Compositions for the treatment of dry eye, conjunctivitis and wound healing

Abstract

The present invention relates to compositions, which are suitable for the treatment of dry eye syndrome related and / or conjunctivitis related signs and symptoms and / or to promote corneal wound healing and / or to protect the eye from the UV and blue light. The compositions comprise Carboxymethyl cellulose (CMC), and Silk Proteins (SP) in defined weight ratios. The invention further relates to the use of CMC, and SP in a method of treatment for the dry eye syndrome related and / or conjunctivitis related signs and symptoms and / or to promote corneal wound healing and / or to protect the eye from the UV and blue light. The invention further relates to CMC, and SP for use in a method to treat corneal wound due to dry eye or surgery or injury derived. The invention further relates to an ophthalmic kit of parts comprising comprise CMC and SP. In addition, CMC of the present invention can be cross-linked.

Description

[0001] Compositions for the treatment of dry eye, conjunctivitis and wound healing

[0002] The present invention relates to compositions suitable for ophthalmic administration, containing carboxymethylcellulose (CMC), possibly cross-linked, and silk proteins (SP) aimed at treating the signs and symptoms of dry eye syndrome and / or conjunctivitis and / or promoting the healing of corneal wounds and / or protecting the eye from UV rays and blue light.

[0003] Background Art

[0004] THE TEAR FILM

[0005] The tear film is a unique thin fluid layer approximately 3pm thick and 3pl in volume that covers the outer mucosal surfaces of the eye. As such, it is the interface of the ocular surface with the environment. The human tear film is essential for the protection of the corneal epithelium against drying and inflammation. This film is transparent and it is composed of three layers:

[0006] 1 . The lipid layer, produced by the meibomian glands in the eyelids, forms the outermost surface of the tear film. Its primary purpose is to smooth the tear surface and reduce evaporation of tears. The lipid layer of the tear film is thin, in the order of 50 to 100nm, yet contains many different lipid species including non-polar lipids such as cholesterol and wax esters which make up it bulk, and polar lipids such as (O-acyl)-co-hydroxy fatty acids and phospholipids which interact with the aqueous layer. The majority of these lipids, with the possible exception of the phospholipids, are secreted from meibomian glands located at the lid margin. The most important role of the lipid layer is to delay the evaporation of tears from the ocular surface in order to keep the volume of the tear film constant and thus ensure the hydration and lubrication of the eye.

[0007] 2. The intermediate aqueous layer forms the majority of the tears. Most of the aqueous fluid is secreted from the lacrimal glands, which also secretes a specific variety of proteins, electrolytes, and water. The conjunctival epithelium is a second source of electrolytes and water in the tears. The aqueous phase provides oxygen and nutrients to the underlying avascular corneal tissue and flushes away epithelial debris, toxins and foreign bodies.

[0008] 3. The inner layer consists of mucus produced by the conjunctiva. Mucus allows the watery layer to spread evenly over the surface of the eye and helps the eye remain moist. Without mucus, tears would not stick to the eye. Many of the mucins are secreted by specialised goblet cells in the conjunctival epithelium, and some transmembrane mucins are released into the tear film from corneal and conjunctival epithelial cells. When anchored into the epithelial cells, these transmembrane mucins extend into the aqueous phase and help stabilise the tear film.

[0009] While traditionally understood as three separate and distinct layers, new studies suggest that the mucin and aqueous layers integrate to create a gradient of decreasing mucin concentration outwards to the aqueous layer. Tears lost by evaporation and drainage are replaced by tear secretion. Tear turnover rate is 1- 3pl / minute under normal circumstances but can increase greatly upon stimulation by irritants or in response to emotion. Indeed, three different types of tears have been described; basal tears that are normally present on the ocular surface, reflex tears produced upon stimulation and closed-eye tears which bathe the eye during sleep.

[0010] One of the primary functions of the tear film is to protect the ocular surface from potentially pathogenic microbes, and it appears to be extremely effective in this function.

[0011] Mucins can have an antimicrobial function by acting as decoy receptors for microbes, preventing them from adhering to the underlying tissues. Many of the major tear film proteins, such as lysozyme and lactoferrin, are either directly toxic to bacteria (lysozyme which is an enzyme that hydrolyses the peptidoglycan cell walls of bacteria) or reduce nutrients needed for microbial growth (lactoferrin chelates iron). Secretory IgA prevents the adhesion of microbes to the epithelia and enhances phagocytosis by neutrophils that enter the tear film during sleep to protect the eye. These molecules can act in synergy to enhance their antimicrobial actions. In addition, tears contain a rich spectrum of antimicrobial peptides.

[0012] DRY EYE SYNDROME

[0013] Dry eye disease (also known as keratoconjunctivitis sicca) is a very common yet frequently underrecognized clinical condition whose aetiology and management challenge both clinicians and researchers. In the last ten years, advances in the understanding of the epidemiology, pathogenesis, clinical manifestation and possible therapy of dry eye disease have been made.

[0014] In 2007, the first International Dry Eye Workshop developed the following definition of dry eye disease: “Dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface”.

[0015] Several factors contribute to the risk of developing the dry eye syndrome

[0016] Dry eye is caused by a disturbance of the lacrimal function unit (LFU), an integrated system comprising the lacrimal glands, ocular surface and lids, and the sensory and motor nerves that connect them. Tear hyperosmolarity and tear film instability play a key role in the pathogenesis of dry eye.

[0017] Tear hyperosmolarity causes damage to the surface epithelium by activating a cascade of inflammatory events at the ocular surface and a release of inflammatory mediators into the tears. Epithelial damage involves cell death by apoptosis (i), a loss of goblet cells (ii), and disturbance of mucin expression (iii), leading to tear film instability (iv). This instability exacerbates ocular surface hyperosmolarity (v) and completes the vicious circle. Tear film instability can be initiated, without the prior occurrence of tear hyperosmolarity, by several aetiologies, including xerophthalmia, ocular allergy, topical preservative use and contact lens wear. The epithelial injury caused by dry eye stimulates corneal nerve endings, leading to symptoms of discomfort, increased blinking and, potentially, compensatory reflex lacrimal tear secretion. Loss of normal mucins at the ocular surface contributes to symptoms by increasing frictional resistance between the lids and ocular globe.

[0018] The major causes of tear hyperosmolarity are reduced aqueous tear flow, resulting from lacrimal failure, and / or increased evaporation from the tear film. Increased evaporative loss is favoured by environmental conditions of low humidity and high airflow, and it may be caused by meibomian gland dysfunction (MGD), which leads to an unstable tear film lipid layer. Reduced aqueous tear flow is due to impaired delivery of lacrimal fluid into the conjunctival sac. It is unclear whether this is a feature of normal aging, but certain systemic drugs, such as antihistamines and anti- muscarinic agents may induce it. The most common cause is inflammatory lacrimal damage, seen in autoimmune disorders such as Sjogren syndrome dry eye (SSDE) and in non-Sjogren syndrome dry eye (NSSDE). Inflammation causes both tissue destruction and a potentially reversible neurosecretory block. Tear delivery may be obstructed by conjunctival scarring or reduced by a loss of sensory reflex drive to the lacrimal gland from the ocular surface. Eventually, the chronic surface damage of dry eye leads to a fall in corneal sensitivity and a reduction of reflex tear secretion.

[0019] Tear supplementation and stabilization, control of inflammation of the lacrimal glands and ocular surface, and possible stimulation of tear production are treatment options that are used according to the character and severity of dry eye disease.

[0020] RED EYE AND EYESTRAIN

[0021] The red eye syndrome is a symptom of inflammatory eye irritation, which is usually caused by toxins, pathogens, viruses or bacteria in the ocular environment. This is an acute reaction that is usually associated with continuous use of contact lenses. It may also be an accompanying phenomenon of certain eye diseases including glaucoma, conjunctivitis, keratitis or uveitis. During continuous wear, lenses accumulate metabolites and other harmful substances produced by bacteria. These substances, together with bacteria, irritate the eye and may eventually cause inflammation.

[0022] Eyestrain, also known as asthenopia, is an eye condition that manifests through nonspecific symptoms such as fatigue, pain in or around the eyes, blurred vision, headache, and occasional double vision. Symptoms often occur after reading, computer work, or other close activities that involve tedious visual tasks. COMPUTER VISION SYNDROME

[0023] Computer vision syndrome (CVS) is the term used to describe the collection of visual, ocular and musculoskeletal (neck and shoulder pain) symptoms that result from prolonged computer use. Symptoms reported by computer users include eyestrain, eye fatigue, burning and irritation of the eyes, tired eyes, dry eyes, ache in and around the eyes, blurred vision at near, blurred vision when looking from near to far, headache, neck ache and shoulder pain. Symptoms such as eye fatigue, ache in and around the eyes, blurred vision and headache, collectively referred to as asthenopia, have also been reported by other workers who perform tasks that require staying close to a screen, like accountants and book keepers. However, in a recent study the symptoms experienced immediately after a sustained near task performed at a computer display were found to be greater than the mean symptom score obtained following a task performed using hard copy material.

[0024] Symptoms that constitute computer vision syndrome can be broadly classified into ocular, visual and musculoskeletal (Table 1 ). The severity and the specific type of symptoms experienced are related to the duration of exposure, nature of the demanding visual task, environmental factors in the workplace and the individual’s visual abilities.

[0025] Table 1: Symptoms associated with computer use classified as ocular, visual and musculoskeletal symptoms.

[0026] Many studies have evaluated the severity and frequency of symptoms associated with the use of computers across different working populations. A survey of computer users and non-users showed that visual complaints were reported by 75% of computer users who work 6-9 hours in front of their screens compared to 50% of nonusers. A recent survey of 419 computer users in India showed that about 46.3% of the users experienced two or more of the following symptoms either during or after computer work: gritty feeling, itching in the eyes, aches, sensitivity, redness, tears in excess, dryness, discomfort in seeing, blurring of vision and discoloration of objects. The severity of symptoms depends on the hours of use of the screen, increasing significantly with longer duration of computer use. An epidemiologic study of 1545 Massachusetts workers found that individuals who spent longer than 4 hours working at a computer display experienced greater incidence of ocular symptoms. More recently, a study that involved 520 New York City office workers confirmed the existence of a significant positive correlation between the symptom score reported and the number of hours spent working on the computer. A survey of optometrists showed that almost 14.5% of patients schedule the examination primarily because of problems related to work with computers. The symptoms of computer workers reported in a recent survey conducted in India at different institutions, included eyestrain (53.8%), itching (47.6%) and burning of the eyes (66.7%). In addition to the discomfort experienced during and immediately after computer use, the presence of CVS has been associated with lower quality of life and work productivity.

[0027] Dry eye like symptoms are common in people working at computer screens. Reduced blink rate is proposed to be a contributing factor to the dry eye like symptoms experienced during, or immediately after prolonged computer work. Computer users are also advised to blink often, take frequent breaks and use artificial eye drops to reduce symptoms.

[0028] Summary of the invention

[0029] Technical Problem

[0030] Tear substitutes play a fundamental role in the clinical management of the various pathologies listed above, for example: dry eye syndrome, signs and symptoms of conjunctivitis, as they provide patients with the form of immediate relief.

[0031] Ocular lubricants are characterized by hypotonic or isotonic buffer solutions containing electrolytes, surfactants and various types of viscosity agents. Viscosity (i.e. thickening) agents are added to ocular lubricants to increase their residence time and contact with the ocular surface, ensuring a longer interval of comfort for the patient and increasing the duration of the action and permanence of the lubricant. At the same time, there are no preparations that, in addition to resolving the signs and symptoms related to dry eye syndrome and conjunctivitis, are able to protect the eye from UV radiation and blue light or improve the healing process of a corneal wound. This is because tear substitutes are mainly based on thickening agents such as carboxymethylcellulose or hyaluronic acid. However, none of these combined the viscosity agent with silk proteins.

[0032] In fact, it is important to note that, in order to improve the management of symptoms and signs related to dry eye syndrome and conjunctivitis, it is desirable that a preparation also protects the ocular surface from UV radiation and blue light (photophobia) and promotes the healing process of a corneal wound. Solution to Problem

[0033] In addressing the problem of developing compositions, uses and methods for the treatment of dry eye syndrome or to protect the eye from UV and blue light and to promote corneal wound healing, the inventor surprisingly found that cross-linked carboxymethyl cellulose (CX-CMC) and silk proteins (SP) are able to treat the signs and symptoms of dry eye and conjunctivitis, absorb UV and blue light, and synergistically promote wound healing. Until now, it was well known that high- carboxymethylcellulose, possibly in combination with other substances, was able to improve dry eye symptoms, but it was not known or suspected that the combination of CX-CMC and SP could be effective in treating dry eye syndrome and / or conjunctivitis and / or to promote corneal wound healing and / or to protect against UV and blue light, and the inventor was not aware of any experimental findings of this nature. Evidence of this synergistic action represents an entirely new and advantageous effect that can be achieved using the composition.

[0034] Accordingly, one aspect of the invention provides a composition, suitable for ophthalmic administration (i.e. eye drops, ointments, gel, spray, contact lens), comprising, as functional or active ingredients, carboxymethyl cellulose (CMC) and silk proteins (SP), wherein carboxymethyl cellulose and silk proteins are present in a w / w% in the final product:

[0035] Table 2 To the above-mentioned quantities well known and commonly used pharmaceutical excipients can be added. In particular, one or more of the following excipients can be added: one or more osmotic regulators; one or more pH stabilising agents; one or more chelating; one or more preservative agents; one or more emulsifiers; one or more thickening agents; one or more antioxidant; one or more colouring agent. It has to be understood that the above pharmaceutical excipient may or may not be present in the formulation and only one, more than one or all of them can be used.

[0036] A further aspect of the invention provides a composition suitable for ophthalmic administration (i.e. eye drops, ointments, gel, spray, contact lens), comprising cross-linked carboxymethyl cellulose (CX-CMC) and silk proteins (SP), wherein cross-linked carboxymethyl cellulose and silk proteins are present in a w / w% in the final product:

[0037] Table 2

[0038] To the above-mentioned quantities well known and commonly used pharmaceutical excipients can be added. In particular, one or more of the following excipients can be added: one or more osmotic regulators; one or more pH stabilizing agent; one or more chelating; one or more preservative agents; one or more emulsifiers; one or more thickening agents; one or more antioxidant; one or more colouring agent. It has to be understood that the above pharmaceutical excipient may or may not be present in the formulation and only one, more than one or all of them can be used.

[0039] A further aspect of the invention provides a combination of carboxymethyl cellulose (possibly cross-linked) and silk proteins for use as a medicament.

[0040] A further aspect of the invention provides for the treatment of dry eye syndrome signs and symptoms and / or conjunctivitis signs and symptoms and / or promotes corneal wound healing and / or protects the eye from UV and blue light.

[0041] It has to be specified that the need of corneal wound healing might have been generated by an accident, by a pathology or surgery. The causes of corneal injury listed above are for illustrative purposes only and are not intended to be exhaustive.

[0042] In a particular embodiment, the invention comprising a combination of carboxymethyl cellulose (eventually cross-linked) and silk proteins in the form of eye drops produced either in a multidose bottle or a unite dose vial.

[0043] A further aspect of the invention provides a kit of parts comprising carboxymethyl cellulose (CMC, eventually cross-linked) and silk proteins (SP) in two or more containers.

[0044] Advantageous Effects of Invention

[0045] As mentioned above, it is important that, in order to improve the management of symptoms and signs related to dry eye syndrome and conjunctivitis, it is desirable that a preparation also protects the ocular surface from UV radiation and blue light (photophobia) and promotes the healing process of a corneal wound.

[0046] The inventor is not aware of any experimental findings of this nature. Evidence of this synergistic action represents an entirely new and advantageous effect that can be achieved using the composition.

[0047] Indeed, the data described clearly shows that a composition containing carboxymethyl cellulose (CMC, eventually cross-linked) and silk proteins (SP) effectively improve dry eye signs and symptoms, to promote corneal wound healing and to protect form UV and blue light.

[0048] Brief Description of Drawings

[0049] Figure 1 - Graph representing TBUT (Tear Break-Up Time) and OSDI (Ocular Surface Disease Index) in subjects with dry eye syndrome before and after instillation of eye drops containing CX- CMC and SP: **** =P<0.0001. Figure 2 - Graph representing the wound healing rate. In particular, a synergistic action between CX-CMC and SP is evident: *=P<0.05; **=P<0.01 ; ***=P<0.001.

[0050] Description of Embodiments

[0051] In the preset paragraph, we aim to define a number of terms used to describe the invention in the current application. Some terms are used interchangeably when describing several elements or inventive embodiments. It is important to note that any feature of the invention described in this document that uses a word or expression that is defined elsewhere in this document also applies to that aspect. This holds true independent of the portion of the current application in which such concepts are defined or addressed.

[0052] The term "comprising" as used in this document means "including", "encompassing", or "containing" in its broadest sense. It comprises the element(s) that have been explicitly recited as well as any other element(s) that may be present but are not necessary. In addition to this wide definition, the word "comprising" as used in this context also includes the limiting definition "consisting of," which denotes the presence of only the expressly recited element(s) and none other(s). Additionally, the word "comprising" also has the meaning of "consisting essentially of", which allows for the presence of elements other than those that have been explicitly listed as long as they do not change the technical outcome that has been achieved by the explicitly listed elements.

[0053] When referring to a specific value, such as an amount represented in weight or percentage, the phrase "about" as employed here incorporates and reveals, in addition to the value expressly recited itself, a specific fluctuation surrounding the specifically recited value. Such a variance may, for instance, result from common measurement variability, such as when different substances are weighed or distributed according to techniques that are known to the expert person. The word "about" should be taken to mean a range of variability above and below the specified value, with the percentage values being relative to the recited value itself, as seen in the following. The phrase "about" may include and reveal variation of up to 15.0% of the specified amount as generally accepted by the expert person.

[0054] As used herein, the term “ophthalmic composition” encompasses and discloses any physical entity comprising or consisting of the respective recited substances. The physical form of the ophthalmic composition is not restricted. As example the term “ophthalmic composition” also encompasses and discloses a liquid solution in which the recited substances are present in solubilized form, it also encompasses and discloses an emulsion or a suspension. As a further example, the term “composition” also encompasses and discloses a gel or an ointment in which the recited substances are present. As a further example, the term “composition” also encompasses and discloses a spray in which the recited substances are present.

[0055] Indeed, the term “ophthalmic composition” encompasses and discloses also contact lenses.

[0056] In particular, the term “ophthalmic composition” may be a “pharmaceutical composition” as defined herein below, which is formulated for a desired route of administration (i.e. ophthalmic). As used and disclosed herein, the term “composition” also includes the possible pharmaceutical forms suitable for ophthalmic use well known by a skilled person, e.g. eye drops in multidose bottle; eye drops in unit dose vials, ophthalmic gel, ointment or spray or contact lenses.

[0057] As used herein, the term “carboxymethyl cellulose” (CMC) encompasses and discloses a cellulose derivative with carboxymethyl groups (-CH2-COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. It is often used as its sodium salt, sodium carboxymethyl cellulose.

[0058] R = H or CH2CO2H

[0059] In a particularly preferred embodiment of the present invention in any of the various aspects set out herein, the crosslinked carboxymethyl cellulose refers to CMC that has been further modified in order to be cross-linked.

[0060] As used herein, the term silk proteins means and discloses the known fiber, composed of two different proteins, i.e. fibroin and sericin, and produced by several larvae to form the cocoons. In particular, a hydrolyzed mixture of protein derived either by fibroin, sericin or a mixture of both can be used in the present invention.

[0061] Being a hydrolysed mixture of proteins, free amino acids can be present for instance: Alanine, Glycine, Serine and tyrosine. The above list has to be taken only as an example, as it is not exhaustive, nor binding. Although the present application mentions discrete embodiments, it is to be understood that any embodiment, and the features therein, may be freely combined with any other embodiment and the features therein, even in the absence of an explicit statement to this effect. Such combinations of one embodiment with another, or of one or more features in any one embodiment with one or more features in any other embodiment, thus belong to the disclosure of the present application as filed as understood by the skilled person.

[0062] Compositions comprising cross-linked CMC and silk proteins

[0063] In addressing the problem of providing improved compositions, uses and methods for alleviating medical conditions such as dry eye disease, signs and symptoms of conjunctivitis, promoting corneal healing and protecting the eye from UV and Blue light, the inventors have surprisingly found the advantageous effect that is due to the topical use of a composition comprising crosslinked CMC (possibly cross-linked) and silk proteins. For instance, as described herein and shown in the later examples (FIG. 1 , FIG. 2, Table 5), the present invention has been able to improve the ocular surface disease index (OSDI) score and the TBUT. In addition, in In vitro experiment the composition comprising cross-linked CMC and silk proteins is able to absorb UV light and to promote wound healing.

[0064] In particular, the OSDI score is a well-recognized and objective method to evaluate symptoms linked to ocular discomfort of different origin including dry eye disease and conjunctivitis. Higher score values represent higher symptoms severity while, on the other hand, lower values represent lower severity symptoms. Concomitantly, the tear break-up time (TBUT) provides a reliable measure of the stability of the tear film. In particular, lower TBUT values indicate reduced stability and volume of the tear film typical of subjects affected by dry eye disease, while higher values of TBUT depict a more stable and healthy tear film.

[0065] On the other hand, the in vitro data on UV absorption have been collected using a Spectrophotometer providing the absorbance values (A). Higher A values depict the ability of the solution to absorb a specific wavelength. On the other hand, the data on wound healing have been collected using a cell culture system and a wound healing test. The test provides results expressed as the speed of healing. The higher values underline quicker wound healing.

[0066] The surprising finding is that combined administration of cross-linked CMC and silk proteins entails important therapeutic advantages in the treatment of dry eye disease, signs and symptoms of conjunctivitis, in promoting corneal healing and protecting the eye from UV and Blue light.

[0067] Accordingly, one aspect of the present invention provides a composition comprising cross-linked CMC (CX-CMC) and silk proteins (SP), wherein cross-linked CMC and silk proteins are present in the inventive solution in % of weights (%W / W) of about 0.1 % CX-CMC and about 0.05% or 0.3% or 0.5% or 1.0% SP. In one embodiment, cross-linked CMC and silk proteins are present in the inventive solution in % of weights (%W / W) of about 0.3% CX-CMC and about 0.05% or 0.3% or 0.5% or 1 .0% SP.

[0068] In another embodiment, CX-CMC and SP are present in the inventive solution in % of weights (%W / W) of about 0.5% CX-CMC and about 0.05% or 0.3% or 0.5% or 1 .0% SP.

[0069] In another embodiment, CX-CMC and SP are present in the inventive solution in % of weights (%W / W) of about 1 .0% CX-CMC and about 0.05% or 0.3% or 0.5% or 1 .0% SP.

[0070] Additional embodiments of the invention CX-CMC and SP are present in the inventive solution in % of weights (%W / W) respectively of about: a) 0.1 % e 0.05%. b) 0.3% e 0.5%. c) 0.5% e 0.5%. d) 1.0% e 0.5%. e) 1.0% e 1.0%.

[0071] Table 4

[0072] It is understood that:

[0073] 1 ) the CMC described above does not have to be cross-linked, so the same examples can be applied to the CMC commonly used by the person experienced in the branch in ophthalmology.

[0074] 2) establishing a certain amount of CX-CMC in the inventive composition is sufficient to automatically establish a corresponding amount of SP in the inventive composition, in accordance with the above weight ratios of CX-CMC: SP. Adhering to the CX-CMC : SP weight ratios set out above ensures that the composition of the invention can achieve an advantageous technical effect in treating dry eye disease, signs and symptoms of conjunctivitis, protecting the eye from UV and blue light and promoting corneal healing.

[0075] 3) establishing a certain amount of SP in the inventive composition is sufficient to automatically establish a corresponding amount of CX-CMC in the inventive composition, in accordance with the above weight ratios of SP.

[0076] Formulation of the inventive composition for suitability for topical formulations The inventive composition comprising CX-CMC and SP may also be formulated to be especially suitable for topical administration. Suitable dosage forms to this end may include any form suitable for application to the eye surface, and may comprise, for example, a solution, ointment, cream, gel, and / or may be prepared so as to contain liposomes, micelles, and / or microspheres sold in the form of multidose bottles, single-dose vials, plastic or aluminum tubes, sprays, contact lenses and topical wipes.

[0077] Well-known and commonly used pharmaceutical excipients may be added to make a formulation suitable for ophthalmic administration. In particular, one or more of the following excipients may be added: one or more osmotic regulators; one or more pH stabilisers; one or more chelators; one or more preservatives; one or more emulsifiers; one or more thickeners; one or more antioxidants; one or more colourants; one or more plastic resins; one or more plasticisers. It is understood that the above pharmaceutical excipients may or may not be present in the formulation and that only one, more than one or all of them may be used.

[0078] A multidose eye drop comprising CX-CMC and SP can, for example, be made by dissolving CX- CMC and SP in water, possibly with one or more pharmaceutically acceptable ingredient(s) and excipients commonly used in the manufacture of multidose eye drops. In particular, the excipients may be one or more osmotic regulators, one or more pH stabilisers, one or more chelators, one or more preservatives, one or more emulsifiers, one or more thickeners, one or more antioxidants, one or more colourants. It is understood that the aforementioned pharmaceutical excipients may or may not be present in the formulation, and only one, more than one or all of them may be used. In particular, depending on the bottle used, preservatives may or may not be present.

[0079] Therapeutic uses and methods

[0080] The inventors have discovered that combining CX-CMC and SP can effectively treat dry eye and conjunctivitis signs and symptoms, protect the eyes from UV and blue light, and promote corneal healing. Notably, there is currently no known therapeutic use of these two substances combined for any of these purposes.

[0081] Within the given context, the term "combination" or "in combination with" pertains to the utilization of CX-CMC and SP jointly in order to treat an individual. A given treatment regimen will typically comprise multiple, repeated coadministrations of CX-CMC and SP over a predetermined duration of therapy, e.g. over 1 month, over 2 months, over 3 months, over 4 months or over 5 months or more, a duration of therapy over at least 3 months being preferred. The coadministration of CX- CMC and SP may be repeated multiple times daily, for example once, twice, 3 times, 4 times, 5 times or more, a repetition of about 3-4 times a day being preferred. That is, the coadministration is repeated over a predetermined length of time, and the sum of the instances of coadministration over this predetermined length of time constitutes the treatment regimen. As used herein, the term “subject or patient or individual” means a female or male subject affected by dry eye syndrome, dry eye disease or conjunctivitis of any origin. In addition, the subjects the inventor refers at might have the need of protecting the eye from UV and blue light wavelength (without restriction on the subtype of wavelengths). Furthermore, the invention can be applied to subjects with corneal injury, by injury inventors refer to both injury that might have been accidental or induced by surgery.

[0082] Adhering to the CX-CMC and SP combined administration set out above ensures that the composition of the invention can achieve the advantageous technical effect described above.

[0083] EXAMPLE 1

[0084] Example 1 with results: Improvement of the OSDI score and TBUT: CX-CMC and SP

[0085] The present example describes an efficacy study administering an eye drop to subject affected by dry eye with an increased OSDI score and a reduced TBUT. The study aims to determine whether the combination of CX-CMC and SP is able to reduce the signs and symptoms linked to dry eye i.e. reducing the OSDI score and to improve the TBUT.

[0086] The study involved 20 subjects affected by dry eye. Subjects were evaluated based on medical history, physical examination. At enrollment, OSDI score and TBUT were taken; subjects who were in poor general health were excluded.

[0087] Subjects received the eye drop containing 0.1 % CX-CMC and 0.05% SP and were counselled to apply 3 times a day for 3 months.

[0088] At the end of the three months, OSDI and TBUT were reevaluated.

[0089] All the enrolled subjects completed the trial. Data showed that CX-CMC and SP are indeed able to significantly improve the OSDI score (i.e. reduced values) therefore improving dry eye signs and symptoms. In addition, the eye drop containing 0.1 % CX-CMC and 0.05% SP was also able to improve TBUT, therefore increasing the tear film stability (FIG.1 ).

[0090] EXAMPLE 2

[0091] Example 2 with results: UV and blue wavelength absorption

[0092] The present study has been conducted using a specific spectrophotometer (UV mini-1280 Shimadzu) able to detect the ability of a solution to absorb specific wavelengths. In particular, the absorption spectra has been studied in the interval ranging from 200 to 500nm. The tested solution were

[0093] Sol A) 0.3% CX-CMC 0.5% SP

[0094] Sol B) 0.5% CX-CMC 0.5% SP

[0095] SOL C) 0.1% CX-CMC 0.05% SP All values were normalised against a negative control containing everything but CX-CMC and SP.

[0096] The table below describes the absorption values (Abs) of UV rays and blue light for eye drops that have the following final concentration of functional ingredients:

[0097] Table 5 Absorbance values clearly demonstrate that the different formulation studied are able to absorb UV and Blue light wave length.

[0098] EXAMPLE 3

[0099] Example 3 with results: Wound healing promotion activities.

[0100] For a wound assay, cells were seeded at 5.8 x 104per well of a 24-well plate and cultivated for 36 h to achieve complete spreading of cells and 100% of monolayer confluency. The monolayer wounding was performed by gentle cross scratching with a 200-pl tip applied to every well. Following the scratching, different groups were defined one control group and eight different treated groups. The control group consisted of cells cultured in a standard medium with PBS buffer solution while the eight different treated groups were: 1 ) 0,1 % CX-CMC 2) 0.05% SP 3) 0,1 % CX-CMC and 0.05% SP; 4) 0,3% CX-CMC 5) 0. 5% SP 6) 0,3% CX-CMC and 0.5% SP

[0101] 7) 0,5% CX-CMC; 8) 0.5% CX-CMC and 0.5% SP.

[0102] Samples were checked during the time interval 5-11 h after scratching in order to verify the cell edge and estimate the average velocity.

[0103] The in invitro study demonstrated the synergistic action of CX-CMC and SP in improving the wound healing speed in all tested concentrations (FIG. 2).

Claims

AMENDED CLAIMS received by the International Bureau on 04 march 2025 (04.05.2025)Claims1. A pharmaceutical formulation, suitable for ophthalmic administration, characterized by a composition comprising cross-linked carboxymethylcellulose (also known as reticulated CMC) and silk proteins (SP).

2. A pharmaceutical formulation, suitable for ophthalmic administration, characterized by a composition comprising cross-linked carboxymethylcellulose (also known as reticulated CMC) and silk proteins (SP) wherein the composition comprises about:

3. A formulation of either of claim 1 or 2 in the composition comprising about:

4. A composition according to any of the preceding claims 1-3, wherein the composition further comprises a well-known pharmaceutical excipient.

5. A composition according to any of the preceding claims, wherein the composition is indicated in the treatment of dry eye and dry eye-related signs and symptoms.

6. A composition according to any of the preceding claims 1-4, wherein the composition is indicated in the treatment of conjunctivitis and conjunctivitis-related signs and symptoms.

7. A composition according to any of the preceding claims 1-4, wherein the composition is indicated for promoting corneal healing.

8. A composition, according to claim 7, wherein the composition is indicated for promoting corneal healing resulting from an injury and / or surgery.

9. Cross-linked carboxymethylcellulose (also known as reticulated CMC) and silk proteins (SP), for use in a method of treating or preventing dry eye syndrome-related signs and symptoms and / or conjunctivitis-related signs and symptoms and / or to promote corneal wound healing and / or protect from UV and Blue light.Taking into account the amendments to the claims, it is clear that the inventive step is present. This is further supported by the data shown in Figure 2, which highlights a synergistic interaction between crosslinked carboxymethylcellulose (CX-CMC) (also known as reticulated CMC) and silk proteins (SP) that could not have been theoretically anticipated. This synergistic action is related to the ability of reticulated carboxymethylcellulose to create a tridimensional (3D) network where SP binds, facilitating wound healing and improving dry eye symptoms. Recently, we collected clinical data from 40 subjects undergoing refractive surgery, which required the complete removal of the corneal epithelium. Among these subjects, 20 were treated with a combination of CX-CMC and SP, while the other 20 received treatment with linear carboxymethylcellulose (CMC) alone.The data clearly indicate that subjects treated with reticulated CMC and SP achieved complete re- epithelialization within 48 hours, whereas those treated with CMC took nine days to fully re-epithelialize. Despite the significant difference in re-epithelialization times, both groups exhibited a comparable epithelial thickness of approximately 75 μm.Additionally, a separate data set on subjects affected by dry eye showed that TBUT improved by about 111% in the CX-CMC and SP groups, while it only improved by 55% in the CMC 0.1% group.

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