Spirolactone eye drop preparation

Abstract

The present invention relates to an aqueous ophthalmic composition comprising spirolactone, cyclodextrin and tromethamine, preferably for topical application to the human eye. The present inventors advantageously dissolve spirolactone in the HP-gamma cyclodextrin excipient at a concentration of 0.1%, which dose has been demonstrated to be effective in the treatment of ocular surface defects. They also have successfully demonstrated that the spirolactone eye drops are stable for up to 9 months at 4 DEG C and achieve its efficacy within at least 6 months, and the formulation is well tolerant after multiple daily instillations within 7 days. In addition, the spirolactone eye drops provided by the invention accelerate healing of corneal wounds of rats, reduce corneal edema and inflammation, enhance epithelial integrity and improve nerve regeneration, and prompt recovery of corneal homeostasis. Thus, the invention also relates to the use of an aqueous ophthalmic composition for the treatment of ocular diseases and disorders.

Description

Technical Field

[0001] This invention belongs to the medical field, particularly ophthalmology. Background Technology

[0002] Mineralocorticoid receptor (MR) activation is pathogenic to the retina because it promotes the development of retinal edema, retinal inflammation, microglia activation, and retinal angiogenesis (Behar-Cohen F, Zhao M. Mineralocorticoid pathway in retinal health and diseases. Br J Pharmacol 2022;179:3190–204. https: / / doi.org / 10.1111 / bph.1577). Mineralocorticoid activation is also pathogenic to the cornea because it contributes to corneal neovascularization and loss of corneal transparency [Mineralocorticoid receptor antagonism limits experimental choroidal neovascularization and structural changes associated with neovascular age-related macular degeneration. Zhao M, Mantel I, Gelize E, Li X, Xie X, Arboleda A, Seminel M, Levy-Boukris R, Dernigoghossian M, Prunotto A, Andrieu-Soler C, Rivolta C, Canonica J, Naud MC, Lechner S, Farman N, Bravo-Osuna I, Herrero-Vanrell R, Jaisser F, Behar-Cohen F. Nat Commun. 2019 Jan 21;10(1):369. doi: 10.1038 / s41467-018-08125-6].

[0003] Mineralocorticoid receptor antagonists (MRAs) have shown potential to alleviate pathogenic mechanisms involved in major eye diseases, including retinal edema, pathological choroid, retinal angiogenesis, retinal cell death, and inflammation of the retina and choroid, corneal neovascularization and inflammation, ocular surface inflammation, and fibrosis. In transgenic mice that are specifically ineffective against MRs in endothelial cells, there is a reduction in corneal neovascularization due to limbal defects, suggesting that MRs may also help maintain corneal avascularity [Nat Commun. 2019 Jan 21;10(1):369. doi: 10.1038 / s41467-018-08125-6]. Glucocorticoids (GCs) act by binding to both glucocorticoid receptors (GRs) and mineralocorticoid receptors, both of which are expressed in the cornea. In the cornea, GC-induced delay in wound healing is mediated by MR activation [Topical Administration of Spironolactone-Loaded Nanomicelles Prevents Glucocorticoid-Induced Delayed Corneal Wound Healing in Rabbits. Dahmana N, Mugnier T, Gabriel D, Kaltsatos V, Bertaim T, Behar-Cohen F, Gurny R, Kalia YN. Mol Pharm. 2018 Mar 5;15(3):1192-1202. doi: 10.1021 / acs.molpharmaceut.7b01028], demonstrating that MR antagonism can prevent GC-induced corneal wound healing defects, which is an important side effect of GC.

[0004] Many retinal, choroidal, and ocular surface diseases can benefit from MR antagonists.

[0005] Spironolactone (SPL) is a more potent mineralocorticoid receptor antagonist. However, spironolactone is extremely poorly water-soluble (0.02 mg / mL), has a short half-life (less than 1 h) in the ocular media, and does not cross the intact ocular barrier even at high intravenous doses due to its binding to efflux proteins expressed at the internal and external retinal barriers [Zhao M, Rodríguez-Villagra E, Kowalczuk L, Le Normand M, Berdugo M, Levy-Boukris R, El Zaoui I, Kaufmann B, Gurny R, Bravo-Osuna I, Molina-Martínez IT, Herrero-Vanrell R, Behar-Cohen F. J Control Release. 2017 Nov 28;266:187-197. doi:10.1016 / j.jconrel.2017.09.029].

[0006] Although some of spironolactone's effects can be achieved via a systemic route, particularly when the ocular blood-blood barrier is disrupted, the optimal efficacy of spironolactone requires direct administration to the eye, as seen in diabetic retinopathy in rats, where the beneficial effects of the MR antagonist were only observed when the drug was administered intraocularly, and not when administered orally or via systemic injection [Mineralocorticoid Receptor Pathway and Its Antagonism in a Model of Diabetic Retinopathy. Zhao M, et al. Diabetes. 2021. PMID: 34426510][Zhao M, Rodríguez-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. JControl Release 2017;266:187–97.] [https: / / doi.org / 10.1016 / j.jconrel.2017.09.029].

[0007] To treat ocular surface diseases and reach the posterior part of the eye, particularly the choroid, after local instillation, spironolactone requires appropriate formulations to increase its water solubility and prolong its contact time on the ocular surface, thereby obtaining therapeutic and stable concentrations of spironolactone for intraocular use.

[0008] We demonstrated that spironolactone micelles (0.1%, w / v) with a mean number-weighted diameter of 20 nm prepared using mPEG-poly(octanoic acid) polymer (mPEG-hexPLA) prevented glucocorticoid-induced delayed corneal wound healing in New Zealand white rabbits. These 0.1% spironolactone micelle formulations were administered via multiple daily instillations over 5 days. [Dahmana N, Mugnier T, Gabriel D, Kaltsatos V, Bertaim T, Behar-Cohen F, Gurny R, Kalia YN. MolPharm. 2018 Mar 5;15(3):1192-1202. doi: 10.1021 / acs.molpharmaceut.7b01028].

[0009] Although micelles exhibit good safety properties, the preparation of polymer-based formulations involves the use of organic solvents (ethanol, acetone), which can be harmful and may limit the preparation process. Extensive safety studies have been conducted on the novel polymers used at a very high cost. Finally, the empty polymers whose degradation products can locally lower the pH may cause irritation and inflammation.

[0010] For all these reasons, there is a need for polymer-free eye drop formulations. Invention Overview In preclinical studies, the ocular surface tolerability and efficacy of a novel polymer-free, hydroxypropyl-γ-cyclodextrin-based eye drop containing 0.1% spironolactone were investigated in a rat model of corneal wound healing. First, the inventors advantageously dissolved spironolactone at a concentration of 0.1% in an HP-γ-cyclodextrin excipient, a dose proven effective in treating ocular surface defects. They then successfully demonstrated that the spironolactone eye drops were stable at 4°C for up to 9 months, achieved their efficacy within at least 6 months, and were well-tolerated after multiple daily instillations over 7 days. Indeed, after multiple topical instillations over a week, the spironolactone eye drops showed no signs of incompatibility. No morphological changes, inflammation, oxidative stress, or increased cell death were observed in the rat cornea, indicating good tolerability.

[0012] Therefore, the present invention relates to an aqueous ophthalmic composition comprising spironolactone, cyclodextrin, and tromethamine. Preferably, the aqueous ophthalmic composition is intended for topical application to the human eye.

[0013] Furthermore, the inventors have demonstrated that the spironolactone eye drops according to the present invention accelerate corneal wound healing in rats, reduce corneal edema and inflammation, enhance epithelial integrity, and improve nerve regeneration, indicating the restoration of corneal homeostasis, while potassium canrate (an active and soluble metabolite of SPL) has no effect.

[0014] Therefore, the present invention relates to the use of aqueous ophthalmic compositions for the treatment of eye diseases and conditions.

[0015] SPL eye drops can be beneficial for patients with impaired corneal wound healing, including those associated with glucocorticoid therapy. Therefore, this invention also relates to the administration of aqueous ophthalmic compositions in conjunction with glucocorticoid therapy.

[0016] In another aspect of the invention, a method for preparing an aqueous ophthalmic composition is provided.

[0017] In another aspect of the invention, an apparatus for dispensing aqueous ophthalmic compositions dropwise is also provided. Attached Figure Description

[0018] Figure 1 The morphology of the cornea and corneal epithelium in rats after 7 days of local instillation of spironolactone-containing eye drops.

[0019] 1A. After 7 days of instillation of eye drops containing 0.1% spironolactone (SPL) or placebo (PBO) once or three times daily, histological sections, similar to the untreated control cornea (Ctrl), showed normal corneal structure and a layered corneal epithelium (Ep), a regular stroma (St), and an intact corneal endothelium (En) monolayer. Double-headed arrows indicate the thickness of the cornea and corneal epithelium. St, stroma; En, endothelium. Scale bar: 20 μm.

[0020] 1B. Quantitative analysis of corneal thickness on tissue sections showed no significant differences among all groups. Data are expressed as mean ± SD, n = 3–4 rat corneas.

[0021] 1C. Quantitative analysis of corneal epithelial thickness showed no significant differences among all groups. Data are presented as mean ± SD, n = 3–4 rat corneas.

[0022] 1D.ZO-1 immunofluorescence showed cell membrane localization primarily in the superficial layer of the corneal epithelium. No disruption of immunostaining was observed after topical treatment with SPL and PBO eye drops. Dashed lines indicate the internal boundaries of the corneal epithelium. Scale bar: 20 μm. n = 3–4 rat corneas.

[0023] Immunofluorescence of 1E.E-cadherin was used to show its localization on the cell membrane in all epithelial cells. No disruption of immunostaining was observed after topical treatment with SPL or PBO eye drops. Dashed lines indicate the internal boundary of the corneal epithelium. Scale bar: 20 μm. n = 3-4 rat corneas.

[0024] Figure 2 Immunostaining of inflammatory cells in the cornea of ​​rats 7 days after local instillation of spironolactone-containing eye drops.

[0025] 2A and B. No IBA1- or ED1-positive inflammatory cells were observed in corneas treated once or three times daily with eye drops containing spironolactone (SPL) or placebo (PBO) for 7 days. Positive control corneas (Ctrl+) from the inflammation model showed IBA and ED1 staining in the inflamed cornea. Ep, epithelium; St, stroma; En, endothelium. Scale bar: 20 μm. n = 3–4 rat corneas.

[0026] Figure 3 : Nitrification stress and cell death markers in the cornea of ​​rats after 7 days of local instillation of spironolactone-containing eye drops.

[0027] 3A. No nitrotyrosine (NT) positive cells were observed in corneas treated once or three times daily with eye drops containing 0.1% spironolactone (SPL) or placebo (PBO) for 7 days. Positive control corneas (Ctrl+) from an inflammation model show NT staining. Ep, epithelium; St, stroma; En, endothelium. Scale bar: 20 μm. n = 3–4 rat corneas.

[0028] 3B.TUNEL-positive apoptotic cells were found only in the superficial corneal epithelium of untreated control eyes (Ctrl) and eyes treated with SPL or PBO eye drops three times daily for 7 days. Scale bar: 20 μm.

[0029] 3C. Quantitative analysis of TUNEL-positive cells throughout the corneal epithelium showed no significant difference among the Ctrl, PBO, and SPL groups. Data are presented as mean ± SD, n = 3 rat corneas.

[0030] Figure 4 SPL eye drops containing spironolactone improve corneal reepithelialization in rats.

[0031] 4A. Fluorescein staining shows a 4-mm central corneal epithelial wound at 0 hours, and healing progress at 6, 24, and 48 hours. Spironolactone (SPL) eye drops improved corneal epithelial regeneration compared to placebo (PBO) and potassium canelate solution (KCAN).

[0032] 4B. Quantitative analysis showed that SPL eye drops significantly increased epithelial healing rates at 6 and 24 hours compared with PBO eye drops and KCAN solution, while there was no difference between the KCAN and PBO groups. Data are presented as mean ± SD, n = 7–9 rat corneas. p<0.05; , p<0.01.

[0033] Figure 5 SPL eye drops reduced corneal edema in rats after corneal deepenthesization.

[0034] 5A. In vivo B-scan optical coherence tomography (OCT) images (left) showing cross-sectional views of the central cornea through the green line (right) before and 24 and 48 hours after corneal deepithelialization. Spironolactone (SPL) eye drops reduced corneal edema at 48 hours compared to placebo eye drops (PBO) and potassium canileate solution (KCAN). Rectangles of the same size were used as a reference for quantitative analysis of corneal edema.

[0035] 5B. Corneal edema is expressed as a percentage of the area of ​​the reference rectangle occupied by the cornea. Compared with the PBO and KCAN groups, SPL eye drops significantly reduced corneal edema at 48 hours. Data are presented as mean ± SD, n = 7–9 rat corneas. , p<0.05.

[0036] Figure 6 SPL eye drops containing spironolactone restored corneal epithelial integrity in rats after corneal wound healing.

[0037] Immunostaining of E-cadherin revealed discontinuities (arrows) and tissue disruption in the placebo-treated (PBO) corneal epithelium. Spironolactone eye drops (SPL) restored E-cadherin continuity and the layered structure of the corneal epithelium compared to the undamaged control cornea (Ctrl). Dashed lines indicate the inner boundary of the corneal epithelium. Scale bar: 20 μm. n = 4–5 rat corneas.

[0038] 6B. Immunostaining of cytokeratin 12 (K12) showed reduced K12 fluorescence and disruption of the corneal epithelial basal layer in PBO treatment (arrows). The inner boundary of the epithelium (dashed lines) was irregular. Compared with control corneas, SPL eye drops enhanced K12 expression in the corneal epithelium and improved the regularity of the inner boundary of the epithelium. Scale bar: 20 μm. n = 4–5 rat corneas.

[0039] Figure 7 SPL eye drops reduced inflammatory cell infiltration in the cornea of ​​rats after corneal deepenthesization.

[0040] Immunostaining with 7A and B. IBA1 and ED1 showed inflammatory cell infiltration throughout the stroma of placebo-treated (PBO) corneas, while spironolactone eye drops (SPL) limited cell infiltration in the anterior stroma. Ep, epithelium; St, stroma; En, endothelium. Scale bar: 20 μm.

[0041] 7C and D. Compared with PBO-treated corneas, SPL eye drops significantly reduced the number of IBA1- and ED1-positive cells in the cornea. Data are presented as mean ± SD, n = 5–6 rat corneas. , p<0.05.

[0042] Figure 8 SPL eye drops promote corneal renervation in the deepithelialized central cornea of ​​rats.

[0043] 8A. TUBB3 immunostaining on whole corneal slides reveals dense and linear subbasal plexuses in undamaged control corneas (Ctrl). Subbasal plexuses were mechanically removed by corneal deepithelialization. Spironolactone (SPL) eye drops improved the morphology of regenerated subbasal plexuses compared to placebo-treated (PBO) corneas.

[0044] 8B. The density of regenerated subbasal nerves was 54.95% of the control cornea in the PBO-treated group (n=3), while the density of regenerated subbasal nerves increased to 70.32% in the SPL-treated group (n=2). Data are presented as mean ± SD; ns, not significant. Invention Details This invention relates to an aqueous ophthalmic composition comprising: (A) Spironolactone, (B) Cyclodextrin, (C) Tromethamine.

[0046] Spironolactone (SPL) Spironolactone (C 24 H 32 O4S (CAS No. 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 represented as SC-9420; NSC-150339; 7a-acetylthiospironolactone; 7a-acetylthio-17a-hydroxy-3-oxopregn-4-ene-21-carboxylic acid g-lactone.

[0047] In one embodiment, the aqueous ophthalmic composition contains 0.01% to 10% by weight, preferably 0.05% to 5% by weight, and preferably 0.1% to 1% by weight of spironolactone, based on the total weight of the composition.

[0048] In pharmaceutical applications, 1% typically corresponds to 1g / 100mL (or 10mg / mL).

[0049] In a preferred embodiment, the composition contains 0.1% by weight of spironolactone based on the total weight of the composition.

[0050] Cyclodextrin (CD) Cyclodextrins are natural cyclic oligosaccharides. Their molecules are ring-shaped with a hydrophilic outer surface and a hydrophobic inner cavity. The most common cyclodextrins are α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, composed of 6, 7, and 8 α-1,4-linked glucose units, respectively. The number of these units determines the size of the cavity. Cyclodextrins can form inclusion complexes with a variety of hydrophobic molecules by absorbing the entire molecule or parts thereof into the cavity. The stability of the formed complex depends on the degree of coordination between the guest molecule and the cyclodextrin cavity. Therefore, cyclodextrins increase drug solubility and stability, enhance drug bioavailability, and reduce drug toxicity.

[0051] Cyclodextrins used in this invention include natural cyclodextrins and their derivatives, including alkylated and hydroxyalkylated derivatives and branched cyclodextrins. Besides α-, β-, and γ-cyclodextrins, ether and mixed ether derivatives, as well as those containing sugar residues, are of particular interest. Especially useful in this invention are hydroxyethyl, hydroxypropyl (including 2- and 3-hydroxypropyl), and dihydroxypropyl ethers, their corresponding mixed ethers, and other mixed ethers having methyl or ethyl groups, such as methyl hydroxyethyl, ethyl hydroxyethyl, and ethyl hydroxypropyl ethers of α-, β-, and γ-cyclodextrins; and maltodextrin, glucosyl, and maltotriose derivatives of α-, β-, and γ-cyclodextrins, which may contain one or more sugar residues, such as glucosyl or dist-glucosyl, maltodextrin or dist-maltodextrin, and various mixtures thereof, such as mixtures of maltodextrin and dist-maltodextrin derivatives. The specific cyclodextrin derivatives used in this article include hydroxypropyl-β-cyclodextrin (or 2-hydroxypropyl-β-cyclodextrin), hydroxyethyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin (or 2-hydroxypropyl-γ-cyclodextrin), hydroxyethyl-γ-cyclodextrin, dihydroxypropyl-β-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, disaccharosyl-β-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, maltotrisyl-β-cyclodextrin, maltotrisyl-γ-cyclodextrin, and disaccharosyl-β-cyclodextrin, maltosyl-β-cyclodextrin / disaccharosyl-β-cyclodextrin, methyl-β-cyclodextrin, and mixtures thereof. Methods for preparing such cyclodextrin derivatives are well known, for example, by Bodor U.S. Patent 5,024,998, dated June 18, 1991, and the references cited therein.

[0052] In one embodiment, the cyclodextrin is selected from 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD).

[0053] In a preferred embodiment, the cyclodextrin is 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD).

[0054] In one embodiment, the aqueous ophthalmic composition contains 1% to 5% by weight, preferably 1.5% to 3% by weight, and more preferably 2% to 3% by weight of cyclodextrin, based on the total weight of the composition.

[0055] In a preferred embodiment, the aqueous ophthalmic composition contains 2.75% or 3% cyclodextrin based on the total weight of the composition.

[0056] Tromethamine Tromethamine, also known as aminobutane glycerol or tris(hydroxymethyl)aminomethane buffer, is a tertiary amine with a pKa of 8.1 at 25°C.

[0057] In one embodiment, the aqueous ophthalmic composition contains 0.1 to 5% by weight, preferably 0.1 to 2% by weight, and more preferably 0.4 to 0.8% by weight of tromethamine, based on the total weight of the composition.

[0058] In a preferred embodiment, the aqueous ophthalmic composition contains 0.6% by weight of tromethamine based on the total weight of the composition.

[0059] pH adjuster The pH of the composition is preferably 6.8-7.5, or 7.0-7.5, more preferably 7.0-7.4.

[0060] It should be understood that any suitable acid or base can be used to adjust the pH to a suitable value or pH range.

[0061] Therefore, in one embodiment, the composition has a pH adjuster in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 6.8-7.5, preferably 7.0-7.5, more preferably 7.0-7.4.

[0062] The pH of the composition can be adjusted after adding tromethamine buffer.

[0063] Typically, it is necessary to increase the pH of the composition by adding an acid, which may suitably be hydrogen chloride (HCl).

[0064] Therefore, in one implementation, the pH adjuster is HCl 1N.

[0065] In one embodiment, HCl 1N is added as a pH adjuster to the aqueous ophthalmic composition until the optimal pH is reached.

[0066] Those skilled in the art know how to measure the pH of a solution. Typically, a pH meter can be used.

[0067] Tensioner The molar osmolality of the aqueous ophthalmic composition is preferably 260 to 310 mOsmol / kg, more preferably 280 to 300 mOsmol / kg, and even more preferably about 280 mOsmol / kg.

[0068] Advantageously, a substantially isotonic composition is selected.

[0069] Therefore, in one embodiment, the aqueous ophthalmic composition has a tensioning agent in an amount sufficient to adjust the tension of the ophthalmic composition to 280-310 mOsmol / kg, preferably 280-300 mOsmol / kg, more preferably about 280 mOsmol / kg.

[0070] It should be understood that any suitable tensioning agent can be used to adjust the molar osmotic concentration to a suitable value or range.

[0071] Molar osmolality is a measure of the total number of particles in a solution per unit volume, typically expressed as molar osmolality per liter of solution (Osmol / L). Osmolality is calculated based on the concentration of individual solutes and their contribution to the total osmolality of the solution. This method assumes that all solutes in the solution are completely dissociated in water.

[0072] Molar osmotic pressure concentration can be determined using common methods known to those skilled in the art, such as freezing point depression, vapor pressure, conductivity, or calculation. Typically, molar osmotic pressure concentration can be calculated based on the concentration of individual solutes in the solution using the following formula: Molar osmotic pressure concentration = (total [solutes] x number of particles per molecule) / solution volume.

[0073] In one implementation, osmotic pressure is measured using freezing point depression. For example, osmotic pressure is measured using an osmoremeter such as the Osmomat 3000 (Gonotec®).

[0074] In a preferred embodiment, the tensioning agent is sodium chloride.

[0075] In one embodiment, the aqueous ophthalmic composition contains 0.25 to 0.75% by weight, preferably 0.4 to 0.6% by weight, and more preferably 0.4 to 0.5% by weight of a tensioning agent, based on the total weight of the composition.

[0076] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.01% to 10% by weight, preferably 0.05% to 5% by weight, and preferably 0.1% to 1% by weight of spironolactone. - Based on the total weight of the composition, 1% to 5% by weight, preferably 1.5% to 3% by weight, preferably 2% to 3% by weight of cyclodextrin. - Based on the total weight of the composition, 0.1% to 5% by weight, preferably 0.1% to 2% by weight, preferably 0.4% to 0.8% by weight of tromethamine.

[0077] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.01% to 10% by weight, preferably 0.05% to 5% by weight, and preferably 0.1% to 1% by weight of spironolactone. - Based on the total weight of the composition, 1% to 5% by weight, preferably 1.5% to 3% by weight, preferably 2% to 3% by weight of cyclodextrin. - Based on the total weight of the composition, 0.1% to 5% by weight, preferably 0.1% to 2% by weight, preferably 0.4% to 0.8% by weight of tromethamine, - HCl, in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 6.8-7.5, preferably 7.0-7.5, more preferably 7.0-7.4. - Sodium chloride, in an amount sufficient to adjust the tension of the aqueous ophthalmic composition to 280-310 mOsmol / kg, preferably 280-300 mOsmol / kg, more preferably 280 mOsmol / kg.

[0078] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.1% to 1% spironolactone, - Based on the total weight of the composition, 2% to 3% cyclodextrin, - 0.4% to 0.8% wt% tromethamine based on the total weight of the composition.

[0079] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.1% to 1% spironolactone, - Based on the total weight of the composition, 2% to 3% cyclodextrin, - Based on the total weight of the composition, 0.4% to 0.8% wt% of tromethamine, - HCl, in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4. - Sodium chloride, in an amount sufficient to adjust the tension of the aqueous ophthalmic composition to 280 mOsmol / kg.

[0080] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.1% by weight of spironolactone, - Based on the total weight of the composition, 3% by weight of cyclodextrin, - 0.6% by weight of tromethamine based on the total weight of the composition.

[0081] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.1% by weight of spironolactone, - Based on the total weight of the composition, 3% by weight of cyclodextrin, - Based on the total weight of the composition, 0.6% by weight of tromethamine, - HCl, in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4. - Sodium chloride, in an amount sufficient to adjust the tension of the aqueous ophthalmic composition to 280 mOsmol / kg.

[0082] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.1% by weight of spironolactone, - Based on the total weight of the composition, 2.75% by weight of cyclodextrin, - 0.6% by weight of tromethamine based on the total weight of the composition.

[0083] In one embodiment, the aqueous composition according to the invention comprises: - Based on the total weight of the composition, 0.1% by weight of spironolactone, - Based on the total weight of the composition, 2.75% by weight of cyclodextrin, - Based on the total weight of the composition, 0.6% by weight of tromethamine, - HCl, in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4. - Sodium chloride, in an amount sufficient to adjust the tension of the aqueous ophthalmic composition to 280 mOsmol / kg.

[0084] Topical application Topical application of aqueous ophthalmic compositions. When used herein to characterize the delivery, application, or use of the compositions of the present invention, the terms "topical" or "locally" are intended to specify the direct delivery, application, or use of the composition at the site of interest (i.e., the eye) to obtain a local effect. Examples of topical formulations include solutions, suspensions, ointments, and gels.

[0085] In a preferred embodiment, the composition is a solution.

[0086] Preferably, the composition is applied directly to the ocular surface, conjunctival fornix, or bulbar conjunctiva.

[0087] The aqueous ophthalmic composition of the present invention can be available in unit dosage form.

[0088] Preferably, the composition is sterile and / or disinfected.

[0089] eye drops The aqueous ophthalmic compositions described herein can be in the form of eye drops for use in the eyes.

[0090] Therefore, in one embodiment, the composition is in the form of an eye drop for application to the eye.

[0091] In one embodiment, the viscosity of the composition of the present invention is 0.5 to 3 mPa·s at 35°C.

[0092] Viscosity was measured using a planar rotational viscometer.

[0093] Flow measurements were performed using an Anton Paar MCR 102 rheometer (Austria) with a planar geometry (flow diameter: 50 mm, air gap: 0.375 mm). Dynamic viscosity was measured for 10 s at 35°C (application site temperature) under constant shear.

[0094] treat Spironolactone was successfully dissolved in HP-γ-CD excipient at a concentration of 0.1%, a dose that proved effective in treating GC-induced delayed corneal wound healing in a rabbit model [Dahmana, N.; Mugnier, T.; Gabriel, D.; Kaltsatos, V.; Bertaim, T.; Behar-Cohen, F.; Gurny, R.; Kalia, YN Topical Administration of Spironolactone-Loaded Nanomicelles Prevents Glucocorticoid-Induced Delayed Corneal Wound Healing in Rabbits. Mol. Pharm. 2018, 15, 1192–1202, doi:10.1021 / acs.molpharmaceut.7b01028].

[0095] Advantageously, the pH of the eye drops according to the invention is adjusted to 7.0-7.4 to reduce irritation. The osmotic pressure of the SPL eye drops is close to that of the tear film osmotic pressure of a normal, non-dry eye [Tomlinson, A.; Khanal, S.; Ramaesh, K.; Diaper, C.; McFadyen, A. Tear Film Osmolarity: Determination of a Referent for Dry Eye Diagnosis. Invest. Ophthalmol. Vis. Sci. 2006, 47, 4309–4315, doi:10.1167 / iovs.05-1504]. Therefore, the compositions according to the invention can be advantageously applied topically to the eye and are effective in treating eye diseases.

[0096] Therefore, in another aspect, the present invention relates to compositions according to the invention for treating eye diseases and conditions, particularly eye diseases and conditions affecting the ocular surface.

[0097] The inventors have demonstrated that the aqueous ophthalmic composition according to the present invention can be used to restore epithelial integrity, improve corneal re-epithelialization, improve epithelial cell adhesion and cell layer differentiation, restore the optical function of the anterior cornea, reduce inflammation, reduce stromal edema, restore corneal transparency, or improve corneal renervation.

[0098] Corneal wound healing Following corneal injury, the epithelium must heal promptly to rebuild barrier function and regenerate a normal epithelial basement membrane, thereby terminating the fibrotic stromal wound healing response and promoting regeneration and repair (Wilson, SE Corneal Wound Healing. Exp. Eye Res. 2020, 197, 108089, doi:10.1016 / j.exer.2020.108089).

[0099] The inventors have advantageously demonstrated that SPL eye drops not only accelerate corneal re-epithelialization, but also improve epithelial cell adhesion and cell layer differentiation. Restoration of epithelial integrity can block the entry of epithelial TGFβ and PDGF into the corneal stroma and prevent corneal cells from transforming into corneal fibroblasts and myofibroblasts (Singh, V.; Santhiago, MR; Barbosa, FL; Agrawal, V.; Singh, N.; Ambati, BK; Wilson, SE Effect of TGFβ and PDGF-B Blockade on Corneal Myofibroblast Development in Mice. Exp. Eye Res. 2011, 93, 810–817, doi:10.1016 / j.exer.2011.09.012, Singh, V.; Jaini, R.; Torricelli, AAM; Santhiago, MR; Singh, N.; Ambati, BK; Wilson, SETGFβ and PDGF-B Signaling Blockade Inhibits Myofibroblast Development from Both Bone Marrow-Derived and Keratocyte-Derived Precursor). Cells in Vivo. Exp.Eye Res. 2014, 121, 35–40, doi:10.1016 / j.exer.2014.02.013).

[0100] In one embodiment, the present invention relates to an aqueous ophthalmic composition according to the invention for restoring epithelial integrity.

[0101] In one embodiment, the present invention relates to a method for restoring epithelial integrity in a subject in need, comprising administering an effective amount of an aqueous ophthalmic composition according to the invention.

[0102] "Restoring epithelial integrity" means that the corneal surface is regular and smooth, and the epithelial-stromal interface is regular without any scars, which is an important factor for optimal visual recovery.

[0103] The restoration of epithelial integrity can be evaluated by measuring the immunostaining of E-cadherin and cytokeratin K12 (markers of differentiated corneal epithelium). Typically, such measurements are performed using immunostaining of E-cadherin and cytokeratin K12.

[0104] In one embodiment, the aqueous ophthalmic composition according to the invention restores the continuity of E-cadherin and the multilayered structure of the corneal epithelium.

[0105] In another embodiment, the aqueous ophthalmic composition according to the invention enhances K12 expression and restores the structure of the corneal epithelium.

[0106] It has been demonstrated that in superficial corneal stromal scarring, disordered surface irregularities such as higher-order aberrations affect final visual acuity [Curr Eye Res 2023 Jun;48(6):536-545. Doi: 10.1080 / 02713683.2023.2173786. Epub 2023 Feb 20. Relationship of Density, Depth, and Surface Irregularity of Superficial Corneal Opacification with Visual Acuity.DOI: 10.1080 / 02713683.2023.2173786]. Therefore, restoration of epithelial integrity is essential for visual recovery. Furthermore, restoration of proper epithelial barrier function prevents potential infection and inflammation.

[0107] In one embodiment, the present invention relates to an aqueous ophthalmic composition according to the invention for improving corneal re-epithelialization, improving epithelial cell adhesion and cell layer differentiation, and for restoring the optical function of the anterior cornea.

[0108] In one embodiment, the present invention relates to a method for improving corneal re-epithelialization, epithelial cell adhesion and differentiation of cell layer stratification in subjects in need, and for restoring the optical function of the anterior cornea, comprising administering an effective amount of the aqueous ophthalmic composition according to the invention.

[0109] "Improving corneal reepithelialization" refers to the restoration of the geometry and optical properties of the epithelium.

[0110] Typically, corneal reepithelialization can be evaluated using fluorescein staining.

[0111] "Improving epithelial cell adhesion and differentiation through cell layer stratification" refers to the restoration of corneal barrier function.

[0112] Typically, it can be evaluated by immunostaining of E-cadherin, ZO-1, and cytokeratin K12.

[0113] "Optimal restoration of the optical function of the anterior cornea" refers to improving visual acuity.

[0114] Typically, it can be assessed through visual acuity measurements.

[0115] Inflammation decreased, stromal edema reduced, and corneal transparency restored. SPL eye drops also significantly reduce inflammation, a pathogenic factor involved in the wound healing process. In fact, damaged epithelial cells immediately release pro-inflammatory IL1α and IL1β. Their upregulation triggers corneal cells, epithelial cells, and fibroblasts to secrete cytokines / chemokines and growth factors, which subsequently infiltrate bone marrow-derived inflammatory cells and remodel stromal cells into the extracellular matrix (Wilson, SE Corneal Wound Healing. Exp. Eye Res. 2020, 197, 108089, doi:10.1016 / j.exer.2020.108089, Liu, C.-Y.; Kao, WW-Y. Corneal Epithelial Wound Healing. Prog. Mol. Biol. Transl. Sci. 2015, 134, 61–71, doi:10.1016 / bs.pmbts.2015.05.002).

[0116] In cases of extensive damage or delayed epithelial closure (where the inflammatory response is amplified), growth factors and cytokines continue to infiltrate the corneal stroma, leading to chronic inflammation and abnormal fibrosis (Ljubimov, AV; Saghizadeh, M. Progress in Corneal Wound Healing. Prog. Retin. Eye Res. 2015, 49, 17–45, doi:10.1016 / j.preteyeres.2015.07.002). By reducing inflammation, SPL eye drops reduce stromal edema, which helps restore corneal transparency.

[0117] In one embodiment, the present invention relates to an aqueous ophthalmic composition according to the invention, which is used to reduce inflammation, decrease stromal edema, and restore corneal transparency.

[0118] In one embodiment, the present invention relates to a method for reducing inflammation, decreasing stromal edema, and restoring corneal transparency in subjects in need, comprising administering an effective amount of the aqueous ophthalmic composition according to the invention.

[0119] The reduction of inflammation can be evaluated by measuring inflammatory cells, for example, by IBA1 and ED1. Typically, the anti-inflammatory effect of SPL eye drops can be assessed by IBA1 and ED1 immunostaining.

[0120] Clinically, corneal edema is assessed by measuring the reduction of corneal stroma using SD-OCT and evaluating corneal transparency using slit-lamp examination and corneal / anterior segment slit-lamp photography.

[0121] "Reducing stromal edema" refers to reducing corneal thickness.

[0122] "Restoring corneal transparency" refers to achieving optimal visualization of the anterior segment.

[0123] Corneal nerve reinnervation The integrity of nerve fibers is essential for normal corneal function by sensing thermal, mechanical, and chemical stimuli, which lead to the release of essential neurotrophic proteins for corneal homeostasis and wound healing (Al-Aqaba, MA; Dhillon, VK; Mohammed, I.; Said, DG; Dua, HS CornealNerves in Health and Disease. Prog. Retin. Eye Res. 2019, 73, 100762, doi:10.1016 / j.preteyeres.2019.05.003). Corneal diseases such as diabetic keratopathy and neurotrophic keratopathy are associated with impaired corneal nerve function and epithelial rupture, resulting in delayed epithelial wound healing and neurotrophic corneal ulcers (NaPier, E.; Camacho, M.; McDevitt, TF; Sweeney, AR Neurotrophic Keratopathy: Current Challenges and Future Prospects. Ann. Med. 2022, 54,666–673, doi:10.1080 / 07853890.2022.2045035). Our results suggest that SPL eye drops may contribute to corneal renervation, which is essential for restoring homeostasis.

[0124] In one embodiment, the present invention relates to an aqueous ophthalmic composition according to the invention for improving corneal nerve reinnervation.

[0125] In one embodiment, the present invention relates to a method for improving corneal nerve reinnervation in a subject in need, comprising administering an effective amount of an aqueous ophthalmic composition according to the invention.

[0126] "Improving corneal nerve reinnervation" refers to improving subbasal nerve density and obtaining longer and more parallel nerves.

[0127] Corneal nerve reinnervation can usually be assessed by TUBB3 immunostaining or by analyzing corneal nerves using confocal microscopy.

[0128] As demonstrated by the inventors, in a rat model of corneal deepenthesization, 0.1% SPL eye drops exhibited significant beneficial effects on corneal wound healing by accelerating wound closure, reducing corneal edema, improving epithelial integrity, and reducing inflammatory responses. Following corneal injury, the epithelium must heal promptly to rebuild barrier function and regenerate a normal epithelial basement membrane, thereby terminating the healing response of fibrotic stromal wounds and promoting regenerative repair. SPL eye drops not only accelerate corneal epithelial regeneration but also improve epithelial cell adhesion and cell layer differentiation. SPL eye drops also significantly reduced the infiltration of ED1 and IBA1 positive cells and their migration in the deep stroma. It cannot be determined whether the reduced cell infiltration is due to faster recovery of the epithelial barrier or whether it is the primary effect of SPL; however, it confirms that SPL is released at an effective concentration in the cornea, as SPL does indeed reduce IBA-1 positive cells in a rat model of laser-induced choroidal neovascularization and diabetic retina.

[0129] In summary, these results lead to the conclusion that the ophthalmic composition of the present invention can be used to treat diseases or conditions selected from the following: corneal ulcers with or without inflammation, postoperative corneal wounds, delayed corneal re-epithelialization after corneal transplantation, corneal transplant rejection, vernal keratitis, ocular erythema, epithelial keratitis secondary to dry eye syndrome or meibomian gland dysfunction, corneal ulcers following corneal cross-linking, delayed corneal wound healing due to refractive surgery, corneal surgery, or anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungal, parasitic, or viral infection or infection by unconventional agents, corneal opacity or corneal epithelial defects due to neurotrophic keratopathy, corneal opacity due to corneal scarring, corneal edema, and corneal lesions due to vernal keratitis. Inflammation or neovascularization, corneal inflammation due to ocular erythema, corneal neovascularization, corneal neovascularization secondary to inflammation or meibomian gland dysfunction or infection or limbal defects or corneal transplant rejection, recurrent corneal erosion, delayed wound healing, postoperative treatment after corneal transplantation or refractive surgery or any other corneal surgery, glucocorticoid-induced delayed epithelial wound healing after corneal physical trauma (e.g., surgical trauma including corneal transplantation, laser induction, UV exposure including crosslinking, refractive surgery), hereditary and hereditary corneal diseases, aniridia-induced corneal diseases, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and related diseases such as dry eye syndrome and blepharitis, and corneal diseases selected from diabetic, neurotrophic, toxic, iatrogenic, and preservative-induced corneal diseases.

[0130] In one implementation, delayed wound healing may be, but is not solely, due to the use of glucocorticoids.

[0131] As used in this article, refractive surgery can be followed by laser-assisted or surgical procedures.

[0132] In one implementation, corneal neovascularization is inhibited due to the anti-angiogenic effect of spironolactone.

[0133] In another aspect, the present invention also relates to a method of treating a disease or condition, the method comprising administering the aqueous ophthalmic composition of the present invention to a patient in need, the disease or condition being selected from corneal ulcers with or without inflammation, postoperative corneal trauma, delayed corneal re-epithelialization after corneal transplantation, corneal transplant rejection, vernal keratitis, ocular erythema, epithelial keratitis secondary to dry eye syndrome or meibomian gland dysfunction, corneal ulcers following corneal cross-linking, delayed corneal wound healing due to refractive surgery, corneal surgery, or anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungal, parasitic, or viral infection or infection by unconventional agents, corneal opacity or corneal epithelial defects due to neurotrophic keratopathy, corneal opacity due to corneal scarring, corneal edema, and vernal keratitis. Corneal inflammation or neovascularization caused by keratitis, corneal inflammation due to ocular erythema, corneal neovascularization, corneal neovascularization secondary to inflammation or meibomian gland dysfunction or infection or limbal defects or corneal transplant rejection, recurrent corneal erosion, delayed wound healing, postoperative treatment after corneal transplantation or refractive surgery or any other corneal surgery, glucocorticoid-induced delayed epithelial wound healing after corneal physical trauma (e.g., surgical trauma including corneal transplantation, laser induction, UV exposure including crosslinking, refractive surgery), hereditary and hereditary corneal diseases, aniridia-induced corneal diseases, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and related diseases such as dry eye syndrome and blepharitis, and corneal diseases selected from diabetic, neurotrophic, toxic, iatrogenic, and preservative-induced corneal diseases.

[0134] In another aspect, the present invention relates to the use of the aqueous ophthalmic composition of the present invention for the treatment of diseases or conditions selected from: corneal ulcers with or without inflammation, postoperative corneal trauma, delayed corneal re-epithelialization after corneal transplantation, corneal transplant rejection, vernal keratitis, ocular erythema, epithelial keratitis secondary to dry eye syndrome or meibomian gland dysfunction, corneal ulcers following corneal cross-linking, delayed corneal wound healing due to refractive surgery, corneal surgery, or anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungal, parasitic, or viral infection or infection by unconventional agents, corneal opacity or corneal epithelial defects due to neurotrophic keratopathy, corneal opacity due to corneal scarring, corneal edema, and corneal inflammation due to vernal keratoconjunctivitis. Or neovascularization, corneal inflammation due to ocular erythema, corneal neovascularization, corneal neovascularization secondary to inflammation or meibomian gland dysfunction or infection or limbal defects or corneal transplant rejection, recurrent corneal erosion, delayed wound healing, postoperative treatment after corneal transplantation or refractive surgery or any other corneal surgery, glucocorticoid-induced delayed epithelial wound healing after corneal physical trauma (e.g. surgical trauma including corneal transplantation, laser induction, UV exposure including crosslinking, refractive surgery), hereditary and hereditary corneal diseases, aniridia-induced corneal diseases, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and related diseases such as dry eye syndrome and blepharitis, and corneal diseases selected from diabetic, neurotrophic, toxic, iatrogenic, preservative-induced corneal diseases.

[0135] In another aspect, the present invention relates to the use of the aqueous ophthalmic composition of the present invention in the preparation of a medicament for treating diseases or conditions selected from: corneal ulcers with or without inflammation, postoperative corneal trauma, delayed corneal re-epithelialization after corneal transplantation, corneal transplant rejection, vernal keratitis, ocular erythema, epithelial keratitis secondary to dry eye syndrome or meibomian gland dysfunction, corneal ulcers following corneal cross-linking, delayed corneal wound healing due to refractive surgery, corneal surgery, or anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungal, parasitic, or viral infection or infection by unconventional agents, corneal opacity or corneal epithelial defects due to neurotrophic keratopathy, corneal opacity due to corneal scarring, corneal edema, and vernal keratitis. Corneal inflammation or neovascularization, corneal inflammation due to ocular erythema, corneal neovascularization, corneal neovascularization secondary to inflammation or meibomian gland dysfunction or infection or limbal defects or corneal transplant rejection, recurrent corneal erosion, delayed wound healing, postoperative treatment after corneal transplantation or refractive surgery or any other corneal surgery, glucocorticoid-induced delayed epithelial wound healing after corneal physical trauma (e.g., surgical trauma including corneal transplantation, laser induction, UV exposure including crosslinking, refractive surgery), hereditary and hereditary corneal diseases, aniridia-induced corneal diseases, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and related diseases such as dry eye syndrome and blepharitis, and corneal diseases selected from diabetic, neurotrophic, toxic, iatrogenic, and preservative-induced corneal diseases.

[0136] On the other hand, the present invention relates to pharmaceutical compositions for treating diseases or conditions, said pharmaceutical compositions comprising spironolactone, cyclodextrin, and tromethamine, said diseases or conditions being selected from corneal ulcers with or without inflammation, postoperative corneal trauma, delayed corneal re-epithelialization after corneal transplantation, corneal transplant rejection, vernal keratitis, ocular erythema, epithelial keratitis secondary to dry eye syndrome or meibomian gland dysfunction, corneal ulcers following corneal cross-linking, delayed corneal wound healing due to refractive surgery, corneal surgery, or anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungal, parasitic, or viral infection or infection by unconventional agents, corneal opacity or corneal epithelial defects due to neurotrophic keratopathy, corneal opacity due to corneal scarring, corneal edema, and vernal keratitis. Corneal inflammation or neovascularization due to conjunctivitis, corneal inflammation due to ocular erythema, corneal neovascularization, corneal neovascularization secondary to inflammation or meibomian gland dysfunction or infection or limbal defects or corneal transplant rejection, recurrent corneal erosion, delayed wound healing, postoperative treatment of corneal transplantation or refractive surgery or any other corneal surgery, glucocorticoid-induced delayed epithelial wound healing after corneal physical trauma (e.g., surgical trauma including corneal transplantation, laser induction, UV exposure including crosslinking, refractive surgery), hereditary and hereditary corneal diseases, aniridia-induced corneal diseases, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and related diseases such as dry eye syndrome and blepharitis, and corneal diseases selected from diabetic, neurotrophic, toxic, iatrogenic, and preservative-induced corneal diseases.

[0137] For the avoidance of doubt, the term “treatment” as used herein includes curative, remittent, and preventative treatment. “Treatment” aims to reverse, alleviate, or inhibit the progression of the disorder or condition to which the term applies, or to prevent one or more symptoms of the disorder or condition to which the term applies.

[0138] Apply with GC Topical corticosteroids (GCs), which are extremely effective at controlling harmful inflammation, are part of most treatment regimens, but they should be used with caution because they may delay corneal epithelial wound healing (Kadmiel, M.; Janoshazi, A.; Xu, X.; Cidlowski, JA Glucocorticoid Action in Human Corneal Epithelial Cells Establishes Roles for Corticosteroids in Wound Healing and Barrier Function of the Eye. Exp. Eye Res. 2016, 152, 10–33, doi:10.1016 / j.exer.2016.08.02). Side effects such as increased intraocular pressure and glaucoma, cataract formation, and increased susceptibility to eye infections are also not insignificant.

[0139] In a rabbit model of corneal wound healing delayed by dexamethasone, polymeric nanomicelles loaded with spironolactone significantly accelerated corneal reepithelialization, while potassium canricate solution (a soluble active metabolite of SPL) showed no effect (Dahmana, N.; Mugnier, T.; Gabriel, D.; Kaltsatos, V.; Bertaim, T.; Behar-Cohen, F.; Gurny, R.; Kalia, YN Topical Administration of Spironolactone-LoadedNanomicelles Prevents Glucocorticoid-Induced Delayed Corneal Wound Healing in Rabbits. Mol. Pharm. 2018, 15, 1192–1202, doi:10.1021 / acs.molpharmaceut.7b01028).

[0140] Since the use of spironolactone micelles counteracts delayed wound healing caused by glucocorticoid therapy, SPL eye drops are believed to be beneficial for impaired corneal wound healing, including in patients associated with glucocorticoid therapy.

[0141] Therefore, in one embodiment, the present invention relates to an aqueous ophthalmic composition according to the invention, which is administered in conjunction with glucocorticoid therapy.

[0142] "To be used with" means that SPL eye drops can be used before, after, or concurrently with glucocorticoid therapy.

[0143] application In one embodiment of the invention, a method for treating a human eye is provided, the method comprising applying the aqueous ophthalmic composition described herein to the eye. The aqueous ophthalmic composition may be instilled into the eye. It may be applied using a dropper or poured into the eye.

[0144] Method for preparing aqueous ophthalmic compositions In another embodiment, the present invention relates to a method for preparing the aqueous ophthalmic composition as described above, comprising the following steps: (i) Dissolve the cyclodextrin in water, (ii) Add spironolactone while stirring until completely dissolved. (iii) Add tromethamine and a tensioning agent, the amount of which is sufficient to adjust the tension of the composition to 280 to 310 mOsmol / kg, preferably 280 to 300 mOsmol / kg, and more preferably 280 mOsmol / kg. (iv) Add a pH adjuster in an amount sufficient to adjust the pH of the composition to 6.8 to 7.5, preferably 7 to 7.5, and more preferably 7.0 to 7.4.

[0145] The composition can be prepared under sterile and / or disinfectant conditions. Aqueous ophthalmic compositions can be autoclaved to make them sterile, or by any method that allows for sterility.

[0146] Device for dispensing composition dropwise In one aspect of the invention, an apparatus for dispensing a composition dropwise is provided, the apparatus comprising a container holding the composition as described herein.

[0147] Example Materials and methods 1. Physicochemical stability of eye drop formulations Spironolactone (SPL) and placebo (PBO) eye drops were stored in sterile eye droppers at 4°C for up to 9 months. Their appearance was observed at 1, 3, 6, and 9 months. pH and molar osmolality were measured at 1, 3, and 9 months.

[0148] 2. animal All experiments were conducted in accordance with European Community Council Directive 8 / 609 / EEC and French national regulations, and approved by the local ethics committee (#23478-2020010317557546 v4, Charles Darwin). Adult male Lewis rats (6–8 weeks old, 200–250 g, Janvier, Le Genest-Saint-Isle, France) were used to evaluate the ocular tolerability of the eye drop formulation. Adult male Sprague-Dawley rats (12–13 weeks old, 450–500 g, Janvier) were used to evaluate the therapeutic effects of SPL eye drops. Animals were kept under pathogen-free conditions, with free access to food and water, and were housed under a 12-hour light / 12-hour dark cycle. Anesthesia was induced by intraperitoneal injection of ketamine 100 mg / kg and toluidine 10 mg / kg, and rats were euthanized by Euthasol® Vet 300 mg / kg.

[0149] 3. Ocular tolerance of eye drop formulations 3.1. Processing PBO or SPL (0.1%, w / v) eye drops were instilled into both eyes of rats. Animals were divided into four treatment groups: 1) SPL, once daily for 7 days; 2) PBO, once daily for 7 days; 3) SPL, three times daily for 7 days; and 4) PBO, three times daily for 7 days. Two drops (approximately 30 μl) were administered each time, with 5-minute intervals between administrations. Untreated rats served as controls. Rats were then euthanized on day 8, and the eyes were enucleated for histological and immunofluorescence analysis.

[0150] 3.2. Corneal Histology Eyes were fixed in 4% paraformaldehyde (PFA) and 0.5% glutaraldehyde for 2 hours, dehydrated in a gradient series of alcohols, and embedded in tissue resin (Lecia, Heidelberg, Germany). 5 μm cross-sections were obtained using a Leica Jung RM2055 Microtome and stained with 1% toluidine blue. Corneal morphology was observed using a microscope (Olympus BX51, Rungis, France) equipped with a CCD camera (Olympus DP70). For quantification, continuous imaging of eye sections through the optic nerve head was performed from the limbus on one side of the cornea to the other. Fiji Image J software (ver. 1.54b; Wayne Rasband, National Institutes of Health, USA) was used. Twenty measurements were taken for each cornea, with 3–4 corneas used per group.

[0151] 3.3. Immunofluorescence Immediately after removal, the eyes were rapidly frozen in a Tissue-Tek-OCT compound (Bayer Diagnostics, Puteaux, France). Cryostat sections (10 μm) were fixed in 4% PFA, washed with PBS, and infiltrated with 0.1% Titron X-100 in PBS for 30 min. Non-specific binding sites were blocked with 5% normal goat serum for 30 min. Sections were then incubated with primary antibody at room temperature (RT) for 1 h, washed with PBS, and further incubated with secondary antibody at RT for 1 h. After washing, sections were stained with 4',6-diamidinyl-2-phenyl-indole (DAPI; 1:10000) for 2 min, washed again with PBS, and mounted with gel (Dako, Agilent, Les Ulis, France). Negative control slides were stained without primary antibody. Positive controls were corneal sections from rats with corneal inflammation. Images were taken using an Olympus fluorescence microscope. The following primary antibodies were used: rabbit anti-ZO-1 (1:200, Invitrogen ref. 40-2200, Waltham, USA), mouse anti-E-cadherin (1:400, Abcam ref. ab231303, Cambridge, UK), rabbit anti-IBA1 (1:400, Wakoref. 019-19741, Richmond, USA), mouse anti-ED1 (1:200, Bio-Rad ref. MCA341R, Colmar, France), and rabbit anti-nitrotyrosine (1:200, ThermoFisher Scientific ref. BS-8551, Saint Aubin, France). The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Thermo Fisher Scientific ref. A 11008) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:200; Thermo Fisher Scientific ref. A 21202). Three to four corneas were used in each group.

[0152] 3.4. TUNEL Measurement TUNEL assays were performed on frozen sections of the eye according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany). Cell nuclei were counterstained with DAPI. TUNEL-positive cells were counted on sections at the level of the optic nerve head across the entire cornea. Three corneas were used in each group.

[0153] 4. The therapeutic effect of SPL eye drops in a rat model of corneal wound healing 4.1. Corneal deepithelialization and treatment Following general and local anesthesia (1% tetracaine, Sigma-Aldrich, Saint-Quentin-Fallavier, France), a 4-mm central corneal trephine was marked. The corneal epithelium was then gently removed from the marked area using a scrubber without damaging the underlying corneal stroma. One of the following treatments was administered three times daily for three days to both corneas of rats: 1) eye drops containing 0.1% SPL, 2) PBO eye drops, and 3) a PBS solution containing 0.1% potassium canrenate (KCAN, Sigma-Aldrich), the water-soluble precursor of which is the active metabolite of SPL. Corneas of rats without surface wounds or treatment were used as controls.

[0154] 4.2. In vivo optical coherence tomography and slit-lamp examination Corneal morphology was assessed in vivo by optical coherence tomography (OCT, Micron III, Phoenix-Micron Inc., Bend, OR, USA) before corneal deepithelialization and at 24 and 48 hours after corneal deepithelialization. To measure central corneal thickness, rectangles were used as references on all corneal images. Figure 5 A). Results are expressed as the percentage of the area occupied by the cornea within the reference rectangle. Corneal epithelial defects were observed under a slit lamp by fluorescein staining immediately after deepithelialization (0h) and at 6, 24, and 48 hours. At each time point, the healing rate was calculated as the percentage reduction in fluorescein-stained area compared to the wound at 0h. Images of 7–9 corneas per group were analyzed at each time point. Immunofluorescence was performed on corneal sections and whole-mount corneas. Five days after corneal epithelialization, rats were sacrificed. Cryosections and corneal planarization were performed using the eyes. Immunofluorescence was performed on the corneal sections as previously described. The primary antibodies used were mouse anti-E-cadherin, rabbit anti-cytokeratin 12 (1:400, Abcamref. ab185627, Cambridge, UK), rabbit anti-IBA1, and mouse anti-ED1. The secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated donkey anti-mouse IgG, and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200; Thermo Fisher Scientific ref. A 11012). The number of IBA1- and ED1-positive inflammatory cells was counted on the entire corneal section. One section was prepared from each cornea, and 5-6 corneas were analyzed per group.

[0155] For whole-body corneal preparation, the eye was fixed in 1.3% PFA at room temperature for 45 minutes. After washing with PBS, the cornea was excised along the limbus into four to five flaps, blocked and infiltrated with PBS containing 0.5% normal goat serum and 0.5% Triton X-100 for 1 hour. The cornea was then incubated for 5 days at 4°C with mouse anti-tubulin β III (TUBB3) antibody diluted in PBS containing 0.5% normal goat serum, 0.1% Triton X-100, and 0.1% Tween 20 (Sigma Aldrich) (1:500, Biolegend ref. 801202, San Diego, USA). After washing, the cornea was incubated overnight at 4°C with Alexa 488-conjugated donkey anti-mouse IgG (1:200, Thermo Fisher Scientific ref. A 21202). Images stained with TUBB3 were observed and captured using a confocal microscope (Zeiss LSM 710 Oberkochen, Germany). For nerve quantification, 10 z-stackings of the subbasal nerve in the re-innervated corneal region were used. Corneal nerve density was measured using Fiji Image J software. The percentage of subbasal nerve density in PBO- (n=3) or SPL-treated corneas (n=2) to that in normal control corneas was calculated.

[0156] 5. statistics Literature review and estimation using the G-power algorithm were conducted to estimate the required number of animals. Data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). The Mann-Whitney test was used to compare two groups. The Kruskal-Wallis test followed by the Dunn test was used to compare more than two groups. For grouped data, two-way ANOVA was used, followed by the Tukey test. A p-value less than 0.05 was considered statistically significant.

[0157] Example 1: Spironolactone dissolution Solubility in water failed after pH and temperature adjustments: at 0.1%, spironolactone was insoluble in water at 20-25°C. After stirring for 15 minutes, the resulting solution was milky white. Undissolved particles were present at the bottom, surface, and top of the beaker when at rest. The appearance of the solution remained unchanged after gradual heating to 80°C. The appearance of the solution remained unchanged after adding 1N hydrochloric acid (until pH=1.17). The appearance of the solution remained unchanged after adding 1N sodium hydroxide (until pH=12.59).

[0158] The inventors have demonstrated (Table 1) that cyclodextrins advantageously improve the bioavailability of spironolactone, while other conventional systems known to improve the bioavailability of poorly soluble or insoluble active ingredients have failed.

[0159] Table 1. Comparison of spironolactone solubility

[0160] Spironolactone dissolution protocol in HP-γ-CD: Add HP-γ-CD to approximately 70 ml of deionized water. After dissolution, add spironolactone while stirring. After stirring for 12 minutes, dissolution is almost complete. Add 1 g of HP-γ-CD (total concentration 3%). After stirring for 22 minutes, dissolution is complete: a clear, colorless solution. Use softened water to achieve a final qs = 100 ml and homogenize for 15 minutes. The solution is clear, colorless, pH = 5.53, and molar osmolality = 20 mOsmol / kg. The appearance remains unchanged after standing for 17.5 hours. No change in appearance is observed after 6 months.

[0161] Stability study of autoclaving Two solutions capable of dissolving spironolactone were prepared: one using 2.75% HP-γ-CD and the other using 3% HP-γ-CD. Both solutions were autoclaved (15-minute cycles, >121°C) and their evolution was compared with other solutions over 3 months at room temperature.

[0162] After autoclaving, the solution showed no change in appearance (no opalescence or precipitation), indicating that spironolactone is heat-stable. After 3 months at room temperature (without special protection), only the autoclaved solution showed a slight change (a very slight pale yellow appearance).

[0163] in conclusion Spironolactone dissolves effectively only in HP-γ-CD. In fact, other systems besides cyclodextrin result in unstable dissolution, with white precipitates appearing after several hours. In contrast, spironolactone dissolves completely in HP-γ-CD: the solution is clear and colorless. No change in appearance was observed after 6 months.

[0164] Example 2: Compatibility of spironolactone with buffers The compatibility of different buffer solutions (0.50 g tromethorphan, 0.50 g sodium citrate and 1.00 g boric acid) with 0.1% spironolactone and 3% HP-γ-CD was tested and their thermal stability (1 month at 50 °C) was evaluated.

[0165] For both buffer solutions, HP-γ-CD was dissolved in approximately 70 ml of deionized water, and then spironolactone was stirred until completely dissolved. The buffer solution was then added, and the pH was measured and adjusted to 7.04–7.4 with 1N HCl (for tromethamine), citric acid and sodium citrate (for citrate buffer), or sodium borate (for borate impressions). Then, 100 ml of the final solution was obtained with softened water and homogenized for 15 minutes to obtain a clear and colorless solution. The solution was stored in a 50°C oven for one month. The results are shown in Table 2 below: Table 2. Buffer compatibility and thermal stability at 50°C

[0166] Borate impressions and citrate buffers will develop a yellow tint over time, which may be a sign of degradation. Therefore, they cannot be stored. In contrast, solutions containing tromethamine / HCl buffer remain clear. Therefore, tromethamine / HCl buffer is a favorable candidate, but it needs to be adjusted with sodium chloride and its isotonicity checked.

[0167] Therefore, the inventors have identified suitable and compatible compositions that are thermally stable, colorless and isotonic, and are described in Table 3 below.

[0168] Table 3. Compositions according to the present invention

[0169] Example 3: Preparation of eye drop formulation Pharmaceutical-grade SPL was purchased from Azelis Pharma (Courbevoie, France), and HP-γ-CD was purchased from Wacker Chemie (Lyon, France). SPL and PBO eye drops were prepared according to their compositions shown in Table 4. After dissolving HP-γ-CD in distilled water, SPL was added at 20–25°C with stirring for at least 1 hour until completely dissolved. Then, tromethamine and NaCl were added, the pH was measured, and adjusted to 7.0–7.4 with 1N HCl. The molar osmolality of the eye drop formulation was approximately 280 mOsmol / kg. The PBO formulation was obtained using the same protocol, without the addition of SPL.

[0170] Table 4. Composition of SPL and PBO eye drops.

[0171] Example 4: The eye drop formulation exhibited good physicochemical stability at 4°C for 9 months. Eye drops containing 0.1% SPL and placebo (PBO) eye drops were prepared according to the formulations in Table 4. Both eye drop formulations remained clear and colorless after 9 months of storage in sterile eye droppers at 4°C. No particulate precipitation was observed. The molar osmolality of the SPL eye drops did not change significantly over time when measured at 1, 2, and 9 months and remained close to 280 mOsmol / kg. The pH of the SPL eye drops was 7.189 on day 0 and varied only slightly between 7.0 and 7.4 over the extended 9-month period.

[0172] Example 5: SPL eye drops did not show signs of corneal integrity disruption. To evaluate the tolerability of the eye drop formulation, Lewis rats were treated with eye drops containing 0.1% SPL or placebo (PBO) once or three times daily for 7 days. Corneal morphology and barrier integrity were evaluated by histology and immunofluorescence on day 8. Untreated rat eyes served as controls.

[0173] On histological sections, corneas treated with PBO or SPL formulations once or three times daily for 7 days exhibited a normal structure with layered epithelium, regular stromal tissue, and a single layer of endothelial cells, similar to untreated corneas. Figure 1 A). Total corneal thickness ( Figure 1 B) and corneal epithelial thickness ( Figure 1 The quantification of C) showed no significant difference among all groups (p = 0.3762 and p = 0.2527, respectively). No corneal edema or inflammatory cell infiltration was observed 7 days after treatment.

[0174] On day 8, the corneal epithelial barrier was evaluated by immunostaining of tight junction molecule ZO-1 and adhesion protein E-cadherin. ZO-1 staining was predominantly located in the superficial layer of the corneal epithelium. We did not detect any disruption of staining in any treatment group, and no difference in localization was observed between the PBO and SPL groups. Figure 1 D). E-cadherin is expressed in the cell membrane of all epithelial cells and forms bridges between adjacent epithelial cells. There was no disruption or difference in its localization between the PBO and SPL groups. Figure 1 E). The inner boundary of the epithelium was regular, and the epithelial cell layers were well defined by E-cadherin staining in all groups. Figure 1 E).

[0175] in conclusion After multiple local instillations over a week, SPL eye drops showed no signs of incompatibility. Therefore, spironolactone eye drops are well tolerated.

[0176] Example 6: SPL eye drops do not induce corneal inflammation, oxidative stress, or apoptotic cell death. IBA1 and ED1 immunostaining was performed on frozen corneal sections to evaluate potential inflammatory cell infiltration. No IBA1- or ED1-positive inflammatory infiltration was observed in the cornea on day 8 following PBO or SPL treatment. Figure 2 Nitrotyrosine (NT) is a marker of cellular damage and nitrosogenic stress. The absence of NT-positive immunostaining indicates the absence of nitrosogenic stress in either the PBO or SPL groups. Figure 3 A). TUNEL assay to detect DNA fragmentation in apoptotic cells. Corneas were treated with eye drops three times daily for 7 days. TUNEL-positive apoptotic cells were found only in untreated normal corneas and in the superficial layer of the corneal epithelium in corneas treated with PBO or SPL. Figure 3 B). The number of apoptotic cells spanning the entire epithelium was similar across groups (control: 25.33±3.06, PBO: 22.67±4.62, SPL: 22.33±5.86, p = 0.7214). Figure 3 C).

[0177] In summary, these results demonstrate the excellent tolerability of SPL eye drops on the ocular surface of rats.

[0178] Example 7: SPL eye drops improve corneal reepithelialization and reduce corneal keratitis in a rat model of corneal wound healing. edema To investigate the therapeutic effects of SPL eye drops, we used a rat model of corneal wound healing via mechanical injury, a model for studying delayed corneal wound healing. After central corneal deepithelialization, both eyes were treated with 0.1% SPL eye drops or PBO three times daily for three days. A group of rats were treated with 0.1% topical potassium canelate (KCAN) in PBS to evaluate the effect of the soluble active metabolites of SPL alone without the eye drop formulation.

[0179] like Figure 4As shown in Figure A, the healing process was monitored immediately after corneal epithelial removal (0 h) and at 6, 24, and 48 hours to highlight the detached areas. The corneal healing rate after SPL treatment was 16.91% ± 10.18 at 6 h, 73.41% ± 11.69 at 24 h, and 98.37% ± 2.25 at 48 h. In contrast, the healing rate after PBO eye drops was 2.70% ± 4.89 at 6 h, 54.75% ± 21.49 at 24 h, and 91.56% ± 12.38 at 48 h. Furthermore, the healing rate after KCAN solution was 3.89% ± 3.99 at 6 h, 57.33% ± 13.23 at 24 h, and 87.78% ± 12.38 at 48 h. Compared with PBO (p = 0.021) and KCAN (p = 0.045), a significant improvement in corneal epithelial wound healing was observed in the SPL treatment group at 6 hours, and a significant improvement in corneal epithelial wound healing was observed in the SPL treatment group at 24 hours compared with the PBO- (p = 0.002) and KCAN- (p = 0.009) treatment groups. Figure 4 B), indicating that the SPL eye drop formulation was superior to PBO and unprepared KCAN solution. No difference was observed between PBO and topical KCAN at 6 hours (p = 0.970) and 24 hours (p = 0.865). Figure 4 B). The corneal epithelial wounds in all groups closed on day 3.

[0180] Corneal edema was assessed in vivo by OCT before corneal deepensis and at 24 and 48 hours after corneal deepensis, and denoted as [missing information]. Figure 5 In Figure A, the percentage of the area occupied by the central cornea within the reference rectangle. Prior to epithelial debridement, corneal thickness was comparable across all groups ( Figure 5 A and B). Corneal deepithelialization resulted in corneal edema. In the SPL eye drop treatment group, the corneal surface occupied 48.68% ± 3.86 of the reference rectangle at 24 hours and 44.85% ± 8.52 at 48 hours. In the PBO treatment group, the relative percentages were 45.73% ± 4.13 at 24 hours and 57.47% ± 7.57 at 48 hours, and in the KCAN treatment group, they were 52.07% ± 6.23 at 24 hours and 58.26% ± 8.67 at 48 hours. Compared with PBO (p = 0.0234) and topical KCAN (p = 0.254) treatments, SPL eye drops significantly reduced corneal thickening at 48 hours. Figure 5 There were no differences between the KCAN and PBO groups at 24 hours (p = 0.074) and 48 hours (p = 0.979). Figure 5 B).

[0181] Example 8: SPL eye drops restore epithelial integrity in a rat model of corneal wound healing. To evaluate the quality of epithelial healing, we performed immunostaining on corneal sections on day 5 post-wound induction of E-cadherin and cytokeratin K12, markers of differentiated corneal epithelium. Although wound healing occurred in PBO-treated corneas, tissue disruption and localized destruction of E-cadherin were observed in the basal layer of the corneal epithelium. Figure 6 A). The structure of the epithelium is not well defined, and the inner boundary of the corneal epithelium is irregular. Figure 6 A). SPL eye drops restored the continuity of E-cadherin and the multilayered structure of the corneal epithelium, comparable to the undamaged control cornea. Figure 6 A). The intraepithelial border is more regular than that of corneas treated with PBO.

[0182] In the cornea of ​​normal rats, K12 is expressed in the corneal epithelium, with more pronounced expression in the superficial layer. Figure 6 B). In PBO-treated corneas, we observed a general decrease in K12 expression and focal disruption in the basal epithelium. The inner boundary of the epithelium was irregular ( Figure 6 B). Topical SPL eye drops enhance K12 expression and restore corneal epithelial structure ( Figure 6 B).

[0183] Example 9: SPL eye drops reduce corneal inflammatory cell infiltration in a rat model of corneal wound healing. IBA1 and ED1 immunostaining was performed to evaluate the anti-inflammatory effect of SPL eye drops. In the PBO-treated group, numerous IBA1- and ED1-positive cells were detected throughout the corneal stroma, while SPL eye drops limited infiltration in the anterior stroma. Figure 7 (A and B). The number of IBA1-positive cells in the PBO-treated group was 327±91, while that in the SPL-treated group was 179±34. The number of ED1-positive cells in the PBO-treated group was 327±107, while that in the SPL-treated group was 164±42. SPL eye drops significantly reduced the number of inflammatory cells in the rat cornea (p = 0.0195 for IBA1 and p = 0.0303 for ED1). Figure 7 (C and D).

[0184] Example 10: SPL eye drops improve corneal nerve reinnervation in a rat model of corneal wound healing. We evaluated corneal nerve reinnervation 5 days after topical PBO and SPL eye drops. In undamaged control corneas, TUBB3 immunostaining revealed dense linear structures of the subbasal nerve on corneal planar slides. Corneal deepithelialization mechanically removed the subbasal neural network (…). Figure 8 A). Until day 5, slow subbasal neurogenesis was observed from the peripheral cornea to the deepithelialized central cornea in both the PBO and SPL treatment groups. Figure 8A). In areas of nerve regeneration, SPL eye drops appeared to improve subbasal nerve density in the re-innervated corneal regions compared to PBO-treated corneas. Furthermore, regenerated nerves appeared longer and more parallel-oriented in SPL-treated corneas than in PBO-treated corneas. Figure 8 A). The density of the subbasal plexus of the cornea in the PBO-treated group was 54.95% ± 2.51 compared to the undamaged control cornea, and 70.32% ± 5.75 in the SPL-treated group, but the difference was not significant due to the small sample size.

Claims

1. An aqueous ophthalmic composition comprising: - Spironolactone, - Cyclodextrin, - Tromethamine.

2. The aqueous ophthalmic composition according to claim 1, wherein the cyclodextrin is selected from 2-hydroxypropyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, hydroxyethyl-γ-cyclodextrin, dihydroxypropyl-β-cyclodextrin, glucosyl-α-cyclodextrin, glucosyl-β-cyclodextrin, disaccharosyl-β-cyclodextrin, maltosyl-α-cyclodextrin, maltosyl-β-cyclodextrin, maltosyl-γ-cyclodextrin, maltotrisyl-β-cyclodextrin, maltotrisyl-γ-cyclodextrin, disaccharosyl-β-cyclodextrin, maltosyl-β-cyclodextrin / disaccharosyl-β-cyclodextrin, methyl-β-cyclodextrin, and mixtures thereof, preferably selected from 2-hydroxypropyl-β-cyclodextrin and 2-hydroxypropyl-γ-cyclodextrin.

3. The aqueous ophthalmic composition according to claim 1 or 2, wherein, based on the total weight of the composition, the composition comprises 1% to 5% by weight, preferably 1.5% to 3% by weight, and preferably 2% to 3% by weight of cyclodextrin.

4. The aqueous ophthalmic composition according to any one of claims 1 to 3, wherein the composition comprises 2.75% by weight or 3% by weight of cyclodextrin based on the total weight of the composition.

5. The aqueous ophthalmic composition according to any one of claims 1 to 4, wherein, based on the total weight of the composition, the composition comprises 0.01% to 10% by weight, preferably 0.05% to 5% by weight, and preferably 0.1% to 1% by weight of spironolactone.

6. The aqueous ophthalmic composition according to any one of claims 1 to 5, wherein the composition comprises 0.1% by weight of spironolactone based on the total weight of the composition.

7. The aqueous ophthalmic composition according to any one of claims 1 to 6, wherein, based on the total weight of the composition, the composition comprises 0.1% to 5% by weight, preferably 0.1% to 2% by weight, and preferably 0.4% to 0.8% by weight of tromethamine.

8. The aqueous ophthalmic composition according to any one of claims 1 to 7, wherein the composition comprises 0.6% by weight of tromethamine based on the total weight of the composition.

9. The aqueous composition according to any one of claims 1 to 8, wherein the composition comprises: - Based on the total weight of the composition, 0.1% by weight of spironolactone, - Based on the total weight of the composition, 3% by weight of cyclodextrin, - 0.6% by weight of tromethamine based on the total weight of the composition.

10. The aqueous composition according to claim 9, wherein the composition further comprises: - HCl, in an amount sufficient to adjust the pH of the ophthalmic composition to 7.0-7.

4. - Sodium chloride, in an amount sufficient to adjust the tension of the ophthalmic composition to 280 mOsmol / kg.

11. The aqueous ophthalmic composition according to any one of claims 1 to 10, for restoring epithelial integrity, for improving corneal re-epithelialization, for improving epithelial cell adhesion and cell layer differentiation, for restoring the optical function of the anterior cornea, for reducing inflammation, for reducing stromal edema, for restoring corneal transparency, or for improving corneal renervation.

12. The aqueous ophthalmic composition according to any one of claims 1 to 10, for treating diseases or conditions selected from: corneal ulcers with or without inflammation, postoperative corneal wounds, delayed corneal re-epithelialization after corneal transplantation, corneal transplant rejection, vernal keratitis, ocular erythema, epithelial keratitis secondary to dry eye syndrome or meibomian gland dysfunction, corneal ulcers following corneal cross-linking, delayed corneal wound healing due to refractive surgery, corneal surgery, or anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungal, parasitic, or viral infection or infection by unconventional agents, corneal opacity or corneal epithelial defects due to neurotrophic keratopathy, corneal opacity due to corneal scarring, and corneal edema. Corneal inflammation or neovascularization due to vernal keratokeratitis, corneal inflammation due to ocular erythema, corneal neovascularization, corneal neovascularization secondary to inflammation or meibomian gland dysfunction or infection or limbal defects or corneal transplant rejection, recurrent corneal erosion, delayed wound healing, postoperative treatment after corneal transplantation or refractive surgery or any other corneal surgery, glucocorticoid-induced delayed epithelial wound healing after corneal physical trauma, hereditary and genetic corneal diseases, aniridia-induced corneal diseases, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and related diseases such as dry eye syndrome and blepharitis, and corneal diseases selected from diabetic, neurotrophic, toxic, iatrogenic, and preservative-induced corneal diseases.

13. The aqueous ophthalmic composition according to any one of claims 1 to 10, which is administered in combination with glucocorticoid therapy.

14. A method for preparing an aqueous ophthalmic composition according to any one of claims 1 to 10, comprising the following steps: (i) Cyclodextrin is soluble in water. (ii) Add spironolactone while stirring until completely dissolved. (iii) Add tromethamine and a tensioning agent, the amount of which is sufficient to adjust the tension of the composition to 280 to 310 mOsmol / kg, preferably 280 to 300 mOsmol / kg, and more preferably 280 mOsmol / kg. (iv) Add a pH adjuster in an amount sufficient to adjust the pH of the composition to 6.8 to 7.5, preferably 7 to 7.5, and more preferably 7.0 to 7.

4.

15. An apparatus for dispensing a composition dropwise, the apparatus comprising a container holding the composition according to any one of claims 1 to 10.

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