WO2025062471A1

WO2025062471A1 - Compositions for treatment of dry eye syndrome or corneal wound healing

Info

Publication number: WO2025062471A1
Application number: PCT/IT2024/050188
Authority: WO
Inventors: Fabio SCARINCI; Viviana TREZZA
Original assignee: D&v Farma Srl
Current assignee: D&v Farma Srl
Priority date: 2023-09-21
Filing date: 2024-09-19
Publication date: 2025-03-27
Anticipated expiration: 2026-03-21
Also published as: IT202300019488A1

Abstract

The present invention relates to compositions suitable for ophthalmic administration, which are suitable for the treatment of dry eye syndrome-related signs and symptoms to improve corneal wound healing or corneal sensitivity. The compositions suitable for ophthalmic administration, comprise hyaluronic acid, chondroitin sulphate and inositol in defined weight ratios. The invention further relates hyaluronic acid and chondroitin sulphate in combination with inositol for use in a method of dry eye syndrome-related signs and symptoms, to promote corneal wound healing, or to improve corneal sensitivity. The invention further relates to a combination of hyaluronic acid, chondroitin sulphate and inositol for use as a medicament. The invention further relates to hyaluronic acid, chondroitin sulphate and inositol for use in a method to treat corneal wounds due to dry eye or surgery or injury derived. The invention further relates to an ophthalmic kit of parts comprising hyaluronic acid, chondroitin sulphate and inositol.

Description

Compositions for treatment of dry eye syndrome or corneal wound healing

The present invention relates to compositions suitable for ophthalmic administration, aimed at treating the signs and symptoms of dry eye syndrome and/or promoting the healing of corneal wounds and/or restoring and improving corneal sensitivity.

Background Art

TEAR FILM

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:

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.

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.

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.

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.

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.

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.

CORNEAL INNERVATION

The corneal nerves derive from both the ophthalmic branch of the trigeminal nerve (therefore of sensory in origin) and autonomic nerves. Nerve bundles enter the cornea from the limbus, then invade both the corneal surface and the dept. Indeed, once nerve fivers penetrate the limbus they move toward the central cornea at the level of stroma and penetrate the Bowman’s membrane to create the sub-basal nerve plexus beneath the basement membrane of the epithelium.

The density and number of nerves in the subbasal epithelial nerve plexus are significantly greater than the density and number of nerves in the remaining corneal layers. Sub-basal fibres subsequently form branches that turn upward and enter the epithelium.

The cornea is one of the most densely innervated tissues in the body. Studies performed in rabbits have demonstrated that corneal epithelium nerve density is about 300-600 times higher than the skin and 20-40 times the one of the dental pulp. It is estimated that there are approximately 7000 sensory receptors per mm in the human corneal epithelium, implying that injuries to individual epithelial cells may be adequate to give a pain perception.

Corneal nerve fibres mediate not only sensation but also influence corneal epithelium homeostasis by producing trophic factors and, therefore, play a vital role in preserving a healthy ocular surface. Patients diagnosed with neuropathy (e.g., degeneration of corneal nerves) experience diminished sensation and/or increased pain (hyperalgesia), characterized by chronic discomfort and irritation. Since the corneal nerves regulate lacrimation, corneal neuropathy (loss or damaged corneal nerve tissue or decreased length of corneal nerve fibers) leads to tear deficiency.

Impairment of corneal innervation and related neuropathic pathology produces a degenerative condition known clinically as “neurotrophic keratitis” which renders the corneal surface vulnerable to occult injury and delayed healing of established corneal epithelial injuries. Most clinical cases of neurotrophic keratitis are caused by herpes simplex or Zoster keratitis, diabetes mellitus, or trigeminal nerve damage associated with orbital or head Surgery, head trauma, aneurysms, or intracranial neurologic disease. Absent or reduced corneal sensation may be congenital in origin. Keratorefractive procedures such as photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) can sever Stromal and Subbasal corneal nerves plexus and produce a transient mild to severe neuropathologic condition or neurotrophic dry eye. This form of “neurotrophic dry eye” characterised by nerve loss and associated dryness, is also seen in non- surgical conditions

DRY EYE SYNDROME

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.

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”.

Several factors contribute to the risk of developing the dry eye syndrome

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.

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 etiologies, including xerophthalmia, ocular allergy, topical preservative use and contact lenses 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.

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.

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.

RED EYE AND EYESTRAIN

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.

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

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.

Symptoms that constitute computer vision syndrome can be broadly classified into ocular, visual and musculoskeletal (Table 1 ). The severity and the specific type of symptom 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.

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

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 [6]. 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 is depends on the hours of use of the computer, 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.

The major factors associated with CVS can be broadly classified into the individual’s visual ability and environmental factors. Compromises in the visual abilities of the user that significantly contribute to CVS include uncorrected refractive error, binocular vision anomalies, accommodative disorders, and ocular surface or tear film abnormalities. The presence of uncorrected refractive error, especially uncorrected hyperopia, under or over-corrected myopia and uncorrected astigmatism, is typically associated with symptoms in computer users.

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.

CORNEAL ALTERATION IN DIABETIC PATIENTS

Diabetes mellitus is a complex metabolic disease characterized by hyperglycemia resulting from either insulin deficiency or resistance. It is associated with various complications, including retinopathy, neuropathy, and nephropathy. Recently, researchers have reported significant alterations in the cornea of diabetic patients.

In diabetic patients, chronic hyperglycemia leads to numerous biochemical changes, including advanced glycation end products (AGEs) formation, and activation of protein kinase C (PKC). These mechanisms contribute to corneal endothelial cell and corneal nerve dysfunction, leading to decreased corneal sensitivity, epithelial defects, and delayed wound healing. Diabetic corneal alterations present with various clinical manifestations. Reduced corneal sensitivity is a prominent feature and is associated with an increased risk of corneal injuries and infections. Epithelial defects and corneal erosions occur more frequently in diabetic individuals due to diminished regenerative capacity and impaired tear film stability. Additionally, patients may experience corneal thickening and endothelial cell loss, affecting visual acuity and leading to corneal oedema.

Diagnosis of diabetic corneal alterations involves a combination of clinical evaluation and specialised tests. Slit-lamp biomicroscopy aids in visualising corneal epithelial defects and alterations in thickness. Corneal sensitivity can be assessed using the Cochet-Bonnet aesthesiometer, while corneal endothelial cell density can be measured using specular microscopy. Non-invasive imaging techniques like anterior segment optical coherence tomography (OCT) and confocal microscopy offer valuable insights into corneal structural changes.

The corneal alterations in diabetes can significantly impact visual function. Reduced corneal sensitivity affects blink reflex and tear production, leading to dry eye syndrome. Epithelial defects and erosions cause discomfort and visual disturbances. Corneal oedema may result in blurred vision and decreased contrast sensitivity. In severe cases, diabetic keratopathy can lead to vision loss, severely impacting the patient's quality of life.

The management of diabetic corneal alterations focuses on glycemic control and symptomatic relief. Optimizing blood glucose levels through lifestyle modifications and medication is crucial for preventing and slowing down corneal complications. No effective and specific topic treatment has been developed yet.

REFRACTIVE SURGERY

Refractive surgery, including techniques such as LASIK (Laser-Assisted In Situ Keratomileusis) and PRK (Photorefractive Keratectomy), has revolutionised the field of ophthalmology by offering a permanent solution to refractive errors such as myopia, hyperopia, and astigmatism. While these procedures are generally safe and effective, some patients may experience corneal alterations post-surgery.

Refractive surgery involves removing or reshaping corneal tissue to correct vision problems. The cornea's biomechanical and structural properties are pivotal in its stability and refractive function. Following surgery, corneal tissue may undergo remodelling processes to adapt to the new shape, leading to corneal curvature, thickness, and biomechanics alterations.

Despite its high success rates, some patients experience dry eye symptoms and reduced corneal sensitivity post-surgery. These complications can significantly impact visual comfort and quality of life.

Refractive surgery induces changes in corneal nerve density, distribution, and function, and affect corneal surface regularity, all of which contribute to dry eye development. Additionally, postoperative use of topical medications and eye shields can further exacerbate dry eye symptoms. The severing of corneal nerves during flap creation or stromal ablation disrupts the neural signaling between the ocular surface and the lacrimal glands, leading to reduced tear production. In addition, the altered corneal surface following surgery can lead to increased tear evaporation, aggravating dry eye symptoms.

Overall we can say that refractive surgery reduces corneal sensitivity. Corneal sensitivity plays a vital role in maintaining corneal health and visual comfort. The cornea’s rich innervation contributes to tear production, blink reflex, and protection against foreign bodies. Following refractive surgery, corneal nerve fibers are disrupted, leading to a temporary or even permanent loss of corneal sensitivity. This can predispose patients to corneal injuries, delayed wound healing, and reduced corneal protective responses.

Considering that corneal nerves exert exocrine gland-like functions, it is easier to understand all the above-mentioned refractive surgery-induced corneal alteration.

Corneal nerves provide trophic support to various corneal cells by secreting neurotrophins, neuropeptides, and neurotransmitters. These neuromediators maintain both neuronal health, as well as promote neuronal regeneration and wound healing. While corneal sensory innervation secretes neurotrophins and neuropeptides, autonomic innervation produces catecholamines and acetylcholine.

Preventive measures and proactive management play a crucial role in reducing the incidence and severity of dry eye and corneal sensitivity loss after refractive surgery. Preoperative screening for dry eye and optimizing ocular surface health are vital. Post-operative management often includes lubricating eye drops, punctal plugs, and anti-inflammatory medications to alleviate dry eye symptoms. Neurotrophic factors, such as nerve growth factor (NGF), have shown promise in promoting nerve regeneration and restoring corneal sensitivity.

In addition, proper patient education about the potential for dry eye and corneal sensitivity changes is essential. Nevertheless, it is prominent providing patients with realistic expectations and post-operative care instructions can lead to better adherence and improved outcomes. Regular follow-up visits allow for monitoring of symptoms and timely intervention when necessary.

Summary of the invention

Technical Problem

Tear substitutes play a key role in the clinical management of the various conditions mentioned above, e.g. dry eye syndrome, as they provide patients with immediate relief.

Ocular lubricants are characterized by hypotonic or isotonic buffered solutions containing electrolytes, surfactants, and various types of viscosity agents. Viscosity agents are added to ocular lubricants in order to increase their residence time and ocular surface contact, providing a longer interval of patient comfort and increasing the duration of action and penetration of the lubricant.

Currently, there are no treatments that can not only relieve the symptoms of dry eye syndrome but also promote the healing of corneal wounds caused by conditions like diabetes or surgical procedures like refractive surgery, or enhance corneal sensitivity.

This is because currently known tear substitutes are mainly based on thickening agents such as carboxymethylcellulose or hyaluronic acid. However, none of these combined the thickening agents with chondroitin sulphate and inositol.

Indeed, it is important to note that, in order to improve the management of patients suffering from dry eye syndrome, or diabetics or patients who have undergone ophthalmic surgery procedures, it is desirable that a preparatory agent both improves the signs and symptoms of dry eye and promotes the healing process of a corneal wound and improves corneal sensitivity.

Furthermore, until now, the ophthalmic administration of Inositol had been suggested as a possible support for subjects affected by presbyopia (European Patent No. EP3130335 - OPHTHALMIC COMPOSITION COMPRISING INOSITOL FOR IMPROVING VISUAL ACCOMODATION -Applicant: Sooft Italia S.p.A.]). In particular, the application explicitly refers to Myo-inositol as the only stereoisomer present in the formulation alone or in association with hydroxypropyl methylcellulose (HPMC) alone. No evidence and suggestion of a possible association of inositol with cross-linked hyaluronic acid and chondroitin sulfate is mentioned.

In addition, also the scope of the invention is extremely different since the preparation described in the above-mentioned patent cannot induce all the clinical beneficial effects of the present invention.

Solution to Problem

In addressing the problem of developing compositions, uses and methods for the treatment of dry eye syndrome or to promote corneal wound healing or enhance corneal sensitivity, the inventor surprisingly found that cross-linked hyaluronic acid (CX-HA), chondroitin sulphate (CS) and inositol (Ins) are able to treat the signs and symptoms of dry eye, promote wound healing and restore and improve corneal sensitivity.

Until now, it was well known that hyaluronic acid, possibly in combination with other substances, was able to improve dry eye symptoms. Still, it was not known or suspected that the combination of cross-linked hyaluronic acid (CX-HA), chondroitin sulphate (CS) and inositol (Ins) could enable the treatment of signs and symptoms related to dry eye, and/or to promote the healing of corneal wounds and/or improve corneal sensitivity. This beneficial effect occurs in patients suffering from dry eye syndrome as well as in diabetic patients or subjects who have undergone ophthalmic surgery procedures or accidents.

Accordingly, one aspect the invention provides a composition, suitable for ophthalmic administration (e.g. eye drops, ointments, gel, spray, contact lens), comprising cross-linked hyaluronic acid (CX-HA) and chondroitin sulphate (CS) and inositol (Ins), wherein cross-linked hyaluronic acid and chondroitin sulphate and inositol are present in about a %w/w in the final product:

Table 2

To the above-mentioned quantities, well know 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, one or more plasticisers one or more plastic resins. 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.

A further aspect of the invention provides a combination of CX-HA, chondroitin sulphate and inositol for use as a medicament.

A further aspect of the invention provides for treating dry eye syndrome-related symptoms or promoting corneal wound healing. It has to be specified that the need of corneal wound healing might have been generated by an accident, by a pathology or surgery.

Afurther aspect of the invention involves the restoration and/or improvement of corneal sensitivity.

In addition, the described compositions can be used to treat dry eye related signs and symptoms and/or to treat corneal wound and/or to restore corneal sensibility in diabetic patients.

In particular, one embodiment the invention comprising a combination of cross-linked hyaluronic acid (CX-HA) chondroitin sulphate (CS) and inositol (INS) in the form of eye drops produced eider in a multidose bottle or a unite dose vial.

A further aspect of the invention provides a kit of parts comprising hyaluronic acid chondroitin sulphate and inositol in two or more containers.

Advantageous Effects of Invention

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.

Brief Description of Drawings

Figure 1 - Graph representing TBUT and OSDI in subjects with dry eye syndrome before and after instillation of an eye drop containing CX-HA 0.1 % and CS 0.1 % and 0.1 Ins: **** =P<0,0001.

Figure 2 - Graph representing the improvement in corneal sensitivity in diabetic subjects treated with CX-HA 0.1 % and CS 0.1 Ins.

Figure 3 - Graph representing the speed of wound healing. In particular, there is a synergistic action between CX-HA 0.1 % and CS 0.1 % and 0.1 Ins: *=P<0.05; **=P<0.01 ; ***=P<0.001.

Description of Embodiments

In the present paragraph provides definitions of several terms and expressions used in the present application to describe the invention. Some terms are declaimed identically in the description of different aspects or embodiments of the invention. It is to be understood that definitions of terms and expressions used in more than one aspect of the invention apply equally to any aspect of the invention described herein in which those terms and expressions appear. This applies equally regardless of where, i.e. which section, within the present application such terms are defined or discussed.

In this application, the use of the singular (e.g. “a” or “the”) may include the plural unless specifically stated otherwise. Further, the use of the term “including” as well as other grammatical forms such as “includes” and “included”, is not limiting.

As used herein the term “comprising” has the broad standard meaning “including”, “encompassing”, or “containing”. It includes the explicitly recited element(s), and also allows, but does not require, the presence of other or another element(s) not recited. In addition to this broad meaning, as used herein, the term “comprising” also encompasses the limiting meaning “consisting of”, according to which only the explicitly recited element(s), and no other(s) are present. In addition, the term “comprising” also includes the meaning of “consisting essentially of’, which means that other element(s) may be present beyond those explicitly recited, provided the additionally present element(s) does not alter the technical effect achieved by the explicitly recited element(s).

As used herein the term “about” when referring to a particular value, e.g. an endpoint or endpoints of a range, encompasses and discloses, in addition to the specifically recited value itself, a certain variation around the specifically recited value. Such a variation may for example arise from normal measurement variability, e.g. in the weighing or apportioning of various substances by methods known to the skilled person. The term “about” shall be understood as encompassing and disclosing a range of variability above and below and indicated specific value, said percentage values being relative to the specific recited value itself, as follows. The term “about” may encompass and disclose variability of ± 15.0% of the declared value.

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.

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

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.

As used herein, the term “hyaluronic acid” encompasses and discloses the substance having chemical (C14H21 NO11 )n (abbreviated HA; conjugate base hyaluronate), also called hyaluronan, is an anionic, nonsulfated and unbranched glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. Hyaluronic acid is a disaccharide, which is composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating p-(1— >4) and p-(1— >3) glycosidic bonds, whose chemical structure is reported below.

In a particularly preferred embodiment of the present invention in any of the various aspects set out herein, the hyaluronic acid refers to hyaluronic acid that has been further modified in order to be cross-linked.

The skilled person in the field may also refer to cross-linked hyaluronic acid as ‘reticulate hyaluronic acid’ as the two definitions refer to the same type of raw materials.

As used herein, the term “chondroitin sulphate” (CS) encompasses and discloses a polymer with a wide molecular weight range composed of an alternating sequence of sulfated and/or unsulfated d-glucuronic acid (GlcA) and N-acetyl- d-galactosamine (GalNAc) residues linked through alternating p-(1 — > 3) and p-(1 — > 4) bonds. The presence of sulfate groups makes it extremely hydrophilic. Predominant sources of chondroitin sulfate raw materials are bovine trachea, porcine skin and rib cartilage, and shark cartilage. CS is used for the treatment of osteoarthritic conditions. It is an essential component of cartilage and plays an important role in the elasticity and function of articular cartilage, where it is mainly attached covalently to core proteins in the form of proteoglycans.

In particular, the term “chondroitin sulphate” refers to all the possible sulphurated form of chondroitin sulphate:

Table 3

In addition, in a particular and non-binding embodiment of the present invention, CS can have a biotechnological origin.

As used herein, the term “inositol” encompasses and discloses the substance having chemical formula C6H12O6 and a structure according to any of the 9 known isomers of inositol, that is:

myo-inositol scyllo-inositol muco-inositol chiro-inositol

neo-inositol allo-inositol epi-inositol cis-inositol It is understood that the present invention can disclose an ophthalmic formulation that can contain one isomers or a mixture of one or more isomers, in any ratio, of myo-inositol and/or schyllo- inositol and or D-chiro-inositol. As used herein, the term “ratio” as applies to the relative amounts of two substances in e.g. composition, denotes a concentration ratio, that is the ratio of the concentration of one compound relative to the concentration of another compound in the respective relative final concentration in the composition.

In a particular non-binding embodiment, the ratio among the concentration, e.g [myo-lns]/[scyllo- Ins], can range from about 1 :1 to about 9:1 .

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.

Compositions comprising CX-HA and CS and Ins

In addressing the problem of providing improved compositions, uses and methods for alleviating medical conditions such dry eye disease or promoting corneal healing or restoring/im proving corneal sensitivity, in both healthy and diabetic subjects, the inventors have surprisingly found the advantageous effect due to the topical use of a composition comprising CX-HA and CS and Ins. For instance, as described herein and shown in the later examples (FIG. 1 , FIG. 2, FIG. 3), the present invention has been able to improve the Ocular surface disease index (OSDI) score and the TBUT therefore directly improving sign and symptoms of dry eye and to restore/improve the corneal sensitivity.

In particular, the OSDI score is a well-recognized and objective method to evaluate symptoms linked to ocular discomfort of different origins 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.

In addition, the authors have unanticipatedly found that the invention was also able to improve cornel sensitivity in diabetic subjects. Additional in vitro data, further strengthen the novelty and the surprising found of the invention demonstrating a synergistic action among CX-HA and CS and Ins on wound healing.

The surprising finding that combined administration of CX-HA, CS and inositol entails important therapeutic advantages in the treatment and prevention signs and symptoms of dry eye disease, in promoting corneal wound healing and in improving corneal sensitivity. For instance, while many several hyaluronic acid based tears substitute are present and routinely used on the market they have shown minor efficacy in treating dry eye sing and symptoms, they have limited to no efficacy in improving corneal wound healing or in restoring corneal sensitivity. Importantly, the present inventors have found that combined administration CX-HA, CS and inositol can more effectively treat dry eye sing and symptoms and provide a solid solution in improving corneal wound healing or in restoring corneal sensitivity. This has the ultimate effect of widening the scope of treating and preventing dry eye sing and symptoms and/or in improving corneal wound healing and/or corneal sensitivity in diabetic and non-diabetic subject. The above-mentioned diabetic and nondiabetic subject are affected by the dry eye syndrome, have a damaged cornea due to pathology, surgery or injury and have a reduced corneal sensitivity due to pathology (e.g. diabetes), surgery or injury.

Accordingly, one aspect of the present invention provides a composition comprising CX-HA, CS and Ins, wherein CX-HA, CS and Ins are present in about a respective %w/w in the final product:

Table 4

Various example of a composition comprising of CX-HA, CS and Ins have been set out above. Especially preferred is a combination of CX-HA, CS of biotechnological origin and Ins in any of the above concentration ratios. Also preferred combination is a mixture of CX-HA, CS of biotechnological origin and Ins, wherein the Ins can be myoinositol or D-chiro-inositol or schyllo- inositol.

An additional embodiment of the invention is a mixture of CX-HA, CS of biotechnological origin and Ins in which Ins derives from a mixture of two of more Ins stereoisomers chosen among myoinositol or D-chiro-inositol or schyllo-i nositol in any concentration ratio among them. An additional embodiment of the invention is a mixture of CX-HA, CS of biotechnological origin and Ins in which Ins derives from a mixture of myoinositol and schyllo-i nositol in a concentration ratio ranging from about 1 :1 to 9:1.

It is understood that establishing a certain amount of CX-HA in the inventive composition is sufficient to automatically establish a corresponding amount of CS and Ins in the inventive composition, in accordance with the above concentration ratios of CX-HA:CS:lns.

Similarly, it is understood that establishing a certain amount CS in the inventive composition is sufficient to automatically establish a corresponding amount of CX-HA and Ins in the inventive composition, in accordance with the above concentration ratios of CX-HA:CS:lns. Likewise, establishing a certain amount Ins in the inventive composition is sufficient to automatically establish a corresponding amount of CX-HA and CS in the inventive composition, in accordance with the above concertation ratios of CX-HA:CS:lns.

Adhering to the CX-HA:CS:lns concetration ratios set out above ensures that the composition of the invention can achieve an advantageous technical effect in treating or preventing dry eye signs and symptoms in improving corneal wound healing and corneal sensitivity in diabetic and nondiabetic subjects. The above-mentioned diabetic and non-diabetic subjects are affected by the dry eye syndrome, have a damaged cornea due to pathology, surgery or injury and have a reduced corneal sensitivity due to pathology (e.g. diabetes), surgery or injury.

In certain embodiments, the composition of the invention may advantageously comprise about the respective w/w % in the final product:

Table 5

In one embodiment, the inventive composition is a pharmaceutical composition, further comprising at least one pharmaceutically acceptable ingredient. While the inventive composition may itself be administered to a subject, it will be understood that adding one or more pharmaceutically acceptable ingredients beyond CX-HA and CS and Ins may be advantageous in rendering the composition more suitable for ophthalmic administration to a subject. Accordingly, as used herein, a “pharmaceutical composition” is any composition which, in addition to CX-HA and CS and Ins, is formulated to comprise a pharmaceutically acceptable ingredient which renders the composition more suitable for ophthalmic administration to a subject, without further workup.

Formulation of the inventive composition for suitability for ophthalmic administration

A composition of the invention suitable for ophthalmic administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including multidose bottle single dose vials, plastic or alluminium tube, spray, contact lenses and topical wipes.

In order to produce a formulation suitable for ophthalmic administration, well-known and commonly used pharmaceutical excipients may be added. In particular, one or more of the following excipients may be added: one or more osmotic regulators; one or more pH stabilizers; 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-mentioned pharmaceutical excipients may or may not be present in the formulation and that it is possible to use only one, more than one or all of them.

A multidose eye drop comprising CX-HA, CS and Ins may, for example, be made by dissolving CX-HA, CS and Ins 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 be present or absent in the formulation, and may be used only one, more than one or all. In particular, depending on the bottle used, preservatives may or may not be present.

Therapeutic uses and methods

The inventors have discovered that combining CX-HA and CS and Ins can effectively treat or preventing dry eye related sing and symptoms can effectively promote corneal healing can effectively improve corneal sensitivity. Notably, there is currently no known therapeutic use of these three substances combined for any of these purposes.

Accordingly, a further aspect of the invention provides a combination of CX-HA and CS and Ins for use of a medicament.

As used herein, the term “combination”, or the expression “in combination with” with reference to CX-HA and CS and Ins, refers to the coupling of CX-HA and CS and Ins for the purpose of their coadministration to a subject within a given treatment regimen. A given treatment regimen will typically comprise multiple, repeated coadministrations of CX-HA and CS and Ins 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-HA and CS and Ins may be repeated multiple times daily, for example once, twice, 3 times, 4 times, 5 times or more, a repetition of about 3 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.

The coupling of CX-HA and CS and Ins meant by a “combination” of these three substances need not be physical, nor need it imply chronological simultaneity. Reference to a “combination” of CX- HA and CS and Ins therefore encompasses multiple possibilities regarding the timing and the route of administration of the respective substances. Encompassed in the meaning of “combination” is for example, the coupling CX-HA and CS and Ins for simultaneous administration by the same route.

As used herein, the term “subject or patient or individual” means a female or male subject affected by dry eye syndrome. In addition, 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 furthermore the invention applies also to subject affected by a reduced cornel sensitivity due to a pathological condition, a surgery or an injury.

In the above-mentioned subject can also be subject affected by diabetes.

The following embodiments apply equally to the above CX-HA and CS and Ins for use in in a method of treatment or prevention out above.

Adhering to the CX-HA and CS and Ins combined administration set out above ensures that the composition of the invention can achieve the advantageous technical effect described above.

EXAMPLE 1

Examples are given below for illustrative and non-limiting purposes only.

Example 1 with results - Improvement in OSDI score, TBUT and corneal sensitivity: CX-HA and CS and Ins

The present example describes an efficacy study administering an eye drop to diabetic subjects affected by dry eye with an increased OSDI score a reduced TBUT and a reduced corneal sensitivity. The study aims to determine whether the combination of CX-HA and CS and Ins is able to reduce the signs and symptoms linked to dry eye i.e. reducing the OSDI score and to improve the TBUT and to improve corneal sensitivity.

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

Subjects received the eye drop containing 0.1 % CX-HA and 0.1 % CS and 0.1 % Ins and were counselled to apply 3 times a day for 3 months.

At the end of the three months, OSDI and TBUT and corneal sensitivity were evaluated.

All the enrolled subjects completed the trial. Data showed that CX-HA and CS and Ins 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-HA and 0.1 % CS and 0.1 % Ins was also able to improve TBUT, therefore increasing the tear film stability (FIG. 1 ).

In addition, subjects treated with CX-HA and CS and Ins displayed an improved corneal sensitivity (FIG. 2).

EXAMPLE 2

Example 2 with results Wound healing promotion activities.

For a wound assay, cells were seeded at 5.8 x 10⁴ per 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 twelve different treated groups. The control group consisted of cells cultured in a standard medium with PBS buffer solution while the twelve different treated groups were: 1 ) 0.1 % Ins 2) 0.1 % CX-HA; 3) 0.1 % CS; 4) 0.1 % CX-HA and 0.1 % CS and 0.1 % Ins; 5) 0.2% Ins; 6) 0.2% CX-HA; 7) 0.2% CS; 8) 0.2% CX-HA and 0.2% CS and 0.2% Ins; 9) 0.3% Ins; 10) 0.3% CX-HA; 11 ) 0.3% CS and 12) 0.3% CX-HA and 0.3% CS and 0.3% Ins.

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

Collected data clearly demonstrate the synergistic action of CX-HA and CS and Ins in improving the wound healing speed in all tested concentrations.

Indeed, the samples treated with the combination of the three molecules displayed a wound healing speed greater than all the tested groups.

Noteworthy, the difference was statistically significant (FIG. 3).

Claims

1. Pharmaceutical formulation, suitable for ophthalmic administration, characterised by a composition comprising cross-linked hyaluronic acid (CX-HA) and chondroitin sulphate (CS) and inositol (Ins).

2. Pharmaceutical formulation according to claim 1 , suitable for ophthalmic administration, characterised by a composition comprising CX-HA and CS and Ins, wherein the composition comprises, in the final product, about:

3. Pharmaceutical formulation according to claim 1 , suitable for ophthalmic administration, characterised by a composition comprising CX-HA and CS and Ins, wherein the composition comprises, in the final product, about:

4. A composition of any one of claims 1-3, wherein the composition further comprising a well known pharmaceutical excipient.

5. A composition of any one of claims 1-4, wherein the composition is indicated in the treatment of dye eye and dry eye related sign and symptoms.

6. A composition of any one of claims 1-4, wherein the composition is indicated for promoting corneal healing.

7. A composition, according to claim 6, wherein the composition is indicated for promoting corneal healing resulting from a pathology, an injury and/or surgery

8. A composition of any one of claims 1-4, wherein the composition is indicated to restore corneal sensitivity.

9. A composition of any one of claims 1-8, wherein the composition is indicated for diabetic patients.

10. Formulation of any one of claims 1-9, for use in a method of treating or preventing signs and symptoms related to dry eye syndrome and/or promoting corneal wound healing and/or restoring corneal sensitivity.

11. Formulation according to one of claims 1-10 in which the concordance of Ins (%w/w, i.e. % w/w of inositol in the final product) is given by myo-inositol or D-chiro-inositol or scyllo-inositol.

12. Formulation according to any one of claims 1-10 wherein Ins is a mixture of two or more inositol stereoisomers.