at concentrations of 1 μM to 1 mM in vitro; IKE (HY-114481, MedChemExpress, Monmouth, USA) at concentrations of 200 nM to 25 μM, e.g., 3 μM in purified cone photoreceptor cells and 20 μM in ex vivo and in vitro experiments; and RSL3 (HY-100218A, MedChemExpress, Monmouth, USA) at concentrations of 10 nM to 10 μM, e.g., 20 nM, 200 nM, and 10 μM in cells, ex vivo, and in vivo experiments, respectively. For inhibition of ferroptosis, FST-1 (HY-100579, MedChemExpress, Monmouth, USA) was used at a concentration of 50 nM against purified cones or 400 nM against retinal explants. DF (HY-B0568, MedChemExpress, Monmouth, USA) was effective at a concentration of 50 μM against pure cones, and ZEN (HY-14164, MedChemExpress, Monmouth, USA) was effective at 20 μM against purified cones (see Figures 1K, 5I, and 7M).

[0154] CellTiter Glo (CTG) Cell viability assays were performed on RPE cells, ARPE-19 cells, and cone photoreceptor cells using CellTiter Glo (G7573, Promega, Madison, WI). Viability was measured according to the manufacturer's instructions.

[0155] result IKE and RSL3 do not alter primary RPE cells in vitro. The inventors further investigated the sensitivity threshold of retinal pigment epithelial cells (RPE) after administration of a ferroptosis-promoting agent. It is well known in the literature that RPE cells may experience death via ferroptosis (Lee et al. (2022) Oxid. Med. Cell. Longev. 2022:1792894). However, the problem is determining the most sensitive cell type between RPE cells and cone PR. The inventors therefore used the ARPE-19 cell line, which has been used in all studies to characterize RPE cells, and employed various concentrations of IKE, RSL3, and glutamate. Treatment of cells with IKE showed a weak effect on ARPE-19, with 34.3% viable cells at 25 μM, compared to 0% for cones (n=3, p=0.0001) (Figure 11A). Similarly, treatment with ARPE-19 in RSL3 resulted in a higher mortality rate, but at considerably higher concentrations than those used to kill cones. At 10 μM RSL3, only 5.3% of ARPE-19 cells remained alive, while at 5 μM, only 50% survived (n=3, p=0.0008) (Figure 11B), while the EC50 of RSL3 for cones was 1.58 nM, demonstrating a much higher sensitivity to GPX4 inhibition in cones. Finally, treatment with glutamate had no effect on ARPE-19, unlike the cone PR at 1 mM, which resulted in complete cell death (n=3, p=0.0050) (Figure 11C).

[0156] However, the inventors were also interested in comparing the effects of ferroptosis-promoting molecules on ARPE-19 cell lines and primary porcine RPE cells. There was a significant difference with IKE treatment, which had no effect on primary cells even at 25 μM (n=3, p=0.0019) (Figure 11A). Survival trends were similar with RSL3 treatment and glutamate treatment (Figures 11B, C).

[0157] conclusion These results indicate that cone photoreceptor cells are more sensitive to ferroptosis-promoting agents than RPE cells. Therefore, lower levels of oxidative stress can cause death of cone photoreceptor cells without causing death of RPE cells. Furthermore, it should be noted that the literature describing ferroptosis in RPE cells uses cell lines that divide readily and are therefore immortalized (Kozlowski et al. (2015) Curr. Eye Res. 40:501). However, ferroptosis is very well described in RPE cells.

[0158] Example 3 Ferroptosis rescued cones from degeneration in RD1 mice both ex vivo and in vivo. Materials and methods Animal model: Mouse The mouse experiments and procedures were approved by the local animal ethics committee Charles Darwin CEEACD #5 and conducted at an accredited facility associated with the Institut de la Vision (Paris, France) in accordance with European Guideline 2010 / 63 / UE. All experimental work was carried out in accordance with institutional policies regarding biosecurity and safety procedures. The mice were confined to a controlled environment, maintained in a reversed half-day dark / light cycle, and had free access to food and water except during surgery.

[0159] The C3H / HeNRj strain (rd1 obtained by mating a Bagg Albino female with a DBA male) was purchased from Janvier Laboratories (Le Genest Saint-Isle, France, ISO 9001 certified) for production and breeding.

[0160] Retinal explants of rd1 mice Eyes were collected at P15, cleaned, and incubated for 20 minutes at 37°C and 5% CO2 in a CO2-independent medium containing 1 / 10 of 3.5 mg / 10 mL of L-cysteine ​​(Millipore Sigma, Burlington, MA) in glucose (6.5 g / L) diluted 1 / 50 with papain (LSO 3124, Worthington). Then, the eyes were immersed on ice for 5 minutes in neurobasal medium (10888022, Thermo Fisher Scientific, Waltham, MA, USA) = NBAg and 10% SVF (A31605-01, Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 1% L-glutamine (G3126, Millipore Sigma, Burlington, MA). Dissection was performed at +4°C to obtain the whole retina with RPE. The entire mounted retina was flattened onto a polycarbonate membrane (140652, Thermo Fisher Scientific, Waltham, Massachusetts). The test components were diluted to 10 μM with NBAg and 1 / 50 B27 supplement (17504044, Thermo Fisher Scientific, Waltham, MA, USA) and replaced twice weekly for 30 days (until P45 was reached). The explant was then fixed with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA) for 1 hour. Finally, PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA) was performed, and the explant was mounted in a Cellvis plate (P12-1.5HN, IBL, Gerasdorf, Austria) using imaging mounting medium.

[0161] Intraperitoneal injection in rd1 mice Rd1 mice were intraperitoneally injected daily from P15 to P45 with either 2 mg / kg of GW 5074 (HY-10542, MedChemExpress, Monmouth, USA) diluted in 40% PEG300 (HY-Y0873, MedChemExpress, Monmouth, USA) or 5% Tween 80 (HY-Y1891, MedChemExpress, Monmouth, USA) in PBS (14190144, Thermo Fisher Scientific, Waltham, MA, USA), or vehicle alone (control group). Eyes were harvested on P45 and fixed with 4% PFA (15714, Electron Microscopy Sciences, Hatfield, PA) for 1 hour. Retinas were harvested for PNA 488 immunolabeling (L21409, Thermo Fisher Scientific, Waltham, MA). The retina of rd1 mice was imaged at ×10 using a CQ1(C) confocal microscope (Yokogawa Electric Corporation, Tokyo, Japan).

[0162] Imaging and Statistics Using Cell Pathfinder analysis software, the number of PNA-labeled objects was quantified in the handwriting-centered portion of retinal explants in ex vivo experiments, and in the entire mounted retina in in vivo experiments. The number of detected objects (=cones) was quantified across the entire area, down to 1 mm. 2 I reported it around there.

[0163] The Mann-Whitney U two-sided test was used to evaluate the difference in cone density between retinal explants and injected mice.

[0164] result To search for protective components that prevent cone loss in retinal degeneration, various ferroptosis inhibitors were screened in isolated porcine cones after initial exposure to 20 nM RSL3 and 3 μM IKE.

[0165] Then, the inventors tested the neuroprotective effects of some of these components in the well-studied retinal degeneration model, rd1 mouse (Leveillard et al. (2004) Nat. Genet. 36:755). In vivo in rd1 mice, cone degeneration is rapid, starting at P15, followed by loss of overall survival (OS) between P15 and P20, with only half of the cell body remaining by P45. Retinas were harvested from rd1 mice at P15, and retinal explants were exposed to ferroptosis inhibitors in culture up to P45. Several treatments protected the cones (Figures 12A-E). However, while this model induces rapid degeneration, the inventors may have identified molecules that can rescue cone photoreceptor degeneration in slower degenerative diseases such as AMD.

[0166] Thanks to automated cone counting, the inventors identified five antioxidant molecules with significant neuroprotective effects: 1,3-dicaffeoylquinic acid (n=3, p=0.0055), HTHQ (1-O-hexyl-2,3,5-trimethylhydroquinone, n=3, p=0.0176), rosmarinic acid (n=4, p=0.0205), norbergenin (n=2, p=0.0256), and tinoridine hydrochloride (n=3, p=0.0055). On the other hand, the inventors identified five molecules that inhibit the ferroptosis process with significant neuroprotective effects. Two of these molecules act directly on 5-LOX (SRS16-86 (n=3, p=0.0001) and caffeate (n=3, p=0.0055)), and two of these molecules play a role in iron metabolism (mangiferin (n=5, p=0.0005) and astilbin (n=3, p=0.0055)). The last molecule involved in specific ferroptosis inhibition (via the Xc pathway) demonstrated a 138.92% neuroprotective effect against pure cone photoreceptor cells and was also effective against retinal explants. For this reason, in vivo injection of this molecule, GW 5074, was performed (n=6, p=0.0001). It was injected intraperitoneally daily at 2 mg / kg from P15 to P45. Immunolabeling of the retina recovered at P45 was GW In rd1 mice injected with 5074, cone rescue was +81.48% compared to controls injected with vehicle alone daily (1 mm). 21,597 atrophic cones vs. 880, n = 5, p = 0.0079 (Figure 12J).

[0167] This finding demonstrates that ferroptosis inhibitors can reverse cone degeneration in animal models of retinal degeneration, ex vivo and in vivo.

[0168] Example 4 Ferroptosis inhibitors do not rescue rods in rd1 mice Materials and Methods Similar to cones, rods were evaluated in retinal explants from rd1 mice after exposure to ferroptosis inhibitors from P15 to P45 in culture, as described in Example 3 above. The inventors also performed the same evaluation after intraperitoneal injection of GW 5074 described in Example 3 above.

[0169] Retinas were labeled with Rho, imaged with CQ1, and rods were counted using Cell Pathfinder.

[0170] Cones were plotted as "fold change", but the results for rods were plotted as density (rods per 1 mm 2 since the changes were not sufficient to account for the change from baseline in each retina (the changes sometimes exceeded the total number of rods).

[0171] Results There was no significant difference between the control and the ferroptosis inhibitor, either in retinal explants or in vivo (Figure 13).

[0172] Example 5 Ferroptosis inducers do not change rods in non - human primates Materials and Methods Rods were labeled with rhodopsin antibody, similar to cone labeling in NHPs injected with 20 μM IKE into the subretinal space.

[0173] Results No significant changes were observed in the level of the outer segment of the rods in the retina injected with 20 μM IKE compared to the control group (Figure 14).

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Claims

1. Compounds for use in the treatment of conditions associated with cone cell degeneration, preferably ferroptosis inhibitors, wherein the treatment involves a decrease in intracellular iron levels in cone cells.

2. The compound for use according to claim 1, wherein the activity of ferritin and / or nuclear receptor coactivator 4 (NCOA4) is reduced or inhibited.

3. The compound for use according to any one of claim 1 or 2, wherein the treatment comprises maintaining or increasing the viability of cone photoreceptor cells.

4. The compound for use according to any one of claims 1 to 3, wherein the treatment comprises inhibiting or reducing glial migration.

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

6. The compound for use according to any one of claims 1 to 5, wherein the treatment comprises maintaining or increasing cone cell activity.

7. A compound for use according to any one of claims 1 to 6, wherein the compound is selected from the group consisting of prominin 2, miR-522, and / or RP11-89 genes; RNA that inhibits FTL, FTH1, and / or NCO4; deferipron, deferoxamine (DFO), gossypol acetate, histochrome, cyclopiroxolamine (CPX), bafilomycin A1, quercitrin, baicalein, dexrazoxane, 2,2'-bipyridine, 1,10-phenanthroline, mangiferin, astilbin, YL-939, deferasirox (DFX), astilbin, CN128 (hydrochloride), ginsenoside Rh3, mangiferin, and tricetin.

8. The compound for use according to any one of claims 1 to 7, wherein the condition is selected from the group consisting of age-related macular degeneration (AMD), cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy (e.g., Stargardt disease), central serous chorioretinopathy, Best's disease and bestrofinopathy, as well as retinal detachment such as rhegmatogenous retinal detachment, serous retinal detachment and tractional retinal detachment, as well as photoretinopathy, laser-induced retinopathy, monochromacy, Stargardt disease, Usher syndrome, Leber's congenital amaurosis (LCA), Alström disease, and Refsum disease, preferably AMD or retinitis pigmentosa, more preferably AMD.

9. The compound for use according to any one of claims 1 to 8, wherein the use comprises administering the compound into the vitreous humor (for example, into the subretinal space, extrachoroidal space, anterior chamber, vitreous fluid, subconjunctival space, or corneal surface), by instillation, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intrafocally, intracranially, intraarticularly, intraprostate, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, locally, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravesically, intramucosa, intrapericardially, intracardiacly, intracardiacly, intracardiacly, intraumbilically, intraocularly, intraorbitally, orally, orally, locally, percutaneously, by inhalation, by injection, by implantation, by infusion, by continuous infusion, directly to target cells by local perfusion bath, by catheter, by lavage, as a cream, or as a lipid composition.

10. The compound for use according to any one of claims 1 to 9, wherein the use comprises administering a further therapeutic agent.

11. The compound for use according to claim 10, wherein the further therapeutic agent is a second ferroptosis inhibitor, pegaptanib, ranibizumab, bevacizumab, brolucizumab, falisimab, AKB-9778, nesbakumab, BI 836880, AKST4290, or aflibercept.

12. The compound for use according to claim 11, wherein administration of the second ferroptosis inhibitor results in activation of the Xc-transporter or GPX4 enzyme in cone photoreceptor cells and / or reduction or inhibition of lipid peroxidation in cone photoreceptor cells.

13. The compound for use according to any one of claims 10 to 12, wherein the compound and further therapeutic agents are administered simultaneously, sequentially, or separately.

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