Ophthalmic formulations

Abstract

The present invention relates to ophthalmic compositions comprising troxerutin and a surfactant selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, and mixtures thereof, wherein the weight ratio of troxerutin to the surfactant is from 0.02:1 to 100:1, wherein the surfactant is present in an amount of up to 7 wt% with respect to the total weight of the composition, and wherein the composition is in the form of a solution, a suspension or a gel. The invention also relates to said composition for use in the treatment of dry eye disease.

Description

[0001] OPHTHALMIC FORMULATIONS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to ophthalmic compositions comprising troxerutin and specific non-ionic surfactants, and to their use in the treatment of dry eye disease.

[0004] BACKGROUND OF THE INVENTION

[0005] The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber. The human cornea has several layers. The outermost layer is the corneal epithelium that keeps moist by the overlying tear film. The cornea is continuous with the conjunctival epithelium. The conjunctiva lines the inside of the eyelids and covers the sclera. It is composed of non-keratinized, stratified columnar epithelium with goblet cells, and also stratified columnar epithelium. The conjunctiva helps lubricate the eye by producing mucins. It also contributes to immune surveillance and helps to prevent the entrance of microbes into the eye.

[0006] The tear film is the interface between the ocular surface epithelium and the environment and consists in three distinct layers, from the most outer surface: lipid layer, aqueous layer and mucous layer. The mucous layer coats the cornea, provides a hydrophilic layer and allows for even distribution of the tear film. The mucins present in the tear film serve to maintain the hydration of the ocular surface and to provide lubrication and antiadhesive properties between the cells of the ocular surface and conjunctiva during the blink and to contribute to the epithelial barrier to prevent pathogens from binding to the ocular surface.

[0007] Dry eye disease is a common condition provoking changes in tear film and ocular surface. Dry eye disease occurs when the eye does not produce tears properly, or when the tears are not of the correct consistency and evaporate too quickly. Untreated dry eye can cause ocular infections, corneal ulcer and blindness. Dry eye is generally associated with inflammation of the surface of the eye, the lachrymal gland or the conjunctiva and any disease that changes the composition of tears. In dry eye syndrome, whatever may be the initial cause, chronic dryness of the surface of the eye leads to neurogenic inflammation, subsequent to activation of T cells and release of inflammatory cytokines into the lachrymal glands, tear fluid and conjunctiva. In later stages, these inflammatory mediators may even cause gradual dysfunction and destruction of the lachrymal glands and impairment of conjunctival epithelium. Thus, lachrymal glands are deprived of normal trophic stimulation required for regular maintenance. Once the disease is initiated, inflammation becomes the key mechanism of ocular surface injury. Recent evidence also suggests a role for oxidative stress in the primary initiating events that lead to the corneal, conjunctival and lachrymal gland injury. Thus, oxidative stress with associated inflammatory process can trigger this disease state.

[0008] Thus, strategies for the management of dry eye disease (including keratoconjunctivitis sicca, xerophthalmia, xerosis and Sjogren's syndrome), include lubrication of the surface of the eye and avoiding excessive tear evaporation from the ocular surface. Lubrication of the surface of the eye and helping tears to remain on the surface of the eye improves the symptoms of dry eye. Currently, artificial tear formulations are used for the treatment of dry eye disease, as well as ophthalmic formulations comprising cyclosporine. Polyphenols, including flavonoids, have been reported as promising natural molecules that are receiving increasing attention for their activity / effects in counteracting the main pathologic mechanisms of dry eye disease and reducing its symptoms [Favero, G. et al, Antioxidants 2021 , 10, 190], Polyphenols present multiple effects against dry eye diseases-related ocular surface injury, in particular, the observed beneficial effects of polyphenols on corneal cells are the reduction of the pathological processes of inflammation, oxidative stress, and apoptosis and modulation of the tear film [Favero, G. et al., Antioxidants 2021 , 10, 190], Topical application of quercetin has been reported to help to improve ocular surface disorders of dry eye not only by decreasing the corneal surface irregularity but also by increasing the tear volume and goblet cell density, as well as due to its antiinflammatory effects on the lacrimal functional unit [Oh, H.N. et al, Cornea, 2015; 34, 9,1130],

[0009] Few new treatment options have been introduced for dry eye disease, despite the fact that it is the most prevalent ophthalmic disease which affects a substantial segment of people worldwide with increasing frequency. Up till now lubricant eye drops or ointments to overcome the symptoms of dry eye are the mainstay treatment, however, they cannot relieve the underline causes of dry eye. The actual drug products (cyclosporine A or corticosteroids) are associated with ocular toxicity or adverse effects after long-lasting topical application.

[0010] Therefore there is a need in the art for alternative treatments for managing dry eye disease, including keratoconjunctivitis sicca, xerophthalmia, xerosis and Sjogren's syndrome.

[0011] SUMMARY OF THE INVENTION

[0012] Ocular administration of compositions is particularly challenging due to the wash-out of the formulation. The inventors have surprisingly found that a mixture of troxerutin and specific non-ionic surfactants have a synergistic effect in mucoadhesion, lower wash-out and have shown to be useful for the treatment of dry eye disease.

[0013] Thus, in the first aspect, the present invention relates to a sterile ophthalmic composition comprising troxerutin and a surfactant selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate , polyoxyethylene (20) sorbitan monolaurate , and mixtures thereof, wherein the weight ratio of troxerutin to the surfactant is from 0.02:1 to 100:1 , wherein the surfactant is present in an amount of up to 7 wt% with respect to the total weight of the composition, and wherein the composition is in the form of a nanoemulsion, a solution, a micellar solution, an emulsion, a microemulsion, a suspension or a gel.

[0014] In a second aspect, the invention relates to a composition as defined in the first aspect for use in medicine.

[0015] In a third aspect, the invention relates to a composition as defined in the first aspect for use in the treatment and / or prevention of dry eye disease.

[0016] DESCRIPTION OF THE FIGURES

[0017] Figure 1 shows the wash-out effect for the system troxerutin (TRX) - Kolliphor® EL (KEL). The amount of released troxerutin (%) for up to 12 hours is represented. Figure 2 shows the wash-out effect for the system troxerutin (TRX) - Kolliphor® RH40 (RH40). The amount of released troxerutin (%) for up to 12 hours is represented.

[0018] Figure 3 shows the wash-out effect for the system troxerutin (TRX) - Tween® 80 (T80). The amount of released troxerutin (%) for up to 12 hours is represented.

[0019] Figure 4 shows the results of the mucoadhesion in vitro cell model. Troxerutin absorbance signal is detected directly on cell surface and expressed as fold signal versus control. The system troxerutin (TRX) - Kolliphor® EL (KEL) is studied after 60 minutes of incubation followed by further cell culture repeated washes.

[0020] Figure 5 shows the results of the mucoadhesion in vitro cell model. Troxerutin absorbance signal is detected directly on cell surface and expressed as fold signal versus control. The system troxerutin (TRX) - Tween® 80 (T80) is studied after 60 minutes of incubation followed by further cell culture repeated washes.

[0021] Figures 6 and 7 show the results of the mucoadhesion in vitro cell model. Troxerutin absorbance signal is detected directly on cell surface and expressed as fold signal versus control. The system 10% troxerutin (TRX) - Kolliphor® EL (KEL) is studied after 60 minutes of incubation followed by further cell culture repeated washes.

[0022] Figure 8 shows the Schirmer test of troxerutin solution (TRX Solution) and composition comprising troxerutin (TRX) and Kolliphor® EL (KEL) (TRX Composition).

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Composition of the invention

[0025] In the first aspect, the present invention relates to a sterile ophthalmic composition comprising troxerutin and a surfactant selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, and mixtures thereof, wherein the weight ratio of troxerutin to the surfactant is from 0.02:1 to 100:1, wherein the surfactant is present in an amount of up to 7 wt% with respect to the total weight of the composition, and wherein the composition is in the form of a nanoemulsion, a solution, a micellar solution, an emulsion, a microemulsion, a suspension or a gel.

[0026] The term “sterile”, when characterizing the compositions of the invention, means that said compositions has been aseptically processed and that is devoid of viable bacteria, fungi or other microorganisms. The sterilization can be performed according to methods well known in the state of the art. In an embodiment, the sterilization step is performed by a method selected from the group consisting of filtration, autoclaving, heating, irradiation, and combination thereof; preferably the sterilization step is performed by filtration. These sterilization methods are well known in the art and correspond to well established pharmaceutical operational procedures.

[0027] The term “ophthalmic”, when characterizing the compositions of the invention, means that said compositions are suitable for their application to the eye. Typically, suitable compositions for application to the eye have a pH from 4.0 to 9.0, preferably from 6.0 to 8.0. Preferably, the ophthalmic compositions of the invention have an osmolality from 100 to 1711 mOsm / kg, from 100 to 500 mOsm / kg, preferably 200 to 400 mOsm / kg.

[0028] The compositions of the invention comprise troxerutin. “Troxerutin” or 2-[3,4-bis(2- hydroxyethoxy)phenyl]-5-hydroxy-7-(2-hydroxyethoxy)-4-oxo-4H-chromen-3-yl 6-O-(6- deoxy-p-D-mannopyranosyl)-p-D-glucopyranoside has CAS number 7085-55-4.

[0029] Troxerutin has been reported to possess antioxidative and anti-inflammatory activity [Fan SH, et al., Int Immunopharmacol, 2009, 9, 91-96; Zhang ZF et al., J Agric Food Chem, 2009, 57, 7731-7736; and Panat, N. A. et al., Food Chemistry, 2016, 194, 32-45],

[0030] Preferably, the amount of troxerutin in the compositions of the invention is from 0.05 to 15%, more preferably 0.15 to 15 wt% with respect to the total weight of the composition, more preferably from 1 to 15 wt%, still more preferably from 1 to 12 wt%, still more preferably from 5 to 15 wt%, still more preferably from 5 to 12 wt%, still more preferably from 8 to 12 wt%, the most preferred about 10 wt%.

[0031] In the context of the present invention, the term “about” refers to the indicated value ± The compositions of the invention comprise a surfactant selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, and mixtures thereof; preferably, a surfactant selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate, and mixtures thereof; more preferably polyoxyl 35 castor oil. In an embodiment, the compositions of the invention comprise this / these surfactant(s) as the only surfactant present in the composition.

[0032] “Polyoxyl 35 castor oil” refers to a nonionic surfactant which is made by reacting castor oil with ethylene oxide in a molar ratio of 1 :35. The main component of polyoxyl 35 castor oil is glycerol polyethylene glycol ricinoleate. Together with fatty acid esters of polyethylene glycol, this forms the hydrophobic part of the product. The smaller hydrophylic part consists of free polyethylene glycols and ethoxylated glycerol Polyoxyl 35 castor oil is commercialized e.g. as Kolliphor® EL (CAS number 61791-12-6).

[0033] “Polyoxyl 40 hydrogenated castor oil” refers to a nonionic surfactant which is obtained by reacting 1 mole of hydrogenated castor oil with 40 moles of ethylene oxide. The main constituent of polyoxyl 40 hydrogenated castor oil is glycerol polyethylene glycol hydroxystearate, which, together with fatty acid glycerol polyglycol esters, forms the hydrophobic part of the product. The hydrophilic part consists of polyethylene glycols and glycerol ethoxylate. Polyoxyl 40 hydrogenated castor oil is commercialized e.g. as Kolliphor ® RH 40 (CAS number 61788-85-0).

[0034] “Polyoxyethylene (20) sorbitan monooleate”, also known as Polysorbate 80, refers to a non-ionic surfactant derived from polyethoxylation of sorbitan oleate esters (sorbitan monooleate). Polyoxyethylene (20) sorbitan monooleate is commercialized e.g. as Tween® 80 (CAS number 9005-65-6).

[0035] “Polyoxyethylene (20) sorbitan monolaurate”, also known as Polysorbate 20, refers to a non-ionic surfactant derived from polyethoxylated sorbitan and fatty acids. Polyoxyethylene (20) sorbitan monolaurate is commercialized e.g. as Tween® 20 (CAS number 9005-64-5). In the compositions of the invention, the surfactant as defined above is present in an amount of up to 7 wt% with respect to the total weight of the composition, preferably, the amount of surfactant in the compositions of the invention is from 0.15 to 7 wt% with respect to the total weight of the composition, more preferably from 0.25 to 7 wt%, more preferably from 0.5 to 7 wt%, more preferably from 0.15 to 6.5 wt%, still more preferably from 0.15 to 5 wt%, the most preferred about 5 wt%.

[0036] In the compositions of the invention, the weight ratio of troxerutin to the surfactant as defined above is from 0.02:1 to 100:1 , preferably from 1.5:1 to 100:1 , more preferably from 1.5:1 to 50:1 , more preferably from 1.5:1 to 25:1 , more preferably from 1.5:1 to 15:1 , more preferably from 1.5:1 to 10:1 , still more preferably from 1.5:1 to 5:1 , the most preferred from 1 :1 to 4:1.

[0037] In the compositions of the invention, the total amount of troxerutin and the surfactant as defined above, i.e. the sum of the amount of troxerutin and the amount of the surfactant as defined above, is not greater than 22 wt% with respect to the total weight of the composition, more preferably not greater than 20 wt%, more preferably not greater than 17 wt%, still more preferably not greater than 15 wt%.

[0038] In one embodiment of the invention the compositions further comprise water. Preferably, the water is present in an amount of at least 40 wt% with respect to the total weight of the composition, more preferably at least 50 wt%, more preferably at least 60 wt %, more preferably at least 70 wt%, more preferably at least 80 wt%, more preferably at least 85 wt%, more preferably at least 90 wt%. In a particularly preferred embodiment, the compositions of the present invention comprise at least 60 wt% of water with respect to the total weight of the composition.

[0039] In another preferred embodiment, the compositions of the present invention may further comprise one or more tonicity adjusting agents. Said tonicity adjusting agents are used for adjusting the osmolality of the compositions of the invention, preferably to an osmolality of from 100 to 1711 mOsm / kg, more preferably from 100 to 500 mOsm / kg, still more preferably from 200 to 400 mOsm / kg. Preferably said one or more tonicity adjusting agent is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate (also known as sodium hydrogen carbonate), calcium carbonate, sodium lactate, sorbitol, mannitol, xylitol, glycerin, dextrose, polyethylene glycol, propylene glycol, dextran, and mixtures thereof. More preferably said one or more tonicity adjusting agent is selected from the group consisting of sodium chloride, glycerin, propylene glycol, and mixtures thereof. Still more preferably said one or more tonicity adjusting agent is glycerin. The tonicity adjusting agent may be absent or present in the compositions of the invention. In a particular embodiment, the tonicity adjusting agent is present in an amount of 0.05 to 15 wt% with respect to the total weight of the composition.

[0040] In another preferred embodiment, the compositions of the present invention may further comprise one or more pH adjusting agents. Said pH adjusting agents are used for adjusting the pH of the compositions of the invention, preferably from 4.0 to 9.0, more preferably from 6.0 to 8.0. Preferably wherein the one or more pH adjusting agent is selected from the group consisting of lactic acid and salts thereof (such as sodium lactate, potassium lactate and calcium lactate), citric acid and salts thereof (such as sodium citrate, potassium citrate, calcium citrate, lithium citrate, trisodium citrate and disodium hydrogen citrate), tartaric acid and salts thereof (such as sodium tartrate potassium tartrate, calcium tartrate and lithium tartrate), acetic acid and salts thereof (such as sodium acetate, potassium acetate and calcium acetate), hydrochloric acid, boric acid and salts thereof (sodium borate), sulphuric acid and salts thereof (such as sodium sulphate and potassium sulphate), nitric acid, hydrochloric acid, phosphoric acid and salts thereof (such as sodium dihydrogen phosphate, sodium monohydrogen phosphate, potassium dihydrogen phosphate lithium phosphate, potassium phosphate and calcium phosphate), carbonic acid and salts thereof (such as sodium carbonate, sodium hydrogen carbonate and potassium hydrogen carbonate), maleic acid and salts thereof (lithium maleate, sodium maleate, potassium maleate and calcium maleate), succinic acid and salts thereof (lithium succinate, sodium succinate, potassium succinate and calcium succinate), sodium hydroxide, potassium hydroxide, triethanolamine, diisopropanolamine, ammonia, tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, and mixtures thereof. More preferably wherein the one or more pH adjusting agent is selected from the group consisting of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and mixtures thereof. The pH adjusting agent may be absent or present in the compositions of the invention. In a particular embodiment, the pH adjusting agent is present in an amount of 0.01 to 5 wt% with respect to the total weight of the composition. The compositions of the present invention may also comprise oils, viscosity increasing agents, preservatives, chelating agents and / or penetration enhancers.

[0041] The term "oil" is used herein in a general sense to identify a wide class of substances typically unctuous, viscous and liquid at room temperature. Oil as here in defined can be from animal, mineral, vegetable or synthetic origin. The term "oils" refers to oil, or a combination of multiple oils. The term "room temperature" refers to a temperature of the environment, without heating or cooling, and is generally from 20 °C to 25 °C.

[0042] In an embodiment, the oil present in the compositions of the invention is a C4-C20 alkyl ester of monoglyceride, diglyceride, or triglyceride and mixtures thereof. In an embodiment, the (C4-C20) alkyl ester of monoglyceride, diglyceride or triglyceride is medium chain triglycerides. The term "medium chain triglycerides" and "MCT" have the same meaning and are used interchangeable and refer to triesters of glycerin and Ce- C12 fatty acids. In an embodiment, the oil is a medium chain triglyceride selected from caproic acid, caprylic acid, capric acid, lauric acid and mixtures thereof; preferably the oil is a caprylic / capric acid triglyceride.

[0043] In an alternative embodiment, the oil present in the compositions of the invention is selected from the group consisting of ethyl oleate, decyl oleate, isopropyl myristate, isopropyl palmitate, isopropyl isostearate, isostearyl isostearate, glyceryl monosterate, myristyl lactate, ethylhexyl hydroxystearate, ethylhexyl pelargonate, triethylhexanoin, isohexadecane, light mineral oil, mineral oil, vegetable oil, triisononanoin, C12-C15 alkyl benzoate, castor oil, monoglyceride, diglyceride or triglyceride esters, and mixtures thereof;

[0044] In an another embodiment, the oil component of the present invention is selected from the group consisting of castor oil, (C4-C20) alkyl ester of monoglyceride, diglyceride or triglyceride (preferably medium chain triglycerides), ethyl oleate, decyl oleate, isopropyl myristate, isopropyl palmitate, isopropyl isostearate, isostearyl isostearate, argan oil, triisononanoin, C12-C15 alkyl benzoate, and mixtures thereof.

[0045] In a particular embodiment, the oil component of the present invention is selected from the group consisting of castor oil, (C4-C20) alkyl ester of monoglyceride, diglyceride or triglyceride, and mixtures thereof, preferably from the group consisting of castor oil, medium chain triglycerides, and mixtures thereof.

[0046] The term "alkyl" refers to a saturated, branched or linear alkyl chain which contains the number of carbon atoms specified in the description or claims.

[0047] The term "vegetable oil" refers to a triglyceride extracted from a plant. Examples of vegetable oils are argan oil, corn oil, palm oil, coconut oil, cottonseed oil, olive oil, peanut oil, rapeseed oil, sunflower oil, sesame oil, soybean oil, safflower oil, castor oil, olive oil, and mixture thereof.

[0048] The oil may be absent or present in the compositions of the invention. In a particular embodiment, the oil is present in an amount of 0.01 to 20 wt% with respect to the total weight of the composition.

[0049] The term “viscosity increasing agent” refers to a substance which can increase the viscosity of the compositions of the invention. Examples of viscosity increasing agents are polyvinylpirrolidones, such as Povidone K 17, Povidone K25, Povidone K 30 and Povidone K 90F; polyvinyl alcohol; xanthan gum; guar gum; welan gum; gellan gum; tragacanth gum; ceratonia gum; agar; methylcellulose; ethylcellulose; hydroxyethyl cellulose; hydroxyethylmethyl cellulose; hydroxypropyl cellulose; hydroxypropylmethyl cellulose; hydroxypropylmethyl cellulose phthalate; hydroxypropylmethyl cellulose acetate succinate; sodium carboxymethylcellulose; calcium carboxymethylcellulose; polyethylene glycol; glycerine; carrageenan; alginic acid; sodium alginate; potassium alginate; propylene glycol alginate; hyaluronic acid; sodium hyaluronate; poly(acrylic acid) derivatives such as carbomer and polycarbol; poloxamers; chitosan and chitosan derivatives; maltodextrin; and mixtures thereof. In a particular embodiment, the compositions of the invention comprise a viscosity increasing agent, preferably selected from the group consisting of sodium alginate, polyvinylpyrrolidone, gellan gum, chitosan or a derivative thereof, and mixtures thereof.

[0050] The viscosity increasing agent may be absent or present in the compositions of the invention. In a particular embodiment, the viscosity increasing agent is present in an amount of from 0.1 to 15 wt% with respect to the total weight of the compositions. The term “preservative” refers to substances formulated in the compositions of the invention to prevent microbial contamination thereof. Examples of preservatives are benzalkonium chloride, bezethonium chloride, chlorhexidine, benzyl alcohol, chlorobutanol, 2-phenylethanol, propylparaben, methylparaben, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, cetyl pyridinium chloride, benzyl bromide, sodium perborate and thimerosal.

[0051] Alternatively, the compositions of the invention are devoid of preservatives; preferably they are devoid of benzalkonium chloride, thimerosal, benzethonium chloride, cetyl pyridinium chloride, benzyl bromide, chlorhexidine and / or sodium perborate; more preferably, the compositions of the invention are devoid of benzalkonium chloride.

[0052] The term “chelating agent” refers to a substance that coordinates with a metal ion. Examples of chelating agents are citric acid, in particular citric acid monohydrate, EDTA (ethylenediaminetetraacetic acid) and its salts, such as dipotassium EDTA, disodium EDTA, calcium disodium EDTA, sodium EDTA and trisodium EDTA, fumaric acid, malic acid, and maltol.

[0053] The compositions of the invention may also comprise further ingredients such as lanolin, white wax and / or petrolatum.

[0054] The term “white wax” refers to a chemically bleached form of yellow wax. Its CAS number is 8012-89-3.

[0055] The term “petrolatum” refers to a purified mixture of semisolid saturated hydrocarbons having the general formula CnH2n+2, which may be obtained from petroleum. Its CAS number is 8009-03-8.

[0056] In a particular embodiment, the compositions of the invention are devoid of gellan gum. In another particular embodiment, the compositions of the invention are devoid of chitosan and chitosan derivatives. In another particular embodiment, the compositions of the invention are devoid of paraffin oil. In another particular embodiment, the compositions of the invention are devoid of shea butter. In another particular embodiment, the compositions of the invention are devoid of sodium pyroglutamate. In another particular embodiment, the compositions of the invention are devoid of octylmethoxycinnamate. In another particular embodiment, the compositions of the invention are devoid of petrolatum. In another particular embodiment, the compositions of the invention are devoid of diisopropyl adipate. In another particular embodiment, the compositions of the invention are devoid of propane. In another particular embodiment, the compositions of the invention are devoid of purine alkaloids. In another particular embodiment, the compositions of the invention are devoid of phenyl benzimidazole tetrasulfonic acid and its salts. In another particular embodiment, the compositions of the invention are devoid of sodium hyaluronate. In another particular embodiment, the compositions of the invention are devoid of dihydroxyacetone. In another particular embodiment, the compositions of the invention are devoid of titanium dioxide. In another particular embodiment, the compositions of the invention are devoid of vitamin E. In another particular embodiment, the compositions of the invention are devoid of proteins. In another particular embodiment, the compositions of the invention are devoid of DNA. In another particular embodiment, the compositions of the invention are devoid of polyacrylic acid. In another particular embodiment, the compositions of the invention are devoid of DNA and polyacrylic acid.

[0057] In another particular embodiment, the compositions of the invention are devoid of gellan gum, chitosan, chitosan derivatives, paraffin oil, shea butter, sodium pyroglutamate, octylmethoxycinnamate, petrolatum, diisopropyl adipate, propane, purine alkaloids, phenyl benzimidazole tetrasulfonic acid and its salts, sodium hyaluronate, dihydroxyacetone, titanium dioxide, vitamin E, and proteins.

[0058] In another particular embodiment, the compositions of the invention are devoid of gellan gum, chitosan, chitosan derivatives, paraffin oil, shea butter, sodium pyroglutamate, octylmethoxycinnamate, petrolatum, diisopropyl adipate, propane, purine alkaloids, phenyl benzimidazole tetrasulfonic acid and its salts, sodium hyaluronate, dihydroxyacetone, titanium dioxide, vitamin E, proteins, polyacrylic acid and DNA.

[0059] In the context of the present invention, the expression “one or more” refers to 1 , 2, 3, 4 or 5, preferably to 1 , 2, 3 or 4, more preferably to 1 , 2 or 3, and even more preferably to 1 or 2. The compositions of the invention are in the form of a nanoemulsion, a suspension, a gel, a solution a micellar solution, an emulsion, or a microemulsion; preferably a nanoemulsion.

[0060] The term “suspension” refers to a heterogeneous mixture of solid particles in a liquid medium (or solution), i.e. the solid particles do not dissolve but remain disperse throughout the liquid medium.

[0061] The term “gel” refers to a non-fluid colloidal system or polymer system that is expanded throughout its whole volume by a fluid. As consequence, a gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are mostly liquid by mass, yet they behave like solids because of a three-dimensional cross-linked network within the liquid. It is the cross-linking within the fluid that gives a gel its structure (hardness) and contributes to the adhesive stick (tack). In this way, gels are a dispersion of molecules of a liquid within a solid medium.

[0062] The term “solution” refers to a liquid homogeneous mixture in which the minor component (the solute) is uniformly solubilized within the major component or solvent.

[0063] The term “micellar solution” refers to a type of solution wherein one or more surfactants are solubilized in a liquid phase in form of micelles, due to the concentration of the surfactant which is above the critical micellar concentration. Micellar solutions are transparent since the micelle size is approximately less than 5 nm.

[0064] The term “emulsion” refers to a dispersion wherein a liquid (the dispersed phase) is dispersed in another liquid (the continuous phase), and wherein both liquids are immiscible. Emulsions contain both a dispersed and a continuous phase, with the boundary between the phases called the “interface” and they are generally stabilized by surfactant molecules. Emulsions tend to have a milky appearance because the droplet size of the disperse phase is above 0.5 microns, preferably from 0.5 to 500 microns.

[0065] The term “microemulsion” refers to a thermodynamically stable isotropic liquid formed by mixing oils, water, and surfactants together. The interface between continuous and internal phase is stabilized by an appropriate combination of surfactants and / or cosurfactants. Mixtures of oils, water, and surfactants can form a variety of different systems depending on their composition and the environmental conditions (particularly temperature). In such systems, dispersed phase droplet does not refract light; hence the dispersed phase appeared invisible to naked eye. Droplet size from 1 to 200 nm and therefore such system is transparent.

[0066] The term “nanoemulsion” refers to a type of emulsion having an average size of the dispersed droplets from 1 to 500 nm. These type of emulsions differ from the microemulsions in that they are not thermodynamically stable. Thus, microemulsions are spontaneously formed when mixing the ingredients, whereas nanoemulsions are not spontaneously formed. The production of nanoemulsions requires an energy input. Nanoemulsion can be prepared according to methods well known in the state of the art for the preparation of nanoemulsion. Typically, nanoemulsions may be produced using well known in the art, such as combining the different components and mixing, in particular by homogenization at high pressure or ultrasounds. However, nanoemulsions can also be produced using a simple process under mild conditions and without the need of a homogenizing step at high pressure. As an example, they may be formed by phase inversion composition (PIC). In an embodiment, the process for the preparation of the nanoemulsion comprises: (a) preparing the oil phase by mixing the oil components and the surfactants; (b) preparing the aqueous phase; (c) emulsifying the oil phase obtained in step (a) in the aqueous phase obtained in step (b); (d) optionally, adjusting the pH; the osmolality; the pH and the osmolality after step (a), step (b) or step (c); and (e) optionally, adding one or more additional pharmaceutically acceptable excipients or carriers in step (a), step (b) or step (c).

[0067] The terms "average size" and "mean size" have the same meaning and are used interchangeable. They refer to average diameter of the droplets. The average size of these systems can be measured by standard processes known by persons skilled in the art such as Dynamic Light Scattering (DLS). By "average size" and "mean size" is understood a D(n,50) droplet average size in number, or a zeta average size (Zave) in intensity. The D(n,50) droplet average size is the median diameter, where 50% of the droplets are composed of droplets larger than the stated value, and 50% of the droplets are composed of droplets smaller than the stated value. In the present invention, the measurement of the average size of the droplets was performed by Dynamic Light Scattering (DLS). DLS makes use of two common characteristics of colloids, the Tyndall effect (scattering) and the Brownian motion which cause light to be scattered at different intensities. Analyses of the time depend on the intensity fluctuations using mathematical models, allows the determination of the zeta average size (cf. Hassan, P. et al, "Making sense of Brownian motion: colloid characterization by dynamic light scattering", Langmuir, 2015, vol. 31 , pp.3-12). The droplets are constantly moving due to Brownian motion and the relationship between the size of a droplets and its speed due to Brownian motion is defined in the Stokes-Einstein equation. As the droplets move around, the scattered light will cause intensity fluctuations. Furthermore, the signal intensity is compared at different times with itself in order to obtain the correlation function. This information can then be used to calculate the size distribution by intensity and it can be converted to a volume or a number size distribution. Particularly, the diameter of the droplets (i.e. the mean size of the droplets) is determined using a Zetasizer Nano ZS (Malvern Instruments). In the present invention the measurement of the average size (Zave or D(n,50)) of the droplets was directly measured (without dilution) by dynamic light scattering (DLS) with Zetasizer Nano ZS (Malvern Instruments) performing the calculations explained above in the present application.

[0068] In a preferred embodiment, the compositions of the invention are in the form of a nanoemulsion.

[0069] In a particular embodiment, the compositions of the invention are in the form of a nanoemulsion and further comprise an oil, preferably an oil selected form the group consisting of castor oil, medium chain triglyceride and mixtures thereof.

[0070] In a particular embodiment, the composition of the invention comprises:

[0071] 1 to 15 wt% of troxerutin, and

[0072] 0.15 to 7 wt% of polyoxyl 35 castor oil.

[0073] In another particular embodiment, the composition of the invention comprises

[0074] 1 to 15% of troxerutin, and

[0075] 0.15 to 7% of polyoxyl 35 castor oil 0.01% to 20% castor oil

[0076] In another particular embodiment, the composition of the invention comprises 1 to 15% of troxerutin, and 0.15 to 7% of polyoxyl 35 castor oil 0.05% to 5% castor oil

[0077] In another particular embodiment, the composition of the invention comprises

[0078] 1 to 14% of troxerutin, and

[0079] 0.15 to 6.5 % of polyoxyl 35 castor oil

[0080] 0.05% to 3% medium chain trygliceride

[0081] The compositions of the invention may be prepared by combining the different components described above and mixing.

[0082] An example for the preparation of composition as defined in the first aspect of the invention, which comprises: (a) providing the surfactant or mixture of surfactants; (b) optionally adding water (when water is present in the composition); (c) optionally, adjusting the pH and / or the osmolality; (d) optionally, adding one or more additional pharmaceutically acceptable excipients or carriers (such as tonicity adjusting agents, pH adjusting agents, oils, viscosity increasing agents, preservatives .chelating agents and / or penetration enhancers) in step (a), step (b) and / or step (c); (d) adding troxerutin in step

[0083] (a), step (b), step (c) and / or step (d).

[0084] Another example for the preparation of the composition as defined in the first aspect of the invention, which comprises: (a) preparing an oil phase by mixing the oil and the surfactants; (b) emulsifying the oil phase by adding water in a stepwise procedure (c) optionally, adjusting the pH, the osmolality, the pH and the osmolality after step (a), or step (b); (d) optionally, adding one or more additional pharmaceutically acceptable excipients or carriers (such as tonicity adjusting agents, pH adjusting agents, oils, viscosity increasing agents, preservatives, chelating agents and / or penetration enhancers) in step (a), step (b), and / or step (c); and (e) adding troxerutin in step (a), step

[0085] (b), step (c), and / or step (d).

[0086] The compositions of the present invention can be prepared according to well known methods widely described in the state of the art for the preparation of colloidal systems. In an embodiment, the process for the preparation of the nanoemulsions of the present invention is performed by phase inversion composition. In an embodiment, the process for the preparation of the oil-in-water nanoemulsion composition as defined above comprises: (a) preparing an oil phase by mixing the oil and the surfactants; (b) emulsifying the oil phase by adding water in a stepwise procedure; (c) optionally, adjusting the pH; the osmolality; the pH and the osmolality after step (a) or step (b); (d) optionally, adding one or more additional pharmaceutically acceptable excipients or carriers (such as tonicity adjusting agents, pH adjusting agents, oils, viscosity increasing agents, preservatives, chelating agents and / or penetration enhancers) in step (a), step (b) and / or step (c); and (e) adding troxerutin after step (a), step (b), step (c) and / or step (d).

[0087] In an embodiment, step (a) is performed by mixing in a suitable container until a homogenous mixture is obtained.

[0088] In an embodiment, step (c) is performed by emulsifying the oil phase with the water keeping a continuous mixing procedure at temperatures between 10 °C to 60 °C. In an embodiment, step (c) is performed when the temperature of the oil phase obtained in step (a) is close to the temperature of the water added in step (b). The expression "the temperature of the oil phase is close to the temperature of the water" means that the temperature value is "approximate" due to the measurement error. It should be understood that "close" corresponds to a given temperature value ± 10 °C. The variability of the values is due to the inherent sensibility of the method.

[0089] The composition of the invention can be performed by a simple process under mild conditions and without the need of a homogenizing step at high pressure. Methods related to high energy procedures (for instance high pressure and ultrasounds) may negatively impact on shelf life of active ingredient and the stability of the final dosage form.

[0090] Therefore, the process for preparing the composition of the present invention is advantageous for the stability of the active ingredient.

[0091] Medical uses of the compositions of the invention

[0092] MLIC5AC and MLIC5B are among the major gel-forming mucins expressed in the ocular surface [Matsuzawa, M. et al., Nature Communications, 2023, 14:1417, pages 1-15], MLIC5AC is believed to play a pivotal role in maintaining the rheological properties of the tear film. As shown in the examples of the present application, the compositions of the invention show mucoadhesion when contacted with mucin both in physico-chemical (non-cellular) and cellular models. Moreover, the compositions of the invention have shown a protective effect against dry eye when evaluated using an aqueous teardeficient animal model. This evidence proves that the compositions of the invention are suitable for their use in the treatment and / or prevention of dry eye disease.

[0093] Thus, in a second aspect, the present invention relates to a composition as defined in the first aspect for use in medicine.

[0094] This aspect may also be formulated as the use of a composition as defined in the first aspect for the manufacture of a medicament, or as a pharmaceutical composition comprising a composition as defined in the first aspect for use in medicine.

[0095] In a third aspect, the present invention relates to a composition as defined in the first aspect, for use in the treatment and / or prevention of dry eye disease.

[0096] This aspect may also be formulated as the use of a sterile ophthalmic composition as defined in the first aspect for the manufacture of a medicament for the treatment and / or prevention of dry eye disease.

[0097] Alternatively, this aspect may also be formulated as a method of treatment and / or prevention of dry eye disease comprising administering to a subject in need thereof a composition as defined in the first aspect.

[0098] In particular embodiments of the above aspects, the dry eye disease is selected from the group consisting of keratoconjunctivitis sicca, xerophthalmia, xerosis and Sjogren's syndrome.

[0099] The term “prevention”, as used herein, refers to the administration of the composition of the invention in an initial or early stage of a disease, or to also prevent its onset.

[0100] The term “treatment” is used to designate the administration of the composition of the invention to control disorder progression before or after the clinical signs had appeared. By control of the disorder progression it is meant to designate beneficial or desired clinical results including, but not limited to, reduction of symptoms, reduction of the length of the disorder, stabilization pathological state (specifically avoidance of further deterioration), delay in the disorder’s progression, improvement of the pathological state and remission (both partial and total). In a particular embodiment of the invention the composition of the invention is used to control the disorder progression once at least one of the disorder’s clinical signs has appeared.

[0101] The term “medicament”, as used herein, refers to a composition of the invention. The medicament may be administered by ocular topical route. It is prepared by conventional means with pharmaceutically acceptable excipients.

[0102] The term “subject”, as used herein, refers to any animal or human that is suffering from one of the diseases disclosed above. Preferably, the subject is a mammal. The term “mammal”, as used herein, refers to any mammalian species, including but not being limited to domestic and farm animals (cows, horses, pigs, sheep, goats, dogs, cats or rodents), primates, and humans. Preferably, the mammal is a human being.

[0103] The following examples represent specific embodiments of the present invention. They do not intend to limit in any way the scope of the invention defined in the present description.

[0104] EXAMPLES

[0105] Example 1. Preparation of the compositions

[0106] The compositions of Example 2 of the present invention were prepared following the process as defined below:

[0107] Step 1 : In a suitable reactor the surfactant (in the amount described in the example), water and troxerutin (in the amount described in the example) were solubilized, in order to obtain a homogenous mixture. Water was added in quantity sufficient to 100 wt% and temperature was kept constant around 25 °C.

[0108] Step 2: In a separate reactor, a homogeneous mixture of 15 wt% the mucin and 85 wt% of composition obtained in step 1 was prepared. Temperature was kept constant around 25 °C. The compositions of Example 3 of the present invention were prepared following the process as defined below:

[0109] In a suitable reactor the surfactant (in the amount described in the example), water and troxerutin (in the amount described in the example) were solubilized, in order to obtain a homogenous mixture. Water was added to the mixture in quantity sufficient to 100 wt% and the temperature was kept constant around 25 °C.

[0110] The compositions of Example 4 of the present invention were prepared following the process as defined below:

[0111] In a suitable reactor the surfactants (in the amount described in the example), hank’s balanced salt solution (HBSS) and troxerutin (in the amount described in the example) were solubilized, in order to obtain a homogenous mixture. HBSS was added to the mixture in quantity sufficient to 100 wt% and temperature was kept constant around 25 °C.

[0112] The compositions with oil of the present invention were prepared following the process as defined below:

[0113] Step 1 : In a suitable reactor a mixture of oil (in the amount described in the example) and surfactant (in the amount described in the example) was prepared. Temperature was kept constant around 25 °C

[0114] Step 2: The hank’s balanced salt solution (HBSS) was added stepwise with stirring to the mixture of step 1. HBSS was added in quantity sufficient to 100 wt% and temperature was kept constant around 25 °C.

[0115] Step 3: Troxerutin (in the amount described in the example) was added to the composition of step 2 and it was stirred until a homogenous mixture was obtained. Temperature was kept constant around 25 °C.

[0116] The compositions of Example 5 of the present invention were prepared following the process as defined below:

[0117] TRX Solution Group:

[0118] In a suitable reactor a mixture of 10 wt% troxerutin, 0.46 wt% tris(hidroxymethyl)- aminomethane / tris(hidroxymethyl)-aminomethane HCI and q.s. 100 wt% of water was prepared. Temperature was kept constant around 25 °C.

[0119] TRX Composition Group: The composition contains 10.0 wt% troxerutin (TRX) + 5 wt% Kolliphor® EL (KEL) + 2 wt% castor oil, 0.46 wt% tris(hidroxymethyl)-aminomethane / tris(hidroxymethyl)- aminomethane HCI, 1 wt% glycerin and q.s. 100 wt% of water

[0120] Step 1 : In a suitable reactor a mixture of 2 wt% castor oil and 5 wt% Kolliphor® EL was prepared. Temperature was kept constant around 25 °C

[0121] Step 2: water in q.s. 100 wt% was added stepwise with stirring to the mixture of step 1 . Temperature was kept constant around 25 °C.

[0122] Step 3: 0.46 wt% tris(hidroxymethyl)-aminomethane / tris(hidroxymethyl)- aminomethane HCI was added with stirring to the mixture of step 2. Temperature was kept constant around 25 °C.

[0123] Step 4: 10 wt% troxerutin was added to the composition of step 3 and it was stirred until a homogenous mixture was obtained. Temperature was kept constant around 25 °C.

[0124] Step 5: 1 wt% glycerin was added to the composition of step 4 and it was stirred until a homogenous mixture was obtained. Temperature was kept constant around 25 °C.

[0125] Placebo:

[0126] In a suitable reactor a mixture of 0.46 wt% tris(hidroxymethyl)- aminomethane / tris(hidroxymethyl)-aminomethane HCI and q.s. 100 wt% of water was prepared. Temperature was kept constant around 25 °C.

[0127] Example 2. Mucoadhesion studies

[0128] 2.1. Method

[0129] The term bioadhesion is commonly defined as adhesion between two materials where at least one of the materials is of biological origin. The term mucoadhesion can be considered to refer to a subgroup of bioadhesion and, more specifically, to the case when the formulation interacts with the mucus layer that covers a mucosae tissue (Edsman et al., Journal of Pharmacy and Pharmacology, 2005, 57: 3-22). The force of bioadhesion / mucoadhesion is the interfacial force which holds together the adhesive material and the biological tissue / mucus.

[0130] The force of bioadhesion or bioadhesion strength, F, was determined by (Hassan et al., Pharmaceutical Research, 1990, Vol. 7; No.5, 491 - 495): F = r]b. CJ were “a” is the rate of shear per second and “r|b” is the viscosity component due to bioadhesion determined by: were “r|t” is the viscosity coefficient of the system, and “i"|m” and “nP” are the individual coefficients of mucin and the product.

[0131] Considering these bibliographic methods and references, the in vitro bioadhesion / mucoadhesion of different compositions were assayed. The preparation of test samples was performed by dispersing a 15 wt% of mucin (from porcine stomach Type II, Sigma-Aldrich ref. M2378) in formulations, by magnetic stirring at room temperature. Viscosities of the mixture mucin / formulation and mucin dispersion were measured at 25 °C using a Brookfield viscometer. The viscosity of the formulations was measured at 25 °C using a vibro viscometer.

[0132] A relative force of bioadhesion was defined as the force of bioadhesion referred to the flavonoid force of bioadhesion, and it has been determined by:

[0133] Fr=F' / rp troxerutin were “Fr” is the relative force of bioadhesion of the sample, “F” is the force of bioadhesion of the sample and “Ftroxerutin” is the force of bioadhesion of troxerutin.

[0134] A synergistic effect was considered when the relative bioadhesion force of the mixture was higher than the sum of the relative bioadhesion force of flavonoid and surfactant:

[0135] When a test sample is adhered to the mucin, the resulting system has an increased viscosity when compared to that of the test sample alone and the mucin alone. Thus, a positive value will be obtained in the test. When a negative value is obtained, it means that the viscosity of the final system is less than that of the mucin alone. This implies that there is no mucoadhesion.

[0136] 2.2. Results

[0137] First, a study on the mucoadhesive properties of different combinations of troxerutin and surfactants was carried out. The results are provided in the table below:

[0138] As it can be seen in the table above, synergistic mucoadhesion is only obtained when troxerutin (TRX) is provided in a composition wherein the surfactant is Kolliphor® EL, Kolliphor® RH40, Tween®80 or Tween®20.

[0139] Subsequently, a study on the mucoadhesive properties of the combinations of 10 wt% of troxerutin (TRX) and Kolliphor® EL at varying concentrations was carried out. The results are provided in the table below: As it can be seen in the tables above, the synergistic effect is obtained when the concentration of surfactant is lower than 8.75 wt%.

[0140] Also, a study on the mucoadhesive properties of the combinations of 5 wt% of Kolliphor ® EL and troxerutin (TRX) at varying concentrations was carried out. The results are provided in the table below:

[0141] As it can be seen in the table above, the synergistic effect is maintained throughout all the compositions studied. Example 3. In vitro wash-out studies

[0142] 3.1. Method description

[0143] The drug release of the samples was studied by means of a vertical diffusion cell (VDC) or Franz cell system, being the acceptor medium Milli-Q water. The drug release profile of each sample was obtained by plotting the median drug release, in pg, versus time.

[0144] The amount of drug released was determined by high-performance liquid chromatography with UV detection. The acceptor medium of the VDC tests was injected into the chromatographic system without any previous treatment. Chromatographic separation was carried out using a Kromasil C18 column (5 pm, 150 x 4.6 mm). Mobile phase consisted of a mixture of 20 % acetonitrile and 80 % of an aqueous solution containing 17.9 g / l of sodium dihydrogenphosphate monohydrate at pH 4.4. The column was kept at 25 °C, and 1 pl of sample was injected. The UV detection wavelength was set at 350 nm.

[0145] 3.2. Results

[0146] This study was carried out for a composition comprising 10 wt% of troxerutin and 3.2 wt% of Kolliphor® EL (10 % TRX + 3.2 % KEL) and is compared to a composition comprising 10 wt% of troxerutin (10 % TRX) and to a composition comprising 10 wt% of troxerutin and 8.75 wt% Kolliphor® EL (10 wt% TRX + 8.75 wt% KEL). The results are shown in Figure 1.

[0147] This study was carried out for a composition comprising 10 wt% of troxerutin and 5 wt% of Kolliphor ® RH40 (10 % TRX + 5 % RH40) and is compared to a composition comprising 10 wt% of troxerutin (10 % TRX) and to a composition comprising 10 wt% of troxerutin and 8 wt% of Kolliphor® RH40 (10 % TRX + 8 % RH40). The results are shown in Figure 2.

[0148] This study was carried out for a composition comprising 10 wt% of troxerutin and 5 wt% of Tween®80 (10 % TRX + 5 % T80) and is compared to a composition comprising 10 wt% of troxerutin (10 % TRX) and to a composition comprising 10 wt% of troxerutin and 9 wt% of Tween®80 (10 % TRX + 9 % T80). The results are shown in Figure 3. As it can be seen in Figures 1-3, a lower wash-out effect is obtained for the compositions comprising the surfactant and having less than 8 wt% of said surfactant, allowing the active ingredient troxerutin to remain for a longer time on the ocular surface and generating an in situ release system. The compositions of the invention also bind or interact with the ocular mucins providing a shield effect which decreases tear evaporation.

[0149] Example 4. In vitro mucoadhesion studies

[0150] 4.1. Method description

[0151] The goblet cell is a specialized epithelial cell type that plays a role in the first-line defense on the wet surfaces of the body including the ocular surface. Goblet cells provide hydration and lubrication of the mucosal surface by secreting high molecular weight mucins. The major gel-forming mucins expressed in the ocular surface include MLIC5AC and MLIC5B [Matsuzawa, M. et al., Nature Communications, 2023, 14:1417, pages 1- 15].

[0152] Human mucus-secreting cell line HT29-MTX-E12 is a homogenous subpopulation of HT29 human colon carcinoma cells selected by adaptation to methotrexate. This cell line expresses and produces mucins, in particular MLIC2, MLIC5AC and MLIC5B providing some similar function to goblet cells in mucin secretion. HT29-MTX-E12 cell line is commonly used in permeability assays as well as to test in vitro mucoadhesion (Adamczak et al., International Journal of Pharmaceutics, 2016, vol. 498, pages 225- 233).

[0153] Troxerutin is a flavonoid that shows a maximum of absorbance at 348 nm and this property can be used to determine the presence of troxerutin in a sample.

[0154] The aim of this study is to characterize the effect in the mucoadhesive properties of troxerutin when it is associated to surfactants in a cellular model.

[0155] HT29-MTX-E12 cells (SIGMA ECACC 12040401) were seeded in sterile 12-well with

[0156] Dulbecco’s Modified Eagles Medium (DMEM) with 10 % fetal bovine serum, 1 % non- essential amino acids and 1 % penicillin / streptomycin and incubated at 37 °C under 5 % CO2 atmosphere until confluence. The medium was changed every two days and monolayers were differentiated over 21 days. Monolayers were then washed and equilibrated in hank’s balanced salt solution (HBSS) at 37 °C and 5 % CO2 for 30 min. Test samples were added onto monolayers and incubated at 37 °C and 5 % CO2 for 60 min with agitation. Test samples were then removed, and monolayers washed twice with HBSS. The absorbance at 348 nm was measured directly on the surface of the cells to determine de presence of adhered troxerutin. Signal fold versus control group (HBSS) was calculated and data are represented as mean ± S.E.M.

[0157] Test samples:

[0158] - “5 % TRX”: 5 wt% TRX + q.s. 100 wt% HBSS

[0159] - “10 % TRX”: 10 wt% TRX + q.s. 100 wt% HBSS

[0160] - “3.2 % KEL”: 3.2 wt% Kolliphor® EL, (KEL) + q.s. 100 wt% HBSS

[0161] - “3.2 % T80”: 3.2 wt% Tween® 80 (T80) + q.s. 100 wt% HBSS

[0162] - “5 % KEL”: 5 wt% Kolliphor® EL (KEL) + 1.5 wt% castor oil + q.s. 100 wt% HBSS

[0163] - “4 % KEL”: 4 wt% Kolliphor® EL (KEL) + 0.5 wt% castor oil + q.s. 100 wt% HBSS

[0164] - “2 % KEL”: 2 wt% Kolliphor® EL (KEL) + 0.2 wt% castor oil + q.s. HBSS

[0165] - “10 % TRX + 3.2 % KEL”: 10 wt% TRX + 3.2 wt% Kolliphor® EL (KEL) + q.s. 100 wt% HBSS

[0166] - “10 % TRX + 3.2 % T80”: 10 wt% TRX + 3.2 wt% Tween® 80 (T80) + q.s. 100 wt% HBSS

[0167] - “5 % TRX + 5 % KEL”: 5 wt% TRX + 5 wt% Kolliphor® EL (KEL) + 1 .5 wt% castor oil + q.s. 100 wt% HBSS

[0168] - “10 % TRX + 5 % KEL”: 10 wt% TRX + 5 wt% Kolliphor® EL (KEL) + 1.5 wt% castor oil + q.s. 100 wt% HBSS

[0169] - “10 % TRX + 4 % KEL”: 10 wt% TRX + 4 wt% Kolliphor® EL (KEL) + 0.5 wt% castor oil + q.s. 100 wt% HBSS

[0170] - “5 % TRX + 2 % KEL”: 5 wt% TRX + 2 wt% Kolliphor® EL (KEL) + 0.2 wt% castor oil + q.s. 100 wt% HBSS

[0171] 4.2. Results No signal of absorbance at 348 nm was detected for any of the compositions without troxerutin, therefore, the detected signal is specific of the presence of troxerutin over the cell surface.

[0172] Troxerutin group exhibited a significant increase of absorbance signal proving a certain adhesive capacity of the molecule to the HT29-MTX-E12 cell culture (Figures 4-7).

[0173] The combination of troxerutin with Kolliphor® EL showed a significantly rise of the absorbance signal indicating that the presence of troxerutin on the cell monolayer considerably increased (Figure 4). The same effect was observed with the combination of troxerutin with Tween® 80 (Figure 5)

[0174] The adhesion of troxerutine on cell culture was also exacerbated by using compositions that combine different concentrations of troxerutin with different concentrations of Kolliphor® EL and castor oil (Figures 6 and 7). An increase of absorbance was observed in all the assayed samples what reinforces the adhesion of the claimed compositions to the mucous cell layer.

[0175] Example 5. In vivo efficacy study in a dry eye model

[0176] 5.1. Method description (Schirmer test)

[0177] The protective effect of troxerutin against dry eye was evaluated using an aqueous teardeficient model induced by repetitive topical ophthalmic administration of atropine (Colircusi atropina 1 %) for 16 days in rabbits, maintaining the relative humidity (RH) environmental condition around 40 % throughout the experiment.

[0178] After a general clinic and ophthalmologic signs supervision, rabbits were randomly divided into three groups:

[0179] TRX Solution Group; n=5

[0180] TRX Composition Group; n=5

[0181] Placebo; n=4 TRX formulations and placebo were topical ophthalmic administered in each eye (0.3 mL), TID (every 4 hours) for 16 days. TRX formulations were administered after atropine administration. The progression of dry eye was evaluated with clinical tests used commonly in humans. Schirmer’s test was used to evaluate the loss of tear volume using sterile ophthalmic strips (l-DEW Tearstrips, Entod Research Cell). Statistically significant differences were found between groups (TRX Solution vs TRX Composition; both days) using II of Mann- Whitney test with a p-value < 0.05.

[0182] 5.2. Results

[0183] Results corresponding to TRX Solution Group and TRX Composition Group have been normalized to the placebo group and represented in Figure 8. As it can be seen, the composition of the invention comprising troxerutin and Kolliphor® EL (TRX Composition Group) provides an improved tear volume when compared to a composition comprising only troxerutin (TRX Solution Group).

Claims

CLAIMS1. Sterile ophthalmic composition comprising troxerutin and a surfactant selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, and mixtures thereof, wherein the weight ratio of troxerutin to the surfactant is from 0.02:1 to 100:1 , wherein the surfactant is present in an amount of up to 7 wt% with respect to the total weight of the composition, and wherein the composition is in the form of a nanoemulsion, a solution, a micellar solution, an emulsion, a microemulsion, a suspension or a gel.

2. Composition according to claim 1 , wherein the surfactant is selected from the group consisting of polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyethylene (20) sorbitan monooleate, and mixtures thereof.

3. Composition according to claim 1 or 2, wherein the weight ratio of troxerutin to the surfactant is from 1.5:1 to 100:1 , preferably from 1.5:1 to 5:1.

4. Composition according to any one of the preceding claims, wherein troxerutin is present in an amount of from 0.15 to 15 wt% with respect to the total weight of the composition, preferably from 1 to 15 wt%, more preferably from 5 to 15 wt%.

5. Composition according to any one of the preceding claims, wherein the surfactant is present in an amount of from 0.05 to 7 wt% with respect to the total weight of the composition, preferably from 0.25 to 7 wt%, more preferably from 0.5 to 7 wt%.

6. Composition according to any one of the preceding claims, further comprising water.

7. Composition according to claim 6, wherein the water is present in an amount of at least 60 wt% with respect to the total weight of the composition.

8. Composition according to any one of the preceding claims, further comprising one or more tonicity adjusting agents, preferably wherein the one or more tonicity adjusting agent is selected from the group consisting of sodium chloride, glycerin, propylene glycol, and mixtures thereof.

9. Composition according to any one of the preceding claims, further comprising one or more pH adjusting agents, preferably wherein the one or more pH adjusting agent is selected from the group consisting of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane hydrochloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and mixtures thereof.

10. Composition according to any one of the preceding claims, further comprising one or more viscosity increasing agents, preferably wherein the one of more viscosity increasing agents is selected form the group consisting of sodium alginate, polyvinylpyrrolidone, gellan gum, chitosan or a derivative thereof, and mixtures thereof.

11. Composition according to any one of the preceding claims in the form of a nanoemulsion.

12. Composition as defined in any one of the preceding claims for use in medicine.

13. Composition as defined in any one of claims 1 to 11 , for use in the treatment and / or prevention of dry eye disease.

14. Composition for use according to claim 13, wherein the dry eye disease is selected from the group consisting of keratoconjunctivitis sicca, xerophthalmia, xerosis and Sjogren's syndrome.

15. Use of a composition as defined in any one of claims 1 to 11 for the manufacture of a medicament.

16. Use of a composition as defined in any one of claims 1 to 11 for the manufacture of a medicament for the treatment and / or prevention of dry eye disease.

17. The use according to claim 16, wherein the dry eye disease is selected from the group consisting of keratoconjunctivitis sicca, xerophthalmia, xerosis and Sjogren's syndrome.

18. Method of treatment and / or prevention of dry eye disease comprising administering to a subject in need thereof a composition as defined in any one of claims 1 to 11.

19. The method according to claim 18, wherein the dry eye disease is selected from the group consisting of keratoconjunctivitis sicca, xerophthalmia, xerosis and Sjogren's syndrome.

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