Methods of inhibiting retinal degeneration

Abstract

Disclosed are novel methods for the therapeutic treatment of retinal degeneration, dry age-related macular degeneration (AMD) and geographic atrophy (GA). The present disclosure describes the use of agents to selectively inhibit the redox function of APE1 / Ref-1.

Description

METHODS OF INHIBITING RETINAL DEGENERATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 642,293, filed on May 3, 2024. the disclosure of which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under R01EY031939 awarded by National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates generally to the fields of molecular biology, biochemistry, and pathology. More specifically, in certain aspects, the disclosure relates to the use of APEl / Ref-1 redox inhibitors in the treatment and prevention of retinal degeneration, dry age-related macular degeneration (AMD) and geographic atrophy (GA).BACKGROUND OF THE DISCLOSURE

[0004] Age-related macular degeneration (AMD) affects over 200 million older adults worldwide and prevalence is increasing as global populations age. The disease can be categorized into two types: '‘dry” and “wef ’ (neovascular). Dry AMD is a chronic disease characterized by atrophy of the retinal pigment epithelium (RPE), the monolayer of cells that underlie the light-sensing photoreceptors. The RPE plays a crucial role in the visual cycle and maintenance of photoreceptor integrity and function. As such, photoreceptor death in the macula and subsequent central visual loss are characteristic of dry AMD, which over time can progress to an end-stage disease process associated with profound vision loss, called geographic atrophy. Wet AMD can develop from dry AMD and is characterized by macular neovascularization leading to rapid central vision loss. Antiangiogenic drugs that target vascular endothelial growthfactor (VEGF) signaling have revolutionized care for wet AMD patients. However, therapies for dry AMD remain scarce, with the first two biologies for geographic atrophy approved only in 2023; these agents slow progression but do not halt the disease.

[0005] This limited pharmacological success for dry AMD is due partly to incomplete understanding of the drivers of this disease. Inflammation and oxidative stress play an important role - the existing drugs, pegcetacoplan and avacincaptad pegol, both target the complement system, which is hyperactive in dry7AMD. Animal models of various oxidative and inflammatory stressors in the eye reflect some of the degenerative characteristics of the disease. One such model involves the systemic injection of sodium iodate. At appropriate doses, this oxidant causes RPE damage without systemic effects and has therefore become a favored model for retinal degeneration due to RPE damage, as seen in dry AMD and geographic atrophy. It is also reflective of genetic dry AMD-like diseases such as Sorsby fundus dystrophy.

[0006] Given the dearth of therapies for dry7AMD, discovery7of targets amenable to systemic therapy (rather than invasive intravitreal injections as currently- used) is critical. Redox effector factor 1 (Ref-1) is one such target. Ref-1 is one function of a bifunctional protein, APE1, that also contains an endonuclease essential for base excision repair. Although part of the same protein, the Ref-1 functionality is biochemically separable from this endonuclease activity7and is involved in modulating the function of transcription factors by chemically reducing them. Ref-1 reduces and therefore activates transcription factors important for proliferation, angiogenesis, and inflammation such as AP-1, STAT3, p53, HIF-la and others. Ref-1 inhibitors such as APX3330 (E3330), APX2009, and APX2014 have therefore found utility' in cancer, inflammatory bowel disease, and neovascular eye disease, including in mouse models with features of neovascular AMD. However, Ref-1 inhibition has not previously been linked to therapy of retinal degeneration. Here, it has been surprisingly found that Ref- 1 inhibition has therapeutic effects in models of retinal degeneration.SUMMARY OF THE DISCLOSURE

[0007] Targeted inhibition of the redox function of apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) is a novel approach to the treatment of retinal degeneration. In one embodiment, the present disclosure is directed to the use of therapeutic agents that inhibit the redox function of APE 1 / Ref- 1. In another embodiment, the present disclosure is directed to inhibitors of the redox function of APEl / Ref-1.

[0008] In one particular aspect, the present disclosure is directed to a method of inhibiting retinal degeneration in a subject in need thereof. The method includes administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

[0009] In another aspect, the present disclosure is directed to a method of treating dry' age-related macular degeneration (AMD) in a subject in need thereof. The method includes administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

[0010] In another aspect, the present disclosure is directed to a method of treating geographic atrophy (GA) in a subject in need thereof. The method includes administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depicts effect ofNalOs dosages (25 mg / kg - 35 mg / kg) on retinal conditions based on fundus, optical coherence tomograph (OCT), and fluorescein angiography (FA) images. Representative fundus, OCT and FA images on day 14 after i.p. NalOs, treatment at the indicated dosages. FA images were collected 2 mins afterfluorescein i.p. injection. Arrows indicate the damage under the retina. Scale bar = 100 pm.

[0012] FIG. 2 depicts hyper reflective foci (HRF)-like areas in a representative OCT image of the 30 mg / kg NalO? treatment group. Arrows indicate HRF. Scale bar = 100 pm.

[0013] FIGS. 3A - 3D depict retina thickness of the NalOs treatment groups based on the segmentation of OCT images by InSight software. The thickness graphs of the retina on days 0 and 14 in mice treated with 25 mg / kg NalO? (FIG. 3 A), 30 mg / kg NalOs (FIG. 3B), or 35 mg / kg NalO? (FIG. 3C) are shown (n = 6). Retina thickness comparison among treatment groups at -300 pm from the optic nerve head is shown in (FIG. 3D), data are presented as mean ± SEM. Statistical significance was analyzed by two-way ANOVA with Tukey’s post hoc test (6 eyes per group) and is shown by * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (those comparisons with p > 0.05 not shown for simplicity).

[0014] FIGS. 4A-4F show representative ERGs at different NalCh dosages. FIG. 4A shows a scotopic ERG response. FIG. 4D shows a photopic ERG response. Statistical significance (FIGS. 4B, 4C, 4E, 4F) was analyzed by one-way ANOVA with Tukey’s post hoc tests (6 eyes per group) and is shown by ns p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

[0015] FIG. 5 depicts representative fundus, OCT, and FA images demonstrating APX2009 ameliorated NaIO?-induced retinal structural damage. Scale bar = 100 pm.

[0016] FIGS. 6A & 6B depict APX2009 reduction of retinal degeneration after NalOs, treatment. The thickness graph of the retina among treatment groups is show n in FIG. 6A. Statistical comparison of the retina thickness at -300 pm away from the optic nerve center is shown in FIG. 6B. Eight (8) biological replicates per treatment group, each replicate contains two eyes (n = 16), mean ± SEM. Statistical significance was analyzed by one-way ANOVA with Tukey’s post hoc test and is shown by ** p < 0.01 and **** p < 0.0001.

[0017] FIGS. 7A and 7B depict APX2009 reduced NF-KB expression in the central and peripheral regions of the ganglion cell layer in the retina following NalCh- induced damage. Quantification of NF-KB mean fluorescence intensity (MFI) of immunostained tissue (not shown) with NF-KB p65 in the central (FIG. 7A) and peripheral (FIG. 7B) regions of the ganglion cell layer indicated that NF- B was upregulated in NalOs-induced retinal damage, with decreased amounts in the APX2009 treatment group. Three animals were used per treatment group, with 5-10 sections analyzed per animal. Data are presented as mean ± SEM. Statistical significance was analyzed by one-way ANOVA with Tukey’s post hoc test and is shown by ns p > 0.05,* p < 0.05, *** p < 0.001, and **** p < 0.0001.

[0018] FIGS. 8A - 8F depict APX2009 efficacy in representative scotopic and photopic responses. APX2009 maintained retinal function after NalCh-induced damage. Representative scotopic (FIG. 8A) and photopic (FIG. 8D) ERGs and group analyses (FIGS. 8B, 8C, 8E, and 8F). 8 biological replicates per treatment group, each replicate contained two eyes (n = 16), Mean ± SEM. Statistical significance was analyzed by one-way ANOVA with Tukey’s post hoc test and is shown by ns p > 0.05,* p < 0.05, *** p < 0.001, and **** p < 0.0001.

[0019] FIGS. 9A and 9B depict protection of RPE barrier function by APX2009 after NalO? treatment. TEER values corrected to baseline (t = 0) are shown in (FIG. 9A), solid and dotted lines indicate mean ± SEM. Each treatment group has 5 replicates (n = 5). Statistical significance in TEER at 46.6 hrs (FIG. 9B) was analyzed by oneway ANOVA with Tukey’s post hoc test and is shown by * p < 0.05, and **** p < 0.0001 (those comparisons with p > 0.05 not shown for simplicity).

[0020] FIG. 10 depicts the effect of APX2009 on RPE cell morphology in vitro. Human iPSC-RPE cells were treated with NaIO3 and APX2009 accordingly. Scale bars = 200 pm; the black area in some of the images is the electrode on the bottom of the plate. There are 5 replicates per treatment group. Images were taken at the end of the experiment shown in FIG. 9.DETAILED DESCRIPTION

[0021] The present disclosure is directed to the use of inhibitors that selectively inhibit the redox function of APEl / Ref-1. Such selective inhibition includes specific inhibition, or, in other words, where there is no or no appreciable effect on the base excision repair (BER) function of APE1 / Ref-1, as well as where the predominant effect is on the redox function, vis-a-vis the BER function. Also encompassed by the disclosure is the use of such inhibitors in combination with additional therapeutic agents and complement inhibitors. It is desired that the other agents work on a subject in a different way to that of the inhibitors which selectively inhibit the redox function of APE 1 / Ref- 1.

[0022] The term subject includes vertebrate animals, and preferably, is a human subject. The term inhibit, and derivatives thereof, includes its generally accepted meaning, which includes prohibiting, preventing, restraining, and slowing, stopping, or reversing progression or severity. Thus, the present methods include both medical therapeutic and prophylactic administration, as appropriate. As such, a subject in need thereof, as it relates to the therapeutic uses herein, is one identified to require or desire medical intervention. An effective amount is that amount of an agent necessary to inhibit the pathological diseases and disorders herein described. When at least one additional therapeutic agent is administered to a subject, such agents may be administered sequentially, concurrently, or simultaneously, in order to obtain the benefits of the agents.

[0023] The redox function of APEl / Ref-1 w as found to be selectively inhibited by 3-[(5-(2,3-dimethoxy-6-methyl l,4-benzoquinoyl)]-2-nony 1-2-proprionic acid, below- (hereinafter "APX3330", also referred to as "RN3-3" or "E3330" or "3330" in this application).

[0024] Other suitable selective redox APEl / Ref-1 inhibitors for use in the present disclosure include analogues of APX3330, including for example, [(2E)-2-[(3- methoxy- 1.4-dioxo- 1 ,4-dihy dronaphthalen-2-yl)methylidene]-N,N- diethylpentanamide] (hereinafter "APX2009"), (2E)-2-[(3-methoxy-l,4-dioxo-l,4- dihydronapthalen-2-yl)methylidene]-N,N-dimethylpentanamide] (hereinafter"APX2007"), (2E)-2-[(3-methoxy-l,4-dioxo-l,4-dihydronapthalen-2-yl)methylidene]- N-methoxypentanamide] (hereinafter "APX2014"), (2E)-2-(3-methoxy-l,4-dioxo-l,4- dihydronaphthalen-2-yl)-N,N,2-trimethylprop-2-enamide (hereinafter "APX2032")). Additional suitable analogs are shown below and in Table 1. Further information on APX3330 may be found in Abe et al., U.S. Pat. No. 5,210,239, and information on APX2009 may be found in Kelley et al., J Pharmacol Exp Ther. 2016 Nov, 359(2): 300- 309, each incorporated herein by reference to the extent they are consistent herewith.APX2032

[0025] Still other suitable additional inhibitors have the formula:Formula (I)wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R3 and Rg are independently selected from the group consisting of an alkoxy and aryl; R4and R5 are independently selected from the group consisting of an alkoxy and aryl, or both R4 and R5 taken together form a substituted or unsubstituted naphthoquinone;X is selected from the group consisting of CH=CR2 and NCH. wherein R2 is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting of N(RZ)R2 or NRAORA, wherein each Rzis independently selected from the group consisting of Ci-Cg alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rzand R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheleroalk l. each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle. Yet other suitable selective redox APEl / Ref-1 inhibitors for use in the present disclosure include those compounds of Table 1 :

[0026] Where subject applications are contemplated, particularly in humans, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of impurities that could be harmful to a subject.

[0027] The agents can be administered orally, intravenously, intramuscularly, intrapleurally, topically to the eye. intravitreally, subconjunctivally, suprachoroidally, or intraperitoneally at doses based on the body weight and degree of disease progression of the subject, and may be given in one, two or even four daily administrations. Intravitreal administration can be less than daily (e.g. monthly).

[0028] One will generally desire to employ appropriate salts and buffers to render agents stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the agent, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as innocuously. The phrase pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a subject. As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary7active ingredients also can be incorporated into the compositions.

[0029] Compositions for use in the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intravitreal, subconjunctival, suprachoroidal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.

[0030] For example, the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, eye drops, ointments, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

[0031] The active compounds may also be administered parenterally, intravitreally, subconjunctivally, suprachoroidally, or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0032] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity7can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0033] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-dry ing and freeze-dry ing techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0034] For oral administration agents of the present disclosure may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

[0035] In some examples, ophthalmic solutions, such as for use in administration through eye drops and ophthalmic ointments, may be made to be sterile, are packaged, and treated so that they remain sterile. The ophthalmic solutions including the active compounds may further include one or more of pharmaceutically acceptable excipients and additives known to the person skilled in the art, for example,carriers, stabilizers, solubilizers, tonicity enhancing agents, buffer substances, preservatives, thickeners, complexing agents and other excipients.

[0036] Examples of buffer substances are acetate, ascorbate, borate, hydrogen carbonate / carbonate, citrate, gluconate, lactate, phosphate, propionate and TRIS (tromethamine) buffers. Tromethamine and borate buffer are preferred buffers. The amount of buffer substance added is. for example, that amount necessary to ensure and maintain a physiologically tolerable pH range. The pH range is typically in the range of from 5 to 9, preferably from 6 to 8.2 and more preferably from 6.8 to 8.1.

[0037] Tonicity enhancing agents are. for example, ionic compounds, such as alkali metal or alkaline earth metal halides, such as, for example, CaCh, KBr, KC1, LiCl, NaBr, NaCl, or boric acid. Non-ionic tonicity enhancing agents are, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. For example, sufficient tonicity enhancing agent is added to impart to a ready-for-use ophthalmic composition an osmolality of approximately from 50 to 1000 mOsmol, suitably from 100 to 400 mOsmol, more suitably from 200 to 400 mOsmol and even more suitably from 280 to 350 mOsmol.

[0038] Furthermore, eye droppers may be designed to deliver a single precisely calibrated dosage, which is sufficient to flood the eye without running off. In some examples, it may be desirable that a technique for dispensing eye drops may be to apply a fingertip immediately below the lower eyelid, and with slight pressure, draw the finger down toward the cheek, which will pull the edge of the lower lid down and out to form a crescent pouch. The calibration of the dropper will deliver one drop sufficient to fill the sack which will hold the solution when the eyelid is released, allowing the single dose to be optimally distributed over the surface of the eye.

[0039] The compositions for use in the present disclosure may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0040] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, eye drops, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intravitreal, subconjunctival, suprachoroidal, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet steri 1 i ty, general safety and purity standards as required by FDA and foreign counterpart agencies.

[0041] Further description of the present disclosure is found in the Examples below.

[0042] EXAMPLES

[0043] Sodium iodate murine model. All mouse experiments were approved by the Institutional Animal Care and Use Committee, Indiana University7School of Medicine (protocol 22017, approved 5 / 23 / 2022) and followed the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Visual Research. 8-week-old wild-type C57BL / 6Jfemale mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed under standard conditions in the Indiana University Laboratory Animal Resource Center (LARC). On day 0, after at least a 48-hour acclimation period, after a single i.p. injection of a certain dose of NalCL, mice in the treatment study started receiving APX2009 at desired dosage intraperitoneally, twice a day for 14 days. OCT scans on days 0, 7. and 14 and FA on day 14 were carried out as described in the “in vivo retinal imaging" section below, while ERG was performed on day 12. After euthanasia at the end of day 14, mouse eyes were enucleated and fixed in 4% PFA (Thermo Fisher Scientific, #43368) for 2 hours at room temperature. Dissected posterior cup (retina and choroid) were embedded in optimal cutting temperature (OCT) compound (Fisher Scientific. Hampton, NH, USA, cat # 23-730-573).

[0044] In vivo retinal imaging. Mice were anesthetized by 80 mg / kg ketamine and 10 mg / kg xylazine intraperitoneally. The pupils of both eyes were dilated using 1% tropicamide and 2.5% phenylephrine. Gonak (2.5% hypromellose solution, Akom Inc, lake Forest, IL, USA) was used to lubricate corneas throughout the experiments. The Micron IV ocular imager (Phoenix-Micron, Bend, OR, USA) was used to collect in vivo retinal images including bright-field fundus, OCT, and fluorescein angiography. Horizontal and vertical OCT scans through optic nerves were taken to assess the retinal physiological conditions. Fluorescein angiography was performed to assess vascular leakage induced by NalCL by injecting 5 pl / g body weight of 2.5% fluorescein sodium (Fisher Scientific) intraperitoneally and imaging with a GFP filter set after 2 minutes. All images were captured and processed by the manufacturer’s software. InSight software was used for OCT segmentation. The automatic segmentation algorithm combined with manual adjustment was used for delineating the retinal layers. Total retina thickness was calculated by InSight and Microsoft Excel software and plotted with GraphPad Prism (GraphPad, San Diego, CA, USA).

[0045] Electroretinogram. Mice were dark-adapted overnight. As described above, mice were anesthetized, both pupils were dilated, and corneas were lubricated. Both scotopic and photopic ERG recording were performed on the LKC NGIT-100 recording machine (LKC Technologies, Inc, Gaithersburg, MD, USA). The a- and b- wave amplitudes and ERG curves were obtained by the manufacturer’s software.

[0046] Immunofluorescence. Mouse eyes were sectioned on a cry ostat (Leica Biosystems, Wetzlar, Germany) at - 20 °C and mounted on SuperFrost Plus Slides (Fisher Scientific, cat # 12-550-15 S24). Slides were stored at - 80 °C. For staining, the sections were rehydrated with PBS and blocked with 5% bovine serum albumin (BSA; Bioshop, #ALB001) in PBS for 1 hour at room temperature. Then sections were incubated with primary mouse anti-Rhodopsin (Abeam, Cambridge. UK, cat # ab3267), primary rabbit anti-RPE65 (Abeam. Cambridge, UK, cat # ab231782), primary' rabbit anti-NF-KB p65 (Cell Signaling Technology7, Boston, MA, USA, cat # 8242) and corresponding IgG control antibodies (Santa Cruz Biotechnology7, Dallas, TX, USA, cat # sc-8204; R&D systems, Minneapolis, MN, USA, cat # AB-105-C) prepared in 1.5% BSA overnight at 4 °C. followed by three 10-minute washes in lx PBS. After that, sections were incubated with Alexafluor 488-conjugated goat antimouse (Invitrogen, Waltham, MA, USA, cat # Al 1001) and Alexafluor 555-conjugated goat anti-rabbit (Invitrogen, Waltham, MA, USA, cat # A21428) secondary antibodies for 1.5 hours in a dark humidified chamber at room temperature, followed by three 10- minute washes in lx PBS. At the end, the sections were mounted with Vectashield mounting medium containing the nuclear stain, DAPI (Vector Laboratories, Inc., Newark, CA, USA, cat # H-1200-10). Immunostaining was imaged using a 20 x objective of an inverted fluorescence microscope (Axio Observer 5, Carl Zeiss, Thornwood, NY. USA).

[0047] TEER measurement. iCell RPE cells were purchased from FUJIFILM Cellular Dynamics (cat # R1113). These cells were seeded and cultured according to the manufacturer's user guide. Briefly, 5xl04cells were seeded in each well on a 96- well CytoView-Z plate (Axion Biosystems, Atlanta, GA, USA). The plate was precoated with 100 pl of 2.5 pg / ml vitronectin (STEMCELL Technologies, Vancouver, BC) per well for at least 1 hour at room temperature and the baseline calibrated by replacing the coating solution with culture medium (MEM alpha with 5% FBS. 1% N- 2 supplement, 55 nM hydrocortisone, 250 pg / ml taurine, 14 pg / ml triiodo-L-thyronine, 25 pg / ml gentamicin, recipe provided by7FUJIFILM Cellular Dynamics). RPE cells were grown for a minimum of 28 days until ready to use, by replacing the culture medium every 2-3 days. Cell morphology was checked by an optical microscope everyweek to confirm the maturity. The TEER values were recorded by Axion Maestro Edge multiwell microelectrode array (MEA) system using the Axis Z software. The desired amount of APX2009 or 0.1% DMSO was added into the wells accordingly at the beginning of the experiment (t = 0). After a day, 3 mM NalOs was added. The experiment was terminated when a decreasing trend was observed in the 3 mM NalOs + 10 pM APX2009 treatment group. Images of every' well were taken by a 20 x objective of an Evos optical microscope.

[0048] Statistical Analysis. Data are expressed as mean ± SEM and were analyzed by GraphPad Prism: One-way ANOVA with Tukey’s post hoc tests was applied to the retina thickness comparison in the APX2009 efficacy experiment, the p65 immunostaining, the ERG a & b-wave amplitude comparison, and the TEER experiment. Two-way ANOVA with Tukey’s post hoc test was applied to the retina thickness comparison in the NalOs dose dependence experiment.

[0049] EXAMPLE 1

[0050] In this Example, the sodium iodate (NalOs) murine model was used to study dry AMD.

[0051] The NalOs murine model is widely used for studying dry AMD. NalOs induces systemic oxidative stress that causes the retina to undergo degeneration initiating with the retinal pigment epithelial cells, which mimics some characteristics of advanced dry AMD.

[0052] For this study, 8-week-old female mice from Jackson Labs were used. NalOs was ordered from Sigma (St. Louis, MO USA). There was at least 48 hours of acclimation time before experiments were started. Four groups of three 8-week-old C57BL / 6J female mice were injected with PBS (control), 25 mg / kg, 30 mg / kg, or 35 mg / kg NalOs intraperitoneally. Funduscopy and optical coherence tomography (OCT) of both eyes in vivo were carried out on days 0, 7 and 14. Electroretinograms (ERGs) of mice were recorded on day 12. Wave forms and amplitudes of the a- and b-waves were analyzed to assess the retinal physiological conditions.

[0053] As shown in FIG. 1, retinal conditions were assessed based on fundus, OCT. and FA images. The 35 mg / kg NalOs treatment group had the most severe damage, evidenced by the most depigmented fundus, the thinnest retina, and the leakiest fluorescein background. The 25 mg / kg NalCh treatment group had a similar condition compared to the PBS control group, which indicated that the retinal damage caused by oxidative stress was minimal at this dosage. The 30 mg / kg NalOa treatment group showed an intermediate degree of damage, which was not as severe as the 35 mg / kg group but was more visible than the 25 mg / kg group. The fundus of the 30 mg / kg group was partially brighter than normal. Its OCT image also indicated damage (outer retinal discontinuities indicated by arrows in FIG. 1) between retina and choroid.

[0054] As shown in FIG. 2, areas similar to hyper reflective foci (HRF) in humans were visible at 30 mg / kg NalOa treatment. HRF serves as a biomarker for intermediate human AMD. Its presence is highly associated with subretinal drusen deposits in humans. Although the NalOs murine model did not develop drusen, HRF- like dots were visible, which is consistent with other published work.

[0055] Retinal thickness was assessed (FIG. 3). The retina thickness of the 25 mg / kg NalO? treatment group did not change significantly on day 14 compared to day 0. However, 30 and 35 mg / kg NalO? treatment groups had significantly reduced retina thickness (FIG. 3D).

[0056] ERG results were consistent with what was observed in OCT and FA (FIG. 4). The 35 mg / kg NalCh treatment group developed the most damage evidenced by the loss of regular ERG wave forms, resulting minimal a- and b-waves, in both scotopic and photopic responses (FIGS. 4A - 4F). 25 and 30 mg / kg NalOa treatment groups had similar wave forms compared to the untreated group (FIGS. 4A & D). These results provide support for determining a moderate dosage of NalCh. In particular, too high of a dosage resulted in photoreceptors being completely destroyed and showed no ERG response. Too low of a dosage resulted in negligible damage. Therefore, 30 mg / kg NalOa was used for subsequent experiments.

[0057] EXAMPLE 2

[0058] In this Example, APX2009 treatment in the NalOs murine model was assessed for efficacy.

[0059] 14-day treatment of twice-per-day 25 mg / kg APX2009 via i.p. injection was used in the NalOs murine model in this efficacy study. There were three treatment groups as shown in Table 2.Table 2. APX2009 Treatment Plan in NalO- Murine Model.

[0060] Similar to Example 1, funduscopy and OCT of both eyes in vivo were carried out on days 0, 7 and 14. ERGs of mice were recorded on day 12. Wave forms and amplitudes of the a- and b-waves were analyzed to assess the retinal physiological conditions.

[0061] Representative fundus, OCT and FA images are shown in FIG. 5. 30 mg / kg NalO? induced similar moderate retinal damage and leakage compared to what was observed in Example 1 (see, FIG. 1). With APX2009 treatment, less retinal damage was observed: reduced fundus pigmentation changes, less damage under retina and fewer HRFs in the retina observed in the OCT image, and less leaky fluorescein observed in the FA image (FIG. 5).

[0062] Retina thickness in the efficacy study of APX2009 (FIG. 6) was assessed in the same way as mentioned above. 30 mg / kg NalCfi significantly reduced the retina thickness compared to the control group (FIG. 6B). There was a significant protection of retina thickness in the NalCh + APX2009 treatment group compared to the NalCh only treatment group (FIG. 6B).

[0063] Immunohistochemistry (1HC) staining was used to assess the treatment effectiveness of APX2009 with NalOs induced oxidative stress in the mouse eye (images not shown). In Rhodopsin antibody was used to identify rod photoreceptor cells of the retina; RPE65 is an RPE specific protein, w hich is essential for regenerating visual pigment: DAPI was used here as a marker of nuclei. The RPE65 signal in the 30 mg / kg NalO? treatment group indicated a more damaged morphology of RPE cells when comparing to the control and the NalOs + APX2009 treatment groups. This staining also confirmed a protective effect of APX2009 with NalCh treatment.

[0064] A hallmark of the NalCh model is oxidative stress and inflammation in the retina. One marker of inflammation is the p65 subunit of NF-KB. Consistent with literature data, this marker was increased in the ganglion cell layer in mice treated with NalOs compared with untreated controls (immunofluorescent images not shown). However, p65 was notably less present in the ganglion cell layer of mice treated with APX2009 after NalOs. This was confirmed with quantification of the fluorescence signal between treatments (FIGS. 7A and 7B). This finding indicated a reduction in inflammation with APX2009 treatment, potentially contributing to the therapeutic effect of this Ref- 1 inhibitor against retinal degeneration.

[0065] To assess functional effects of therapy, scotopic and photopic ERGs were performed on day 12. Both scotopic and photopic responses in the NalCh treatment group (vehicle) showed decreased amplitudes of a- and b-waves compared to those of the vehicle control group (FIGS. 8A and 8D). And the NaTOs + APX2009 treatment group showed higher scotopic a- and b-waves compared to those of the NalCh + Vehicle treatment group, but not 100% recovery of those in the vehicle control group (FIGS. 8 A - 8C). The photopic a and b-wave in the NalCh + APX2009 treatment groupshowed a trend of higher w ave amplitude compared to the NalOs + Vehicle treatment group, but not significantly (FIGS. 8D - 8F).

[0066] EXAMPLE S

[0067] In this Example, the effect of APX2009 on NalOs-treated iPSC-RPE in vitro on cellular barrier function was analyzed.

[0068] Human induced pluripotent stem cell-derived (iPSC)-RPE (iCell-RPE) were cultured for at least 4 weeks on a 96-well Axion plate for trans -epitheli al electrical resistance (TEER) measurement by an Axion Edge system.

[0069] iPSC-RPE were treated with 1 to 10 pM of APX2009 at the beginning of the experiment. Cells in the groups without APX2009 received the same amount of DMSO at the same time. After 26 hours, 3 mM NalOs was added accordingly. Cells in the control group (“DMSO”) received the same amount of medium at the same time.

[0070] TEER values were normalized to t = 0 (FIG. 9A). In FIG. 9A, the TEER in the 3 mM NalO? group decreased after NalOs addition, meaning that N al Os-induced oxidative stress damaged the cellular barrier function. TEER values also dropped after NalOs addition when treated with 1 or 3 pM APX2009 but showed either a delayed trend or a more gradual drop compared to the TEER of NalOs only (FIG. 9A).

[0071] The 10 pM APX2009 treatment group behaved differently from the other APX2009 groups: its TEER value decreased slightly in the first few hours after the NalOs addition, then recovered and stayed above the TEER of the “DMSO” control group (FIG. 9A. black line). Its decreased response firstly could be due to the fast change in the environment after adding NalO . However, the high concentration APX2009 helped the cells survive under this oxidative stress and TEER recovered later.

[0072] Eventually the TEER started to decrease at t = 42 hr. meaning that 10 pM APX2009 protected cells from 3 mM NalO for about 16 hours (26 to 42 hr) in this system. A similar trend was observed in the 3 pM APX2009 treatment group (FIG. 9A), but with a much shorter time window (about 5 hours). Endpoint analysis at t =46.6 hr is shown in FIG. 9B. The recovery7effect in 3 pM or 10 pM APX2009 treatment group was significant compared to the 3 mM NalCh group.

[0073] FIG. 10 shows the cell morphology at the end of the TEER experiment. With 3 mM NalCh and DMSO, RPE cells detached from the plate and lost their polygonal shape, as seen clearly in the DMSO control group. As the APX2009 concentration increased, the morphological change became less obvious. At 10 pM APX2009, most cells maintained the polygon shape and did not come off the plate. This observation matched the TEER value changes in FIG. 9 and confirmed the APX2009 protective effect against 3 mM NalO in iPSC-RPEs.

[0074] The results show that inhibition of Ref-1 with a well-characterized, specific inhibitor of this protein ameliorated the degenerative effects of sodium iodate in the murine retina. The preventative effect of APX2009 treatment indicates that it can be used to prevent progression of dry AMD from early to intermediate or intermediate to advanced (geographic atrophy). Long-term Ref-1 inhibitor treatment can be considered for impeding the progression of AMD, or even be used prophylactically in individuals with high AMD genetic risk or in patients predisposed to Sorsby fundus dystrophy.

[0075] The mechanism of Ref-1 inhibition’s protective effect appears to involve partial maintenance of RPE after sodium iodate exposure, as evidenced by retained immunostaining for the RPE marker RPE65 in APX2009-treated eyes and also the in vitro finding of maintained RPE barrier function with APX2009 treatment initiated prior to sodium iodate challenge. Based on the p65 immunostaining findings, this is likely mediated by an anti-inflammatory effect. APX2009 may also have an independent or related effect on protecting the photoreceptors themselves. Based on the scotopic ERG data, rods in particular seemed well-protected by systemic APX2009 treatment; cone ERGs were less responsive to APX2009 treatment, but were also less impacted by sodium iodate.

[0076] The Ref-1 inhibitor, APX2009, maintained the structure, morphology, and function of the retina undergoing degeneration in response to the oxidative stressor sodium iodate. These results provide a novel class of dry AMD therapies.

Claims

CLAIMSWhat is claimed is:

1. A method of inhibiting retinal degeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

2. The method as set forth in claim 1, wherein the APEl / Ref-1 inhibitor has the formula:Formula (I) wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R? and Re are independently selected from the group consisting of an alkoxy and aryl; R4 and R5 are independently selected from the group consisting of an alkoxy and aryl, or both R4and R5 taken together form a substituted or unsubstituted naphthoquinone;X is selected from the group consisting of CH=CR2 and NCH, wherein R2 is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting of N(RZ)R2 or NRAORA, wherein each Rzis independently selected from the group consisting of Ci-Ce alkyl, heteroalkyl, cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rzand R2 taken together with the attached nitrogen form an optionallysubstituted heterocycle; where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.

3. The method as set forth in claim 1, wherein the APEl / Ref-1 inhibitor is selected from the group consisting of (2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa- 1,4- dien-l-yl)methylidene]undecanoic acid (APX3330), [(2E)-2-[(3-methoxy-l,4- dioxo-1,4- dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide](APX2009), (2E)-2-[(3- methoxy-l,4-dioxo-l,4-dihydronapthalen -2-yl)methylidene]- N-methoxypentanamide] (APX2014), pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof, and combinations thereof.

4. The method as set forth in claim 3, wherein the APEl / Ref-1 inhibitor is APX3330 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX3330 per day.

5. The method as set forth in claim 3, wherein the APEl / Ref-1 inhibitor is APX2009 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX2009 per day.

6. The method as set forth in claim 3, wherein the APEl / Ref-1 inhibitor is APX2014 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX2014 per day.

7. The method as set forth in claim 1, wherein the APEl / Ref-1 inhibitor is an inhibitor as shown in Table 1.

8. The method as set forth in claim 1 further comprising administering at least one additional therapeutic agent to the subject.

9. The method as set forth in claim 8. wherein the additional therapeutic agent is selected from the group consisting of complement inhibitors.

10. The method as set forth in claim 9, wherein the complement inhibitor treatment is selected from the group consisting of pegcetacoplan, avacincaptad pegol, IONIS-FB- LRx, GT005, ANX-007, danicopan, JNJ- 81201887 and combinations thereof.

11. The method as set forth in claim 1, wherein the subject has a disease selected from the group consisting of retinal degeneration, dry’ age-related macular degeneration (AMD), geographic atrophy. Sorsby fundus dystrophy', and combinations thereof.

12. A method of treating dry age-related macular degeneration (AMD) in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic / apynmidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

13. The method as set forth in claim 12, wherein the APEl / Ref-1 inhibitor has the formula:Formula (I) wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R3 and Re are independently selected from the group consisting of an alkoxy and aryl; R4 and R5 are independently selected from the group consisting of an alkoxy and aryl, or both R4 and R5 taken together form a substituted or unsubstituted naphthoquinone;X is selected from the group consisting of CH=CR2 and NCH, wherein R2is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting of N(RZ)R2 or NRAORA, wherein each Rzis independently selected from the group consisting of Ci-Ce alkyl, heteroalkyl,cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rzand R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.

14. The method as set forth in claim 12, wherein the APEl / Ref-1 inhibitor is selected from the group consisting of (2E)-2-[(4.5-dimethoxy-2-methyl-3.6- dioxocyclohexa-l,4-dien-l-yl)methylidene]undecanoic acid (APX3330), [(2E)-2- [(3 - methoxy - 1 ,4-dioxo- 1 ,4-dihy dronaphthalen-2-y l)methylidene] -N,N- diethylpentanamide] (APX2009), (2E)-2-[(3-methoxy-l,4-dioxo-l,4- dihydronapthalen - 2-yl)methylidene]-N-methoxypentanamide] (APX2014), pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof, and combinations thereof.

15. The method as set forth in claim 14, wherein the APEl / Ref-1 inhibitor is APX3330 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX3330 per day.

16. The method as set forth in claim 14, wherein the APEl / Ref-1 inhibitor is APX2009 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX2009 per day.

17. The method as set forth in claim 14, wherein the APEl / Ref-1 inhibitor is APX2014 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX2014 per day.

18. The method as set forth in claim 12, wherein the APEl / Ref-1 inhibitor is an inhibitor as shown in Table 1.

19. The method as set forth in claim 12 further comprising administering at least one additional therapeutic agent to the subject.

20. The method as set forth in claim 19, wherein the additional therapeutic agent is selected from the group consisting of complement inhibitors.

21. The method as set forth in claim 20, wherein the complement inhibitor treatment is selected from the group consisting of pegcetacoplan, avacincaptad pegol, IONIS-FB- LRx, GT005. ANX-007, danicopan, JNJ- 81201887 and combinations thereof.

22. A method of treating geographic atrophy (GA) in a subject in need thereof, the method comprising administering to the subject an effective amount of an apurinic / apyrimidinic endonuclease 1 redox factor 1 (APEl / Ref-1) inhibitor, pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.

23. The method as set forth in claim 22, wherein the APEl / Ref-1 inhibitor has the formula:Formula (I) wherein Ri is selected from the group consisting of alkyl, alkoxy, hydroxyl, and hydrogen; R3and Re are independently selected from the group consisting of an alkoxy and ary l; R4 and R5 are independently selected from the group consisting of an alkoxy and aryl, or both R4 and R5 taken together form a substituted or unsubstituted naphthoquinone;X is selected from the group consisting of CH=CR2 and NCH, wherein R2is selected from the group consisting of C1-C10 alkyl and CF3CH2CH2; andY is selected from the group consisting of N(RZ)R2or NRAORA, wherein each Rzis independently selected from the group consisting of Ci-Ce alkyl, heteroalkyl,cycloalkyl and cycloheteroalkyl, straight or branched chain or optionally substituted, or both Rzand R2 taken together with the attached nitrogen form an optionally substituted heterocycle; where each RAis independently selected from the group consisting of hydrogen, alkyl, heteroalkyl, cyclohexyl, and cycloheteroalkyl, each of which is optionally substituted, or both RAare taken together with the attached nitrogen and oxygen to form an optionally substituted heterocycle.

24. The method as set forth in claim 22, wherein the APEl / Ref-1 inhibitor is selected from the group consisting of (2E)-2-[(4.5-dimethoxy-2-methyl-3.6- dioxocyclohexa-l,4-dien-l-yl)methylidene]undecanoic acid (APX3330), [(2E)-2- [(3 - methoxy - 1 ,4-dioxo- 1 ,4-dihy dronaphthalen-2-y l)methylidene] -N,N- diethylpentanamide] (APX2009), (2E)-2-[(3-methoxy-l,4-dioxo-l,4- dihydronapthalen - 2-yl)methylidene]-N-methoxypentanamide] (APX2014), pharmaceutically acceptable salts and pharmaceutically acceptable solvates thereof, and combinations thereof.

25. The method as set forth in claim 24, wherein the APEl / Ref-1 inhibitor is APX3330 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX3330 per day.

26. The method as set forth in claim 24, wherein the APEl / Ref-1 inhibitor is APX2009 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX2009 per day.

27. The method as set forth in claim 24, wherein the APEl / Ref-1 inhibitor is APX2014 and the subject is administered from about 1 mg / kg to about 20 mg / kg APX2014 per day.

28. The method as set forth in claim 22, wherein the APEl / Ref-1 inhibitor is an inhibitor as shown in Table 1.

29. The method as set forth in claim 22 further comprising administering at least one additional therapeutic agent to the subject.

30. The method as set forth in claim 29, wherein the additional therapeutic agent is selected from the group consisting of complement inhibitors.

31. The method as set forth in claim 30, wherein the complement inhibitor treatment is selected from the group consisting of pegcetacoplan, avacincaptad pegol, IONIS-FB- LRx, GT005. ANX-007, danicopan, JNJ- 81201887 and combinations thereof.

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