Intravitreal spironolactone suspension

Abstract

There was a need, in the state of the art, to develop simpler spironolactone formulations, with known ingredients authorized by the pharmacopeia, for intravitreal administration. Hence, the inventors develop an intraocular suspension comprising spironolactone, carboxymethylcellulose and polysorbate 80, that can be injected into the vitreous and release spironolactone for at least a month. The formulation was well-tolerated since the vitreous was clear, no sign of retinal inflammation or edema was observed. The inventors also demonstrate that the formulation significantly reduces the CNV activity and the fibrosis, shows a significant effect on the ability of RPE cells to cover the laser burn site, and increases by almost 5 times the % of impacts with complete RPE coverage. These results support the use of aqueous ophthalmic composition according to the invention for use in the treatment of ophthalmic pathogenic mechanisms and diseases.

Description

[0001] INTRA VITREAL SPIRONOLACTONE SUSPENSION

[0002] FIELD OF THE INVENTION

[0003] The present invention is in the field of medicine, in particular ophthalmology.

[0004] BACKGROUND OF THE INVENTION

[0005] Mineralocorticoid receptor activation is pathogenic for the retina as it favours the development of retinal oedema, retinal inflammation, activation of microglial cells and retinal angiogenesis (Behar-Cohen F, Zhao M. Mineralocorticoid pathway in retinal health and diseases. Br J Pharmacol 2022; 179:3190-204).

[0006] Mineralocorticoid receptor antagonists have shown potential to alleviate pathogenic mechanisms involved in major retinal diseases, including retinal oedema, pathological choroidal, retinal angiogenesis, retinal cell death as well as retinal and choroidal inflammation.

[0007] Spironolactone is the more potent mineralocorticoid receptor antagonist, but it has a very poor intraocular bioavailability due to its binding to efflux proteins, expressed at the inner and outer retinal barriers (Zhao M, Rodriguez-Villagra E, Kowalczuk L, Le Normand M, Berdugo M, Levy-Boukris R, et al. Tolerance of high and low amounts of PLGA microspheres loaded with mineralocorticoid receptor antagonist in retinal target site. J Control Release 2017;266: 187-97). Although some effects of spironolactone can be achieved using the systemic route, particularly when the ocular blood barriers are disrupted, the optimal effect of spironolactone requires its administration directly into the vitreous (Zhao M, Gelize E, Levy R, Moulin A, Azan F, Berdugo M, et al. Mineralocorticoid Receptor Pathway and its Antagonism in a Model of Diabetic Retinopathy. Diabetes 202 l:db210099 and Daruich A, Matet A, Dirani A, Gallice M, Nicholson L, Sivaprasad S, et al. Oral Mineralocorticoid-Receptor Antagonists: Real-Life Experience in Clinical Subtypes of Nonresolving Central Serous Chorioretinopathy With Chronic Epitheliopathy. Transl Vis Sci Technol 2016;5:2).

[0008] Spironolactone half-life in the vitreous is very short (<lhour) and even at high intravenous dose, it does not cross the intact ocular barriers. It is thus necessary to develop long-lasting intraocular formulations to achieve therapeutic and stable concentrations of spironolactone inside the eye for a sustained period of time (at least a month).

[0009] Various formulations of spironolactone have been tested for intravitreous administration such as biodegradable spironolactone-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (Zhao M, Rodriguez-Villagra E, Kowalczuk L, Le Normand M, Berdugo M, Levy-Boukris R, et al. Tolerance of high and low amounts of PLGA microspheres loaded with mineralocorticoid receptor antagonist in retinal target site. J Control Release 2017;266: 187-97) that allows the slow release of efficient concentrations of spironolactone inside the eye.

[0010] Although the microspheres formulations showed good safety profile, their preparation and sterilization make them difficult to develop.

[0011] Dahmana et al., explores the development and evaluation of a sustained-release formulation of spironolactone using a biodegradable polymer, hexyl-substituted poly-lactic acid (hexPLA). The study investigates the ocular biodistribution and tolerability of SPL and its metabolites in rats over one month following a single intravitreal injection (Naoual Dahmana, Laura Kowalczuk, Doris Gabriel, Francine Behar-Cohen, Robert Gumy, Yogeshvar N Kalia "Ocular Biodistribution of Spironolactone after a Single Intravitreal Injection of a Biodegradable Sustained-Release Polymer in Rats" Mol Pharm. 2020 Jan 6; 17(l):59-69. doi: 10.1021 / acs.molpharmaceut.9b00707. Epub 2019 Dec 5). This formulation presented however several issues for human use, as mentioned in the result section of Dahmana, such as:

[0012] - Inflammation and Healing Issues: The high viscosity of the hexPLA formulation led to inflammation at the injection site. The polymer remained in the injection hole, preventing closure and healing, which resulted in an inflammatory reaction. Inflammation was also observed by the presence of inflammatory cells at the surface of the retina on spectral domain optical tomography and on histology.

[0013] - Visual Obstruction: The polymeric gel stayed in the visual axis, obstructing vision for several weeks and did adhere to the posterior face of the lens, with risk of cataract induction.

[0014] - Unknown Degradation Products: The hexPLA polymer has unknown degradation products, making it unsuitable for human use.

[0015] Moreover, the exact fate of polymers within the eye after intraocular injection is unknown and potential accumulation in cells such as the retinal pigment epithelium with subsequent toxicity cannot be excluded.

[0016] For all these reasons, there is a need to develop simpler spironolactone formulations, with known ingredients authorized by the pharmacopeia, for intravitreal administration. These formulations should notably avoid visual obstructions and exclude poorly understood polymers with uncertain degradation products to prevent, particularly, costly and extensive safety studies. Additionally, no inflammatory reaction should be induced including at the injection site allowing for proper healing and minimizing adverse effects.

[0017] SUMMARY OF THE INVENTION The inventors advantageously develop an intraocular suspension of spironolactone that can be injected into the vitreous and release spironolactone for at least a month. The formulation was well-tolerated since the vitreous was clear, no sign of retinal inflammation or oedema was observed. Advantageously, the formulation avoids visual obstruction. Additionally, it excludes poorly understood polymers with uncertain degradation products, enhancing safety and reducing the need for extensive safety studies.

[0018] Hence, the present invention relates to an aqueous ophthalmic composition comprising spironolactone, carboxymethylcellulose and polysorbate 80.

[0019] The efficacy of this spironolactone formulation has been ascertained on 2 impact laser-induced choroidal neovascularization (CNV) to evaluate both the anti-angiogenic effect of spironolactone and its effect on fibrosis and on the restoration of the retinal pigment epithelium integrity. The inventors advantageously demonstrate that the spironolactone formulation according to the invention significantly reduces the CNV activity and the fibrosis. They also highlight that the spironolactone formulation influences retinal pigment epithelium (RPE) integrity. Indeed, the inventors demonstrate that spironolactone suspension induces wound healing and allows restoration and normalization of the retinal pigment epithelium. This restoration prevents subretinal fibrosis and epithelia to mesenchymal transition (EMT).

[0020] Furthermore, spironolactone formulation according to the invention is more efficient than systemic administration to promote the regeneration of normal RPE cells after injury. Indeed, and surprisingly, effects on wound healing, restoration and normalization of the retinal pigment epithelium are only obtained when spironolactone is delivered inside the eye (i.e. by intravitreal administration), and not when spironolactone or the more specific MR antagonist, eplerenone, is administered systemically. These effects are all the more unexpected, since the retinal pigment epithelium does not divide and does not regenerate spontaneously.

[0021] Moreover, the inventors demonstrate that the spironolactone formulation according to the invention has a superior effect on fibrosis compared to oral eplerenone, which is another MR antagonist, on a one single laser bum impact model.

[0022] Furthermore, the suspension according to the invention was compared to the Spironolactone-hexPLA formulation described in Dahmana et al.

[0023] The spironolactone suspension according to the invention showed excellent ocular tolerance with proper dispersion in the vitreous and no signs of inflammation throughout the follow-up period, as confirmed by fundus examinations, histological analysis, and inflammatory markers (GFAP and IBA-1). In contrast, the Spironolactone-hexPLA formulation exhibited poor tolerance, characterized by inflammatory cell infiltration visible on OCT and on histology and major retinal structure disorganization due to the persistent polymer at the injection site, resulting in retinal detachment and inflammation. These findings demonstrate the superior safety and clinical tolerance profile of the spironolactone suspension compared to the S-hexPLA formulation.

[0024] Furthermore, the stability results are outstanding, showing exceptional consistency in Spironolactone concentration over an extended period of approximately 18 months. The measurements demonstrate excellent stability with only a negligible 0.05% decrease from the initial concentration when measured in May 2024 (initial measure on November 2022). This is especially significant considering that Spironolactone is a prodrug, which typically raises concerns about potential metabolization into various metabolites during long-term storage. The fact that the suspension maintained such stable concentrations over this extended period is a strong indicator of a well-formulated preparation, effectively preventing any significant degradation or chemical transformation of the active compound. This stability profile is quite an achievement for a prodrug formulation and suggests optimal formulation parameters were achieved.

[0025] Hence, the present invention also relates to an aqueous ophthalmic composition for use in the treatment of ophthalmic pathogenic mechanisms and diseases, for reducing the choroidal neovascularization activity and the fibrosis and to promote the restoration of retinal pigment epithelium integrity.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGURE 1: 2 impact laser-induced choroidal neovascularization (CNV) experiment

[0028] FIGURE 2: Collagen (Colli) immunostaining grading scale

[0029] FIGURE 3: Fibrosis evaluation at day 30 on flat-mounted RPE / choroid using collagen (Colli) immunostaining after administration of spironolactone suspension according to the invention and a vehicle.

[0030] FIGURE 4: Fibrosis evaluation at day 30 on flat-mounted RPE / choroid using collagen (Colli) immunostaining after administration of eplerenone and a vehicle.

[0031] FIGURE 5: Retinal epithelium integrity grading scale

[0032] FIGURE 6: Comparison of % impact per grading severity between vehicle treated eyes (Pl formulation) and spironolactone formulation treated eyes (SI formulation)

[0033] FIGURE 7: Expression of RPE65, a marker specific of RPE cells FIGURE 8: Percentage of impacts per grading severity in a single impact laser model in the rats after administration of eplerenone and a vehicle.

[0034] FIGURE 9: Experiment scheme of RNA sequencing in the two-impact model

[0035] FIGURE 10: Transgenic mouse over expressing MR in the RPE vs Control WT mouse at 6 months.

[0036] FIGURE 11: In-vitro dissolution profiles of spironolactone from the formulations Fl with 0.75% CMC and F2 with 1.5%. Average results (n=3) represented by curves and standard deviations by error bars.

[0037] DETAIEEED DESCRIPTION

[0038] The present invention relates to an aqueous ophthalmic composition comprising:

[0039] (a) Spironolactone,

[0040] (b) Carboxymethylcellulose,

[0041] (c) Polysorbate 80.

[0042] Spironolactone (SPL)

[0043] Spironolactone (C24H32O4S - Cas Number 52-01-7 - Molecular weight = 416.6 g / mol) is a highly hydrophobic molecule. Its solubility in water is less than 0.1 g / L. Spironolactone is also denoted SC- 9420; NSC-150339; 7a- Acetylthiospirolactone; 7a- Acetylthio-17a-hydroxy-3-oxopregn-4-ene-21- carboxylic acid g-lactone.

[0044] In an embodiment, the aqueous ophthalmic composition according to the invention comprises from 0.2 to 20 % by weight, preferably from 0.5 to 10 % by weight, preferably from 1 to 5 % by weight, based on the total weight of the composition, of spironolactone.

[0045] As conventionally used in the pharmaceutical field, 1% corresponds to 1 g / lOOmL (or lOgm / mL).

[0046] In a preferred embodiment, the composition according to the invention comprises 2% by weight, based on the total weight of the composition, of spironolactone.

[0047] Typically, spironolactone can be purchased from AZELIS FARMA.

[0048] In an embodiment, spironolactone is in micronized form. Spironolactone in “micronized form" refers to spironolactone that has been processed into extremely small particles, enhancing its surface area. This form improves the drug's solubility and bioavailability.

[0049] Indeed, after intravitreous injection, the particles will stay into the vitreous and form a reservoir of drug, they will not migrate into the retina since their size >500 nm retain them into the vitreous. But the very small size will prevent visual disturbance. Typically, triamcinolone size is 26.7pm and forms aggregates of more than 200pm, which is responsible for visual disturbance. The use of a micronized form of spironolactone with low dispersion of particle size allows a better control of the particle size.

[0050] In an embodiment, the average particle diameter of spironolactone is comprised between 3.5 and 6.0 pm, preferably between 4.0 and 5.2pm. The average particle diameter is expressed as the d4, 3 value, which is the volume weighted mean diameter. The volume-based particle size equals the diameter of the sphere that has the same volume as a given particle.

[0051] In an embodiment, the volume weighted mean diameter (d4s) of spironolactone particles is around 4.61pm.

[0052] In an embodiment, the volume diameter d( 10) particles of spironolactone particles is comprised between 0.5 and 1.5pm, preferably between 0.7 and 1.1pm and is preferably around 0.88pm.

[0053] In an embodiment, the volume diameter d(30) of spironolactone particles is comprised between 1.8 and 3.3pm, preferably between 2.2 and 2.9 pm and preferably around 2.54pm.

[0054] In an embodiment, the volume diameter d(40) of spironolactone particles is comprised between 2.5 and 4.0pm, preferably between 3.0 and 3.6pm and preferably around 3.29pm.

[0055] In an embodiment, the volume diameter d(50) of spironolactone particles is comprised between 3.2 and 4.8pm, preferably between 3.8 and 4.3pm and preferably around 4.06pm.

[0056] In an embodiment, the volume diameter d(60) of spironolactone particles is comprised between 4.0 and 5.8pm, preferably between 4.6 and 5.2pm and preferably around 4.90pm.

[0057] In an embodiment, the volume diameter d(70) of spironolactone particles is comprised between 4.8 and 7.0pm, preferably between 5.5 and 6.3pm and preferably around 5.89pm.

[0058] In an embodiment, the volume diameter d(90) of spironolactone particles is comprised between 7.5 and 11.0pm, preferably between 8.5 and 9.7pm and preferably around 9.09pm.

[0059] In an embodiment, the volume diameter d(100) of spironolactone particles is comprised between 13.0 and 18.0pm, preferably between 14.5 and 16.5 pm and preferably around 15.69pm. In this context, "around" means plus or minus 0.2pm (e.g., "around 4.61 m" means 4.61 ± 0.2pm)

[0060] The term d(X) refers to a parameter in the particle size distribution. d(X) corresponds to the theoretical opening of a sieve such that X% of the particles, by volume, have a diameter smaller than d(X).

[0061] Hence, the term d(10) refers to a parameter in the particle size distribution. d(10) corresponds to the theoretical opening of a sieve such that 10% of the particles, by volume, have a diameter smaller than d(10).

[0062] The term d(30) refers to a parameter in the particle size distribution. d(30) corresponds to the theoretical opening of a sieve such that 30% of the particles, by volume, have a diameter smaller than d(30).

[0063] The term d(40) refers to a parameter in the particle size distribution. d(40) corresponds to the theoretical opening of a sieve such that 40% of the particles, by volume, have a diameter smaller than d(40).

[0064] The term d(50) refers to a parameter in the particle size distribution. d(50) corresponds to the theoretical opening of a sieve such that 50% of the particles, by volume, have a diameter smaller than d(50).

[0065] The term d(70) refers to a parameter in the particle size distribution. d(70) corresponds to the theoretical opening of a sieve such that 70% of the particles, by volume, have a diameter smaller than d(70).

[0066] The term d(90) refers to a parameter in the particle size distribution. d(90) corresponds to the theoretical opening of a sieve such that 90% of the particles, by volume, have a diameter smaller than d(90).

[0067] The term d(100) refers to a parameter in the particle size distribution. d(100) corresponds to the theoretical opening of a sieve such that 100% of the particles, by volume, have a diameter smaller than d(100).

[0068] The surface weighted mean diameter d(3,2), also known as the Sauter mean diameter, represents the diameter of a sphere that has the same volume / surface area ratio as the entire population of particles.

[0069] In an embodiment, the surface weighted mean diameter (d3,2) of spironolactone particles is comprised between 1.5 and 2.5 pm, preferably between 1.8 and 2.2 pm and around 2.068 pm. The surface weighted mean diameter represents the diameter of a sphere that has the same volume / surface area ratio as the entire population of particles.

[0070] The specific surface area (SSA) represents the total surface area of particles per unit mass and is typically expressed in square meters per milligram (m2 / mg). It characterizes the surface availability of the particles. In an embodiment, the specific surface area of spironolactone particles is comprised between 2.0 and 3.8 m2 / mg, preferably between 2.5 and 3.3 m2 / mg and around 2.9 m2 / mg. The specific surface area represents the total surface area of particles per unit mass.

[0071] The particle size parameters (volume weighted mean diameter d(4,3), surface weighted mean diameter d(3,2), volume diameters d(X)) and the specific surface area were measured using laser light scattering technique with a particle size analyzer. Carboxymethylcellulose (CMC)

[0072] In an embodiment, the aqueous ophthalmic composition according to the invention comprises from 0.2 to 7.5 % by weight, preferably from 0.1 to 5 % by weight, preferably from 0.75 to 1.50% by weight, based on the total weight of the composition, of carboxymethylcellulose.

[0073] In a preferred embodiment, the aqueous ophthalmic composition according to the invention comprises 0.75 or 1.50 % by weight, based on the total weight of the composition of carboxymethylcellulose.

[0074] Advantageously, the amount of CMC in the invention helps control the release rate of spironolactone and enhances the stability of the suspension by preventing rapid sedimentation and ensuring uniform particle distribution. Additionally, the CMC content provides a consistent texture to the formulation, which is crucial for maintaining the suspension's integrity overtime. Polysorbate 80

[0075] The term “polysorbate” as used herein relates to polyoxyethylene sorbitan fatty acid esters (polysorbates). Polysorbate 80 (“Polyoxyethylene-80-sorbitane monooleate”) is used according to the invention. Commercial reference can be used such as Tween® 80 commercialized by Carl Roth GmbH.

[0076] In an embodiment, the aqueous ophthalmic composition according to the invention comprises from 0.01 to 1 % by weight, preferably from 0.01 to 0.5 % by weight, preferably from 0.02 to 0.05 % by weight, based on the total weight of the composition, of polysorbate 80.

[0077] In an embodiment, the aqueous ophthalmic composition according to the invention comprises 0.02 % by weight, based on the total weight of the composition, of polysorbate 80.

[0078] The formulation excipients benzyl alcohol (preservative) and / or polysorbate 80 are thought to be the cause of non-bacterial endophthalmitis associated with intravitreal injection of active principle. For example, the presence of benzyl alcohol preservative and polysorbate 80 surfactant tends to lead to unnecessary and / or undue cell damage or other toxicities in ocular tissues.

[0079] Advantageously, the composition according to the invention do not cause substantial damage or injury to ocular tissues due to the association of carboxymethylcellulose and polysorbate 80 in proportions according to the invention.

[0080] Advantageously, the amount of polysorbate according to the invention aids stabilizing the formulation and notably aids in flocculation control by reducing the surface tension between particles, promoting a stable suspension. This helps prevent caking and ensures easy rehomogenization, maintaining the suspension's effectiveness and usability.

[0081] Trometamol

[0082] In an embodiment, the aqueous ophthalmic composition according to the invention, further comprises trometamol.

[0083] Trometamol is also known as tromethane or tris(hydroxymethyl)aminomethane buffer. It is a tertiary amine with a pKa of 8.1 at 25 °C.

[0084] In an embodiment, the aqueous ophthalmic composition comprises from 0.4 to 1.2 % by weight, preferably from 0.6 to 1 % by weight, preferably from 0.7 to 0.9 % by weight, based on the total weight of the composition, of trometamol.

[0085] In a preferred embodiment, the aqueous ophthalmic composition comprises 0.8% by weight, based on the total weight of the composition of trometamol.

[0086] Advantageously the amount of trometamol according to the invention enhances formulation stability, helps maintain the consistency an integrity of the suspension over the time and a stable pH over a month. pH adjusting agent

[0087] The pH of the composition is preferably from 7 to 7.4, preferably from 7 to 7.2.

[0088] It will be understood that any suitable acid or base may be used to adjust the pH to the appropriate value or pH range.

[0089] Hence, in an embodiment, composition has a pH adjusting agent in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to about 7.0 - 7.4, preferably to about 7.0-7.2.

[0090] The pH of the composition may be adjusted after addition of the trometamol buffer.

[0091] Typically, the pH of the composition will need to be raised by the addition of an acid, which suitably may be hydrogen chloride (HC1) orNaOH.

[0092] Hence, in an embodiment, the pH adjusting agent is HC1 IN.

[0093] In an embodiment, the aqueous ophthalmic composition HCL IN added as pH adjusting agent until the optimal pH is reached.

[0094] The one skilled in the art knows how to measure pH of a solution. Typically, a pH-meter can be used.

[0095] Tonicity agent The osmolarity of the aqueous ophthalmic composition is preferably from 260 to 320 mOsmol / kg, preferably from 275 to 285 mOsmol / kg, preferably from 280 to 300 mOsmol / kg, more preferably still to an osmolarity of about 280 mOsmol / kg.

[0096] Advantageously, the composition is chosen to be substantially isotonic.

[0097] It will be understood that any suitable tonicity agent may be used to adjust the tonicity of the aqueous ophthalmic composition to the appropriate value if needed.

[0098] Hence, in an embodiment, the aqueous ophthalmic composition has a tonicity agent in an amount sufficient to adjust the tonicity of the aqueous ophthalmic composition from 260 to 320 mOsmol / kg, preferably from 275 to 285 mOsmol / kg, preferably from 280 to 300 mOsmol / kg and preferably of about 280 mOsmol / kg.

[0099] Osmolarity is a measure of the total number of particles in a solution per unit of volume of solution, typically expressed as osmoles per liter of solution (Osmol / L). Osmolarity is calculated based on the concentrations of individual solutes and their contributions to the overall osmotic pressure of the solution. This method assumes that all solutes in the solution are fully dissociated in water.

[0100] Osmolarity can be measured according to commonly used method known by the one skilled in the art such as freezing point depression, vapor pressure, conductivity or calculation. Typically, osmolarity can be calculated based on the concentrations of individual solutes in a solution, using the formula: Osmolarity = (sum of [solute] x number of particles per molecule) / volume of solution.

[0101] In an embodiment, the osmolarity is measured with the freezing point depression. For example, osmolarity has been measured with an osmometer such as Osmomat 3000 (Gonotec®).

[0102] In an embodiment, Suspension osmolarity is measured using a type6 Loser messtechnik® microosmometer.

[0103] In an embodiment, the aqueous composition according to the invention comprises:

[0104] From 0.2 to 20 % by weight, preferably from 0.5 to 10 % by weight, preferably from 1 to 5 % by weight, based on the total weight of the composition, of spironolactone, from 0.2 to 7.5 % by weight, preferably from 0. 1 to 5 % by weight, preferably from 0.75 to 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose. from 0.01 to 1 % by weight, preferably from 0.01 to 0.5 % by weight, preferably from 0.02 to 0.05 % by weight, based on the total weight of the composition, of polysorbate 80.

[0105] In an embodiment, the aqueous composition according to the invention comprises:

[0106] From 0.2 to 20 % by weight, preferably from 0.5 to 10 % by weight, preferably from 1 to 5 % by weight, based on the total weight of the composition, of spironolactone, from 0.2 to 7.5 % by weight, preferably from 0.1 to 5 % by weight, preferably from 0.75 to 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose. from 0.01 to 1 % by weight, preferably from 0.01 to 0.5 % by weight, preferably from 0.02 to 0.05 % by weight, based on the total weight of the composition, of polysorbate 80.

[0107] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0108] In an embodiment, the aqueous composition according to the invention comprises:

[0109] From 0.2 to 20 % by weight, preferably from 0.5 to 10 % by weight, preferably from 1 to 5 % by weight, based on the total weight of the composition, of spironolactone, from 0.2 to 7.5 % by weight, preferably from 0.1 to 5 % by weight, preferably from 0.75 to 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose. from 0.01 to 1 % by weight, preferably from 0.01 to 0.5 % by weight, preferably from 0.02 to 0.05 % by weight, based on the total weight of the composition, of polysorbate 80.

[0110] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4. from 0.4 to 1.2 % by weight, preferably from 0.6 to 1 % by weight, preferably from 0.7 to 0.9 % by weight, based on the total weight of the composition, of trometamol.

[0111] In an embodiment, the aqueous composition according to the invention comprises:

[0112] From 1 to 5 % by weight, based on the total weight of the composition, of spironolactone, from 0.75 to 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose . from 0.01 to 1 % by weight, based on the total weight of the composition, of polysorbate 80.

[0113] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0114] In an embodiment, the aqueous composition according to the invention comprises:

[0115] From 1 to 5 % by weight, based on the total weight of the composition, of spironolactone, from 0.75 to 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose . from 0.01 to 1 % by weight, based on the total weight of the composition, of polysorbate 80. HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4. from 0.7 to 0.9 % by weight, based on the total weight of the composition, of trometamol.

[0116] In an embodiment, the aqueous composition according to the invention comprises:

[0117] 2 % by weight, based on the total weight of the composition, of spironolactone,

[0118] 0.75 % by weight, based on the total weight of the composition, of carboxymethylcellulose.

[0119] 0.02 % by weight, based on the total weight of the composition, of polysorbate 80.

[0120] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0121] In an embodiment, the aqueous composition according to the invention comprises:

[0122] 2 % by weight, based on the total weight of the composition, of spironolactone,

[0123] 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose.

[0124] 0.02 % by weight, based on the total weight of the composition, of polysorbate 80.

[0125] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0126] In an embodiment, the aqueous composition according to the invention comprises:

[0127] 2 % by weight, based on the total weight of the composition, of spironolactone,

[0128] 0.75 % by weight, based on the total weight of the composition, of carboxymethylcellulose.

[0129] 0.02 % by weight, based on the total weight of the composition, of polysorbate 80.

[0130] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0131] 0.8 % by weight, based on the total weight of the composition, of trometamol.

[0132] In an embodiment, the aqueous composition according to the invention comprises:

[0133] 2 % by weight, based on the total weight of the composition, of spironolactone,

[0134] 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose.

[0135] 0.02 % by weight, based on the total weight of the composition, of polysorbate 80.

[0136] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0137] 0.8 % by weight, based on the total weight of the composition, of trometamol. Treatment

[0138] Mineralocorticoid receptor activation is pathogenic for the retina as it favors the development of retinal edema, retinal inflammation, activation of microglial cells and retinal angiogenesis (Behar-Cohen F, Zhao M. Mineralocorticoid pathway in retinal health and diseases. Br J Pharmacol 2022; 179:3190-204). It acts, at least in part through the expression and localization of ion and water channels in retinal glial Muller cells (Zhao M, Valamanesh F, Celerier I, Savoldelli M, Jonet L, Jeanny J-C, et al. The neuroretina is a novel mineralocorticoid target: aldosterone up-regulates ion and water channels in Muller glial cells. FASEB J 2010;24:3405-15).

[0139] In the diabetic retina, mineralocorticoid pathway activation contributes to diabetic retinopathy, including macular edema, retinal inflammation, and excitotoxic ganglion cell death and spironolactone intraocular formulations exerted beneficial effects (Zhao M, Gelize E, Levy R, Moulin A, Azan F, Berdugo M, et al. Mineralocorticoid Receptor Pathway and its Antagonism in a Model of Diabetic Retinopathy. Diabetes 202 l:db210099). In the diabetic retina, spironolactone beneficial effects were not mediated by VEGF but exerted anti-edematous and neuroprotective effects through other molecular pathways including VLDLR and caveolin in example. Glaucoma-induced ganglion cell death has been rescued in animal models using mineralocorticoid receptor antagonists suggesting its potential to exert neuroprotection in glaucomatous eyes (Nitta E, Hirooka K, Tenkumo K, Fujita T, Nishiyama A, Nakamura T, et al. Aldosterone: a mediator of retinal ganglion cell death and the potential role in the pathogenesis in normal-tension glaucoma. Cell Death Dis 2013;4:e711).

[0140] In the choroid and retinal pigment epithelial cells, mineralocorticoid receptor activation induces choroidal vessel dilation, choroidal inflammation and epithelial to mesenchymal transition of retinal pigment epithelial cells. Mineralocorticoid receptor antagonists, administered systemically or intraocularly, encapsulated in microspheres have reduced choroid neovascularization in the laser- induced model and oral eplerenone has reduced the signs of exudation in patients treated with monthly intravitreal anti-VEGF, demonstrating that at least partial anti-edematous effects can be achieved using systemic administration (Zhao M, Mantel I, Gelize E, Li X, Xie X, Arboleda A, et al. Mineralocorticoid receptor antagonism limits experimental choroidal neovascularization and structural changes associated with neovascular age-related macular degeneration. Nat Commun 2019;10:369, Canonica J, Mehanna C, Bonnard B, Jonet L, Gelize E, Jais J-P, et al. Effect of acute and chronic aldosterone exposure on the retinal pigment epithelium-choroid complex in rodents. Exp Eye Res 2019;187: 107747. and Canonica J, Zhao M, Favez T, Gelize E, Jonet L, Kowalczuk L, et al. Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium. Int J Mol Sci 2021;22:9618. https: / / doi.org / 10.3390 / ijms22179618).

[0141] Mineralocorticoid receptor pathway activation has been involved in the pathogenesis of central serous chorioretinopathy (CSCR) (Zhao M, Celerier I, Bousquet E, Jeanny J-C, Jonet L, Savoldelli M, et al. Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy. J Clin Invest 2012;122:2672-9).

[0142] Furthermore, to support the pathogenic role of MR overactivation in the induction of RPE loss of integrity and differentiation, inventors generated a mouse model that over express MR specifically in the RPE. They show that at 6 months of age, the RPE in this mouse is altered and has lost its normal organization and shape, demonstrating loss of integrity and differentiation (Figure 10).

[0143] In summary, mineralocorticoid receptor antagonists have shown potential to alleviate pathogenic mechanisms involved in major retinal diseases, including retinal edema, pathological choroidal and retinal angiogenesis and neovascularization, retinal cell death and ganglion cell death as well as retinal and choroidal inflammation, retinal pigment epithelium dedifferentiation, subretinal fibrosis, diabetic retinopathy, age-related macular degeneration, choroidal neovascularization, ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity, retinal vein occlusion, macular edema, central serous chorioretinopathy .

[0144] The inventors also advantageously demonstrate that the formulation according to the invention significantly reduces the CNV activity, significantly reduces the fibrosis, shows a significant and beneficial effect on the ability of RPE cells to cover the laser bum site, and increases by almost 5 times the % of impacts with complete RPE coverage.

[0145] Altogether, these results and the involvement of mineralocorticoid receptor pathway activation in the pathogenesis of major ophthalmic disorders, argue in favor of and support the use of aqueous ophthalmic composition according to the invention for use in the treatment of ophthalmic pathogenic mechanisms and diseases.

[0146] In an embodiment, ophthalmic pathogenic mechanisms and diseases are chosen among:

[0147] Retinal edema

[0148] Pathological choroidal and retinal angiogenesis and neovascularization

[0149] Retinal cell death

[0150] Ganglion cell death

[0151] Retinal and choroidal inflammation

[0152] Retinal pigment epithelium dedifferentiation

[0153] Retinal pigment epithelium cell death

[0154] Subretinal fibrosis

[0155] Diabetic retinopathy

[0156] Age-related macular degeneration (AMD)

[0157] Wet Age-related macular degeneration

[0158] Dry Age-related macular degeneration Choroidal neovascularization

[0159] Choroidal neovascularization secondary to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid

[0160] Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity

[0161] Retinal vein occlusion

[0162] Macular edema,

[0163] Central serous chorioretinopathy

[0164] Myopia

[0165] Inflammation

[0166] Pachychoroid associated epitheliopathy

[0167] Subretinal fibrosis that complicates choroidal neovascularization

[0168] Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration

[0169] Myopic macular neovascularization

[0170] High myopia with alteration of RPE cells and / or staphyloma

[0171] Pachychoroid neovasculopathy

[0172] Subretinal fibrosis that reduces the vision in patients with wet AMD

[0173] Loss of RPE cells that reduce vision in AMD

[0174] Oxidative stress induced damages

[0175] Complement activation induced damages in RPE

[0176] Inflatnmasome-induced damages

[0177] Rupture of the bruch membrane due to RPE detachments

[0178] CNV (choroidal neovascularization)

[0179] MNV (macular neovascularization)

[0180] Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease Staphyloma

[0181] Geographic atrophy

[0182] Posterior uveitis

[0183] Ocular posterior trauma

[0184] Bruch membrane rupture

[0185] Photic injury

[0186] Pachychoroid epitheliopathy

[0187] Pachychoroid pigment epitheliopathy

[0188] Toxic epitheliopathy such as pentosan polysulfate retinopathy

[0189] Myopic degeneration

[0190] RPE atrophy secondary to any type of macular neovascularization Subretinal fluid.

[0191] In an embodiment, the present invention relates to the ophthalmic composition according to the invention for use in the treatment of ophthalmic pathogenic mechanism and diseases chosen among Retinal edema; Pathological choroidal and retinal angiogenesis and neovascularization; Retinal cell death; Ganglion cell death; Retinal and choroidal inflammation; Retinal pigment epithelium dedifferentiation; Retinal pigment epithelium cell death; Subretinal fibrosis; Diabetic retinopathy; Age- related macular degeneration; Wet Age-related macular degeneration; Dry Age-related macular degeneration; Choroidal neovascularization; Choroidal neovascularization secondary to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid; Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity; Retinal vein occlusion; Macular edema; Central serous chorioretinopathy; Myopia; Inflammation; Pachychoroid associated epitheliopathy; Subretinal fibrosis that complicates choroidal neovascularization; Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration; Myopic macular neovascularization; High myopia with alteration of RPE cells and / or staphyloma; Pachychoroid neovasculopathy; Subretinal fibrosis that reduces the vision in patients with wet AMD; Loss of RPE cells that reduce vision in AMD; Oxidative stress induced damages; Complement activation induced damages in RPE; Inflammasome- induced damages; Rupture of the bruch membrane due to RPE detachments; CNV (choroidal neovascularization); MNV (macular neovascularization); Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease; Staphyloma; Geographic atrophy; Posterior uveitis; Ocular posterior trauma; Bruch membrane rupture; Photic injury; Pachychoroid epitheliopathy; Pachychoroid pigment epitheliopathy; Toxic epitheliopathy such as pentosan polysulfate retinopathy; Myopic degeneration; RPE atrophy secondary to any type of macular neovascularization andSubretinal fluid.

[0192] In another aspect, the invention also concerns a method of treating pathogenic mechanism and diseases chosen among Retinal edema; Pathological choroidal and retinal angiogenesis and neovascularization; Retinal cell death; Ganglion cell death; Retinal and choroidal inflammation; Retinal pigment epithelium dedifferentiation; Retinal pigment epithelium cell death; Subretinal fibrosis; Diabetic retinopathy; Age- related macular degeneration; Wet Age-related macular degeneration; Dry Age-related macular degeneration; Choroidal neovascularization; Choroidal neovascularization secondary to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid; Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity; Retinal vein occlusion; Macular edema; Central serous chorioretinopathy; Myopia; Inflammation; Pachychoroid associated epitheliopathy; Subretinal fibrosis that complicates choroidal neovascularization; Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration; Myopic macular neovascularization; High myopia with alteration of RPE cells and / or staphyloma; Pachychoroid neovasculopathy; Subretinal fibrosis that reduces the vision in patients with wet AMD; Loss of RPE cells that reduce vision in AMD; Oxidative stress induced damages; Complement activation induced damages in RPE; Inflammasome- induced damages; Rupture of the bruch membrane due to RPE detachments; CNV (choroidal neovascularization); MNV (macular neovascularization); Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease; Staphyloma; Geographic atrophy; Posterior uveitis; Ocular posterior trauma; Bruch membrane rupture; Photic injury; Pachychoroid epitheliopathy; Pachychoroid pigment epitheliopathy; Toxic epitheliopathy such as pentosan polysulfate retinopathy; Myopic degeneration; RPE atrophy secondary to any type of macular neovascularization and Subretinal fluid in a subject in need thereof comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0193] In another aspect, the present invention relates to the use of an aqueous ophthalmic composition according to the invention for the treatment of ophthalmic pathogenic mechanisms and diseases chosen among Retinal edema; Pathological choroidal and retinal angiogenesis and neovascularization; Retinal cell death; Ganglion cell death; Retinal and choroidal inflammation; Retinal pigment epithelium dedifferentiation; Retinal pigment epithelium cell death; Subretinal fibrosis; Diabetic retinopathy; Age- related macular degeneration; Wet Age-related macular degeneration; Dry Age-related macular degeneration; Choroidal neovascularization; Choroidal neovascularization secondary to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid; Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity; Retinal vein occlusion; Macular edema; Central serous chorioretinopathy; Myopia; Inflammation; Pachychoroid associated epitheliopathy; Subretinal fibrosis that complicates choroidal neovascularization; Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration; Myopic macular neovascularization; High myopia with alteration of RPE cells and / or staphyloma; Pachychoroid neovasculopathy; Subretinal fibrosis that reduces the vision in patients with wet AMD; Loss of RPE cells that reduce vision in AMD; Oxidative stress induced damages; Complement activation induced damages in RPE; Inflammasome- induced damages; Rupture of the bruch membrane due to RPE detachments; CNV (choroidal neovascularization); MNV (macular neovascularization); Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease; Staphyloma; Geographic atrophy; Posterior uveitis; Ocular posterior trauma; Bruch membrane rupture; Photic injury; Pachychoroid epitheliopathy; Pachychoroid pigment epitheliopathy; Toxic epitheliopathy such as pentosan polysulfate retinopathy; Myopic degeneration; RPE atrophy secondary to any type of macular neovascularization and Subretinal fluid

[0194] In another aspect, the present invention relates to the use of an ophthalmic composition according to the invention for the manufacture of a medicament for the treatment of ophthalmic pathogenic mechanisms and diseases chosen among Retinal edema; Pathological choroidal and retinal angiogenesis and neovascularization; Retinal cell death; Ganglion cell death; Retinal and choroidal inflammation; Retinal pigment epithelium dedifferentiation; Retinal pigment epithelium cell death; Subretinal fibrosis; Diabetic retinopathy; Age-related macular degeneration; Wet Age-related macular degeneration; Dry Age-related macular degeneration; Choroidal neovascularization; Choroidal neovascularization secondary to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid; Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity; Retinal vein occlusion; Macular edema; Central serous chorioretinopathy; Myopia; Inflammation; Pachychoroid associated epitheliopathy; Subretinal fibrosis that complicates choroidal neovascularization; Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration; Myopic macular neovascularization; High myopia with alteration of RPE cells and / or staphyloma; Pachychoroid neovasculopathy; Subretinal fibrosis that reduces the vision in patients with wet AMD; Loss of RPE cells that reduce vision in AMD; Oxidative stress induced damages; Complement activation induced damages in RPE; Inflammasome- induced damages; Rupture of the bruch membrane due to RPE detachments; CNV (choroidal neovascularization); MNV (macular neovascularization); Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease; Staphyloma; Geographic atrophy; Posterior uveitis; Ocular posterior trauma; Bruch membrane rupture; Photic injury; Pachychoroid epitheliopathy; Pachychoroid pigment epitheliopathy; Toxic epitheliopathy such as pentosan polysulfate retinopathy; Myopic degeneration; RPE atrophy secondary to any type of macular neovascularization and Subretinal fluid

[0195] In a further aspect, the present invention relates to a pharmaceutical composition for treating ophthalmic pathogenic mechanisms and diseases chosen among Retinal edema; Pathological choroidal and retinal angiogenesis and neovascularization; Retinal cell death; Ganglion cell death; Retinal and choroidal inflammation; Retinal pigment epithelium dedifferentiation; Retinal pigment epithelium cell death; Subretinal fibrosis; Diabetic retinopathy; Age-related macular degeneration; Wet Age-related macular degeneration; Dry Age-related macular degeneration; Choroidal neovascularization; Choroidal neovascularization secondary to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid; Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity; Retinal vein occlusion; Macular edema; Central serous chorioretinopathy; Myopia; Inflammation; Pachychoroid associated epitheliopathy; Subretinal fibrosis that complicates choroidal neovascularization; Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration; Myopic macular neovascularization; High myopia with alteration of RPE cells and / or staphyloma; Pachychoroid neovasculopathy; Subretinal fibrosis that reduces the vision in patients with wet AMD; Loss of RPE cells that reduce vision in AMD; Oxidative stress induced damages; Complement activation induced damages in RPE; Inflammasome-induced damages; Rupture of the bruch membrane due to RPE detachments; CNV (choroidal neovascularization); MNV (macular neovascularization); Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease; Staphyloma; Geographic atrophy; Posterior uveitis; Ocular posterior trauma; Bruch membrane rupture; Photic injury; Pachychoroid epitheliopathy; Pachychoroid pigment epitheliopathy; Toxic epitheliopathy such as pentosan polysulfate retinopathy; Myopic degeneration; RPE atrophy secondary to any type of macular neovascularization and Subretinal fluid, said pharmaceutical composition comprising spironolactone, carboxymethylcellulose and polysorbate 80.

[0196] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of ophthalmic pathogenic mechanisms and diseases (such as retinal edema, pathological choroidal, retinal angiogenesis, retinal cell death and choroidal inflammation), or ameliorate one or more symptoms of such pathogenic mechanisms, disorders or recurring disorders, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0197] Choroidal neovascularization (CNV) activity

[0198] The inventors advantageously showed that the suspension according to the invention significantly reduces the CNV activity at day 14 and day 28.

[0199] Hence, in an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for use for reducing the choroidal neovascularization activity.

[0200] In an embodiment, the present invention relates to a method for reducing the choroidal neovascularization activity in a subject in need thereof, comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0201] By “reducing the choroidal neovascularization activity” we mean, for example, the decrease of the percentage of CNV lesions with high grades of leakage on the late stage of fluorescein angiography (grade 3+4).

[0202] Grade 3+4 impacts can be evaluated based on a two-stages laser impacts model (Little K, Llorian- Salvador M, Tang M, Du X, O’Shaughnessy O, Mcllwaine G, et al. A Two-Stage Laser-Induced Mouse Model of Subretinal Fibrosis Secondary to Choroidal Neovascularization. Transl Vis Sci Technol 2020;9:3). which choroidal neovascularization does not spontaneously regress at day 30 as shown by the persistent leakage of fluorescein injected systemically. Fibrosis

[0203] The inventors demonstrate that spironolactone formulation according to the invention significantly reduces the fibrosis since 53% of impact were in grade 1 in the vehicle treated eyes as compared to 32% in the spironolactone treated eyes.

[0204] In an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for use for reducing fibrosis.

[0205] In an embodiment, the present invention relates to a method for reducing fibrosis in a subject in need thereof, comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0206] By “reducing fibrosis” we mean the labelling of a collagen 1 plug that forms a scare in the center of the laser impact, in place of retinal pigment epithelium.

[0207] Typically, the fibrosis can be evaluated on flat-mounted RPE / choroid using collagen (Colli) immunostaining.

[0208] Effect on retinal pigment epithelium (RPE) integrity

[0209] Inventors demonstrate that spironolactone formulation, injected into the vitreous in the two step-laser CNV induced rat model showed a significant and beneficial effect on the ability of RPE cells to cover the laser bum site. The spironolactone formulation has thus increased by almost 5 times the % of impacts with complete RPE coverage, despite the fact that the model comprised two successive laser bums.

[0210] Hence, in an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for use to promote the restoration of retinal pigment epithelium integrity.

[0211] By promoting the restoration of RPE integrity, we mean the formation of a layer of cells, with the morphological characteristics of RPE cells at the site of RPE and bmch membrane bum by the laser impacts, such as hexagonal shape, complete coverage of the laser bum site, expression of differentiation markers such as RPE 65. In addition, the formulation induces the expression of genes mandatory for RPE differentiation such as YAP1.

[0212] Typically, the ability of drugs to act on the RPE restoration after injury can be evaluated by following the fate of RPE after two step-laser-induced injury and mpture of the Bmch membrane. There is indeed various degree of RPE coverage and proliferation at the site of laser bum.

[0213] In an embodiment, the aqueous ophthalmic composition according to the invention increase the number of polygonal RPE cells on the laser bum site.

[0214] In another embodiment, and based on the above-described results, the present invention relates to the aqueous ophthalmic composition according to the invention for use for reducing subretinal fibrosis in the context of choroidal neovascularization, and / or for promoting the restoration of retinal pigment epithelium integrity in the context of choroidal neovascularization, and / or for promoting the restoration of a differentiated retinal pigment epithelium in the context of choroidal neovascularization associated to age-related macular degeneration.

[0215] In another aspect, the invention also concerns a method for reducing subretinal fibrosis in the context of choroidal neovascularization and / or for promoting the restoration of retinal pigment epithelium integrity in the context of choroidal neovascularization, and / or for promoting the restoration of a differentiated retinal pigment epithelium in the context of choroidal neovascularization associated to age-related macular degeneration, in a subject in need thereof comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0216] Intravitreal administration

[0217] The aqueous ophthalmic composition is administered intravitreally. The term “intravitreal” or “intravitreally”, when used herein to characterize the delivery, administration or application of a composition of the present invention, is meant to specify that the composition is delivered, administered or applied into a posterior segment of an eye of a human, preferably into the vitreous body of the eye.

[0218] Hence, in an embodiment, the present invention relates to an aqueous ophthalmic composition for use in a therapeutic treatment of a patient, wherein said aqueous ophthalmic composition is administered into a posterior segment of an eye of said patient, preferably, into the vitreous body of the eye of said patient.

[0219] In an embodiment, the therapeutic treatment is the treatment of an ophthalmic disease or condition as described hereinbefore.

[0220] Examples of intravitreal formulation include solutions or suspensions or gels comprising a suspension.

[0221] In an embodiment, the viscosity of the solution or of the suspension is from 0.5 to 3 mPa.s at 35°C.

[0222] In a preferred embodiment, the composition is a suspension.

[0223] As used herein, the term suspension may be defined as a coarse dispersion containing finely divided insoluble material suspended in a liquid or gel medium.

[0224] In an embodiment, the suspension is suspended in a liquid medium, wherein the liquid medium is water.

[0225] In an embodiment, the liquid suspension" it is meant a coarse dispersion in which finely divided insoluble material is uniformly suspended in a liquid medium, maintaining transparency and allowing light to pass through without significant scattering or obstruction.

[0226] In an embodiment, the suspension is suspended in a gel medium, wherein the gel medium is chosen among hyaluronic acid gel, polyacrylates hydrogel, Suitable viscosities of the gel medium are in the range of about 10,000-50,000 mPa.s.

[0227] The one skilled in the art knows how to measure viscosity. Typically, viscosity values are based on the measurement with a Brookfield viscometer, at 35°C.

[0228] Preferably, the composition is administered into the vitreous body of the eye through a needle. The one skilled in the art is able to determine the needle to be used for intravitreal injection of the composition according to the invention.

[0229] Typically, the composition according to the invention may be administered through a 27G needle or a 30G needle, preferably a 30G needle.

[0230] Preferably, the composition is sterile and / or aseptic.

[0231] Sustained release

[0232] In an embodiment, the composition of the present invention is administered intravitreally so as to deliver IpM to 1 mM / day, preferably IpM to 100 pM / day, preferably IpM to 50 pM / day and more preferably about 10 pM / day of spironolactone.

[0233] The present invention relates to an aqueous ophthalmic composition according to the invention for use in a therapeutic treatment of a patient, wherein said aqueous ophthalmic composition is administered to deliver IpM to 1 m M / day, preferably IpM to 100 pM / day, preferably IpM to 50 pM / day and more preferably about lOpM / day of spironolactone.

[0234] In an embodiment, the therapeutic treatment is the treatment of an ophthalmic pathogenic mechanism or disease as described hereinbefore.

[0235] In an embodiment, the composition according to the invention releases spironolactone for at least 15 days, preferably at least 1 month, preferably at least 2 months.

[0236] The present invention also relates to an aqueous ophthalmic composition according to the invention for use in a therapeutic treatment of a patient, wherein said aqueous ophthalmic composition release spironolactone for at least 15 days, preferably at least 1 month.

[0237] In an embodiment, the therapeutic treatment is the treatment of an eye disease or condition as described hereinbefore.

[0238] The composition may be prepared under sterile and / or aseptic conditions. The aqueous ophthalmic composition may be autoclaved to sterilise it, or by any method allowing the sterilization. EXAMPLES

[0239] Materials and methods

[0240] The experiments were performed in accordance with the European Communities Council Directive 86 / 609 / EEC and French national regulations and approved by local ethical committees.

[0241] Experimental procedure to evaluate the ocular tolerance of spironolactone suspension

[0242] Healthy adult male Lewis rats (6-8 weeks) weighing 200-250 g purchased from Janvier Labs were used to study the ocular tolerance of spironolactone eye drops. Intravitreal injections of the suspensions were performed using microfine syringes (300 pL) with 30G needles under general anaesthesia. Intravitreous injections (IVT) were performed with five microliters of the 2% spironolactone SI (n=5 rats) and S2 (n=4 rats) suspensions or placebo Pl (n=3 rats) and P2 (n=3 rats). Control rat (n=l) received 5 pL of 0.9% NaCl. Retinal morphology was assessed in vivo at D7 by OCT (optical coherence tomography). The rats were then euthanized on day 30 and the eyes recovered for histological and immunofluorescence analysis.

[0243] SI, S2, Pl and P2 are described hereinafter.

[0244] Histological analysis

[0245] Retinal histology

[0246] Eyes were fixed in 4% paraformaldehyde (PFA) and 0.5% glutaraldehyde for 2 hours, then dehydrated in a series of alcohols and finally embedded in historesin (Leica, Heidelberg, Germany). Cross-sections of 5 pm were obtained with a Leica Jung RM2055 microtome, then stained with 1% toluidine blue. Morphology of the entire retina was observed using a microscope (Olympus BX51, Rungis, France) equipped with a CDD camera (Olympus DP70). For retinal thickness quantification, measurements of peripheral retinal thickness at 1.3 mm from the optic nerve and central retinal thickness at 0.65 mm from the optic nerve at temporal, nasal, superior and inferior levels were performed with Image J. The mean of peripheral and central retinal thickness measurements was calculated and used for comparison between treatment groups.

[0247] Immunofluorescence on cryosections

[0248] Eyes were fixed in 4% paraformaldehyde for 2 hours then rinsed in PBS IX and included in the optimal cutting temperature for cryosectioning. Cryosections were incubated with the following primary antibodies: rabbit anti-IBAl (1:300, Wako, Neuss, Germany) and rabbit anti-GFAP (1:300, Abeam, Cambridge, U.K. Ref: ab7260). The secondary antibody used was: Alexa Fluor 488-conjugated goat anti-rabbit IgG.

[0249] TUNEL labeling

[0250] The TUNEL assay was performed on eye cryosections in accordance with the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Cell nuclei were counterstained with DAPI. TUNEL-positive cells were counted throughout the retina on eye sections at the level of the optic nerve. Statistical analysis

[0251] Graphs represent mean values ± SD (Standard Deviation). Statistics were compiled using Prism - GraphPad software (GraphPad Softaware, version 8, San Diego, CA, USA). A non-parametric Mann- Whitney test was used to compare means between two groups. A non-parametric Kruskal -Wallis test followed by a Dunn's post-test was performed for comparison between several groups. For grouped data, a two-way ANOVA test a followed by a Tukey post-test was used. Results are considered significant if P < 0.05.

[0252] Experimental procedure to evaluate the efficiency of spironolactone suspension

[0253] Healthy adult male Long Evans rats (6-8 weeks) weighing 200-250 g purchased from Janvier Labs (n=16) were used for laser-induced CNV. After anesthesia and dilation of the pupils, coverslips were positioned on the cornea as a contact glass. Six laser bums were performed 2 to 3 optic disc diameters away from the optic nerve on both eyes with an Argon laser (532 nm) mounted on a slit lamp (175 mW, 0.1 s and 50 pm). Both eyes of animals received two laser induction, one at DO and one at D7 in the same position as the first one. The presence of a bubble witnessed the rupture of Bruch’s membrane and confirmed a successful laser impact. Intravitreous injections (IVT) were performed with five microliters of the 2% spironolactone S 1 (n=8 rats) suspensions or placebo Pl (n=8 rats) at two times: DO just after laser lesions were performed, and 14 days after the first laser induction. Retinal morphology was assessed in vivo at DO, D7 and D28 by OCT (optical coherence tomography) and angiographies (indocyanine green (ICG) and fluorescein) was performed at D14 and D28 using Heidelberg Retina Angiograph II (Heidelberg Engineering, Inc., Dossen- 103 heim, Germany) to image choroidal circulation The rats were then euthanized on day 30 and the eyes recovered for histological and immunofluorescence analysis.

[0254] After pupil dilatation, a mix of 0.2 ml ICG (2.5mg / ml INFRACYANINE®, SERB, Paris, France) with 0.1 ml fluorescein (0.2 mb of 10% fluorescein in saline) was injected intravenously in the tail of rats. Early and late phase angiograms were recorded at 0-2 and 5-8 min, respectively, after injection. Simultaneously, infrared images were acquired to detect the site and effective presence of laser bum. For each laser-induced lesion, fluorescein leakage was graded qualitatively by evaluating the increase in size / intensity of dye between the early and late phases. Angiographic scores were established by two blinded observers according to the following criteria: grade 0, no hyperfluorescence; grade 1, slight hyperfluorescence with no increase in intensity nor in size; grade 2, hyperfluorescence increasing in intensity but not in size; grade 3, hyperfluorescence increasing both in intensity and size; grade 4, hyperfluorescence size increase more than 2-diameter of the initial laser bum.

[0255] Histological evaluation

[0256] The RPE-choroid-sclera complex was fixed with 4% PFA, then post-fixed with acetone for 10 min at - 20° and proceeded for flat-mounting by making four radical incisions. Tissues were incubated with the following primary antibodies: rabbit anti-collagen 1 (1:500, Abeam, Cambridge, U.K) and mouse anti- RPE65 (1:500, Abeam, Cambridge, U.K) and the secondary antibodies used was respectively alexa Fluor 488-conjugated goat anti-rabbit IgG and alexa Fluor 488-conjugated donkey anti-mouse IgG. Rhodamin Phalloidin (Life Technologies) at a 1:300 dilution was also used.

[0257] Statistical analysis

[0258] Statistics were compiled using Prism-GraphPad software (GraphPad Softaware, version 8, San Diego, CA, USA). A chi-square test was performed to compare two groups based on the proportion of different grades. Results are considered significant if P < 0.05.

[0259] Experimental procedure to evaluate the efficiency of oral spironolactone in rat CNV

[0260] Healthy adult male Long Evans rats (6-8 weeks) weighing 200-250 g purchased from Janvier Labs (n=16) were used for laser-induced CNV. After anesthesia and dilation of the pupils, coverslips were positioned on the cornea as a contact glass. Six laser bums were performed 2 to 3 optic disc diameters away from the optic nerve on both eyes with an Argon laser (532 nm) mounted on a slit lamp (175 mW, 0.1 s and 50 pm). Both eyes of animals received laser induction. The presence of a bubble witnessed the rupture of Bruch’s membrane and confirmed a successful laser impact.

[0261] Eplerenone oral treatment: Oral eplerenone (INSPRA®, 200 mg / kg / day, 0.2% in chow) was given from day 0 until sacrifice; Control animal received normal chow from day 0 until sacrifice.

[0262] Retinal morphology was assessed in vivo at DO, D7 and D28 by OCT (optical coherence tomography) and angiographies (indocyanine green (ICG) and fluorescein) was performed at D14 and D28 using Heidelberg Retina Angiograph II (Heidelberg Engineering, Inc., Dossen- 103 heim, Germany) to image choroidal circulation The rats were then euthanized on day 30 and the eyes recovered for histological and immunofluorescence analysis.

[0263] After pupil dilatation, a mix of 0.2 ml ICG (2.5mg / ml INFRACYANINE®, SERB, Paris, France) with 0.1 ml fluorescein (0.2 mL of 10% fluorescein in saline) was injected intravenously in the tail of rats. Early and late phase angiograms were recorded at 0-2 and 5-8 min, respectively, after injection. Simultaneously, infrared images were acquired to detect the site and effective presence of laser bum. For each laser-induced lesion, fluorescein leakage was graded qualitatively by evaluating the increase in size / intensity of dye between the early and late phases. Angiographic scores were established by two blinded observers according to the following criteria: grade 0, no hyperfluorescence; grade 1, slight hyperfluorescence with no increase in intensity nor in size; grade 2, hyperfluorescence increasing in intensity but not in size; grade 3, hyperfluorescence increasing both in intensity and size; grade 4, hyperfluorescence size increase more than 2-diameter of the initial laser bum.

[0264] Histological evaluation

[0265] The RPE-choroid-sclera complex was fixed with 4% PFA, then post-fixed with acetone for 10 min at - 20°, and proceeded for flat-mounting by making four radical incisions. Tissues were incubated with the following primary antibodies: rabbit anti-collagen 1 (1:500, Abeam, Cambridge, U.K) and mouse anti- RPE65 (1:500, Abeam, Cambridge, U.K) and the secondary antibodies used was respectively alexa Fluor 488-conjugated goat anti-rabbit IgG and alexa Fluor 488-conjugated donkey anti-mouse IgG. Rhodamin Phalloidin (Fife Technologies) at a 1:300 dilution was also used.

[0266] Example 1: preparation of the suspension

[0267] Suspensions according to the invention were prepared according to its composition shown in Table 1.

[0268] Batches of 20 mb suspension are prepared. In a 50 mb beaker, CMC is gradually dispersed in ultrapure water using a paddle shaker (EUROSTAR digital - IKA® - WERKE) at a stirring speed of 500 rpm for 15 min. After complete dispersion of the CMC, Tween 80 is added and mixed with the CMC for 5 min at the same stirring speed (500 rpm). Spironolactone is then gradually added to the mixture (CMC + Tween 80), followed by NaCl and Trometamol / HCl. The speed is set at 750 rpm for 2 hours to obtain a homogeneous dispersion. The osmolarity of the suspension is measured as described below. The osmolarity should be between 280 and 300 mOsml / Kg (osmolarity of blood plasma).1 M NaOH solution is added dropwise while measuring pH as described below, until a pH between 7.0 and 7.2 (neutral pH) is obtained.

[0269] Table 1. Composition of the suspensions according to the invention.

[0270] Compound Quantities

[0271] Tween 80 0.02 g

[0272] Carboxymethyl Cellulose (CMC) 0.75 g or 1.50 g

[0273] Spironolactone 2.00 g

[0274] Trometamol 0.80 g

[0275] HC1 (IN, q.s) pH = 7.2

[0276] Distilled water (q.s) 100.00 mb

[0277] Example 2: In vitro release profiles of spironolactone from suspensions according to the invention The in-vitro release profiles of spironolactone from suspensions Fl and F2 (respectively at 0.75 and 1.5% CMC) over 30 days shows a release <20% at t = 24h.

[0278] The release rate for the F 1 formulation is slightly higher than for the F2 formulation which is probably due to a higher amount of CMC. The higher the amount of CMC, the higher the viscosity of the suspension and the slower the water penetrates and dissolves the active substance which will be subsequently released into the medium. The release curves are presented in two segments (Figure 11):

[0279] A segment in controlled release up to approximately 50% of the amount of spironolactone released between DO and D14.

[0280] A segment in plateau between D14 and D30 and which could remain constant in time.

[0281] The two release curves of the two formulations Fl and F2 (0.75% and 1.5% CMC respectively) are statistically similar (according to the f2 test of similarity) showing that the two amounts of CMC have no real impact on the release kinetics of spironolactone.

[0282] These results allow to conclude that the formulations according to the invention allow to release spironolactone for at least 1 month.

[0283] Example 3: Stability of the suspension according to the invention

[0284] For intravitreal injection, the pH and osmolarity of the suspension should be adjusted to avoid irritation of the eye. A neutral pH (between 7.00 and 7.20) and an osmolarity close to that of blood plasma (between 280 and 300 mOsml / Kg) are recommended.

[0285] The suspension according to the invention as described in example 1 (with 1.5% CMC) has been evaluated. pH (conforms to between 7 and 7.20) and osmolarity (conforms to between 280 and 300 mOsml / Kg) are consistent.

[0286] Indeed, osmolarity and pH remains at stable values after 1 month.

[0287] Example 4: Tolerance of the spironolactone suspensions in rat eyes

[0288] The suspensions of spironolactone were tested in rat eyes to evaluate their intraocular tolerance after 5pL intravitreous injection (IVT). The rats were divided into 4 groups of treatment (SI, S2, Pl and P2).

[0289] 4 rats (8 eyes) were used per formulation. The tested formulations are as follows:

[0290] The in vivo retinal morphology was evaluated with optical coherence tomography (OCT) at day 7 and rats were euthanized at 1 month for histology and immunofluorescence.

[0291] At day 7, we observed hyperreflective substance in the vitreous of rats injected with SI and S2 on OCT images in contrast to those injected with Pl and P2, indicating small particles of spironolactone suspended in the vitreous. The vitreous was however clear, no sign of retinal inflammation or edema was observed.

[0292] The histology at 1 month after IVT showed normal multi-layer retinal structure in all groups. There was no inflammation cell infiltration. The thickness of the retina and choroid was comparable among the groups.

[0293] Example 5: Efficacy of spironolactone suspension on 2 impact laser-induced choroidal neovascularization (CNV)

[0294] The spironolactone suspension SI was tested on a two-stages laser impacts model in order to evaluate both the anti-angiogenic effect of spironolactone but also its effect on fibrosis, as the fibrosis was shown to be more pronounced with two laser stages and the leakage duration increased as compared to the one-stage laser model (Little K, Llorian-Salvador M, Tang M, Du X, O’Shaughnessy O, Mcllwaine G, et al. A Two-Stage Laser-Induced Mouse Model of Subretinal Fibrosis Secondary to Choroidal Neovascularization. Transl Vis Sci Technol 2020;9:3. https: / / doi.Org / 10.1167 / tvst.9.4.3.).

[0295] Long Evans pigmented rats were used for these experiments.

[0296] The experiment is summarized in Figure 1.

[0297] Effect on CNV activity

[0298] CNV activity was graded in a blind manner at day 14 and day 28 as previously described (Zhao M, Mantel I, Gelize E, Li X, Xie X, Arboleda A, et al. Mineralocorticoid receptor antagonism limits experimental choroidal neovascularization and structural changes associated with neovascular age- related macular degeneration. Nat Commun 2019;10:369).

[0299] The spironolactone suspension significantly reduced the CNV activity (leakage) at day 14 and day 28 as presented in Table 2 hereinafter. Spironolactone significantly decreased the percentage of grade 3+4 impact from 81% to 38% at day 14 [Chi-square test. %2= 43.26; df = 4; P<0,0001] and from 30% to 6% at 4 weeks [Chi-square test. y = 36.63; df = 4; P<0,0001],

[0300] Table 2. CNV activity evaluation

[0301] Effect on fibrosis Fibrosis was evaluated at day 30 on flat-mounted RPE / choroid using collagen (Colli) immunostaining according to the grading scale represented at Figure 2 with the following caption:

[0302] 0: No fibrosis.

[0303] 0.5: Coll 1 positive fibrosis filling the laser bum.

[0304] 1: Coll 1 positive fibrosis beyond the laser bum.

[0305] The spironolactone formulation significantly reduced the fibrosis since 53% of impact were in grade 1 in the vehicle treated eyes as compared to 32% in the spironolactone treated eyes (Chi square p<0.0001) as represented at Figure 3.

[0306] Example 6: Efficacy of eplerenone on one single laser burn impact fibrosis

[0307] Fibrosis was evaluated after eplerenone was administered orally from the day of laser until day 30.

[0308] Eplerenone is also a mineralocorticoid receptor antagonist. Oral eplerenone (INSPRA®, 200 mg / kg / day, 0.2% in chow) from day 0 until sacrifice; Controls are fed with normal chow from day 0 until sacrifice. As shown in Figure 4, the oral eplerenone treatment also significantly increased the proportion of impact with no fibrosis from 33% to 52% and reduced significantly the proportion of impact with large fibrosis from 32% to 17%, but fibrosis is less intense in a one impact laser model as compared to the two-impact laser model, in which intraocular spironolactone efficiently reduced the fibrosis.

[0309] Example 7: Effect on retinal pigment epithelium (RPE) integrity

[0310] To evaluate the ability of dmgs to act on the RPE restoration after injury, inventors have followed the fate of RPE after two step-laser-induced injury and mpture of the Bmch membrane. They indeed observed that at the site of laser bum, there is various degree of RPE coverage and proliferation.

[0311] The following grading as represented at Figure 5 with the following caption has been established to evaluate RPE barrier integrity:

[0312] 1 : full coverage with polygonal RPE cells

[0313] 0.5: partial coverage with polygonal RPE cells 0: no coverage or coverage with abnormal cells.

[0314] Integrity of RPE cells is evaluated at 30 days after laser has been performed.

[0315] Spironolactone formulation (SI formulation), injected into the vitreous in the two step-laser CNV induced rat model showed a significant and beneficial effect on the ability of RPE cells to cover the laser bum site.

[0316] Labelling of RPR by phalloidin shows the restoration of a complete RPE barrier in 7% of impacts in the vehicle treated eyes (Pl formulation) as compared to 34% in the spiro formulation treated eyes (SI formulation). Absence of coverage (grade 0) was observed in 66% of impacts in vehicle treated eyes as compared to 47% of impact in spironolactone formulation treated eyes. The spironolactone formulation has thus increased by almost 5 times the % of impacts with complete RPE coverage, despite the fact that the model comprised two successive laser bums (Figure 6).

[0317] The inventors also verified that the cells that cover the wound express RPE65, a marker specific of RPE cells. These results demonstrate that spironolactone formulation favor the regeneration of a normal RPE layer as shown in Figure 7.

[0318] Furthermore, the effect of spironolactone formulation of RPE coverage was different from the effect of eplerenone administered orally from the laser bum until day 30.

[0319] Indeed, at day 30, in a single impact laser model in the rats, eplerenone failed to improve RPE coverage as shown in Figure 8

[0320] The spironolactone formulation according to the invention is thus more efficient that systemic administration to favor the regeneration of normal RPE cells after injury.

[0321] Example 8: Comparison of tolerance between the suspension according to the invention versus Spironolactone-hexPLA formulation

[0322] The suspension according to the invention was compared to Spironolactone-hexPLA formulation described in Dahmana et al.

[0323] Experimental Procedure

[0324] Spironolactone suspension according to the invention: 2% (m / v) SPL, 0.75 % CMC, 0.02% Polysorbate 80 and 0.8% Trometamol.

[0325] Healthy adult Lewis rats (6-8 weeks old), weighing 200-250 g, were obtained from Janvier Labs. Intravitreal injections of the test suspensions were administered under general anesthesia using microfme syringes (300 pL) with 30G needles. Each rat received a five-microliter intravitreal injection (IVT) of either a 2% spironolactone suspension (n=5 rats) or a 0.75 % CMC placebo (n=3 rats). Retinal morphology was assessed in vivo at D7 by OCT (optical coherence tomography), with a simultaneous fundus examination. The rats were then euthanized on day 30, and the eyes were collected for immunohistochemical analysis.

[0326] Spironolactone-hexPLA: 5% SPL, hexPLA

[0327] Healthy adult Wistar rats (7-10 weeks old) weighing approximately 230-370 g were obtained from Charles-River Laboratories. Intravitreal injections were administered under general anesthesia using microfme syringes (300 pL) with 29G needles. Two groups received five microliters of hexPLA placebo IVT injections and were monitored for 7 (n=l rat) and 31 (n=2 rats) days post-injection. Three groups received five microliters of 5% SPL-hexPLA and were followed for 3 (n=3 rats), 7 (n=4 rats), and 31 (n=5 rats) days after the IVT injection. The rats were then euthanized, and the eyes were collected for histological analysis. Retinal morphology was assessed in vivo at D14 by OCT (optical coherence tomography), with a simultaneous fundus examination.

[0328] Histology (Spironolactone-hexPLA)

[0329] The enucleated eyes were fixed in Bouin’s solution for 24 hours at room temperature and then dehydrated in graded alcohol solutions (70-100%) followed by xylene using an automated processor (Histokinette 2000, Reichert-Jung). The eyes were subsequently embedded in paraffin, sectioned into 5 pm slices using a microtome (Hyrax M55, Carl Zeiss), and mounted on glass slides. Finally, the sections were stained with hematoxylin and eosin and examined under a light microscope (Zeiss, Germany).

[0330] Immunofluorescence on cryosections (Spironolactone suspension according to the invention)

[0331] Eyes were fixed in 4% paraformaldehyde for 2 hours then rinsed in PBS IX and included in the optimal cutting temperature for cryosectioning. Cryosections were incubated with the following primary antibodies: rabbit anti-IBAl (1:300, Wako, Neuss, Germany) and rabbit anti-GFAP (1:300, Abeam, Cambridge, U.K. Ref: ab7260). The secondary antibody used was: Alexa Fluor 488-conjugated goat anti-rabbit IgG. Results

[0332] • Spironolactone Suspension according to the invention

[0333] After injection, spironolactone particles were suspended homogenously in the vitreous without forming masses or droplets and they very rapidly sedimented.

[0334] One week after injection, the fundus examinations showed clear and normal results with no masses or droplets formed and no signs of retinal inflammation or oedema, as observed clinically and on SD-OCT.

[0335] 7 days after the intravitreal injection, no inflammatory cells were observed.

[0336] 30 days after the intravitreal injection, no inflammatory reaction on histological images was observed. No GFAP activation extending into the retina was seen in any of the groups and no difference was seen regarding IBA-1 between the groups, indicating no additional activation related to the injections of spironolactone.

[0337] • Spironolactone-hexPLA Formulation

[0338] Firstly, it was observed that this formulation spontaneously forms a semi-solid droplet in aqueous buffer as the polymer is viscous forming a bubble in solution, consistent with fundus examination findings, where as early as day 7 post-injection, floating droplets were visible in the vitreous and on the posterior face of the lens, sometimes accompanied by clustered bubbles.

[0339] Clinical examinations performed by OCT (Optical coherence tomography) revealed abnormal fundus and hyper-reflective dots, indicating infiltrating cells and an inflammatory reaction. Histological images showed that both the placebo polymer and the SPL-loaded polymer remained at the injection site, preventing proper closure and healing, leading to retinal detachment, severe retinal disorganization and inflammation.

[0340] Overall, the spironolactone suspension according to the invention demonstrated better tolerance without material remaining on the visual axis subsequently preventing vision with no inflammatory reaction compared to the Spironolactone-hexPLA formulation.

[0341] Example 9: Long-term Stability of S Suspension

[0342] In a stability study of a suspension containing Spironolactone, remarkable results were obtained over an 18-month observation period. The initial concentration measured in November 2022 was established at 2%. Follow-up analysis in May 2024 revealed a concentration of 19989.75 ± 205 pg / ml, representing an exceptionally small decrease of only 0.05%.

[0343] This outstanding stability is particularly noteworthy given that Spironolactone, being a prodrug, would typically be expected to undergo metabolic transformation into various metabolites during storage. Such minimal variation in concentration demonstrates that the formulation successfully prevented any significant degradation or chemical transformation of the active compound. These findings validate the formulation strategy selected for this suspension.

Claims

CLAIMS1. An aqueous ophthalmic composition comprising:(a) Spironolactone,(b) Carboxymethylcellulose,(c) Polysorbate 80.

2. The aqueous ophthalmic composition according to claim 1, wherein the composition comprises from 0.2 to 20 % by weight, preferably from 0.5 to 10 % by weight, preferably from 1 to 5 % by weight, based on the total weight of the composition, of spironolactone.

3. The aqueous ophthalmic composition according to claim 1 or 2, wherein the composition comprises 2% by weight, based on the total weight of the composition, of spironolactone.

4. The aqueous ophthalmic composition according to anyone of claims 1 to 3, wherein the composition comprises from 0.2 to 7.5 % by weight, preferably from 0.1 to 5 % by weight, preferably from 0.75 to 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose .

5. The aqueous ophthalmic composition according to anyone of claims 1 to 4, wherein the composition comprises 0.75 or 1.50 % by weight, based on the total weight of the composition, of carboxymethylcellulose.

6. The aqueous ophthalmic composition according to anyone of claims 1 to 5, wherein the composition comprises from 0.01 to 1 % by weight, preferably from 0.01 to 0.5 % by weight, preferably from 0.02 to 0.05 % by weight, based on the total weight of the composition, of polysorbate 80.

7. The aqueous ophthalmic composition according to anyone of claims 1 to 6, wherein the composition comprises 0.02% by weight, based on the total weight of the composition, of polysorbate 80.

8. The aqueous ophthalmic composition according to anyone of claims 1 to 7, wherein said composition further comprises trometamol.

9. The aqueous ophthalmic composition according to anyone of claims 1 to 8, wherein the composition comprises from 0.4 to 1.2 % by weight, preferably from 0.6 to 1 % by weight, preferably from 0.7 to 0.9 % by weight, based on the total weight of the composition, of trometamol.

10. The aqueous ophthalmic composition according to anyone of claims 1 to 5, wherein the composition comprises 0.8% by weight, based on the total weight of the composition of trometamol.

11. An aqueous ophthalmic composition according to anyone of claims 1 to 10, for use in the treatment of ophthalmic pathogenic mechanisms and diseases.

12. The aqueous ophthalmic composition according to claim 11, wherein ophthalmic pathogenic mechanisms and diseases are chosen among Retinal edema; Pathological choroidal and retinal angiogenesis and neovascularization; Retinal cell death; Ganglion cell death; Retinal and choroidal inflammation; Retinal pigment epithelium dedifferentiation; Retinal pigment epithelium cell death; Subretinal fibrosis; Diabetic retinopathy; Age-related macular degeneration; Wet Age-related macular degeneration; Dry Age-related macular degeneration; Choroidal neovascularization; Choroidal neovascularization secondary7to Age-related macular degeneration or myopia or choroidal and / or retinal inflammation or idiopathic or to Central serous chorioretinopathy or to pachychoroid; Ganglion cell death due to glaucoma or ocular hypertension or excitotoxicity; Retinal vein occlusion; Macular edema; Central serous chorioretinopathy; Myopia; Inflammation; Pachychoroid associated epitheliopathy; Subretinal fibrosis that complicates choroidal neovascularization; Subretinal fibrosis that complicates choroidal neovascularization in Age related macular degeneration; Myopic macular neovascularization; High myopia with alteration of RPE cells and / or staphyloma; Pachychoroid neovasculopathy; Subretinal fibrosis that reduces the vision in patients with wet AMD; Loss of RPE cells that reduce vision in AMD; Oxidative stress induced damages; Complement activation induced damages in RPE; Inflammasome-induced damages; Rupture of the bruch membrane due to RPE detachments; CNV (choroidal neovascularization); MNV (macular neovascularization); Diseases affecting the extracellular matrix or elastic fibers such as Ehler-Danlos, Marfan disease; Staphyloma; Geographic atrophy; Posterior uveitis; Ocularposterior trauma; Bruch membrane rupture; Photic injury; Pachychoroid epitheliopathy; Pachychoroid pigment epitheliopathy; Toxic epitheliopathy such as pentosan polysulfate retinopathy; Myopic degeneration; RPE atrophy secondary to any type of macular, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

13. An aqueous ophthalmic composition according to anyone of claims 1 to 10, for use for reducing the choroidal neovascularization activity, for reducing the fibrosis or for promoting the restoration of retinal pigment epithelium integrity.

14. An aqueous ophthalmic composition according to anyone of claims 1 to 10 for use in a therapeutic treatment of a patient, wherein said aqueous ophthalmic composition is administered into a posterior segment of an eye of said patient, preferably into the vitreous body of the eye of said patient.

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