Spironolactone eye drop formulations

Abstract

The present invention relates to an aqueous ophthalmic composition comprising spironolactone, cyclodextrin and trometamol, preferably for topical application to the human eye. The inventors advantageously dissolve spironolactone in HP-γ-cyclodextrin excipient at a concentration of 0.1%, a dose that proved effective in treating ocular surface defects. They also successfully demonstrate that spironolactone eyedrops were stable for up to 9 months at 4°C and dept its efficacy for at least 6 months and that the formulation was well-tolerated after multiple daily instillations over 7 days. Moreover, the inventors show that spironolactone eyedrops according to the invention accelerated rat corneal wound healing, reduced corneal edema and inflammation, enhanced epithelial integrity and improved nerve regeneration, suggesting restoration of corneal homeostasis. Hence, the present invention also relates to the use of aqueous ophthalmic composition for the treatment of eye diseases and conditions.

Description

[0001] SPIRONOLACTONE EYE DROP FORMULATIONS

[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 (MR) activation is pathogenic for the retina as it favours 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. https: / / doi.org / 10.1111 / bph.1577). Mineralocorticoid activation is also pathogenic for the cornea as 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(l):369. doi: 10.1038 / s41467-018-08125-6.]

[0006] Mineralocorticoid receptor antagonists (MRA) have shown potential to alleviate pathogenic mechanisms involved in major ocular diseases, including retinal oedema, pathological choroidal, retinal angiogenesis, retinal cell death as well as retinal and choroidal inflammation, corneal neovascularization and inflammation, ocular surface inflammation and fibrosis. In transgenic mice invalidated specifically for MR in endothelial cells, there was a reduction in corneal neovascularization due to limbal deficiency, showing that MR could also contribute to the maintenance of corneal avascularity [Nat Commun. 2019 Jan 21 ; 10( 1) :369. doi: 10.1038 / s41467-018-08125-6.] Glucocorticoids (GCs) act through binding to the glucocorticoid receptor (GR) and to the mineralocorticoid receptor, both expressed in the cornea. In the cornea, GCs-induced delay in wound healing is mediated by the activation of MR [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 CGs-induced corneal wound healing defect, an important side effect of GCs.

[0007] Number of retinal, choroidal and ocular surface diseases can benefit from MR antagonists.

[0008] Spironolactone (SPL) is the more potent mineralocorticoid receptor antagonist. However, spironolactone is very low water soluble (0.02 mg / mL) with a short half-life in ocular media (less than Ih) and, even at high intravenous dose, it does not cross the intact ocular barriers [ Zhao M, Rodriguez- Villagra E, Kowalczuk L, Le Normand M, Berdugo M, Levy-Boukris R, El Zaoui I, Kaufmann B, Gurny R, Bravo-Osuna I, Molina-Martinez IT, Herrero- Vanrell R, Behar-Cohen F. J Control Release. 2017 Nov 28;266:187-197. doi: 10.1016 / j.jconrel.2017.09.029.] due to its binding to efflux proteins, expressed at the inner and outer retinal barriers.

[0009] 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 eye, like in diabetic retinopathy in rats, in which the beneficial effects of MR antagonists were only observed if the drug was administered inside the eye and not when the drug was given orally or by systemic injections [Mineralocorticoid Receptor Pathway and Its Antagonism in a Model of Diabetic Retinopathy. Zhao M, et al. Diabetes. 2021. PMID: 34426510] [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. https: / / doi.Org / 10.1016 / j.jconrel.2017.09.029],

[0010] For the treatment of ocular surface diseases and to reach the back of the eye, particularly the choroid, after topical instillation, spironolactone requires proper formulation to increase its aqueous solubility and to extent its contact time at the ocular surface to achieve therapeutic and stable concentrations of spironolactone inside the eye.

[0011] It was demonstrated that spironolactone micelles (0.1%, w / v) with a mean number weighted diameter of 20 nm, prepared using (mPEG-poly(caprylic acid) polymer (mPEG-hexPLA) prevented glucocorticoid-induced delayed corneal wound healing in New Zealand white rabbits. These 0.1% spironolactone micellar formulations were administered by multiple daily instillations over 5 days. [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.].

[0012] Although the micelles showed good safety profile, the preparation of polymer-based formulations involves the use of organic solvents (ethanol, acetone), which can be harmful and might expose to manufacturing limitations. The novel polymers used imposes extensive safety studies, at very high cost. Finally, empty polymer, whose degradation products can lower the pH locally, might cause irritation and inflammation.

[0013] For all these reasons, a polymer-free eyedrop formulation is needed.

[0014] SUMMARY OF THE INVENTION

[0015] In a preclinical study, a new polymer-free hydroxypropyl-gamma-cyclodextrin-based eyedrop containing 0.1% spironolactone, was investigated for its ocular surface tolerance and efficacy in a rat model of corneal wound healing. First, the inventors advantageously dissolve spironolactone in HP-y- cyclodextrin excipient at a concentration of 0.1%, a dose that proved effective in treating ocular surface defects. Then, they successfully demonstrate that spironolactone eyedrops were stable for up to 9 months at 4°C and dept its efficacy for at least 6 months and, that the formulation was well-tolerated after multiple daily instillations over 7 days. Indeed, after multiple topical instillations for a week, spironolactone eyedrops did not show any signs of incompatibility. No morphological changes, inflammation, oxidative stress, or increased cell death were observed in rat corneas, suggesting their good tolerability.

[0016] Hence, the present invention relates to an aqueous ophthalmic composition comprising spironolactone, cyclodextrin and trometamol. Preferably the aqueous ophthalmic composition is for topical application to the human eye.

[0017] Moreover, the inventors show that spironolactone eyedrops according to the invention accelerated rat corneal wound healing, reduced corneal edema and inflammation, enhanced epithelial integrity and improved nerve regeneration, suggesting restoration of corneal homeostasis, while potassium canrenoate, an active and soluble metabolite of SPL, had no effect.

[0018] Thus, the present invention relates to the use of an aqueous ophthalmic composition for the treatment of eye diseases and conditions.

[0019] SPL eyedrops could benefit to patients with impaired corneal wound healing, including that associated with glucocorticoid therapy. Hence, the present invention also relates to an aqueous ophthalmic composition to be administered with glucocorticoid therapy.

[0020] In a further aspect of the present invention, there is provided a method of making the aqueous ophthalmic composition.

[0021] In a further aspect of the present invention there is also provided a device for dropwise dispensing of the aqueous ophthalmic composition.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGURE 1: Rat corneal and corneal epithelial morphology after topical instillation of eyedrops containing spironolactone for 7 days.

[0024] 1A. Histological sections show normal corneal structure and stratified corneal epithelium (Ep), well- organized stroma (St) and intact corneal endothelial (En) monolayer after instillation of eyedrops containing 0.1% spironolactone (SPL) or placebo (PBO) once of three times a day for 7 days, similar to that of the untreated control cornea (Ctrl). Double-headed arrows indicate the thickness of the cornea and corneal epithelium. St, stroma; En, endothelium. Scale bars: 20 pm.

[0025] IB. Quantification of the corneal thickness on histological sections shows no significant difference among all groups. Data are expressed as mean + SD, n=3-4 rat corneas.

[0026] IC. Quantification of the corneal epithelial thickness shows no significant difference among all groups. Data are expressed as mean + SD, n=3-4 rat corneas.

[0027] ID. ZO-1 immunofluorescence shows a cell membrane localization mainly in the superficial layers of the corneal epithelium. No disruption of the immunostaining was observed after topical treatment of SPL and PBO eyedrops. Dash lines indicate the inner limit of the corneal epithelium. Scale bars: 20 pm. n=3-4 rat corneas

[0028] IE. E-cadherin immunofluorescence shows a cell membrane localization in all epithelial cells. No disruption of the immunostaining was observed after topical treatment of SPL or PBO eyedrops. Dash lines indicate the inner limit of the corneal epithelium. Scale bars: 20 pm. n=3-4 rat corneas

[0029] FIGURE 2: Immunostaining of inflammatory cells in rat corneas after topical instillation of eyedrops containing spironolactone for 7 days.

[0030] 2A and B. No IB Al- or EDI -positive inflammatory cells were observed in corneas treated with eyedrops containing spironolactone (SPL) or placebo (PBO) once or 3 times a day for 7 days. Positive control corneas (Ctrl+) from an inflammation model show IB A and EDI staining in inflamed corneas. Ep, epithelium; St, stroma; En, endothelium. Scale bars: 20 pm. n=3-4 rat corneas.

[0031] FIGURE 3: Nitrosative stress and cell death markers in rat corneas after topical instillation of eyedrops containing spironolactone for 7 days.

[0032] 3A. No nitrotyrosine (NT) positive cells were observed in in corneas treated with eyedrops containing 0.1% spironolactone (SPL) or placebo (PBO) once or 3 times a day for 7 days. Positive control corneas (Ctrl+) from an inflammation model show NT staining. Ep, epithelium; St, stroma; En, endothelium. Scale bars: 20 pm. n=3-4 rat corneas.

[0033] 3B. TUNEL positive apoptotic cells only present in the superficial layers of the corneal epithelium in untreated control eyes (Ctrl) as well as in eyes treated with SPL or PBO eyedrops 3 times a day for 7 days. Scale bars: 20 pm.

[0034] 3C. Quantification of TUNEL positive cells across the whole corneal epithelium shows no significant difference among Ctrl, PBO and SPL groups. Data are expressed as mean + SD, n=3 rat corneas.

[0035] FIGURE 4: SPL eyedrops containing spironolactone improve rat corneal re-epithelialization.

[0036] 4A.. Fluorescein staining shows the 4-mm central corneal epithelial wound created at 0 h, and healing progress at 6, 24 and 48 hrs. Spironolactone (SPL) eyedrops improve the corneal re-epithelialization compared to the placebo (PBO) and potassium canrenoate solution (KCAN). 4B. Quantitative analysis shows that SPL eyedrops significantly increase the epithelial healing rate at 6 and 24 hrs compared with the PBO eyedrops and KCAN solution while there is no difference between the KCAN and PBO groups. Data are expressed as mean + SD, n=7-9 rat corneas. *, p<0.05; **, p<0.01..

[0037] FIGURE 5: SPL eyedrops reduce rat corneal edema after corneal de-epithelialization.

[0038] 5A. In vivo B-scan optical coherence tomography shows the cross-sectional images (left) of the central cornea through the green line (right) before and 24 and 48 hrs after corneal de-epithelialization. Spironolactone (SPL) eyedrops reduce the corneal edema at 48 hrs as compared to placebo eyedrops (PBO) and potassium canrenoate solution (KCAN). The rectangles are of the same size and were used as a reference for quantitative analysis of corneal edema.

[0039] 5B. Corneal edema is presented as a percentage of the area of the reference rectangle occupied by the cornea. SPL eyedrops decrease significantly corneal edema compared with the PBO and KCAN groups at 48 hrs. Data are expressed as mean + SD, n=7-9 rat corneas. *, p<0.05.

[0040] FIGURE 6: SPL eyedrops containing spironolactone restore corneal epithelial integrity in rats after corneal wound healing.

[0041] 6A. Immunostaining of E-cadherin shows discontinuity (arrows) and disorganization of E-cadherin in the epithelium of placebo-treated (PBO) corneas. Spironolactone eyedrops (SPL) restore E-cadherin continuity and the stratified structure of the corneal epithelium comparable to the control uninjured cornea (Ctrl). Dash lines indicate the inner border of the corneal epithelium. Scale bars: 20 pm. n=4-5 rat corneas.

[0042] 6B. Immunostaining of cytokeratin 12 (K12) shows a decrease in K12 fluorescence and disruption (arrows) in the basal layer of the epithelium of PBO-treated corneas. The inner edge of the epithelium (dash line) is irregular. SPL eyedrops enhance the K12 expression in the corneal epithelium and improve the regularity of the epithelial inner border, comparable to the control cornea. Scale bars: 20 pm. n=4-5 rat corneas.

[0043] FIGURE 7: SPL eyedrop reduces inflammatory cell infiltration in rat corneas after corneal de- epithelialization.

[0044] 7A and B. IB Al and EDI immunostaining shows infiltration of inflammatory cells throughout the stroma of placebo-treated (PBO) corneas, whereas spironolactone eyedrops (SPL) limit the cell infiltration in the anterior stroma. Ep, epithelium; St, stroma; En, endothelium. Scale bars: 20 pm.

[0045] 7C and D. SPL eyedrops significantly decrease the number of IBA1- and EDl-positive cells in the cornea compared with PBO-treated corneas. Data are expressed as mean + SD, n=5-6 rat corneas. *, p<0.05.

[0046] FIGURE 8: SPL eyedrop favors corneal re-innervation in the de-epithelialized central cornea in rat.

[0047] 8A. TUBB3 immunostaining on corneal whole mounts shows dense and linear corneal sub-basal nerves in control uninjured corneas (Ctrl). Corneal de-epithelialization me-chanically removed the sub-basal nerve plexus. Spironolactone (SPL) eyedrops improve the morphology of regenerated sub-basal nerves compared with placebo-treated (PBO) corneas.

[0048] 8B. The density of regenerated sub-basal nerves is 54.95 % relative to control cornea in PBO-treated group (n=3) and increased to 70.32 % in SPL-treated group (n=2). Data are expressed as mean + SD; ns, not significant.

[0049] DETAILLED DESCRIPTION

[0050] The present invention relates to an aqueous ophthalmic composition comprising:

[0051] (a) Spironolactone,

[0052] (b) Cyclodextrin,

[0053] (c) Trometamol.

[0054] Spironolactone (SPL)

[0055] 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.

[0056] In an embodiment, the aqueous ophthalmic composition according comprises from 0.01 to 10 % by weight, preferably from 0.05 to 5 % by weight, preferably from 0.1 to 1% by weight, based on the total weight of the composition, of spironolactone.

[0057] As conventionally used in the pharmaceutical field, 1% corresponds to 1 g / lOOmL (or lOmg / mL).

[0058] In a preferred embodiment, the composition comprises 0.1 % by weight, based on the total weight of the composition, of spironolactone.

[0059] Cyclodextrins (CDs)

[0060] Cyclodextrins are natural cyclic oligosaccharides. Their molecules are donut-shaped with a hydrophilic outer surface and a hydrophobic inner cavity. The most common cyclodextrins are a-cyclodextrin, - cyclodextrin and y-cyclodextrin, consisting of 6, 7 and 8 a-l,4-linked glucose units, respectively. The number of these units determines the size of the cavity. Cyclodextrins are capable of forming inclusion complexes with a wide variety of hydrophobic molecules by taking up a whole molecule, or some part of it, into the cavity. The stability of the complex formed depends on how well the guest molecule fits into the cyclodextrin cavity. Cyclodextrins hence increase drug solubility and stability, enhance drug bioavailability, and reduce drug toxicity. Cyclodextrins for use in the present invention include the natural cyclodextrins and their derivatives, including the alkylated and hydroxyalkylated derivatives and the branched cyclodextrins. In addition to a-, P- and y-cyclodextrins, the ether and mixed ether derivatives and those derivatives bearing sugar residues are of special interest. Especially useful herein are the hydroxyethyl, hydroxypropyl (including 2- and 3-hydroxypropyl) and dihydroxypropyl ethers, their corresponding mixed ethers and further mixed ethers with methyl or ethyl groups, such as methylhydroxyethyl, ethyl-hydroxyethyl and ethylhydroxypropyl ethers of a-, - and y-cyclodextrin; and the maltosyl, glucosyl and maltotriosyl derivatives of a, - and y-cyclodextrin, which may contain one or more sugar residues, e.g. glucosyl or diglucosyl, maltosyl or dimaltosyl, as well as various mixtures thereof, e.g. a mixture of maltosyl and dimaltosyl derivatives. Specific cyclodextrin derivatives for use herein include hydroxypropyl-P- cyclodextrin (or 2-hydroxypropyl-P-cyclodextrin), hydroxyethyl-P-cyclodextrin, hydroxypropyl-y- cyclodextrin ( or 2-hydroxypropyl-y- cyclodextrin), hydroxyethyl-y-cyclodextrin, dihydroxypropyl-P- cyclodextrin , glucosyl-a-cyclodextrin, glucosyl-P-cyclodextrin, diglucosyl-P-cyclodextrin, maltosyl-a- cyclodextrin, maltosyl-P-cyclodextrin, maltosyl-y-cyclodextrin, maltotriosyl-P-cyclodextrin, maltotriosyl-y-cyclodextrin and dimaltosyl-P-cyclodextrin , maltosyl-P-cyclodextrin / dimaltosyl-P- cyclodextrin, methyl-P-cyclodextrin and mixtures thereof. Procedures for preparing such cyclodextrin derivatives are well-known, for example, from Bodor United States Patent No. 5,024,998 dated June 18, 1991, and references cited therein.

[0061] In an embodiment, the cyclodextrin is chosen among 2-hydroxypropyl-P-cyclodextrin (HP-P-CD) and 2-hydroxypropyl-y- cyclodextrin (HP-y-CD).

[0062] In a preferred embodiment, the cyclodextrin is 2-hydroxypropyl-y- cyclodextrin (HP-y-CD).

[0063] In an embodiment, the aqueous ophthalmic composition comprises from 1% to 5 % by weight, preferably from 1.5 to 3 % by weight, preferably from 2 to 3 % by weight, based on the total weight of the composition, of cyclodextrin.

[0064] In a preferred embodiment, the aqueous ophthalmic composition comprises 2.75 or 3 % by weight, based on the total weight of the composition of cyclodextrin.

[0065] Trometamol

[0066] Trometamol is also known as also known as tromethane or tris(hydroxymethyl)aminomethane buffer. It is a tertiary amine with a pKa of 8.1 at 25 °C.

[0067] In an embodiment, the aqueous ophthalmic composition comprises from 0.1 to 5 % by weight, preferably from 0.1 to 2 % by weight, preferably from 0.4 to 0.8 % by weight, based on the total weight of the composition, of trometamol. In a preferred embodiment, the aqueous ophthalmic composition comprises 0.6% by weight, based on the total weight of the composition of trometamol. pH adjusting agent

[0068] The pH of the composition is preferably from 6.8 to 7.5, or from 7.0 to 7.5, more preferably still to a pH of from 7.0 to 7.4.

[0069] It will be understood that any suitable acid or base may be used to adjust the pH to the appropriate value or pH range.

[0070] Hence, in an embodiment, the composition has a pH adjusting agent in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 6.8 to 7.5, preferably to 7.0 to 7.5, and more preferably to 7.0 to 7.4.

[0071] The pH of the composition may be adjusted after addition of the trometamol buffer.

[0072] Typically, the pH of the composition will need to be raised by the addition of an acid, which suitably may be hydrogen chloride (HC1).

[0073] Hence, in an embodiment, the pH adjusting agent is HC1 IN.

[0074] In an embodiment, the aqueous ophthalmic composition HCL IN added as pH adjusting agent until the optimal pH is reached.

[0075] The one skilled in the art knows how to measure pH of a solution. Typically, a pH-meter can be used.

[0076] Tonicity agent

[0077] The osmolarity of the aqueous ophthalmic composition is preferably from 260 to 310 mOsmol / kg, preferably from 280 to 300 mOsmol / kg, more preferably still to an osmolarity of about 280 mOsmol / kg.

[0078] Advantageously, the composition is chosen to be substantially isotonic.

[0079] Hence, in an embodiment, the aqueous ophthalmic composition has a tonicity agent in an amount sufficient to adjust the tonicity of the ophthalmic composition from 280 to 310 mOsmol / kg, preferably from 280 to 300 mOsmol / kg and more preferably of about 280 mOsmol / kg.

[0080] It will be understood that any suitable tonicity agent may be used to adjust the osmolarity to the appropriate value or osmolarity range.

[0081] 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. 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.

[0082] 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®).

[0083] In a preferred embodiment, the tonicity agent is sodium chloride.

[0084] In an embodiment, the aqueous ophthalmic composition comprises from 0.25 to 0.75 % by weight, preferably from 0.4 to 0.6 % by weight, preferably from 0.4 to 0.5 % by weight, based on the total weight of the composition, of a tonicity agent.

[0085] In an embodiment, the aqueous composition according to the invention comprises:

[0086] From 0.01% to 10% by weight, preferably from 0.05 to 5 % by weight, preferably from 0.1 to 1% by weight, based on the total weight of the composition, of spironolactone,

[0087] From 1% to 5% by weight, preferably from 1.5 to 3% by weight, preferably from 2 to 3% by weight, based on the total weight of the composition, of cyclodextrin,

[0088] From 0.1 to 5 % by weight by weight, preferably from 0.1 to 2 % by weight, preferably from 0.4 to 0.8 % by weight, based on the total weight of the composition, of trometamol.

[0089] In an embodiment, the aqueous composition according to the invention comprises:

[0090] From 0.01% to 10% by weight, preferably from 0.05 to 5 % by weight, preferably from 0.1 to 1% by weight, based on the total weight of the composition, of spironolactone,

[0091] From 1% to 5% by weight, preferably from 1.5 to 3% by weight, preferably from 2 to 3% by weight, based on the total weight of the composition, of cyclodextrin,

[0092] From 0.1 to 5 % by weight by weight, preferably from 0.1 to 2 % by weight, preferably from 0.4 to 0.8 % by weight, based on the total weight of the composition, of trometamol.

[0093] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 6.8 to 7.5, preferably to 7.0 to 7.5, and more preferably to 7.0 to 7.4.

[0094] Sodium chloride in an amount sufficient to adjust the tonicity of the aqueous ophthalmic composition from 280 to 310 mOsmol / kg, preferably from 280 to 300 mOsmol / kg and more preferably of 280 mOsmol / kg.

[0095] In an embodiment, the aqueous composition according to the invention comprises: From 0.1 to 1% by weight, based on the total weight of the composition, of spironolactone,

[0096] From 2 to 3% by weight, based on the total weight of the composition, of cyclodextrin,

[0097] From 0.4 to 0.8 % by weight, based on the total weight of the composition, of trometamol.

[0098] In an embodiment, the aqueous composition according to the invention comprises:

[0099] From 0.1 to 1% by weight, based on the total weight of the composition, of spironolactone,

[0100] From 2 to 3% by weight, based on the total weight of the composition, of cyclodextrin,

[0101] From 0.4 to 0.8 % by weight, based on the total weight of the composition, of trometamol.

[0102] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0103] Sodium chloride in an amount sufficient to adjust the tonicity of the aqueous ophthalmic composition to 280 mOsmol / kg.

[0104] In an embodiment, the aqueous composition according to the invention comprises:

[0105] 0.1% by weight, based on the total weight of the composition, of spironolactone,

[0106] 3% by weight, based on the total weight of the composition, of cyclodextrin,

[0107] 0.6 % by weight, based on the total weight of the composition, of trometamol.

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

[0109] 0.1% by weight, based on the total weight of the composition, of spironolactone,

[0110] 3% by weight, based on the total weight of the composition, of cyclodextrin,

[0111] 0.6 % by weight, based on the total weight of the composition, of trometamol.

[0112] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0113] Sodium chloride in an amount sufficient to adjust the tonicity of the aqueous ophthalmic composition to 280 mOsmol / kg.

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

[0115] 0.1% by weight, based on the total weight of the composition, of spironolactone,

[0116] 2.75% by weight, based on the total weight of the composition, of cyclodextrin, 0.6 % by weight, based on the total weight of the composition, of trometamol.

[0117] In an embodiment, the aqueous composition according to the invention comprises:

[0118] 0.1% by weight, based on the total weight of the composition, of spironolactone,

[0119] 2.75% by weight, based on the total weight of the composition, of cyclodextrin,

[0120] 0.6 % by weight, based on the total weight of the composition, of trometamol.

[0121] HC1 in an amount sufficient to adjust the pH of the aqueous ophthalmic composition to 7.0 to 7.4.

[0122] Sodium chloride in an amount sufficient to adjust the tonicity of the aqueous ophthalmic composition to 280 mOsmol / kg.

[0123] Topical administration

[0124] The aqueous ophthalmic composition is administered topically. The term “topical” or “topically”, 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 directly to the site of interest (i.e., to the eye) for a localized effect. Examples of topical formulation include solutions, suspensions, ointments, and gels.

[0125] In a preferred embodiment, the composition is a solution.

[0126] Preferably, the composition is administered directly on the ocular surface, in the conjunctival fornix or on the bulbar conjunctiva.

[0127] The aqueous ophthalmic composition of the present invention may be presented in a unit dosage form.

[0128] Preferably, the composition is sterile and / or aseptic.

[0129] Eye drops

[0130] The aqueous ophthalmic composition as described herein may be in the form of an eye drop for application to the eye.

[0131] Hence, in an embodiment, the composition is in the form of eye drops for application to the eye.

[0132] In an embodiment, the viscosity of the present composition is from 0.5 to 3 mPa.s at 35°C.

[0133] Viscosity is measured by a method using a rotary viscometer of Plan-Plan type.

[0134] Flow measurements were carried out using an Anton Paar MCR 102 rheometer (Austria), with a planar geometry (diameter of mobile: 50mm, air gap: 0.375 mm). Dynamic viscosities were measured at constant shear for 10 s at 35°C (administration site temperature). Treatment

[0135] Spironolactone was successfully dissolved in HP-y-CD excipient at a concentration of 0.1%, a dose that proved effective in treating corneal wound healing delay induced by GCs in a rabbit model [Dahmana, N.; Mugnier, T.; Gabriel, D.; Kaltsatos, V.; Bertaim, T.; Behar-Cohen, F.; Gurny, R.; Kalia, Y.N. 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].

[0136] Advantageously, the pH of the eyedrop solution according to the invention was adjusted to between 7.0 and 7.4 to reduce irritation. The osmolality of SPL eyedrops was close to the tear film osmolality in normal, non-dry eyes [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.]. Hence, the composition according to the invention can be advantageously topically administered to the eye and is effective in the treatment of ocular diseases.

[0137] Hence, in another aspect, the present invention relates to composition according to the invention for use for the treatment of eye diseases and conditions, in particular eye diseases and conditions that affect the surface of the eye.

[0138] The inventors demonstrated that the aqueous ophthalmic composition according to the invention can be used for restoring epithelial integrity, for improving corneal re- epithelialization, for improving epithelial cell adhesion and differentiation with cell layer stratification, for recovering the optical function of the anterior cornea, for reducing of the inflammation, for decreasing stromal edema, for restoring corneal transparency or for improving corneal reinnervation.

[0139] Corneal wound healing

[0140] After corneal injury, the epithelium must heal in a timely manner to re-establish barrier function and regenerate the normal epithelial basement membrane so that the fibrotic stromal wound healing response can be terminated in favour of regenerative repair (Wilson, S.E. Corneal Wound Healing. Exp. Eye Res. 2020, 197, 108089, doi:10.1016 / j.exer.2020.108089.).

[0141] The inventors advantageously showed that SPL eyedrops not only accelerate corneal re- epithelialization, but also improve epithelial cell adhesion and differentiation with cell layer stratification. Restoration of the epithelial integrity can block the entry of epithelial TGF[3 and PDGF into the corneal stroma and avoid the transformation of keratocytes into corneal fibroblasts and myofibroblasts (Singh, V.; Santhiago, M.R.; Barbosa, F.L.; Agrawal, V.; Singh, N.; Ambati, B.K.; Wilson, S.E. Effect of TGF[3 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, A.A.M.; Santhiago, M.R.; Singh, N.; Ambati, B.K.; Wilson, S.E. TGF[3 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).

[0142] In an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for use for restoring epithelial integrity.

[0143] In an embodiment, the present invention relates to a method for restoring epithelial integrity in a subject in need thereof, comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0144] By “restoring the epithelial integrity” we mean that the corneal surface is regular and smooth, and that the epithelia-stromal interface is regular without any scar, which is an important factor for optimal visual recovery.

[0145] The restoration of the epithelial integrity can be evaluated by measuring immunostaining of E-cadherin and cytokeratin K12, markers of differentiated corneal epithelium. Typically, such a measure can be performed by immunostaining of E-cadherin and cytokeratin K12.

[0146] In an embodiment, the aqueous ophthalmic composition according to the invention restores the continuity of E-cadherin and the multilayer stratified structure of the corneal epithelium.

[0147] In another embodiment, the aqueous ophthalmic composition according to the invention enhances the K12 expression and restored the structure of corneal epithelium.

[0148] It has been demonstrated that in superficial corneal stromal scarring, deranged surface irregularity parameters like higher-order aberrations affect the 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]. Hence the restoration of the epithelial integrity is essential for visual recovery. Furthermore, the restoration of proper barrier function of the epithelium prevents potential infection and inflammation.

[0149] In an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for use for improving corneal re-epithelialization, for improving epithelial cell adhesion and differentiation with cell layer stratification and for recovering the optical function of the anterior cornea

[0150] In an embodiment, the present invention relates to a method for improving corneal re-epithelialization, epithelial cell adhesion and differentiation with cell layer stratification and for recovering the optical function of the anterior cornea in a subject in need thereof, comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0151] By “improving corneal re-epithelialization” we mean restoration of the geometry of the epithelium and of its optical properties Typically, the corneal re-epithelialization can be evaluated by fluorescein staining.

[0152] By “improving epithelial cell adhesion and differentiation with cell layer stratification” we mean restoration of the barrier function of the cornea.

[0153] Typically, it can be evaluated by immunostaining of E-cadherin, ZO-1 and cytokeratin K12.

[0154] By “optimal recovery of the optical function of the anterior cornea”, we mean improve visual acuity.

[0155] Typically, it can be evaluated by visual acuity measurement.

[0156] Reduction of the inflammation. Decrease of stromal edema and restoration of corneal transparency

[0157] SPL eyedrops also significantly reduce inflammation, a pathogenic factor involved in wound healing process. Indeed, proinflammatory ILla and ILip are released immediately by injured epithelial cells. Their upregulation triggers secretion of cytokines / chemokines and growth factors by keratocytes, epithelial cells and fibroblasts, followed by infiltration of bone marrow-derived inflammatory cells and remodeling of stromal extracellular matrix (Wilson, S.E. Corneal Wound Healing. Exp. Eye Res. 2020, 197, 108089, doi:10.1016 / j.exer.2020.108089, Liu, C.-Y.; Kao, W.W.-Y. Corneal Epithelial Wound Healing. Prog. Mol. Biol. Transl. Sci. 2015, 134, 61-71, doi:10.1016 / bs.pmbts.2015.05.002.).

[0158] In case of extensive injury or delayed epithelial closure where inflammatory response is amplified, growth factors and cytokines continue to penetrate into the corneal stroma, leading to chronic inflammation and aberrant fibrosis (Ljubimov, A.V.; 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 eyedrops decrease stromal edema, helping to restore corneal transparency.

[0159] In an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for use for reducing of the inflammation, decreasing stromal edema and restoring corneal transparency.

[0160] In an embodiment, the present invention relates to a method for reducing of the inflammation, decreasing stromal edema and restoring corneal transparency in a subject in need thereof, comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0161] The reduction of the inflammation can be evaluated by measuring inflammatory cells such as by IB Al and EDI. Typically, evaluation of the anti-inflammatory effect of SPL eyedrops can be performed by IBA1 and EDI immunostaining.

[0162] Clinically, corneal edema is evaluated by measuring the reduction of corneal stromal using SD-OCT and by evaluating the corneal transparency using slit-lamp examination and corneal / anterior segment slit-lamp photography. By “decreasing stromal edema” we mean reduction of corneal thickness.

[0163] By “restoring corneal transparency” we mean optimal visualization of the anterior segment.

[0164] Corneal reinnervation

[0165] The integrity of nerve fibers is crucial for normal corneal function by sensing thermal, mechanical, and chemical stimuli leading to the release of essential neurotrophins for corneal homeostasis and wound healing (Al-Aqaba, M.A.; Dhillon, V.K.; Mohammed, I.; Said, D.G.; Dua, H.S. Corneal Nerves in Health and Disease. Prog. Retin. Eye Res. 2019, 73, 100762, doi:10.1016 / j.preteyeres.2019.05.003). Corneal diseases, such as diabetic and neurotrophic keratopathies, are associated with impaired function of corneal nerves and epithelial breakdown with the consequence of delayed epithelial wound healing and corneal neurotrophic ulceration (NaPier, E.; Camacho, M.; McDevitt, T.F.; Sweeney, A.R. Neurotrophic Keratopathy: Current Challenges and Future Prospects. Ann. Med. 2022, 54, 666-673, doi: 10.1080 / 07853890.2022.2045035). Our results indicate that SPL eyedrops might help reinnervation of the cornea, which is essential for return to homeostasis.

[0166] In an embodiment, the present invention relates to the aqueous ophthalmic composition according to the invention for improving corneal reinnervation.

[0167] In an embodiment, the present invention relates to a method for improving corneal reinnervation in a subject in need thereof, comprising the administration of an effective amount of an aqueous ophthalmic composition according to the invention.

[0168] By “improving the corneal reinnervation”, it means to improve the sub-basal nerve density and to obtain longer and more parallelly oriented nerves.

[0169] Typically, the corneal reinnervation can be evaluated by TUBB3 immunostaining or by analysis of the corneal nerves in vicio using confocal microscopy.

[0170] As demonstrated by the inventors, in the rat model of corneal de-epithelialization, the 0.1% SPL eyedrops showed a significant beneficial effect on corneal wound healing by accelerating the speed of wound closure, decreasing corneal edema, improving epithelial integrity, and reducing inflammatory response. After corneal injury, the epithelium must heal in a timely manner to re-establish barrier function and regenerate the normal epithelial basement membrane so that the fibrotic stromal wound healing response can be terminated in favor of regenerative repair. SPL eyedrops not only accelerate corneal re-epithelialization, but also improve epithelial cell adhesion and differentiation with cell layer stratification. SPL eyedrops also significantly reduced the infiltration of EDI and IB Al positive cells and their migration in the deep stroma. Whether the reduced cell infiltration results from the quicker restoration on the epithelial barrier or whether it is the primary effect of SPL cannot be determined, but it confirms that SPL is released at efficient concentration in the cornea as SPL did reduce IBA-1 positive cells in the rat model of laser-induced choroidal neovascularization and in the diabetic retina.

[0171] Altogether, these results allow concluding that the ophthalmic composition according to the invention can be used in the treatment of a disease or a disorder selected from the group comprising corneal ulcer with or without inflammation, post operative corneal wound, corneal re epithelialization delay following corneal graft surgery, corneal graft rejection, vernal keratitis, ocular rosacea, epithelial keratitis secondary to dry eye syndrome or to meibomian gland dysfunction, corneal ulcer post corneal cross linking, corneal wound healing delay due to refractive surgery or to corneal surgery or to anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungi, parasite or to viral infection or to non-conventional agent infection, corneal opacification or corneal epithelial defect due to neurotrophic keratopathy, corneal opacification due to corneal scar, corneal edema, corneal inflammation or neovascularization due to vernal keratoconjunctivitis, corneal inflammation due to ocular rosacea, corneal neovascularization, corneal neovascularization secondary to inflammation or to meibomian gland dysfunction or to infection or to limbal deficiency or to corneal graft rejection, recurrent corneal erosions, wound healing delay, post-surgical treatment of corneal graft or refractive surgery, or any other corneal surgery, glucocorticoid-induced epithelial wound healing delay following corneal physical trauma (such as surgical trauma including corneal graft, laser-induced, UV exposure including cross linking, refractive surgery), inherited and genetic keratopathy, aniridia-induced keratopathy, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and associated diseases such as dry eye syndromes and blepharitis and corneal diseases chosen among diabetic, neurotrophic, toxic, iatrogenic, preservatives-induced keratopathies.

[0172] In an embodiment, wound healing delay can be but is not only due to association with the use of glucocorticoids.

[0173] As used herein, post refractive surgery can be a laser assisted or a surgical procedure.

[0174] In an embodiment, corneal neovascularization is inhibited due the anti-angiogenic effects of spironolactone.

[0175] In another aspect, the invention also concerns a method of treating a disease or a disorder selected from the group comprising corneal ulcer with or without inflammation, post operative corneal wound, corneal re epithelialization delay following corneal graft surgery, corneal graft rejection, vernal keratitis, ocular rosacea, epithelial keratitis secondary to dry eye syndrome or to meibomian gland dysfunction, corneal ulcer post corneal cross linking, corneal wound healing delay due to refractive surgery or to corneal surgery or to anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungi, parasite or to viral infection or to non-conventional agent infection, corneal opacification or corneal epithelial defect due to neurotrophic keratopathy, corneal opacification due to corneal scar, corneal edema, corneal inflammation or neovascularization due to vernal keratoconjunctivitis, corneal inflammation due to ocular rosacea, corneal neovascularization, corneal neovascularization secondary to inflammation or to meibomian gland dysfunction or to infection or to limbal deficiency or to corneal graft rejection, recurrent corneal erosions, wound healing delay, post-surgical treatment of corneal graft or refractive surgery, or any other corneal surgery, glucocorticoid-induced epithelial wound healing delay following corneal physical trauma (such as surgical trauma including corneal graft, laser-induced, UV exposure including cross linking, refractive surgery), inherited and genetic keratopathy, aniridia- induced keratopathy, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and associated diseases such as dry eye syndromes and blepharitis and corneal diseases chosen among diabetic, neurotrophic, toxic, iatrogenic, preservatives-induced keratopathies, in a patient in need thereof, the method comprising the administration of an aqueous ophthalmic composition according to the invention.

[0176] In another aspect, the present invention relates to the use of an aqueous ophthalmic composition according to the invention for the treatment of a disease or a disorder selected from the group comprising corneal ulcer with or without inflammation, post operative corneal wound, corneal re epithelialization delay following corneal graft surgery, corneal graft rejection, vernal keratitis, ocular rosacea, epithelial keratitis secondary to dry eye syndrome or to meibomian gland dysfunction, corneal ulcer post corneal cross linking, corneal wound healing delay due to refractive surgery or to corneal surgery or to anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungi, parasite or to viral infection or to non-conventional agent infection, corneal opacification or corneal epithelial defect due to neurotrophic keratopathy, corneal opacification due to corneal scar, corneal edema, corneal inflammation or neovascularization due to vernal keratoconjunctivitis, corneal inflammation due to ocular rosacea, corneal neovascularization, corneal neovascularization secondary to inflammation or to meibomian gland dysfunction or to infection or to limbal deficiency or to corneal graft rejection, recurrent corneal erosions, wound healing delay, post-surgical treatment of corneal graft or refractive surgery, or any other corneal surgery, glucocorticoid-induced epithelial wound healing delay following corneal physical trauma (such as surgical trauma including corneal graft, laser-induced, UV exposure including cross linking, refractive surgery), inherited and genetic keratopathy, aniridia-induced keratopathy, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and associated diseases such as dry eye syndromes and blepharitis and corneal diseases chosen among diabetic, neurotrophic, toxic, iatrogenic, preservatives-induced keratopathies.

[0177] In another aspect, the present invention relates to the use of an aqueous ophthalmic composition according to the invention for the manufacture of a medicament for the treatment of a disease or a disorder selected from the group comprising corneal ulcer with or without inflammation, post operative corneal wound, corneal re epithelialization delay following corneal graft surgery, corneal graft rejection, vernal keratitis, ocular rosacea, epithelial keratitis secondary to dry eye syndrome or to meibomian gland dysfunction, corneal ulcer post corneal cross linking, corneal wound healing delay due to refractive surgery or to corneal surgery or to anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungi, parasite or to viral infection or to non-conventional agent infection, corneal opacification or corneal epithelial defect due to neurotrophic keratopathy, corneal opacification due to corneal scar, corneal edema, corneal inflammation or neovascularization due to vernal keratoconjunctivitis, corneal inflammation due to ocular rosacea, corneal neovascularization, corneal neovascularization secondary to inflammation or to meibomian gland dysfunction or to infection or to limbal deficiency or to corneal graft rejection, recurrent corneal erosions, wound healing delay, post- surgical treatment of corneal graft or refractive surgery, or any other corneal surgery, glucocorticoid- induced epithelial wound healing delay following corneal physical trauma (such as surgical trauma including corneal graft, laser-induced, UV exposure including cross linking, refractive surgery), inherited and genetic keratopathy, aniridia-induced keratopathy, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and associated diseases such as dry eye syndromes and blepharitis and corneal diseases chosen among diabetic, neurotrophic, toxic, iatrogenic, preservatives-induced keratopathies.

[0178] In a further aspect, the present invention relates to a pharmaceutical composition for treating a disease or a disorder selected from the group comprising corneal ulcer with or without inflammation, post operative corneal wound, corneal re epithelialization delay following corneal graft surgery, corneal graft rejection, vernal keratitis, ocular rosacea, epithelial keratitis secondary to dry eye syndrome or to meibomian gland dysfunction, corneal ulcer post corneal cross linking, corneal wound healing delay due to refractive surgery or to corneal surgery or to anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungi, parasite or to viral infection or to non-conventional agent infection, corneal opacification or corneal epithelial defect due to neurotrophic keratopathy, corneal opacification due to corneal scar, corneal edema, corneal inflammation or neovascularization due to vernal keratoconjunctivitis, corneal inflammation due to ocular rosacea, corneal neovascularization, corneal neovascularization secondary to inflammation or to meibomian gland dysfunction or to infection or to limbal deficiency or to corneal graft rejection, recurrent corneal erosions, wound healing delay, post- surgical treatment of corneal graft or refractive surgery, or any other corneal surgery, glucocorticoid- induced epithelial wound healing delay following corneal physical trauma (such as surgical trauma including corneal graft, laser-induced, UV exposure including cross linking, refractive surgery), inherited and genetic keratopathy, aniridia-induced keratopathy, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and associated diseases such as dry eye syndromes and blepharitis and corneal diseases chosen among diabetic, neurotrophic, toxic, iatrogenic, preservatives-induced keratopathies, the pharmaceutical composition comprising spironolactone, cyclodextrin and trometamol.

[0179] For the avoidance of doubt, references herein to "treatment" include references to curative, palliative and prophylactic treatment. A “treatment” aims at reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

[0180] Administration with GCs

[0181] Topical glucocorticoids (GCs), which are extremely efficient to control the deleterious inflammation are part of most therapeutic regimen, but they should be used with caution as they might delay corneal epithelial wound healing (Kadmiel, M.; Janoshazi, A.; Xu, X.; Cidlowski, J. A. 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). Adverse effects like elevated intraocular pressure and glaucoma, cataract formation and increased susceptibility to ocular infections, are also non insignificant.

[0182] In a rabbit model of corneal wound healing delayed by dexamethasone, spironolactone-loaded polymeric nanomicelles significantly accelerated corneal re-epithelialization, whilst potassium canrenoate 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, Y.N. 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).

[0183] As the use of spironolactone micelles countered the delayed wound healing caused by glucocorticoid therapy, it is believed that SPL eyedrops could benefit to patients with impaired corneal wound healing, including that associated with glucocorticoid therapy.

[0184] Hence, in an embodiment, the present invention relates to an aqueous ophthalmic composition according to the invention to be administered with glucocorticoid therapy.

[0185] “To be administered with” means that SPL eye drops can be administered before the administration of the glucocorticoid therapy, after the glucocorticoid therapy or simultaneously to the glucocorticoid therapy.

[0186] Administration

[0187] In one embodiment of the present invention there is provided the method of treating the eye of a human, the method comprising administering the aqueous ophthalmic composition described herein to the eye. The aqueous ophthalmic composition may be administered drop wise to the eye. It may be administered using a dropper or it may be poured into the eye.

[0188] Method for making the aqueous ophthalmic composition

[0189] In a further embodiment, the present invention relates to a method of making the aqueous ophthalmic composition as hereinbefore described, comprising the following steps: (i) Dissolving cyclodextrins in water

[0190] (ii) Adding spironolactone under stirring until complete dissolution,

[0191] (iii) Adding trometamol and a tonicity agent in an amount sufficient to adjust the tonicity of the composition from 280 to 310 mOsmol / kg, preferably from 280 to 300 mOsmol / kg and preferably to 280 mOsmol / kg,

[0192] (iv) Adding a pH adjusting agent in an amount sufficient to adjust the composition of the pH from 6.8 to 7.5, preferably to 7 to 7.5, and more preferably to 7.0 to 7.4.

[0193] 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.

[0194] Device for dropwise dispensing of a composition

[0195] In one aspect of the present invention there is provided a device for dropwise dispensing of a composition, the device comprising a container holding the composition as described herein.

[0196] EXAMPLES

[0197] Materials and methods

[0198] 1. Physicochemical stability of eyedrop formulations

[0199] Eyedrops of Spironolactone (SPL) and placebo (PBO) were stored in sterilized eyedroppers at 4°C until 9 months. Their appearance was observed at 1, 3, 6 and 9 months. The pH and osmolarity were measured at 1 month, 3 months and 9 months.

[0200] 2. Animals

[0201] All experiments were performed in accordance with the European Communities Council Directive 8 / 609 / EEC and French national regulations and approved by local ethical committees (#23478- 2020010317557546 v4, Charles Darwin). Adult male Lewis rats (6-8 weeks, 200-250g, Janvier, Le Genest-Saint-Isle, France) were used to assess the ocular tolerance of eyedrop formulations. Adult male Sprague-Dawley rats (12-13 weeks, 450-500 g, Janvier) were used to evaluate the therapeutic effects of SPL eyedrops. Animals were kept in pathogen-free conditions with ad libitum access to food and water and housed under a 12-h light / 12-h dark cycle. Anaesthesia was induced by intraperitoneal ketamine 100 mg / kg and xylazine 10 mg / kg. Rats were euthanized by Euthasol® Vet 300 mg / kg.

[0202] 3. Ocular tolerance of eyedrop formulations

[0203] 3.1. Treatments PBO or SPL (0.1 %, w / v) eyedrops were instilled in both eyes of rat Animals were divided into four treatment groups: 1) SPL once a day for seven days; 2) PBO once a day for seven days; 3) SPL three times a day for seven days, and 4) PBO three times a day for seven days.. On each time, two drops (~30 pl each) were administered with a 5-minute interval between each administration. Untreated rats were used as controls. Rats were than euthanized on day 8, and eyes enucleated for histology and immunofluorescence.

[0204] 3.2. Corneal histology

[0205] Eyes were fixed in 4% paraformaldehyde (PF A) and 0.5% glutaraldehyde for 2 hours, dehydrated in a graded alcohol series, and embedded in historesine (Lecia, Heidelberg, Germany). Cross sections of 5 pm were obtained with a Leica Jung RM2055 Microtome and then 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, eye sections through the optic nerve head were photographed serially, from the limbus on one side of the cornea to the other. Corneal thickness and corneal epithelial thickness were measured every 200 pm using Fiji Image J software (ver. 1.54b; Wayne Rasband, National Institutes of Health, USA). Twenty measurements were performed for each cornea, 3 to 4 corneas were used for each group.

[0206] 3.3. Immunofluorescence

[0207] Immediately after enucleation, eyes were snap-frozen in Tissue-Tek-OCT-compound (Bayer Diagnostics, Puteaux, France). Cryostat sections (10 pm) were fixed in 4% PFA, washed with PBS and permeabilized with 0.1% Titron X-100 in PBS for 30 min. Unspecific binding sites were blocked with 5% normal goat serum for 30 min. Sections were then incubated with primary antibodies for Ih at room temperature (RT), washed in PBS, and further incubated with secondary antibodies for Ih at RT. After washing, slides were stained for 2 min with 4’,6-Diamidino-2-Phenyl-Indole (DAPI; 1:10000), washed again with PBS, and mounted with gel mount (Dako, Agilent, Les Ulis, France). Negative control slides were stained without primary antibodies. Positive controls were corneal sections from rats with corneal inflammation. Images were taken using 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, Abeam ref. ab231303, Cambridge, U.K), rabbit anti-IBAl (1:400, Wako ref. 019- 19741, Richmond, USA), mouse anti-EDl (1:200, Bio-Rad ref. MCA341R, Colmar, France), and rabbit anti-Nitrotyrosine (1:200, Thermo Fisher Scientific ref. BS-8551, Saint Aubin, France). Secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Thermo Fisher Scientific ref. Al 1008)) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1 :200; Thermo Fisher Scientific ref. A21202). Three to four corneas were used for each group.).

[0208] 3.4. TUNEL Assay TUNEL Assay was performed on eye cryosections according to manufacturer’s instruction (Roche Diagnostics, Mannheim, Germany). Nuclei were counter-stained with DAPI. TUNEL-positive cells were counted across on the whole cornea on the sections at the level of optic nerve head. Three corneas were used for each group.

[0209] 4. Therapeutic effects of SPL eyedrops in a rat model of corneal wound healing

[0210] 4.1. Corneal de-epithelialization and treatments

[0211] After general and local anaesthesia (1% tetracaine, Sigma-Aldrich, Saint-Quentin-Fallavier, France), the central 4 mm of the cornea was marked with a 4-mm trephine. The corneal epithelium was then gently removed by a scarifier in the demarcated area without injuring the underlying corneal stroma. One of the following treatments was administered 3 times a day for 3 days on both corneas of rat: 1) eyedrops containing 0.1% SPL, 2) PBO eyedrops, 3) PBS solution containing 0.1% % potassium canrenoate (KCAN, Sigma-Aldrich), a water-soluble precursor of canrenone which is an active metabolite of SPL. Rat corneas without surface wound and treatment were used as controls.

[0212] 4.2. In vivo Optical coherence tomography and slit lamp examination

[0213] Corneal morphology was assessed in vivo by optical coherence tomography (OCT, Micron III, Phoenix- Micron Inc., Bend, OR, USA) before, and 24 and 48 hours after corneal de-epithelialization. To measure the central corneal thickness, a rectangle was used as a reference on all corneal images (Fig.5A). Results were expressed as a percentage of the area of the reference rectangle occupied by the cornea. Corneal epithelial defect was visualized by fluorescein staining under slit lamp immediately (0 h), and 6, 24, and 48 hours after de-epithelialization. At each time point, the healing rate was calculated as a percentage reduction in the fluorescein-stained area compared to the wound created at Oh. Images from seven to nine corneas were analyzed at each time point for each group. Immunofluorescence on corneal sections and on whole-mounted corneas

[0214] Rats were euthanized 5 days after corneal de-epithelialization. Eyes were used for cryosection and corneal flat mounting. Immunofluorescence on corneal sections were performed as previously described. Primary antibodies used were mouse anti-E-cadherin, rabbit anti-Cytokeratin 12 (1:400, Abeam ref. abl 85627, Cambridge, U.K), rabbit anti-IBAl and mouse anti-EDl. Secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated donkey antimouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200; Thermo Fisher Scientific ref. Al 1012). The number of IB Al - and EDI -positive inflammatory cells was counted on the entire corneal section. One section per cornea, and 5-6 corneas were analysed for each group.

[0215] For corneal whole mounts, eyes were fixed in 1.3% PFA for 45 min at room temperature. After washing with PBS, corneas were excised along the sclerocorneal rim, cut into four to five petals, and blocked and permeabilized with PBS containing 0.5% normal goat serum and 0.5% Triton X-100 for Ih. corneas were then incubated with a mouse anti-Tubulin III (TUBB3) antibody (1:500, Biolegend ref. 801202, San Diego, USA) diluted in PBS containing 0.5% normal goat serum, 0.1% Triton X-100 and 0.1% Tween 20 (Sigma Aldrich) for 5 days at 4 °C. After washing, corneas were incubated with Alexa 488- conjugated donkey anti-mouse IgG (1:200 Thermo Fisher Scientific ref. A21202) for overnight at 4 °C. Images of TUBB3 staining were observed and captured with a confocal microscope (Zeiss LSM 710 Oberkochen, Germany). For nerve quantification, ten z-stacks of the sub-basal nerves in the reinnervated corneal area were used. The density of corneal nerves was measured using the Fiji Image J software. The percentage of sub-basal nerve density in PBO- (n=3) or SPL-treated corneas (n=2) to that in normal control corneas was calculated.

[0216] 5. Statistics

[0217] A study of the literature and an estimation using the algorithm G-power software were carried out to estimate the number of animals required. Data are expressed as mean + SD. Statistical analysis was performed using the GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Mann-Whitney test was used to compare two groups. Kruskal- Wallis test followed by Dunn’s test was used to compare more than two groups. For grouped data, a two-way ANOVA was used followed by Tukey’s test. A p value less than 0.05 was considered statistically significant.

[0218] Example 1: Spironolactone solubilization

[0219] Solubilization in water failed after pH and temperature adjustments: At 0.1%, Spironolactone is insoluble in water at 20-25°C. After 15 minutes’ agitation, the resulting solution is opalescent. At rest, the undissolved particles are present at the bottom of the beaker, on the surface, as well as on the surface. After gradual heating to 80°C, the appearance of the solution remains unchanged. After adding 1 N hydrochloric acid (until pH = 1.17), the appearance of the solution remains unchanged. After addition of 1 N sodium hydroxide (until pH = 12.59), the appearance of the solution remains unchanged.

[0220] The inventors demonstrated (Table 1) that cyclodextrins advantageously enhance the bioavailability of spironolactone while others conventional systems, known to enhance the bioavailability of poorly soluble or insoluble active ingredients failed.

[0221] Table 1. Spironolactone solubilization comparison

[0222] Spironolactone dissolution protocol in HP-Y-CD:

[0223] Add HP-y-CD to approx. 70 ml deionized water. After dissolving, add spironolactone with stirring. After 12 minutes stirring, dissolution is almost complete. Add 1 g HP-y-CD (for a total concentration of 3%). After 22 minutes' stirring, dissolution is complete: clear, colourless solution. Make the final qs to 100 ml with demineralized water and homogenize for 15 minutes. The solution is clear, colourless, pH = 5.53 and osmolality = 20 mOsmol / kg. Unchanged appearance after 17.5 hours' rest. After 6 months, appearance unchanged.

[0224] Autoclave stability study

[0225] Two solutions, able to solubilize spironolactone, has been prepared. One with 2.75% HP-y-CD, the other with 3% HP-y-CD. These 2 solutions have been autoclaved (15 min cycle, > 121 °C) and their evolution compared over 3 months with other solutions at room temperature.

[0226] After autoclaving, the appearance of the solutions was unchanged (no opalescence or precipitate), which suggests that Spironolactone is heat stable. After 3 months at room temperature (without special protection), only autoclaved solutions change slightly (very slightly yellowish appearance).

[0227] Conclusion

[0228] Solubilization of spironolactone was only effective in HP-y-CD. Indeed, apart from cyclodextrins, the other systems lead to unstable solubilization with white deposits appearing after a few hours. In contrast, spironolactone dissolution in HP-y-CD is complete: the solution is clear and colourless. The appearance is unchanged after 6 months.

[0229] Example 2: Spironolactone compatibility with buffers

[0230] Different buffers (0.50g Trometamol, 0.50g sodium citrate and 1.00g of boric acid) were tested for their compatibility with 0.1% spironolactone and 3% HP-y-CD and their heat stability (1 month at 50°c) evaluated. For both buffer, HP-y-CD was dissolved in approximatively 70 ml of deionized water and then stirred in spironolactone until completely dissolved. Buffer is then added, and pH is measured and adjust to 7.04-7.4 with 1 N HC1 for Trometamol, with citric acid and sodium citrate for citrate buffer and with sodium borate of borate stamp. Then, a 100 ml final solution is obtained with demineralized water and homogenize for 15 minutes to obtain a clear and colourless solution. Solutions have been oven 50°C for

[0231] 1 month. The results are presented in the Table 2 hereinafter:

[0232] Table 2. Buffer compatibility and heat stability at 50°C

[0233] For borate stamp and citrate buffer, yellow colour appears over time which is a probable sign of degradation. They cannot therefore be preserved. In contrast, the appearance of the solution comprising trometamol / HCl buffer remains clear. Hence, trometamol / HCl buffer is advantageously the best candidate but needs to be adjusted and isotonicity checked with sodium chloride.

[0234] Thus, the inventors determined the suitable and compatible composition, which is heat stable, colourless, and isotonic and described in the Table 3 hereinafter. Table 3. Composition according to the invention

[0235] Compound Quantities

[0236] HP-y-CD 3.00 g

[0237] Spironolactone 0.10 g

[0238] Trometamol 0.60 g

[0239] HC1 (IN, q.s) pH = 7.2

[0240] NaCl 0.50 g

[0241] Distilled water (q.s) 100.00 mL Example 3: Preparation of eyedrop formulations

[0242] Pharmaceutical grade SPL was purchased from Azelis Pharma (Courbevoie, France) and HP-y-CD was obtained from Wacker Chemie (Lyon, France). SPL and PBO eyedrops were prepared according to their composition shown in Table 4. After dissolving HP-y-CD in distilled water, SPL was added under stirring for at least Ih at 20-25°C until complete dissolution. Trometamol and NaCl were then added, and pH was measured and adjusted to 7.0 - 7.4 with IN HC1. The osmolality of eyedrop formulations was around 280 mOsmol / kg. The PBO formulation was obtained using the same protocol without the addition of SPL.

[0243] Table 4. Composition of SPL and PBO eyedrops.

[0244] Compound Spironolactone Placebo

[0245] HP-y-CD 3.00 g 3.00 g

[0246] Spironolactone 0.10 g

[0247] Trometamol 0.60 g 0.60 g

[0248] HC1 (IN, q.s) pH = 7.2 pH = 7.2

[0249] NaCl 0.50 g 0.50 g

[0250] Distilled water (q.s) 100.00 mL 100.00 mL

[0251] Example 4: Eyedrop formulations showed good physicochemical stability at 4 °C for 9 months

[0252] Eyedrops containing 0.1% SPL and placebo (PBO) eyedrops were respectively prepared according to the formula in Table 4. Both eyedrop formulations remained clear and colourless after 9 months’ storage in sterilized eyedroppers at 4°C. No particle precipitation was observed. The osmolality of SPL eyedrops did not vary significantly over time and was close to 280 mOsmol / kg when measured at 1 month, 2 months and 9 months. The pH of SPL eyedrops was 7.189 on day 0 and varied only slightly between 7.0 and 7.4 over a period of up to 9 months.

[0253] Example 5: SPL eyedrops showed no sign of corneal integrity disruption

[0254] To evaluate the tolerance of eyedrop formulations, both eyes of Lewis rats were treated with eyedrops containing 0.1% SPL or placebo (PBO) once or three times a day for 7 days. The corneal morphology and barrier integrity were evaluated by histology and immunofluorescence on day 8. Untreated rat eyes were used as controls. On histological sections, corneas treated with PBO or SPL formulations once or 3 times / day for 7 days presented a normal structure with stratified epithelium, regularly organized stroma, and a monolayer of endothelial cells, similar to untreated corneas (Fig. 1 A). The quantification of the total corneal thickness (Fig. IB) and corneal epithelial thickness (Fig. 1C) did not show significant difference amongst all groups (p=0.3762 and p=0.2527 respectively). No corneal edema or inflammatory cell infiltration was observed after 7 days of treatment.

[0255] Corneal epithelial barrier was evaluated by immunostaining of tight junction molecule ZO-1 and adhesion protein E-cadherin on day 8. ZO-1 staining was mainly located in the superficial layer of the corneal epithelium. We did not find any disruption of the staining in any treatment groups and no difference in localization was observed between PBO and SPL groups (Fig. ID). E-cadherin was expressed in the cell membrane of all epithelial cells, forming bridges between adjacent epithelial cells. There was no disruption or difference in its localization between PBO and SPL groups (Fig. IE). The inner border of the epithelium was regular, and epithelium layers were well defined by E-cadherin staining in all groups (Fig. IE).

[0256] Conclusion: After multiple topical instillations for a week, SPL eyedrops did not show any signs of incompatibility. Spironolactone eye drops are thus well tolerated.

[0257] Example 6: SPL eyedrops did not induce corneal inflammation, oxidative stress or apoptotic cell death

[0258] IB Al and EDI immunostaining was performed on corneal cryosections to evaluate potential inflammatory cell infiltration. No IB Al- or EDI -positive inflammatory infiltrates were observed in the corneas on day 8 after PBO or SPL treatments (Fig.2). Nitrotyrosine (NT) is a marker of cell damage and nitrosative stress. There was no NT-positive immunostaining suggesting no nitrosative stress in any of the PBO and SPL groups (Fig. 3A). TUNEL assay detects DNA fragmentation of apoptotic cells. It was performed on the corneas treated with eyedrops 3 times / day for 7 days. TUNEL-positive apoptotic cells were located only in the superficial layer of the corneal epithelium in untreated normal corneas as well as in corneas treated with PBO or SPL (Fig. 3B). The number of apoptotic cells across the whole epithelium was similar amongst groups (25.33 + 3.06 in control, 22.67 + 4.62 in PBO and 22.33 + 5.86 in SPL, p=0.7214) (Fig. 3C).

[0259] Altogether, these results demonstrate the excellent tolerance of SPL eyedrop on the rat ocular surface.

[0260] Example 7: SPL eyedrops improved corneal re-epithelialization and reduced corneal edema in a rat model of corneal wound healing

[0261] To investigate the therapeutic effects of SPL eyedrops, we used the rat model of corneal wound healing, by mechanical injury, a model to study delayed corneal wound healing. After central corneal de- epithelialization, both eyes were treated with 0.1% SPL eyedrops or PBO 3 times a day for 3 days. A group of rats was treated with 0.1% topical potassium canrenoate (KCAN) in PBS to assess the effects of the soluble active metabolite of SPL alone without eyedrop formulation.

[0262] The healing progress was monitored with fluorescein staining to highlight denuded area immediately (Oh), and 6, 24 and 48 hrs after corneal de-epithelialization as shown in Fig. 4A. The healing rate of SPL-treated corneas was 16.91 % ± 10.18 at 6 hrs, 73.41 % ± 11.69 at 24 hrs, and 98.37 % ± 2.25 at 48 hrs, while the healing rate of PBO eyedrops was 2.70 % + 4.89 at 6 hrs, 54.75 % + 21.49 at 24 hrs, and 91.56 % ± 12.38 at 48 hrs, and that of KCAN solution was 3.89 % ± 3.99 at 6 hrs, 57.33 % ± 13.23 at 24 hrs, and 87.78 %± 12.38 at 48 hrs. A significant improvement of corneal epithelial wound healing was observed in SPL-treated group at 6 hrs as compared to the PBO (p=0.021) and KCAN (p=0.045), and at 24 hrs as compared to the PBO- (p=0.002) and KCAN- (p=0.009) treated groups (Fig. 4B), suggesting the superiority of SPL eyedrop formulation over PBO and unformulated KCAN solution. No difference was observed between PBO and topical KCAN (Fig. 4B) at 6 hrs (p=0.970) and 24 hrs (p=0.865). Corneal epithelial wound was closed on day 3 in all groups.

[0263] Corneal edema was assessed in vivo by OCT before, and 24 and 48 hrs after corneal de-epithelialization, and expressed as a percentage of the area of the reference rectangle in Fig. 5A occupied by the central cornea. Before epithelial debridement, corneal thickness was comparable amongst all groups (Fig. 5A and B). Corneal edema occurred after corneal de-epithelialization. In SPL eyedrop-treated group, corneal surface occu-pied 48.68 % + 3.86 of the reference rectangle at 24 hrs and 44.85 % + 8.52 at 48 hrs, while in PBO-treated group, the relative percentage was 45.73 % + 4.13 at 24 hrs and 57.47 % + 7.57 at 48 hrs, and in KCAN-treated group, it was 52.07 % ± 6.23 at 24 hrs and 58.26 % ± 8.67 at 48 hrs. SPL eyedrops significantly reduced corneal thickening at 48 hrs compared to the PBO (p=0.0234) and topical KCAN (p=0.254) treatments (Fig. 5A and B). There was no difference between the KCAN and PBO groups at 24 hrs (p=0.074) and at 48 hrs (p=0.979) (Fig. 5B).

[0264] Example 8: SPL eyedrops restored epithelial integrity in the rat model of corneal wound healing

[0265] In order to evaluate the quality of the epithelial healing, we performed immunostaining of E-cadherin and cytokeratin K12, markers of differentiated corneal epithelium on corneal sections on day 5 after wound induction. Although the wound was healed in the PBO-treated corneas, there was a disorganization of E-cadherin and focal disruption in the basal layer of the corneal epithelium (Fig. 6A). The structure of the epithelium was not well defined, and the inner border of corneal epithelium was irregular (Fig. 6A). SPL eyedrops restored the continuity of E-cadherin and the multilayer stratified multilayer structure of the corneal epithelium, comparable to the control uninjured cornea (Fig. 6A). Epithelial inner border was more regular than in PBO-treated corneas. In normal rat corneas, corneal epithelium expressed K12 with more intense expression in the superficial layers (Fig.6B). In PBO-treated corneas, we observed a general decrease in K12 expression and focal disruption in the basal layer of the epithelium. The inner border of the epithelium was irregular (Fig. 6B). Topical SPL eyedrops enhanced the K12 expression and restored the structure of corneal epithelium (Fig. 6B).

[0266] Example 9: SPL eyedrops reduced corneal inflammatory cells infiltration in the rat model of corneal wound healing

[0267] IB Al and EDI immunostaining were performed to evaluate the anti-inflammatory effect of SPL eyedrops. In PBO-treated group, numerous IBA1- and EDl-positive cells were detected throughout the corneal stroma, while SPL eyedrops limited the infiltration in the anterior stroma (Fig. 7A and B). The number of IBAl-positive cells was 327 + 91 in PBO-treated group vs. 179 + 34 in SPL-treated group. The number of EDl-positive cells was 327 + 107 in PBO-treated group vs. 164 + 42 in SPL-treated group. SPL eyedrops significantly reduced the number of inflammatory cells in rat corneas (p=0.0195 for IBA1 and p=0.0303 for EDI) (Fig. 7C and D).

[0268] Example 10: SPL eyedrops improved corneal reinnervation in the rat model of corneal wound healing

[0269] We evaluated corneal reinnervation 5 days after topical PBO and SPL eyedrop treatments. In control uninjured corneas, TUBB3 immunostaining showed a dense linear architecture of the sub-basal nerves on corneal flat mounts. Corneal de-epithelialization mechanically removed the sub-basal nerve network (Fig. 8A). Slow sub-basal nerve regeneration from the peripheral cornea to the de-epithelialized central cornea was observed in both PBO and SPL-treated groups up to day 5 (Fig. 8A). In the reinnervated area, SPL eyedrops seemed to improve the sub-basal nerve density in the reinnervated corneal area compared to PBO-treated corneas. Moreover, the regenerated nerves appeared longer and more parallelly oriented in SPL-treated corneas than in PBO-treated corneas (Fig. 8A). The density of corneal sub-basal nerve plexus was 54.95 % + 2.51 relative to control uninjured corneas in the PBO-treated group, and 70.32 % + 5.75 in the SPL-treated group, although the difference was not significant due to the small sample size.

Claims

CLAIMS1. An aqueous ophthalmic composition comprising:Spironolactone,Cyclodextrin,Trometamol.

2. The aqueous ophthalmic composition according to claim 1 , wherein the cyclodextrin is chosen among 2-hydroxypropyl-P-cyclodextrin, hydroxyethyl- -cyclodextrin, 2-hydroxypropyl-y- cyclodextrin, hydroxyethyl-y-cyclodextrin, dihydroxypropyl-P-cyclodextrin, glucosyl-a- cyclodextrin, glucosyl-P-cyclodextrin, diglucosyl-P-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-P-cyclodextrin, maltosyl-y-cyclodextrin, maltotriosyl-P-cyclodextrin, maltotriosyl-y- cyclodextrin, dimaltosyl-P-cyclodextrin, maltosyl-P-cyclodextrin / dimaltosyl-P-cyclodextrin, methyl-P-cyclodextrin, and mixtures thereof, preferably among 2-hydroxypropyl-P- cyclodextrin and 2-hydroxypropyl-y-cyclodextrin.

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

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

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

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

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

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

9. The aqueous composition according to anyone of claims 1 to 8, wherein the composition comprises:0.1 % by weight, based on the total weight of the composition of spironolactone,3% by weight, based on the total weight of the composition of cyclodextrin,0.6 % by weight, based on the total weight of the composition of trometamol.

10. The aqueous composition according to claim 9, wherein the composition further comprises:HC1 in an amount sufficient to adjust the pH of said ophthalmic composition to 7.0 to 7.4.Sodium chloride in an amount sufficient to adjust the tonicity of said ophthalmic composition to 280mOsmol / kg.

11. An aqueous ophthalmic composition according to anyone of claims 1 to 10, for use for restoring epithelial integrity, for improving corneal re-epithelialization, for improving epithelial cell adhesion and differentiation with cell layer stratification, for recovering the optical function of the anterior cornea, for reducing of the inflammation, for decreasing stromal edema, for restoring corneal transparency, or for improving corneal reinnervation.

12. An aqueous ophthalmic composition according to anyone of claims 1 to 10, for use in the treatment of a disease or a disorder selected from the group comprising corneal ulcer with or without inflammation, post operative corneal wound, corneal re epithelialization delay following corneal graft surgery, corneal graft rejection, vernal keratitis, ocular rosacea,epithelial keratitis secondary to dry eye syndrome or to meibomian gland dysfunction, corneal ulcer post corneal cross linking, corneal wound healing delay due to refractive surgery or to corneal surgery or to anterior segment surgery, corneal trauma, corneal inflammation due to bacterial, fungi, parasite or to viral infection or to non-conventional agent infection, corneal opacification or corneal epithelial defect due to neurotrophic keratopathy, corneal opacification due to corneal scar, corneal edema, corneal inflammation or neovascularization due to vernal keratoconjunctivitis, corneal inflammation due to ocular rosacea, corneal neovascularization, corneal neovascularization secondary to inflammation or to meibomian gland dysfunction or to infection or to limbal deficiency or to corneal graft rejection, recurrent corneal erosions, wound healing delay, post-surgical treatment of corneal graft or refractive surgery, or any other corneal surgery, glucocorticoid-induced epithelial wound healing delay following corneal physical trauma, inherited and genetic keratopathy, aniridia-induced keratopathy, peripheral ulcerative keratitis, corneal neovascularization, meibomian gland dysfunction and associated diseases such as dry eye syndromes and blepharitis and corneal diseases chosen among diabetic, neurotrophic, toxic, iatrogenic, preservatives-induced keratopathies.

13. An aqueous ophthalmic composition according to anyone of claims 1 to 10, to be administered with glucocorticoid therapy.

14. A method of making the aqueous ophthalmic composition according to anyone of claims 1 to 10, comprising the following steps:(i) Dissolving cyclodextrins in water(ii) Adding spironolactone under stirring until complete dissolution,(iii) Adding trometamol and a tonicity agent in an amount sufficient to adjust the tonicity of the composition from 280 to 310mOsmol / kg, preferably from 280 to 300m0smol / kg and preferably of 280 mOsmol / kg,(iv) Adding a pH adjusting agent in an amount sufficient to adjust the pH of the composition from 6.8 to 7.5, preferably from 7 to 7.5, and more preferably from 7.0 to 7.

415. A device for dropwise dispensing of a composition, the device comprising a container holding the composition as claimed in anyone of claims 1 to 10.

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