Ophthalmic Compositions Comprising Resveratrol for Treating Dry Eye Syndrome - Patent application

Abstract

The present invention relates to an ophthalmic composition comprising hyaluronic acid or one of its salts, advantageously with a molecular weight between 100 and 800 kDa; an oligosaccharide, advantageously a diholoside, more advantageously trehalose; and resveratrol, and its use for treating eye diseases such as DED.

Description

[Technical field]

[0001] The present invention relates to an ophthalmic composition for topical application, in particular intended to treat dry eye, which combines hyaluronic acid or a salt thereof, an oligosaccharide, and resveratrol. [Background technology]

[0002] Dry eye syndrome, also known as dry eye or keratoconjunctivitis sicca or keratoconjunctivitis sicca (KCS), causes ocular discomfort, visual disturbances, and tear film instability, accompanied by itching, stinging, and / or burning.

[0003] It is a multifactorial disease: dry eye can result from abnormalities of the tear glands, inflammation of the eyelids, allergy-related eye inflammation, refractive surgery (including laser surgery), a deficiency of certain fats in the daily diet, extended contact lens wear, hormonal changes, autoimmune diseases, or side effects of certain medications.

[0004] The incidence of dry eye increases with age, and aging is associated with increased oxidative stress. Furthermore, environmental factors such as exposure to pollution, blue light, ultraviolet (UV) radiation, and ozone have been implicated in the pathophysiology of dry eye. These factors increase oxidative stress and inflammation of the ocular surface. Oxidative stress generates reactive oxygen species (ROS) that can cause deleterious changes in deoxyribonucleic acid, lipids, and proteins in corneal and conjunctival epithelial cells, which may lead to ocular surface diseases (OSD), including dry eye disease (DED).

[0005] In a preventative manner and / or in the early stages of some eye diseases, daily intake of dietary supplements such as those disclosed in WO2016 / 151269 containing vitamins, trace elements, carotenoids, omega-3 fatty acids, and resveratrol is recommended.

[0006] In treating dry eye, the application of artificial tears, also known as lubricating or moisturizing eye drops, provides localized relief, but for a short period of time. Low to medium viscosity artificial tears are generally based on polyvinyl alcohol or cellulose derivatives, while high viscosity ones contain carbomer or hyaluronic acid.

[0007] Hyaluronic acid (HA) is used in eye drops to moisturize the cornea, and its "barrier" effect on friction reduces the pain caused by dry eye. It forms a transparent, lubricating moist film on the surface of the eye, protecting the cornea from drying out. These properties provide effective relief of functional symptoms and allow patients to apply eye drops less frequently each day.

[0008] Studies have shown that HA eye drops are very well tolerated even by the driest eyes. After application, slight discomfort may occur for a few seconds to a few minutes. Vision may become slightly blurred. Therefore, it is better not to use this product before driving or in an "emergency". The discomfort disappears very quickly and the product lasts much longer than simple eye drops. The duration of action depends on how dry the eyes are. As a general rule, simple eye drops are effective for 10-15 minutes, whereas the action of hyaluronic acid lasts for 45 minutes to an hour instead.

[0009] The conclusion is that hyaluronic acid-based eye drops are effective for mild to moderate dry eye. Patients with more severe dry eye may also find it worthwhile to use these drops, but generally must rely on different products, such as immunosuppressant eye drops such as cyclosporine.

[0010] To improve the efficacy of hyaluronic acid-based eye drops, document WO2015 / 136186 proposes combining HA, advantageously of low molecular weight (100-800 kDa), with an oligosaccharide, advantageously a diholoside, in a topical formulation to increase the persistence, i.e. the retention time, on the ocular surface.

[0011] In addition, VisuFarma has marketed a product to improve the oxidative component of DED: Visu XL® eye drop product contains Vitamin E TPGS along with the antioxidant Coenzyme Q (ubiquinone Q10) and cross-linked sodium hyaluronate.

[0012] Topical ophthalmic compositions comprising resveratrol and hyaluronic acid have also been disclosed, see for example document WO 2009 / 084069.

[0013] However, there is a continuing need to develop ophthalmic formulations with improved efficacy for treating ocular surface diseases, particularly DED. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] WO2016 / 151269 [Patent Document 2] WO2015 / 136186 [Patent Document 3] WO 2009 / 084069 [Patent Document 4] WO2009087229 [Non-patent literature]

[0015] [Non-Patent Document 1] https: / / www.nature.com / articles / s41598-017-18211-2 Summary of the Invention [Means for solving the problem]

[0016] The present invention proposes a composition useful in the treatment of dry eye by combining a lubricious polymer, in particular hyaluronic acid, the use of which is already known in this application, with an oligosaccharide, both of which improve the survivability and bioavailability of resveratrol and protect the ocular surface from oxidative stress induced by the external environment or induced by dry eye disease, by preventing and inhibiting the generation of free radicals, which are the root cause of tissue damage.

[0017] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0018] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0019] As used herein, "about" or "approximately," when referring to a measurable value, such as an amount, temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, as such variations are appropriate for carrying out the disclosed methods.

[0020] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0021] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs from an organism, tissue, cell, or component thereof that exhibits a "normal" (expected) respective characteristic in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.). A characteristic that is normal or expected to be normal in one cell or tissue type may be abnormal in a different cell or tissue type.

[0022] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof that are subject to the methods described herein, whether in vitro or in situ. The subject can be an animal, preferably a mammal. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0023] A "disease" or "pathology" is a state of health in a subject in which the subject is unable to maintain homeostasis, where if the disease is not ameliorated, the subject's health will continue to deteriorate. In contrast, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but the subject's health is no better than it would be without the disorder. If left untreated, the disorder does not necessarily cause a further decline in the subject's health.

[0024] A disease or disorder is "alleviated," "ameliorated," or "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, are reduced. This also includes halting the progression of the disease or disorder. A disease or disorder is "cured" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, are eliminated.

[0025] A "therapeutic" treatment is a treatment administered to a subject who exhibits symptoms of a pathology, for the purpose of reducing or eliminating those symptoms. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit symptoms of a pathology, or whose condition has not yet been diagnosed, for the purpose of preventing or postponing the onset of those symptoms.

[0026] As used herein, "treating a disease or disorder" means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject. Disease and disorder are used interchangeably herein in the context of treatment.

[0027] In the context of this invention, an "active ingredient" or "active compound" or "active agent" or "active substance" or "active ingredient" or "active" is a molecule or component in a pharmaceutical drug that is biologically active.

[0028] An "effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered. The phrase "therapeutically effective amount" as used herein refers to an amount sufficient or effective to prevent or treat (delay or prevent the onset of, impede the progression of, inhibit, reduce, or reverse) a disease or condition, including the alleviation of symptoms of such disease. An "effective amount" of a delivery vehicle is an amount sufficient to effectively bind or deliver a compound.

[0029] Thus, in a first aspect, the application describes an ophthalmic composition comprising a lubricious polymer, an oligosaccharide, and an antioxidant. According to a preferred embodiment of the invention, the composition comprises hyaluronic acid or one of its salts, advantageously having a molecular weight between 100 and 800 kDa; an oligosaccharide, advantageously a diholoside, more advantageously trehalose; and resveratrol.

[0030] In the context of the present application, a lubricating polymer, advantageously a moisturizing polymer, is defined as one capable of forming a transparent film on the ocular surface, in particular due to its water-retaining capacity.

[0031] Advantageously, said polymer is a polysaccharide, i.e. a polymer consisting of several sugars linked together by O-osidic bonds, and even more advantageously, it is a glycosaminoglycan (GAG).

[0032] Moreover, in a privileged manner, the lubricious polymer is a compound of natural, non-synthetic origin.

[0033] According to another preferred embodiment, the polymer is not a cellulose derivative, in particular the polymer is not carboxymethylcellulose (CMC).

[0034] According to certain embodiments, said glycosaminoglycan is selected from the group consisting of hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate (or heparin), or a pharma- ceutically acceptable salt thereof.

[0035] Hyaluronic acid is characterized by the absence of O-sulfation and is used in the present invention, particularly in the form of sodium hyaluronate. Sodium hyaluronate is naturally found in the human eye and retains water to hydrate and lubricate the ocular surface. It maintains solution on the ocular surface, providing long-lasting relief and shortening the healing time of the ocular surface.

[0036] The polymer used actually determines the viscosity of the ophthalmic composition, which depends on both the concentration of the polymer and the molecular weight of this polymer, so these two factors are adjusted so that the composition has an appropriate viscosity that does not cause discomfort to the user (either during or after application).

[0037] Therefore, and in a suitable manner, the percentage concentration of the lubricating polymer in the composition, especially in the case of hyaluronic acid, is 0.5% or less, advantageously 0.2% or less. In other words, the mass of the lubricating polymer advantageously represents at most 0.5g or even 0.2g in 100ml of the composition. Furthermore, and for purposes of efficiency, the percentage concentration of the lubricating polymer is advantageously 0.05% (0.05g / 100ml) or more, or even 0.1% (0.1g / 100ml) or more. In other words, and according to a preferred embodiment, the lubricating polymer, preferably HA or one of its salts, such as sodium hyaluronate, represents 0.05-0.5g per 100ml of the composition, advantageously 0.1-0.2g / 100ml, for example 0.15g / 100ml.

[0038] As already mentioned, the polymer is preferably in a non-reticulated form. Moreover, in the case of hyaluronic acid, the preferred molecular weight (MW) is between 100 kDa and 800 kDa (medium MW).

[0039] Another component of the formulation of the present invention is an oligosaccharide. An oligosaccharide, also called an oligoholoside or oligoside, is an oligomer formed by n sugar (or monosaccharide) units, where n generally ranges from 2 to 10. Oligosides can be linear, branched, or cyclic.

[0040] It is therefore not a glycerol or glycerin type polyol.

[0041] According to a particular embodiment, the oligosaccharide is a diholoside, which can contain two identical (homo) or different (hetero) sugars. Oligosaccharides of interest include, but are not limited to, trehalose, rutinosis (rhamnose alpha (1-6) glucose), kojibiosis, nigerosis, maltose, isomaltose, sophorosis, laminaribiosis, cellobiosis, and gentibiosis, or even trehalulose, sucrose, turanose, maltulose, leucrose, isomaltulose, gentiobiosis, melibiosis, lactulose, lactose, rutinose, inulobiosis, 2-alpha-Mannobiosis, or 3-alpha-Mannobiose.

[0042] It should be noted that these oligosaccharides may possess inherent properties that are of interest in the ophthalmic field, in particular anti-inflammatory, anti-apoptotic or osmolarity-regulating activity, which in addition, and as shown in the examples, contribute to the stability and activity of the compositions according to the invention.

[0043] According to a preferred embodiment, said oligosaccharide is trehalose, a natural substance present in many plants and animals, which is able to survive in extremely dry conditions, providing it with protective and hydrating properties.

[0044] In another embodiment, the percentage concentration of oligosaccharides is less than or equal to 5% by weight of the composition, advantageously less than or equal to 4% by weight. In other words, the content of oligosaccharides advantageously represents at most 5 g or even 4 g in 100 ml of the composition. Furthermore, and for purposes of efficiency, the percentage concentration of oligosaccharides is advantageously greater than or equal to 0.5% (0.5 g / 100 ml) or even greater than or equal to 2% (2 g / 100 ml). In other words, and according to a preferred embodiment, the oligosaccharide, preferably trehalose, represents between 0.5 and 5 g per 100 ml of the composition, advantageously between 2 and 4 g / 100 ml, for example 3 g / 100 ml.

[0045] The third component of the composition according to the present invention is resveratrol, a natural product known for its antioxidant and anti-free radical properties.

[0046] It is derived from stilbene, of the formula: [ka] It is a polyphenol compound.

[0047] There are two isomers, but the trans form is predominantly active, and in the following description the term "resveratrol" can therefore be used instead of trans-resveratrol.

[0048] A high concentration of resveratrol, ideally 0.1% w / v, is preferred for strong antioxidant activity, but any concentration that guarantees good efficacy and ocular tolerance is acceptable. Resveratrol then advantageously represents 0.01-0.1 g / 100 ml of composition, advantageously 0.04-0.06 g / 100 ml, and even more advantageously 0.05 g / 100 ml. In other words, the concentration of resveratrol can be, for example, equal to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 g / 100 ml. According to a specific embodiment, the concentration of resveratrol is equal to or less than 0.08 g / 100 ml.

[0049] According to a preferred embodiment, the composition of the present invention is in liquid form, preferably in solution.In this context, solution generally refers to the liquid pharmaceutical composition in which compound is at least partially dissolved, preferably completely dissolved, and can be administered as liquid.According to a specific embodiment, and in relation to its administration to eye, the composition is advantageously an aqueous solution, i.e. a solution that comprises water as main vehicle.

[0050] Given the hydrophobic nature of resveratrol, the compositions of the present invention advantageously further comprise a solubilizer.

[0051] According to a preferred embodiment, the solubilizing agent is capable of solubilizing resveratrol in water. According to another preferred embodiment, the solubilizing agent is compatible with ocular administration, i.e., non-toxic at the concentrations administered.

[0052] Such a solubilizer is advantageously a macrogol hydroxystearate, more advantageously a macrogol glycerol hydroxystearate (Polyoxyl 40 hydrogenated castor oil or hydrogenated ethoxylated castor oil or PEG-40 hydrogenated castor oil or Cremophor® RH40 or Kolliphor® RH40; CAS number 61788-85-0).

[0053] Other potential candidates include: - Polyoxyl 15 Hydroxystearate (Macrogol 15 Hydroxystearate, or Kolliphor® HS 15, or Solutol® HS 15; CAS number 70142-34-6); - Polysorbate 20 (CAS number 9005-64-5), Polysorbate 60 (CAS number 9005-67-8), or Polysorbate 80 (CAS number 9005-65-6); - macrogol glycerol ricinoleate (or polyoxyl 35 castor oil, or PEG-35 castor oil, or Cremophor® EL, or Kolliphor® EL; CAS number 61791-12-6); - Vitamin E TPGS (TPGS, or Vitamin E polyethylene glycol succinate, or D-α-tocopherol polyethylene glycol (1000) succinate; CAS number 9002-96-4).

[0054] The concentration of the solubilizer in the solution is typically 0.1-20% (w / v), advantageously 0.5-5% (w / v), more advantageously 0.5% or 1% (w / v).

[0055] Of course, the composition according to the invention may also further comprise at least one additive component selected from the group of non-ionic tonicity agents, antioxidants and / or buffer systems.

[0056] Preferably, the composition according to the invention has a slightly acidic pH, advantageously below 7, but advantageously above 5 or even 5.5. The pH of the composition may be 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9. It is advantageously comprised between 5.5 and 6.9, and more advantageously equal to 6.0.

[0057] A weakly acidic pH can be obtained by adding an acid, for example hydrochloric acid (HCl).

[0058] For the desired weakly acidic pH, which prevents the conversion of phenol to phenolate and prevents the appearance of undesirable yellow color, a suitable buffer is advantageously selected from the group consisting of citrate, borate, and phosphate, more advantageously citrate.As an example, trisodium citrate, 2H2O, can be used.Its suitable amount can be determined by those skilled in the art, for example, 0.588g / 100ml for a 20mM solution.

[0059] According to a specific embodiment, such a buffer is not a Tris buffer, such as trometamol, present in the Thealoz® duo formulation disclosed in WO2015 / 136186.

[0060] In an advantageous manner, the composition according to the invention has a viscosity adapted for topical ophthalmic application.

[0061] Thus, the composition according to the invention preferably has a viscosity of less than or equal to 50 mPa.s (cP), or even less than or equal to 20, 15, 10, or even less than or equal to 5 mPa.s. Depending on the application, it is between 2 and 50 mPa.s, advantageously between 2 and 15 mPa.s, and even more advantageously between 2 and 10 mPa.s, for example of the order of 5 mPa.s. These values ​​correspond to those measured at 25° C. by means of a BROOKFIELD RVDV III mobile rotational viscometer.

[0062] In fact, this viscosity is advantageously close to that of human tears, estimated at about 6.5 mPa.s at 20° C., with a velocity gradient close to zero. Application of the composition according to the invention therefore does not cause any sensation of discomfort or irritation. In fact, it has been shown that easy and unhindered clearance is only possible if the viscosity of the ophthalmic composition is less than 50 mPa.s.

[0063] According to another particular embodiment, it corresponds to a hypotonic to isotonic formulation with an osmolality advantageously between 170 and 350 mosmol / kg, more advantageously between 200 and 300 mosmol / kg, for example about 250 mosmol / kg. This characteristic can be obtained, for example, by adding sodium chloride (NaCl). The oligosaccharides in the concentrations used can also contribute to the control of the osmolarity of the solution.

[0064] According to another embodiment, the composition according to the invention does not contain antimicrobial preservatives, in particular quaternary ammonium preservatives, in particular benzalkonium chloride (BAK), which is known for its harmful effects on the ocular surface, in other words, it is advantageously a so-called unpreserved composition or a preservative-free composition.

[0065] In the context of the present invention, "antimicrobial agent or antimicrobial preservative or preservative" refers to a preservative that has antimicrobial properties, i.e. a compound that is capable of protecting the ophthalmic composition from possible microbial contamination.

[0066] Quaternary ammonium compounds, particularly benzalkonium chloride (BAK) or Polyquad®, optionally in combination with EDTA, are commonly used as preservatives. Other preservatives include, for example: - Guanidine-based preservatives (PHMB, chlorhexidine); - benzyl alcohol; - Parabens (methyl, ethyl, propyl, etc.); - Mercury-based preservatives, e.g. thimerosal; - Chlorobutanol; - Benzethonium chloride; - Oxidative preservatives (Purite, etc.).

[0067] In the case of an unpreserved composition, it is advantageously presented in a single-use vial (unidose) or a preservative-free multi-dose vial, e.g., an Abak®, Novelia®, Comod® vial or equivalent, i.e. a vial capable of delivering preservative-free eye drops for several days.

[0068] The present invention therefore also relates to single-use (single-dose) or multi-dose vials adapted for unpreserved compositions, including the ophthalmic compositions previously described. As is known in the art, such vials, containers or drop dispensers can be made of low density polyethylene (LDPE), advantageously of European Pharmacopoeia (EP) quality, and do not contain additives.

[0069] Another aspect of the invention relates to the use of such compositions in the treatment of eye disorders and conditions. In other words, the invention relates to a method of treating an eye disease comprising administering a composition of the invention.

[0070] Thus, the combination of polymeric lubricants, oligosaccharides and resveratrol can be used in the context of dry eye disease (DED or Dry Eye Syndrome (DES)), particularly in mild to moderate forms, and in some cases for the treatment of ocular inflammation.

[0071] Further ocular inflammatory conditions are, for example, non-infectious uveitis (anterior, intermediate, posterior, generalized), non-infectious conjunctivitis, iritis, or scleritis.

[0072] More generally, the ophthalmic compositions of the invention can be used to reduce the effects of oxidative stress at the eye level and for the treatment of the manifestations of pathologies associated with apoptosis on the part of keratocytes.

[0073] Thus, a further aspect of the invention is the use of said composition for the treatment of degenerative conditions including ischemic opticopathy, senile lens opacities, cataracts, uveitis, and familial, metabolic and senile age-related degenerative conditions, as well as neurodegenerative conditions of the retina and optic nerve including glaucoma, age-related macular degeneration, retinitis pigmentosa, Stargardt's disease, vitelliform macular cysts, cone dystrophies, proliferative diabetic retinopathy, hypertensive retinopathy, retinal detachment, retinoblastoma, and neuritis and visual neuropathy of toxic, inflammatory and degenerative origin.

[0074] The compositions of the present invention may contain an additional active agent for treating the same condition or another ocular disease, such as an eye infection, an eye allergy, or glaucoma.

[0075] In other words, the compositions according to the invention may contain a therapeutically effective amount of one or more other active agents. These may include antibiotics, e.g., antibacterial agents, antifungal agents, antiviral agents, immunosuppressants, anti-inflammatory agents, or antiglaucoma agents, e.g., prostaglandins, beta-blockers, carbonic anhydrase inhibitors, or alpha-adrenergic agonists.

[0076] Such active agents may be selected from the following group: aceclidine, acetazolamide, acyclovir, anecortave, apraclonidine, atropine, azapentacene, azelastine, bacitracin, befunolol, betamethasone, betaxolol, bimatoprost, brimonidine, brinzolamide, carbachol, carteolol, celecoxib, chloramphenicol, chlortetracycline, ciprofloxacin, cromoglycate, cyclopentolate, cyclosporine, dapiperidine, tetracycline ... zole, demecarium, dexamethasone, diclofenac, dichlorphenamide, dipivefrin, dorzolamide, echothiophate, emedastine, epinastine, epinephrine, erythromycin, ethoxyzolamide, eucatropine, fludrocortisone, fluorometholone, flurbiprofen, homovirsen, framycetin, ganciclovir, gatifloxacin, gentamicin, homatropine, hydrocortisone, idoxuridine, indomethacin, isoflurophate, ketoconazole, Lac, ketotifen, latanoprost, levobetaxolol, levobunolol, levocabastine, levofloxacin, lodoxamide, loteprednol, medrysone, methazolamide, metipranolol, moxifloxacin, naphazoline, natamycin, nedocromil, neomycin, norfloxacin, ofloxacin, olopatadine, oxymetazoline, pemirolast, pegaptanib, phenylephrine, physostigmine, pilocarpine, pindolol, pirenoxine, polymyxin B, Prednisolone, proparacaine, ranibizumab, rimexolone, scopolamine, sezolamide, squalamine, sulfacetamide, suprofen, tetracaine, tetracycline, tetrahydrozoline, tetrizoline, timolol, tobramycin, travoprost, triamcinolone, trifluoromethazolamide, trifluridine, trimethoprim, tropicamide, unoprostone, vidarabine, xylometazoline, a pharma- ceutically acceptable salt, or a combination thereof.

[0077] The compositions according to the invention can be used in humans and animals, treating a wide range of individuals: adults, children, pregnant or lactating women, contact lens wearers of all kinds.

[0078] According to a preferred embodiment, the compositions of the invention are intended for topical application, preferably by dropping into the eye or spraying onto the ocular surface, in other words, the compositions of the invention are preferably administered ophthalmically to the affected eye.

[0079] The dosage is adapted to the severity of the disease, but generally consists of administering one to several drops per day to the affected eye. Thus, dosages of 10 drops per eye per day, or even 9, 8, 7, 6, 5, 4, 3, 2, or even 1 drop per eye per day are conceivable. According to one embodiment, the composition is administered in the form of drops at least once per day to at least 10 times per day, advantageously 2 to 4 times daily.

[0080] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0081] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present compounds and practice the claimed methods.

[0082] Experimental Example The present invention is further described in detail by reference to the following experimental examples and the accompanying figures, which are provided for illustrative purposes only and are not intended to be limiting. [Brief description of the drawings]

[0083] [Figure 1] FIG. 1 shows the percentage of apoptotic cells vs. total cells at 8 hours + overnight. [Diagram 2] FIG. 1 shows the antioxidant profile (%) of different formulations versus the negative control. [Diagram 3]FIG. 1 shows replicate averages of the percentage of apoptotic cells vs. total cells. [Figure 4] Quantification of IL8 release 24 hours after UV irradiation. NC = negative control: treated with 0.9% NaCl, no irradiation. PC = positive control: treated with 0.9% NaCl, irradiation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0084] Examples of compositions according to the invention 1. Composition [Table 1]

[0085] After preparation, the composition is advantageously sterilized, for example with a 0.2 μm sterile filter.

[0086] After sterilization, such compositions can be packaged in single-dose (or single-use) vials or preservative-free multi-dose vials, such as ABAK® or NOVELIA®. Single doses are traditionally made of low-density polyethylene (LDPE) without additives.

[0087] 2. Characteristics of the composition a / Stability of the composition after 3 months under various storage conditions The results are shown in Table 1 below. [Table 2]

[0088] b / Stability of the composition after 3 months of storage in preservative-free multi-dose vials The results are shown in Table 2 below. [Table 3]

[0089] c / Conclusion In conclusion, the compositions according to the invention exhibit the following characteristics that make them compatible with ocular administration: - Slightly acidic pH; - Hypotonic to isotonic preparations; - Viscosity similar to that of human tears (approximately 6.5 mPa.s at 20°C).

[0090] Moreover, stability studies show that such compositions are stable after at least 3 months. The characteristics of the composition do not change after storage at room conditions. At more extreme conditions, the composition remains suitable, although the resveratrol content is not significantly reduced and there is a slight yellowing.

[0091] In addition, it can be stably stored in a preservative-free delivery system. EXAMPLES

[0092] Study of the efficacy of the composition according to the invention 1.Cytotoxicity / Irritation Potential To further evaluate the protection of tear substitutes against UVA+B light (290–400 nm)-induced oxidative stress damage to the superficial corneal epithelium, initial studies in human corneal epithelium (HCE) were performed to determine the cytotoxicity of selected tear substitutes.

[0093] Preliminary cytotoxicity was measured after 4 h acute exposure to the tear substitute and after 8 h exposure (repeated twice daily with final reading the next day; 0.015 J / cm 2 ) was performed using two assays: the MTT test and the TUNEL assay.

[0094] Preliminary cytotoxicity was tested in vitro in the HCE model for the following tear substitutes compared to benzalkonium chloride (BAK) 0.1% solution (positive control) and saline (NaCl 0.9%; negative control): Composition according to Example I - Visu XL (commercial standard containing 0.1% Coenzyme Q10, 0.1% cross-linked hyaluronic acid (HA), 0.5% Vitamin E TPGS; pH 6.9).

[0095] method 0.5cm 2 Reconstructed human corneal epithelium (HCE) from EPISKIN was used for the evaluation. Human epithelial immortalized cells (HICECs) were placed on polycarbonate filters and cultured at the air-liquid interface in chemically defined medium for 5 days to form a structured epithelium. Inserts containing the 5-day tissue (thickness: 55 μm; 5.5 cell layers) were transferred to and embedded in a multi-well plate filled with agarose nutrient solution at room temperature.

[0096] HCE were placed in 6-well plates with 1 mL of maintenance medium overnight in a CO2 incubator at 37°C and 90% humidity. The tissues were then used the next day following an acute 4-hour exposure and a repeat exposure and overnight protocol for 8 hours.

[0097] 30 μL of tear substitute was applied directly to the human corneal epithelium (HCE), and 30 μL of saline and BAK 0.1% were used as negative and positive controls, respectively.

[0098] Two exposure protocols were followed: Acute Exposure Protocol (4 hours): 4 hour exposure followed by tissue rinsing Repeated exposure and overnight protocol (8 hours + overnight): two exposures each day with at least 4 hours between the two treatments and readout after overnight incubation.

[0099] This protocol was performed in triplicate tissues.

[0100] At the end of exposure, the following endpoints were investigated: Cytotoxicity assay by MTT Immunofluorescence analysis with TUNEL assay to detect apoptotic cells.

[0101] Viability assay: modified MTT test Principle of the method The method allows the assessment of toxic events and associated cytotoxicity by a colorimetric assay. Solutions of MTT in balanced salt solution are yellowish in color. Mitochondrial dehydrogenases in viable cells cleave the tetrazolium ring, resulting in blue / purple MTT crystals that are insoluble in aqueous solutions.

[0102] procedure Residual viability after treatment by MTT test was performed according to SOP M 04 with a modified protocol including evaluation of the surface layer.

[0103] Data Acquisition The precision of the spectrophotometric measurements is in the range of 0-2 OD: <+ / -(1%+10 mOD). Raw OD data are recorded directly on a microplate autoreader (TECAN INFINITE M-200) and further processed by the software Magellan 6.5.

[0104] The blank OD mean was subtracted from each individual OD value. The viability of each biological replicate was calculated by dividing the respective OD mean to the optical density mean of the negative control (NC) biological replicates. The mean % viability of tissues treated with the same substances was calculated.

[0105] Acceptance Criteria NC is accepted if the OD negative control mean of the two tissues is ≧1 and the standard deviation (SD) of % viability is <18%. A test substance meets the acceptance criteria if the standard deviation value (SD) of % cell viability (expressed as a percentage compared to NC) between two tissue replicates is <18%.

[0106] TUNEL assay: apoptotic cell detection Principle of the method Cleavage of genomic DNA during apoptosis can result in double-stranded, low molecular weight DNA fragments (mononucleosomes and oligonucleosomes) and single-strand breaks ("nicks") in high molecular weight DNA. These DNA strand breaks can be identified by labeling the free 3'-OH termini with modified nucleotides in an enzymatic reaction.

[0107] procedure Labeling of DNA strand breaks with terminal deoxynucleotidyl transferase (TdT), which catalyzes the polymerization of labeled nucleotides to free 3'-OH DNA ends in a template-independent manner (TUNEL-reaction), was performed on paraffin sections with the In Situ Cell Death Detection Kit (Roche, Cat. No. 11684795910) according to the manufacturer's instructions. Nuclei were counterstained with DAPI, mounted on microscope slides and prepared for analysis in fluorescence with a LEICA DMi8 THUNDER Imager 3D, Camera K5 and processing with LASX 3.7.1 software. For each sample, whole stained sections were acquired at 20x magnification (12-13 acquisitions per sample) and the number of Tunel (green) positive cells and total cells (blue, DAPI) present in the tissue were counted, as well as the percentage of positive cells vs. total cells assessed.

[0108] result The results are shown in Table 3 below. [Table 4]

[0109] The results of the MTT test showed the following: As expected, BAK 0.1% caused severe cytotoxicity at both exposures. The composition of Example I did not induce significant cytotoxicity at both time points considered. Visu XL did not induce significant cytotoxic effects after 4 h acute exposure, but after 8 h and subsequent overnight incubation, MTT values ​​were borderline the cytotoxicity cut-off (60% cell viability).

[0110] Based on the TUNEL assay and as shown in FIG. 1, it is interesting to note that a reduction in the total number of apoptotic cells was observed with the formulation of Example I even upon prolonged exposure, suggesting a protective efficacy.

[0111] 2. Antioxidant activity 2-1 In vitro assay The objective of this study was to investigate the potential antioxidant properties of different formulations containing the composition of Example I by ABTS assay.

[0112] method Principle of the method When reacted with potassium persulfate, ABTS, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate), forms the ABTS.+ radical, which is blue to green in color. Addition of an antioxidant test article should reduce this radical and cause a color change in the mixture. The color change of the radical, measured spectrophotometrically, was proportional to the antioxidant properties of the test article.

[0113] The assay was performed using the Antioxidant Assay kit (Sigma Aldrich) according to the manufacturer's instructions.

[0114] The method was standardized against the positive control Trolox (Sigma Aldrich).

[0115] Test substances The following formulations were tested: Visu XL in three concentrations (pure, 50% and 25% diluted in assay buffer) Thealoz® duo in three concentrations (pure, 50% and 25% diluted in assay buffer) The composition of Example I (containing 0.05% resveratrol) in three concentrations (pure, 50% and 25% after dilution in assay buffer). The composition of Example I in three concentrations (pure, 50% and 25% after dilution in assay buffer) but containing 0.01% resveratrol Negative control: Assay buffer Positive control: Trolox at five concentrations (23.4, 47, 94, 188, and 375 μg / ml)

[0116] result Data expressed relative to the negative control according to the formula 100-(OD test article / OD negative control x 100) is shown in Figure 2.

[0117] conclusion Visu XL at 25%, 50% and 100% exhibits moderate antioxidant effects.

[0118] Thealoz® duo exhibits no antioxidant effect under the conditions of the assay.

[0119] The compositions of Example I tested at concentrations of 25%, 50%, or 100% showed high levels of antioxidant efficacy, slightly higher for the composition containing 0.05% resveratrol than for the composition containing 0.01% resveratrol.

[0120] 2-2 In vivo assay 5MED equivalent dose 125mJ / cm 2 An experimental model of human corneal epithelium (HCE) was performed to evaluate the protection offered by tear substitutes against corneal epithelial oxidative stress damage induced by UVA + UVB light (290-400 nm) in the setting of UVA+UVB light.

[0121] The formulation of Example I was tested against Visu XL (a commercial standard containing 0.1% Coenzyme Q10, 0.1% cross-linked hyaluronic acid (HA), 0.5% Vitamin E TPGS; pH 6.9).

[0122] UV light-induced oxidative stress damage, as GSH content, apoptosis and DNA damage, pro-inflammatory and cytotoxic responses, were investigated 2 and 24 h after direct irradiation of the HCE surface with 5MED.

[0123] method 0.5cm 2 Reconstructed human corneal epithelium (HCE) from EPISKIN was used for the evaluation. Human epithelial immortalized cells (HICECs) were placed on polycarbonate filters and cultured at the air-liquid interface in chemically defined medium for 5 days to form a structured epithelium. Inserts containing the 5-day tissue (thickness: 55 μm; 5 cell layers) were transferred to and embedded in a multi-well plate filled with agarose nutrient solution at room temperature.

[0124] The HCE was placed in a 6-well plate with 1 mL of maintenance medium overnight in a CO2 incubator at 37°C and 90% humidity.

[0125] This protocol was performed in triplicate tissues.

[0126] The following series were part of the study: Negative control (NC): HCE treated with 30 μL of PBS and stored in the dark (for the same time as the irradiation period). Positive control (IRR): 30 μL of PBS was applied to the HCE surface before UV exposure. HCE was applied at 125 mJ / cm 2 The patient was irradiated with 5 MED, which corresponds to Test Article: The composition of Example I and 30 μL of Visu XL were applied to the HCE surface prior to UV exposure.

[0127] HCE was treated prior to UV exposure according to the following procedure: two treatments of 30 μL with at least 4 hours between the two applications, followed by overnight incubation.

[0128] UVA + UVB irradiation was performed the next day using a xenon arc lamp and filter WG320 [mW / cm 2 125mJ / cm2 measured by an Oriel solar simulator equipped with 2 So I went.

[0129] After UV exposure to the different HCE series, 30 μL of tear substitute was applied again and then the HCEs were incubated at 37° C. and 90% humidity for 2 and 24 hours.

[0130] The following tests were then performed on HCE at one or both time points: I. TUNEL assay-IHC: apoptotic cell detection (at 24 hours) II. IL-8 Release by ELISA: A Pro-inflammatory Chemokine (at 2 and 24 hours) III. Toxilight assay: Measurement of membrane integrity and cytotoxicity by adenylate kinase release (at 2 and 24 hours)

[0131] I / TUNEL assay: apoptotic cell detection See section II.1 above.

[0132] II / TOXILIGHT Bioassay Kit, Catalog No. LT27-060 Principle of the method The ToxiLight™ Bioassay is a non-destructive bioluminescent cytotoxicity assay designed to measure toxicity in mammalian cells and cell lines in culture. The kit quantitatively measures the release of adenylate kinase (AK) from damaged cells. The kit is based on the bioluminescent measurement of AK, which is present in all cells. Loss of cellular integrity through damage to the plasma membrane leads to leakage of several factors from cells cultured in vitro into the surrounding medium. Measurement of the release of AK from cells allows an accurate and sensitive measurement of cytotoxicity and cell lysis.

[0133] procedure At the end of the treatment, 20 μL of medium is transferred to a 96-well plate and 100 μL of adenylate kinase (AK) detection reagent is added to the medium. After 5 minutes of incubation at room temperature, the AK concentration is measured in each well.

[0134] Data Acquisition and Analysis Luminescence data acquisition was performed using a TECAN INFINITE M-200.

[0135] III / IL-8 ELISA quantification, Catalog No. RAB 0319 Principle of the method ELISA (Enzyme-Linked Immunosorbent Assay) kits are in vitro assays for quantitatively measuring target proteins in biological samples. The assay uses a specific capture antibody coated onto a 96-well plate. Standards and samples are pipetted into the wells and the target protein present in the samples is bound to the wells by the immobilized antibody. The wells are washed and a biotinylated detection antibody specific for the target protein is added. After washing away unbound biotinylated antibody, streptavidin conjugated to HRP is pipetted into the wells. The wells are washed again and a TMB solution is added to the wells, which develops color in proportion to the amount of bound target protein. A stop solution changes the color from blue to yellow and the color intensity is measured at 450 nm.

[0136] procedure ELISA quantification of IL-8 was performed on media cultures harvested from protocol A and B treatments. Samples were stored at -20°C. Human IL-8 / CXCL8 Elisa kit was performed according to the instructions of the Millipore Elisa kit (#RAB0319). Samples were diluted 1:20 (2 hours) and 1:35 (24 hours).

[0137] Data Acquisition and Acceptance Criteria OD was obtained using a microplate autoreader (TECAN INFINITE M-200). Standard curves were obtained by four-parameter logistic regression (4PL). The sensitivity range was 0.8-600 pg / ml. The minimal detectable dose of human IL-8 was determined to be 1 pg / ml. Statistical analysis by ANOVA.

[0138] result 1 / Cell membrane damage by TOXILIGHT: Release of adenylate kinase (AK) The Relative Luminescence Unit (RLU) data at 2 hours and 24 hours after UV irradiation are shown in Table 4 below. [Table 5]

[0139] Increased release of AK was quantified in HCE-irradiated controls compared to non-irradiated negative controls at both time points, confirming the cytotoxic effect of 5MED UV.

[0140] The new treatments, performed 24 hours after UV exposure and immediately after UV, made it possible to highlight the actual protective effect of the composition of the invention against UV-induced damage.Indeed, the product induced a significant reduction in AK compared to the IRR control and also compared to the NC, suggesting a clear protective effect on cell membrane integrity.At this point, Visu XL does not show a protective effect against UV-induced damage to cell membranes.

[0141] 2. ELISA quantification of IL-8 Data at 2 and 24 hours after UV exposure are shown in Table 5 below. [Table 6]

[0142] At 2 hours post-UV, no significant IL-8 release due to irradiation effect (IRR) was determined, but release was significantly increased at 24 hours.

[0143] The Visu XL product blocks IL-8 release only for a short time (only observed 2 hours after irradiation), whereas at both time points the composition according to the invention shows a very significant anti-inflammatory activity, with no detectable IL-8 for at least 24 hours after irradiation.

[0144] 3 / TUNEL staining: apoptosis Data 24 hours after UV exposure are shown in FIG.

[0145] This shows approximately 30% apoptotic cells in the presence of the irradiated control (IRR) and the Visu XL product, whereas the composition of Example I (according to the invention) inhibits the formation of apoptotic cells (by approximately 10%) and the associated DNA damage.

[0146] conclusion The data presented above confirm the antioxidant properties of the composition of Example I, not only in vitro but also in vivo. Furthermore, they demonstrate that the composition of Example I: - Protection against inflammation (significant reduction in IL-8 release); - Significantly reduced UV damage (significantly reduced release of adenylate kinase); - shows inhibition of apoptotic cell formation and associated DNA damage (% of TUNEL positive nuclei similar to negative control).

[0147] On the other hand, the Visu XL product was only effective as an anti-inflammatory drug for 2 hours after irradiation, and a significant reduction in IL-8 release was observed. EXAMPLES

[0148] Comparison with other antioxidants Whereas Example II demonstrated that a composition combining Thealoz® duo formulation with resveratrol was superior to the commercial standard Visu XL (0.1% Coenzyme Q10, 0.1% cross-linked hyaluronic acid (HA), 0.5% Vitamin E TPGS; pH 6.9), it was decided to test additional antioxidants against the background of Thealoz® duo.

[0149] This study focused on clarifying the antioxidant potential of four eye drop preparations by the LUCS method (AOP1) in order to evaluate their direct antioxidant effect (neutralization of intracellular free radicals) in living human HepG2 cells.

[0150] method LUCS method (AOP1) Principle of the method AOP's proprietary LUCS method is based on the production of free radical species by cells after the addition of a fluorescent biosensor that targets nucleic acids in the culture medium. The combined effect of light and the presence of the biosensor (photoinduced) leads to the production of singlet oxygen, which in turn triggers the generation of reactive oxygen species (ROS), a cascade of biochemical reactions, resulting in a positive change in the emitted fluorescence.

[0151] The LUCS test measures the ability of antioxidants to neutralize intracellular oxidative stress. This effect is measured by the resulting delay in the kinetics of the increase in fluorescence emission. The method has been standardized to a 96-well cell culture plate format to ensure high-throughput and reliable statistical analysis (WO2009087229; https: / / www.nature.com / articles / s41598-017-18211-2).

[0152] procedure This study was performed on cultured human HepG2 hepatocytes. Cells were seeded in 96-well plates at 75,000 cells per well in complete DMEM medium in the presence of SVF and placed in an incubator at 37°C / 5% CO2 for 24 hours.

[0153] The experiments were performed in DMEM medium without SVF. Cells were incubated with the different samples (8 dilutions obtained by two-step serial dilution) for 4 hours at 37°C / 5%CO2. Two independent experiments were performed in triplicate in 96-well plates. After 4 hours of incubation, HepG2 cells were treated with fluorescent biomarkers for 1 hour, and then measurements (RFU at 535 nm) were performed following a replicate protocol (20 replicates) of illumination and fluorescence reading of the 96-well plate at 480 nm.

[0154] The cellular antioxidant performance index (AOP index) was calculated using the formula:

number

[0155] Dose-response curves obtained by collecting the AOP index as a function of the log(10) of sample concentration were Formula: AOP index = AOP index min +(AOP index max -AOP index min ) / 1+10 (Log(EC50-CE)*(HS) ) The curves are processed by sigmoidal fitting according to the following formula: and the EC50 (the concentration at which the test compound exerts 50% or half of its full effect), EC10, and EC90 are calculated.

[0156] sample The samples tested in this study were: - Sample No. 1 = Thealoz® duo + TPNa (sodium tocopherol phosphate) 0.5% - Sample No. 2 = Thealoz® duo + TPGS VitE (Vitamin E Polyethylene Glycol Succinate) 0.5% - Sample No. 3 = Thealoz® duo + Resveratrol 0.01% - Sample No. 4 = Thealoz® duo + Quercetin 0.01% [Table 7] [Table 8]

[0157] The samples, in liquid form in amber bottles labeled Samples 1, 2, 3 and 4, were kept in the dark at 4°C until the experiment.

[0158] result The results are shown in Table 6 below. [Table 9]

[0159] conclusion The formulations Thealoz® duo+TPNa and Thealoz® duo+TPGS VitE show no antioxidant activity.

[0160] Thealoz® duo + Resveratrol exhibits potent, direct antioxidant activity by neutralizing intracellular free radicals, with an Antioxidant Index (AI) approaching 1000 (theoretical maximum) at dilutions >= 1 / 32.

[0161] Thealoz® duo + Quercetin exhibits strong, direct antioxidant activity, with an Antioxidant Index (AI) approaching 1000 (theoretical maximum) at dilutions >= 1 / 2.

[0162] This activity appears to be more potent in formulations with resveratrol than with quercetin in this model.

[0163] In conclusion, in the context of Thealoz® duo, resveratrol has a better antioxidant effect than quercetin at the same concentration, in other words, resveratrol is the most powerful antioxidant in this context. EXAMPLES

[0164] Contribution of trehalose in the composition according to the invention 1. Stability To study the stability of resveratrol (R) in the presence of trehalose (T) or trehalose + hyaluronic acid (T+HA), the amount of resveratrol was determined after storage at 60°C for 7 days.

[0165] To make the test more sensitive, resveratrol was used at a concentration of approximately 0.08 g / 100 mL of test formulation.

[0166] The composition of the different formulations tested, and the end amount of resveratrol in the formulations, are shown in Table 7 below. [Table 10]

[0167] The data demonstrate that under these storage conditions, resveratrol is significantly unstable in the absence of trehalose (last column).

[0168] 2. Activity To evaluate the effect of trehalose on the activity of the composition according to the invention, the experiment disclosed in Example II-2-2 (Antioxidant activity tested in vivo by IL-8 release quantification - ELISA test) was reproduced using the formulations disclosed in Table 8 below. [Table 11]

[0169] As shown in Figure 4, 24 hours after irradiation, a decrease in IL-8 release is observed in the presence of trehalose alone (P4). Trehalose would then contribute to inhibiting inflammation in addition to resveratrol.

Claims

1. - hyaluronic acid or one of its salts, advantageously with a molecular weight between 100 and 800 kDa; - oligosaccharides, preferably diholosides, more preferably trehalose; - Resveratrol and 1. An ophthalmic composition comprising:

2. 10. The composition of claim 1, which is an aqueous solution.

3. 3. The composition according to claim 1 or 2, further comprising a solubiliser, preferably macrogol hydroxystearate, more preferably polyoxyl 40 hydrogenated castor oil.

4. 4. The composition according to claim 3, wherein the solubilizer represents from 0.1 to 20% (w / v), advantageously from 0.5 to 5% (w / v), more advantageously 0.5% or 1% (w / v).

5. 5. The composition according to claim 1, exhibiting a pH below 7, advantageously between 5.5 and 6.9, and more advantageously equal to 6.

0.

6. 6. The composition according to claim 5, comprising a buffer selected from the group consisting of citrates, borates and phosphates, advantageously citrate.

7. 7. Composition according to any one of claims 1 to 6, wherein hyaluronic acid or one of its salts represents between 0.05 and 0.5 g per 100 ml of composition, advantageously between 0.1 and 0.2 g / 100 ml, and even more advantageously 0.15 g / 100 ml.

8. 8. The composition according to any one of claims 1 to 7, wherein the oligosaccharides represent between 0.5 and 5 g / 100 ml of the composition, advantageously between 2 and 4 g / 100 ml, and even more advantageously 3 g / 100 ml.

9. 9. The composition according to any one of claims 1 to 8, wherein resveratrol represents between 0.01 and 0.1 g / 100 ml of the composition, advantageously between 0.04 and 0.06 g / 100 ml, and even more advantageously 0.05 g / 100 ml.

10. 10. The composition of any one of claims 1 to 9, which is free of preservatives of antimicrobial type.

11. 11. A composition according to any one of claims 1 to 10 for use in the treatment of an ocular disease, advantageously dry eye disease (DED).

12. 12. A composition for use as defined in claim 11, which is administered ocularly.

13. A composition for use thereof as defined in claim 11 or 12, administered to each affected eye of a human or animal at least once per day to at least 10 times per day, advantageously 2 to 4 times daily.

14. 11. A single use or preservative-free multi-dose vial comprising a composition according to any one of claims 1 to 10.

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