Colouring solutions and their use in vitreoretinal surgery

Abstract

The present invention relates to ophthalmic compositions comprising at least one extract of Arthrospira spp. or one or more pigments derived therefrom, Trypan Blue and tocopherol or one or more derivatives thereof, and their use in medicine, specifically as ophthalmic dyes or dyes for vitreoretinal surgery.

Description

[0001] COLOURING SOLUTIONS AND THEIR USE IN VITREORETINAL SURGERY

[0002] DESCRIPTION

[0003] Field of the invention

[0004] The present invention relates to the technical field of colourants for use in medicine, preferably ophthalmic colourants for vitreoretinal surgery.

[0005] State of the art

[0006] The use of dyes in vitreoretinal surgery, particularly in vitrectomy, is well known.

[0007] Chromovitrelectomy is a particular ophthalmic surgical technique that uses dyes during vitrectomy, a procedure involving the removal of the vitreous body, used to treat various diseases of the retina or the posterior segment of the eye. Over the last decade, chromo-vitrectomy has enabled surgeons to visualise transparent intraocular tissues, particularly the posterior hyaloid, epiretinal membranes (ERMs) and the internal limiting membrane (ILM), and has been unanimously established as the standard for macular surgery, as virtually any type of membrane can be visualised and safely removed.

[0008] Removal or peeling of the internal limiting membrane (ILM) is an ophthalmic surgical procedure used to treat traction maculopathies such as macular holes and macular puckers. According to recent publications, ILM peeling in the treatment of idiopathic macular holes (MH) has increased closure rates to approximately 95% in subjects undergoing this procedure, compared to closure rates of 50-90% in untreated subjects. However, as this procedure is highly dependent on the experience of the surgeon performing it, it can cause anatomical and functional damage to the retina, such as visual field defects and damage to the retinal pigment epithelium.

[0009] The use of solutions containing vital dyes in ILM peeling has greatly simplified the removal of this membrane, which is not visible without dyes, thanks to improved visibility of the vitreoretinal interface or retinal surface, making this procedure feasible even for less experienced surgeons.

[0010] There are several solutions available on the market that include vital dyes for ophthalmic surgery, particularly vitreoretinal surgery. For example, the use of 0.18% Trypan Blue solutions allows for a subtle optical distinction between collagen fibres and other morphological structures of the eye, but results in excessively bland staining, far from an optimal and clear optical and morphological distinction between the tissues of interest.

[0011] There are also formulations containing two different colouring compounds, and possible adjuvants, for the concomitant staining of ERM and ILM; these allow surgical time and exposure of retinal tissues to chemicals to be minimised, reducing the amount of dye injected and thenumber of injections. A recent analysis has shown that triamcinolone acetonide and brilliant blue G are the optimal dyes to assist, respectively, the peeling of the ERM and ILM in macular hole surgery and in pathologies such as idiopathic retinal detachments, traction detachments due to diabetic retinopathies, retinal holes, macular puckers, vitreitis and vitreoschisis. Clinical studies have confirmed the efficacy profiles of these dyes, but have also highlighted postoperative complications such as increased ocular pressure, especially when using triamcinolone acetate.

[0012] Several studies have investigated the safety of different dyes used in vitreoretinal surgery, raising doubts about the possible retinal toxicity of some of them. For example, although indocyanine green (ICG) has a long history of safe use when injected intravenously to visualise choroidal perfusion during angiography, some reports have indicated potential toxicity related to this dye after intravitreal injection during macular surgery. Furthermore, no in vivo or in vitro studies have been conducted on the intraocular injection of ICG in humans; therefore, intraocular application still represents an off-label use.

[0013] In conclusion, although several studies have effectively demonstrated the safety profiles of commercially available dyes in intravitreal surgery, surgeons are still cautious in their use and tend to adopt a series of precautions, such as filling the eye with iso-osmolar or diluted solutions, limiting exposure time to the dye to less than 10 seconds and / or thoroughly washing the eye to remove the dye and avoid proximal and prolonged endo-illumination of the stained tissue.

[0014] In light of the above, there is a clear need to develop new dyes, specifically vital dyes, that are effective, in particular by ensuring optimal optical distinction, and safe. Preferably, the dyes should also have an improved toxicity profile compared to those available on the market.

[0015] SUMMARY OF THE INVENTION

[0016] Extensive in vitro and in vivo studies conducted by the Applicant on experimental models have surprisingly demonstrated that the use as an ophthalmic dye of a composition comprising Arthrosplra spp. extract, Trypan Blue, tocopherol or its derivatives and, optionally, riboflavin phosphate and / or hyaluronic acid as an ophthalmic dye allows for optimal optical and morphological distinction of ocular tissues, particularly collagen-based tissues. Advantageously, this composition allows for selective staining of the internal limiting membrane and / or the epiretinal membrane (ERM). Furthermore, the Applicant has found that the use of an aqueous suspension comprising 2% w / v basic riboflavin (or free base) is capable of selectively staining the epiretinal membrane (ERM). The present invention therefore relates to the above composition and suspension, as well as combinations thereof and kits comprising them. The invention alsorelates to their use as ophthalmic dyes in treatment, surgical or diagnostic methods, as well as their use in staining tissues in vitro or ex vivo.

[0017] Glossary:

[0018] The terms used in this description are as generally understood by those skilled in the art, unless otherwise indicated.

[0019] The term 'extract1, in the context of this description, refers to any product derived from biomass of botanical or bacterial origin, including all products derived from mechanical treatments (pulverisation, grinding, mixing and / or other methods) or extractive treatments (solvent extraction, distillation, maceration and / or other specific methods) performed on such biomass, including mother tinctures and / or essential oils.

[0020] In the context of this description, the term 'effective amount' means an amount of active compound, comprising the compounds of the invention, which is high enough to provide the desired benefits and at the same time low enough not to cause serious side effects.

[0021] The term 'vital dyes', in the context of this description, refers to the use in medicine of particular chemical substances capable of reaching the tissues of the human body physiologically and / or mechanically and colouring them.

[0022] In the context of this description, the term "biopotentiator" refers to molecules, substances, compounds or agents that may or may not have their own pharmacological activity but, when used in combination with an active ingredient or colouring agent, are able to increase the activity of the latter in various ways, for example, by increasing its absorption through biological membranes, enhancing the active ingredient through conformational interaction, acting as a receptor for the active ingredient, or making the target cells or tissues more receptive to the active ingredient or colouring agent. A 'biopotentiator' is therefore a molecule, substance, compound or agent capable of enhancing the bioavailability and / or efficacy of a particular active ingredient with which it is combined, by exerting, or even without exer, a pharmacological activity in itself. They can also be functional excipients included in the formulation to increase the absorption of a pharmacologically active molecule. A 'biopotentiator' can therefore be used as an adjuvant in a therapy based on a specific active ingredient or in a method that uses a specific colouring agent that benefits, for example in terms of speed and extent of absorption, bioavailability and / or efficacy, from the presence of said biopotentiator. In the context of this description, the term 'biopotentiator' refers specifically to tocopherol or its derivatives.

[0023] The terms 'tocopherol or its derivatives', 'tocopherol derivatives' or 'tocopherol derivatives', in the context of this description, refer to derivatives or analogues of vitamin E, inparticular pharmaceutically or physiologically acceptable salts and esters of tocopherol as well as substitutes for it, as detailed below.

[0024] The terms 'vitreous body1or 'vitreous humour' in the context of this description refer to a gelatinous, transparent and colourless mass present inside the vitreous chamber, i.e. the space between the posterior surface of the lens and the retina; in the human eye, the vitreous body constitutes 80% of this organ. The vitreous body consists of approximately 99% water and hyaluronic acid, produced within the vitreous body itself by hyalocytes, cells that also have certain phagocytic functions. The main functions of the vitreous body are to provide support, filling the eyeball, and protection, cushioning shocks. In addition, the vitreous body, which is part of the dioptric apparatus, plays an important role in vision. Particular physical or environmental conditions, such as advancing age, prolonged exposure to high temperatures, dehydration, etc., can cause the vitreous body to liquefy and, consequently, the appearance of isolated or confluent opacities that can cause discomfort but do not in themselves affect visual acuity. Distinct and mobile opacities, known as myodesopsia, can cause traction on the retina (especially the peripheral retina), with the appearance of visual manifestations such as sparkles and flashes. Myodesopsia can also occur in myopic individuals due to deformation of the vitreous body caused by excessive eye length. However, flashes, flickers and seeing sticks or holes are not exclusive to vitreous body pathology, as they occur in a multitude of circumstances such as migraine, retinal detachment, infections, inflammation and trauma (e.g. violent blow to the head).

[0025] The reduction in volume of the vitreous body frequently causes it to detach from the back of the eyeball, generally without causing damage. In this case too, bright spots and flashes may appear due to both small fragments of the vitreous body hitting the retina and the traction of the vitreous body as it contracts.

[0026] In the context of this description, the term "hyaluronic acid" refers to an anionic glycosaminoglycan with moisturising and viscoelastic properties. It is mainly found in connective tissue, vitreous humour and aqueous humour. It has gained widespread application in lubricants used for the treatment of dry eye because it effectively binds water, resists dehydration, and exhibits excellent biocompatibility. Previous studies have shown that hyaluronic acid protects corneal epithelial cells, stimulates epithelial migration, and improves retinal image quality (Nakamura M, Hikida M, Nakano T, et al. Characterisation of water retentive properties of hyaluronan. Cornea. 12:433-436, 1993; Johnson ME, Murphy PJ, and Boulton M. Effectiveness of sodium hyaluronate eye drops in the treatment of dry eye. Graefes Arch. Clin. Exp. Ophthalmol.

[0027] 244:109-112, 2006; Montes-Mico R, Cervino A, Ferrer-Blasco T, et al. Optical quality afterinstillation of eye drops in dry eye syndrome. J. Cataract Refract. Surg. 36:935-940, 2010). In eye drops, hyaluronic acid is used to improve the irregularity of the ocular surface, to stabilise the precorneal tear film and to improve and alleviate the symptoms of dry eye. Specifically, "hyaluronic acid" in the context of this description refers to hyaluronic acid or a salt thereof, preferably sodium salt. For the purposes of the present invention, hyaluronic acid, preferably sodium salt hyaluronic acid, having a molecular weight between 100 and 2000 kDa, preferably between 200 kDa and 1800 kDa, more preferably between 300 kDa and 1600 kDa, may be used, for example. The composition may, of course, also comprise, as 'hyaluronic acid1, a mixture of hyaluronic acids having different molecular weights, provided that said molecular weight falls within the above-mentioned range.

[0028] In the context of the present invention, the term 'spirulina algae1refers to a biomass, commonly considered to be a marine algae, comprising one or more cyanobacteria belonging to the genus Arthospira; this 'spirulina algae' contains a series of pigments, in particular phycocyanin and allophycocyanin and their derivatives, which have the characteristic of being moderately soluble in water and having a typical blue colour that does not tend to fade when exposed to light. Spirulina also has a particular affinity for proteins, in particular proteins that have high amounts of hydrophilic groups, such as collagen and gelatin.

[0029] The terms 'free riboflavin', 'riboflavin in free form', 'free riboflavin base' or 'basic riboflavin', in the context of the present invention, refer to a compound of formula I and therefore preferably refer to riboflavin that is not salted and not conjugated.

[0030]

[0031] Formula I

[0032] The term "Trypan Blue" or "Trypan blue" in the context of this description refers to a compound of formula II, usually marketed in solution form.

[0033]

[0034] The term "comprising" in the context of this description also means "consisting essentially of" or "consisting of".

[0035] The term 'Matrigel1, in the context of the present invention, refers to a natural extracellular matrix comprising type IV collagen, laminin, entactin, nidogen, proteoglycans and growth factors, used in the laboratory and employed in cell and tissue biology to recreate a three-dimensional (3D) physiological environment that simulates the extracellular matrix (ECM) in vitro.

[0036] In the context of this invention, the term "epiretinal membrane" refers to a thin fibrotic membrane on the retina, such as an irregular thickening of the posterior hyaloid, which contracts the underlying retina, causing it to wrinkle and thus interfering with visual function. In idiopathic cases, the cause is often unidentifiable, but may be associated with the following risk factors: diabetic retinopathy, uveitis, retinal detachment or tear, eye injury and vitreous schisis. It affects about 12% of the population and typically develops after the age of 50 and is more common in people over the age of 75. Symptoms may include blurred vision or distorted vision (e.g., straight lines may appear wavy) . Diagnosis is based on examination of the fundus and optical coherence tomography.

[0037] In the context of the present invention, the term 'macular hole1refers to a retinal defect affecting the fovea, i.e. the central area of the macula. It occurs after the age of 50 and affects both eyes in 10% of cases.

[0038] In the context of the present invention, the term "fractional retinal detachment" refers to a retinal detachment caused by the formation of scar tissue on the retinal surface, which creates traction that lifts the retina. It is typical in cases of retinal ischaemia, which can be caused by diabetes, venous thrombosis or early retinopathy.

[0039] In the context of this description, "approximately" refers to the experimental error that may occur during conventional measurements. More specifically, when referring to a value, itindicates ± 5% of the indicated value, and when referring to an interval, ± 5% of the extremes of that interval, unless otherwise indicated.

[0040] In the context of this description, unless otherwise indicated, the percentage ratios 'weight / weight' or 'w / w1, 'weight / volume' or 'w / v' and 'volume / volume' or 'v / v' are to be understood as the ratio of the weight or volume of the individual ingredient to the total weight or volume of the composition; for example, 1% w / w means 1 mg per 100 mg of composition.

[0041] In the context of this description, when a range of values is indicated, that range also includes the respective extremes, unless specifically excluded.

[0042] In the context of the present invention, the terms "compound for use in medicine", "compound for use as a medicament" or "compound for use in the treatment of" have the same meaning and represent an alternative formulation of the expressions "use of the compound in medicine", "use of the compound as a medicament", "use of the compound in the treatment of", "use of the compound for the production of a medicament" and "use of the compound for the production of a medicament for the treatment of". Therefore, these terms may be used interchangeably, even where not expressly stated.

[0043] BRIEF DESCRIPTION OF THE FIGURES

[0044] Figure 1 (a-b): injection (a) and visualisation (b) of ERM with REMARK V during dye injection (2% basic riboflavin suspension).

[0045] Figure 2: REmark M dye injection phase.

[0046] Figure 3: visualisation of the ILM and its removal by peeling with REmark M.

[0047] Figure 4: standard three-way pars plana vitrectomy in a pig eye.

[0048] Figure 5: measurement of the percentage of permeation on Matrigel of (from left to right): halo 1 (Arthrospira platensis and riboflavin phosphate) vs matrigel 1 (Arthrospira platensis and riboflavin phosphate after absorption on matrigel); halo 2 (Arthrospira platensis, Trypan blue and riboflavin phosphate) vs (Arthrospira platensis, Trypan blue and riboflavin phosphate after absorption on Matrigel); halo 3 (Trypan blue and riboflavin phosphate) vs (Trypan blue and riboflavin phosphate after absorption on Matrigel).

[0049] Figure 6: Histological section of retina examined under electron microscopyDETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention provides a composition comprising (or consisting essentially of):

[0051] at least one extract of Arthrospira spp. or one or more pigments derived therefrom,

[0052] Trypan Blue; and

[0053] tocopherol or its derivatives

[0054] as defined in the first of the attached claims. This composition may also comprise riboflavin and / or hyaluronic acid as well as one or more acceptable excipients and / or vehicles. The compositions of the invention may be pharmaceutical, nutraceutical, cosmetic, veterinary or food compositions, optionally containing biologically active ingredients. According to one embodiment, the composition comprises said Arthrospira spp. extract or one or more pigments derived therefrom, Trypan Blue and tocopherol or its derivatives as the only ingredients with colouring activity. According to an alternative embodiment, however, the composition may comprise one or more additional colouring agents.

[0055] The Arthrospira spp extract may be any extract commercially available or known to those skilled in the art. Typically, the Arthrospira spp extract may be selected from the group consisting of Arthrospira platensis, Arthrospira fusiformis, Arthrospira maxima, and mixtures thereof; , preferably Arthrospira platensis and / or Arthrospira maxima; more preferably, Arthrospira platensis.

[0056] The Arthrospira spp extract contained in the composition may be an extract of Arthrospira platensis, also known as Spirulina platensis, preferably a dry extract with a protein content of between 50-70%, such as, for example, the extract called "Spirulina Alga" marketed by Farmalabor.

[0057] The extract is preferably an extract for food use, more preferably compliant with the current edition of the European Pharmacopoeia and / or Directive 2009 / 32 / EC.

[0058] As is well known to those skilled in the art, Arthrospira spp. extracts contain one or more pigments preferably chosen from: phycocyanins, for example phycocyanin and / or allophycocyanin, chlorophylls, preferably chlorophyll-a, carotenoids, for example beta-carotene and / or zeaxanthin and / or lutein and / or cryptoxanthin, xanthophylls, for example echinenone and / or astaxanthin, and mixtures thereof; more preferably, the pigments may be phycocyanin and / or allophycocyanin.

[0059] Therefore, the phrase "at least one extract of Arthrospira spp. or one or more pigments derived therefrom" means that the composition of the invention may comprise the extract itselfor also one or more pigments contained therein, such as, for example, phycocyanin and / or allophycocyanin.

[0060] Typically, tocopherol derivatives can be selected from the group consisting of tocopherol esters, e.g. tocopheryl acetate, tocopheryl succinate, tocopheryl nicotinate, tocopheryl laurate, tocopheryl palmitate and / or tocopherol linoleate, tocopherol phosphates, e.g. tocopheryl phosphate, disodium tocopheryl phosphate and / or calcium tocopheryl phosphate, polyoxyethylene glycol esters of tocopherol esters, e.g. alpha-tocopheryl polyoxyethylene glycol (1000) succinate (hereinafter also referred to as Vitamin E TPGS or VE-TPGS). By way of example, but in no way limiting, tocopherol derivatives useful forthe purposes of the present invention may be selected from: polyoxyethylene glycol esters of tocopherol esters with a carboxylic acid, preferably wherein said tocopherol ester with a carboxylic acid is selected from tocopheryl acetate, tocopheryl succinate, tocopheryl nicotinate, tocopheryl laurate, tocopheryl palmitate, tocopherol linoleate and tocopherol phosphate and / or preferably wherein the polyoxyethylene glycol function has a molecular weight of between about 600 Da and about 6000 Da, preferably between about 600 Da and about 1500 Da. The preferred ester is alpha-tocopheryl polyoxyethylene glycol (1000) succinate, i.e. a polyoxyethylene glycol ester of alpha-tocopheryl succinate in which the polyoxyethylene glycol moiety of the molecule has an average molecular weight of about 1000 Da. VE-TPGS can be prepared by any method known in the art, for example, by esterification of alpha-tocopherol succinate with polyethylene glycol 1000 or by any of the methods described in U.S. Patent No. 2,680,749 (Cawley et al.) or even readily available commercially, for example, VE-TPGS from Isochem (Isodel®). In developing the present invention, the Applicant surprisingly found that tocopherol or one or more of its derivatives as defined above, preferably VE-TPGS, is capable of enhancing the colouring effect of the composition of the invention and, in particular, of the Arthrospira spp extract or one or more pigments derived therefrom and / or Trypan Blue contained therein.

[0061] The present invention therefore also relates to the use of tocopherol or its derivatives as an adjuvant or enhancer of the colouring effect in colouring compositions according to any of the embodiments described herein.

[0062] Typically, the weight / volume ratio between at least one extract of Arthrospira spp., or one or more pigments derived therefrom, and tocopherol or one or more derivatives thereof, is between 20:0.001 and 0.001:20, preferably between 10:0.01 and 0.01:15; more preferably between 1:0.1 and 0.1:10; even more preferably 1:10. By way of example, a weight / volume ratioof 1:10 means 1 mg of at least one extract of Arthrospira spp. or one or more pigments derived therefrom per 10 mL of tocopherol or one or more derivatives thereof.

[0063] Typically, the sum of the individual quantities of at least one Arthrospira spp. extract, or one or more pigments derived therefrom, and tocopherol or one or more derivatives thereof, constitutes 0.5-15% w / w of the weight of the entire composition, more preferably 1.5% w / w of the weight of the entire composition, and even more preferably 1% w / w of the weight of the entire composition.

[0064] Typically, the amount of Trypan Blue is in the range of 0.001-5% v / v, preferably in the range of 0.01-2.5% v / v, more preferably in the range of 0.1-1% v / v, and even more preferably in the range of 0.18-0.25% v / v of the total volume of the composition.

[0065] The composition of the invention may comprise one or more additional synthetic or natural dyes or pigments, for example, selected from the group consisting of betaine, indigo, anthocyanins, chlorophyll, curcumin, biliverdin, cresyl violet, rhodamine B, 4',6-diamidino-2-phenylindole, hoechst 33342, fluorescein, giemsa stain, alcain blue, neutral red, acid yellow 73, eosin, brilliant green and methylene blue; preferably methyl blue, fluorescein, rhodamine B, anthocyanins, indigo or mixtures thereof. Alternatively, the composition of the invention may comprise as the sole ingredients with colouring activity Arthrospira spp. extract or pigments derived therefrom, Trypan Blue and tocopherol or its derivatives.

[0066] Typically, the composition of the invention may further comprise riboflavin, preferably riboflavin phosphate. The amount of riboflavin is in the range of 0.0001-5% w / w, preferably in the range of 0.0001-2.5% w / w, more preferably in the range of 0.0001-0.5% w / w, and even more preferably 0.0001% w / w of the total weight of the composition. By way of example, 0.0001% by weight of the entire composition means 0.0001 mg of riboflavin phosphate per 100 mg of composition.

[0067] Typically, the composition of the invention may comprise an additional viscoelastic moisturising ingredient selected, for example, from the group consisting of hyaluronic acid, carboxymethylcellulose, hydroxypropylmethylcellulose, glycerine, povidone, polyethylene glycol, sorbitol, xanthan gum, agarose, sodium chondroitin sulphate and mixtures thereof; preferably hyaluronic acid, glycerine and polyethylene glycol; more preferably hyaluronic acid. When present, the viscoelastic moisturising ingredient is present in an amount in the range of 0.001-5% w / w, preferably in the range of 0.01-2.5% w / w, more preferably in the range of 0.1-1% w / w, and even more preferably equal to 0.3% w / w of the total weight of the composition. The presence of the viscoelastic moisturising ingredient, preferably hyaluronic acid, in the composition isparticularly advantageous: in fact, thanks to this ingredient, once injected into the eye filled with BSS®, the composition immediately precipitates as a cohesive sphere in the fundus, selectively staining the desired tissue without spreading throughout the posterior segment. The presence of hyaluronic acid also gives the composition particular cohesion and stability. Thanks to these characteristics, the surgeon is able to precisely direct one or more drops of the composition of the invention, without dispersion or fogging, onto a specific area to be stained. Furthermore, the cohesion and stability of the drops not only prevent the uncontrolled distribution of the composition within the eye, a phenomenon that would make the removal of the dye more difficult and laborious, but also, in the case of macular hole surgery, prevent the unintentional subretinal migration of the dye through the hole.

[0068] Preferably, the present invention refers to a composition comprising at least one extract of Arthrospira platensis, VE-TPGS, Trypan Blue, hyaluronic acid and riboflavin phosphate, more preferably in the quantities and ratios defined herein.

[0069] The above composition, if in liquid form, may further comprise a solvent suitable for intraocular injection, preferably in an amount sufficient to achieve 100% of the composition. The solvent, for example, may be selected from the group consisting of water, isotonic aqueous solutions (e.g., comprising sodium chloride, potassium chloride, magnesium chloride, sodium acetate and / or sodium citrate, or solutions having a pH in the range of 6.7-7.6, also known as balanced saline solutions or BSS®), physiological solutions (e.g., isotonic aqueous solutions comprising 0.9% sodium chloride), phosphate buffer solution, or similar sterile aqueous saline solutions, e.g., usable for ocular irrigation or for surgical procedures or ophthalmic treatments.

[0070] The composition that is the subject of the invention, in particular when comprising Arthrospira platensis, VE-TPGS, Trypan Blue, hyaluronic acid and riboflavin phosphate, can be identified by the name 'REmark M'. This composition is a blue-coloured solution and, as explained in more detail below and in the attached experimental section, has proven to be particularly effective and advantageous as a dye.

[0071] In fact, the Applicant conducted a spectrophotometric analysis (see graph in Fig. 5), recording the absorbance values at A=607 nm (wavelength of maximum absorption for Arthrospira platensis extract,! and at A=615 nm (wavelength of maximum absorption for Trypan Blue) in order to measure the percentage of permeation on matrigel of a first solution, in the graph 'halo I1, comprising Arthrospira platensis and riboflavin phosphate, a second solution, in the graph 'halo 2', comprising Arthrospira platensis, Trypan blue and riboflavin phosphate, and a third solution comprising Trypan blue and riboflavin phosphate, in graph "alone 3". The same quantities ofriboflavin phosphate, Arthrospira platensis and Trypan blue were used in the three different solutions.

[0072] More specifically, the analysis involved incubating the three different solutions on Matrigel at 37°C for 24 hours. At the end of incubation, the residual solutions (halo 1, halo 2 and halo 3), i.e. those not permeated into Matrigel, were removed and the Matrigel was first washed with PBS and then dissolved using a 2% SDS solution, thus obtaining the solutions 'Matrigel 1', 'Matrigel 2' and 'Matrigel 3' respectively. The absorbance of the different solutions, halo 1-3 and matrigel 1-3, was measured using a Multiskan Go spectrophotometer (Thermo Scientific, Waltham, MA, USA) and spectrophotometric measurements were performed at wavelengths A= 607 nm and = 615 nm. Surprisingly, the absorbance value of the Matrigel 2 solution, i.e. the solution obtained by dissolving Matrigel incubated with a solution containing Arthrospira platensis, Trypan blue and riboflavin phosphate, is greater than the sum of the absorbance values of Matrigel solutions 1 and 3, obtained by dissolving Matrigel incubated with solutions containing Arthrospira platensis and riboflavin phosphate or Trypan blue and riboflavin phosphate, respectively. REmark M has proven to be even more effective as a dye because, in addition to containing the dyeing agents referred to in solution 'alone 2', which act synergistically, it also contains tocopherol or derivatives, for example VE-TGPS, which increase its permeability.

[0073] The present invention also relates to a composition in the form of an aqueous suspension comprising free riboflavin (or free or basic riboflavin base) in an amount greater than 0.5% w / v of the total weight of the suspension, preferably in an amount between 1-3% w / v, more preferably 2% w / v. Typically, the solvent present in the suspension may be any solvent suitable for intraocular injection, for example, as defined above.

[0074] The free-form riboflavin suspension of the invention is a yellow suspension and is also identified below by the name 'REmark V'.

[0075] The compositions according to any of the embodiments of the invention are compositions for topical use in liquid, semi-liquid, semi-solid or solid form, such as, for example, eye drops, solution, sterile solution, suspension, eye drops, spray, ophthalmic bath, gel, ointment, cream,cream, medicated wipes, occlusive dressings (e.g. in the form of medicated gauze), ophthalmic inserts (e.g. controlled-release medicated hydroxypropylcellulose inserts).

[0076] Typically, when in liquid form, the pH of the composition of the invention is in the range 6.7-7.6.

[0077] Typically, when in liquid form, the density of t e composition of the invention is in the range 0.85-1.30 Kg / L; preferably, 0.95-1.19 Kg / L; more preferably, it is equal to 1.01 Kg / L.

[0078] Typically, when in liquid form, the osmolality of the composition of the invention is in the range of 270-369 mOsm / kg H2O.

[0079] The density and osmolality may contribute separately, additively or synergistically to conferring the advantageous characteristics of cohesiveness and stability described above on the composition.

[0080] Suitable excipients include, but are not limited to, diluents such as water or other solvents, solubilising agents and emulsifiers chosen from ethyl alcohol, polyalcohols, propylene glycol, glycerol, polyethylene glycol and sorbitan esters.

[0081] The composition or aqueous suspension according to the present invention may contain no excipients and / or other additives, but only the colouring agents according to any of the embodiments described herein, dissolved or suspended in a suitable vehicle. However, where necessary, suitable excipients may be selected from those normally known in the state of the art and include, but are not limited to: surfactants (e.g. sodium lauryl sulphate and polysorbates), adsorbents (e.g. silica gel, talc, starch, bentonite, kaolin), glidants and anti-adherents (e.g. talc, colloidal silica, corn starch, silicon dioxide), antioxidants, binders (e.g. gums, starch, gelatin, cellulose derivatives, sucrose, sodium alginate), plasticisers (e.g. ethylcellulose and other cellulose derivatives, acrylates and methacrylates, glycerol and sorbitol), preservatives (e.g. parabens, sulphur dioxide, polyhexamethylene biguanide or PHMB), disodium edetate (EDTA), viscosifiers, emulsifiers, humectants, wetting agents, chelating agents and mixtures thereof.

[0082] According to another embodiment, the composition and aqueous suspension of the invention do not contain benzyl alcohol. This ingredient is commonly present as a vehicle in other surgical dyes, known to cause damage to the retina. While performing the same function as existing dyes already on the market, the absence of this ingredient in the composition and suspension of the present invention is an advantageous feature.

[0083] The composition or suspension of the present invention may also be included in, for example, a medical device, a food supplement, a nutraceutical, dietary and nutritionalcomposition, a food product, a beverage, a medicament, a medicated food, a food for special medical purposes, or a pharmaceutical composition.

[0084] Therefore, the present invention also provides a medical device, preferably a pre-filled syringe, comprising a composition or suspension according to any of the embodiments of the invention described herein. The present invention also relates to a kit of parts comprising a container suitable for containing (or containing) the composition of the invention, or to a kit of parts comprising a first container containing at least one extract of Arthrospira spp. or pigments derived therefrom, a second container containing Trypan Blue and a third container containing tocopherol or its derivatives, and optionally, where present, further containers containing (or suitable for containing) riboflavin, preferably riboflavin phosphate, and / or a viscoelastic moisturising agent, preferably hyaluronic acid

[0085] The kit of parts may further comprise, preferably in a separate container, the aqueous suspension according to any of the embodiments described herein, in particular a suspension comprising free riboflavin in an amount of not less than 0.5% w / v, preferably 2% w / v.

[0086] The present invention also relates to a kit of parts comprising a container suitable for containing (or containing) the aqueous suspension according to any of the embodiments described here, preferably REmark V, or a kit of parts comprising at least a first container containing free riboflavin; and at least a second container containing a suitable solvent as defined above. This kit may optionally comprise a further container containing the composition in any of the embodiments described herein, preferably that indicated as REmark M.

[0087] The container may be chosen from any of the suitable containers known to those skilled in the art, for example, the container may be chosen from a syringe, a pre-filled syringe, a vial, an ampoule, a single-use dispenser, bottles, preferably made of transparent glass or amber glass or opaque glass or plastic, tubes, preferably made of aluminium or laminated plastic, sachets, preferably made of laminated material or aluminium, graduated measuring cups, single-dose capsules, blister packs, zip-lock sachets, resealable ampoules. Obviously, if the kit contains two or more containers, they may be the same or different from each other. Typically, the volume of the container is between 0.5 and 5 mL, preferably between 0.5 and 1 mL, and more preferably 0.5 mL In a particularly preferred aspect, the container in the kit is a syringe, a vial, a pre-filled syringe, or combinations thereof.

[0088] Typically, the pre-filled syringe and / or vial has a volume of between 0.5 and 5 mL, preferably between 0.5 and 1 mL, and more preferably 0.5 mL. The present invention also relates to a process for preparing a composition according to any of the embodiments of the invention, in particularcomprising at least: an extract of Arthrospira spp. or pigments derived therefrom, Trypan Blue and tocopherol or one or more derivatives thereof, the process comprising the following steps:

[0089] (i) Dissolving or suspending an extract of Arthrospira spp. or one or more pigments derived therefrom in tocopherol or its derivatives; preferably an extract of Arthospira platensis in VE-TGPS; more preferably an extract of Arthospira platensis in VE-TGPS in a weight / volume ratio of 1:10;

[0090] (ii) Maintaining the solution or suspension referred to in point (i) under agitation for at least 30 minutes and filtering said solution or suspension to give a filtered solution;

[0091] (iii) Dissolving hyaluronic acid, Trypan Blue and riboflavin phosphate, preferably in a weight ratio of 3000:1800:1, in water to give an aqueous solution; (iv) Mixing the filtered solution referred to in point (ii) and the aqueous solution referred to in point (iii).

[0092] The present invention also relates to the composition obtainable from the process of the invention.

[0093] The present invention also relates to the composition, suspension or kit of parts according to any of the embodiments described herein for use in medicine, preferably for use as an ophthalmic dye, more preferably for use in vitreous surgery.

[0094] By way of example, the composition, suspension and / or kit of the invention may be used as ophthalmic dyes for the vitreous, posterior hyaloid, epiretinal membrane (ERM), internal limiting membrane (ILM), dyes for ophthalmic surgery, vitrectomy, chromo-vitrectomy and / or retinal surgery, etc. Advantageously, the composition according to any of the embodiments described herein, comprising at least one extract of Arthrospira spp. or pigments derived therefrom, Trypan Blue and tocopherol or tocopherol derivatives, has been found to be suitable for selective staining of the internal limiting membrane (ILM), while the suspension comprising free riboflavin according to any of the embodiments described herein has been found to be suitable for selective staining of the epiretinal membrane (ERM) .

[0095] The composition, suspension and / or kit according to any of the embodiments described herein have been found to be particularly suitable for use as an ophthalmic dye in surgical procedures such as, for example, the treatment of idiopathic, infectious or traumatic vitreitis, haemorrhagic vitreous opacities, exudative vitreous opacities, endophthalmitis, proliferative diabetic retinopathy, idiopathic retinal detachment, tractional retinal detachment, macular hole, fractional maculopathies, macular puckers and vitreoschisis.

[0096] This means that the present invention also relates to the composition, suspension and / or kit according to any of the embodiments described herein, as well as their use in the diagnosis ortreatment of any of the above-listed pathologies, as well as for the visualisation and / or removal of ocular structures, in particular ERM and ILM.

[0097] Therefore, the present invention also relates to the combination of the composition of the invention with the suspension of the invention.

[0098] Advantageously, the composition of the invention can be used as an adjuvant to the suspension of the invention - and vice versa - in the above-mentioned uses.

[0099] Consequently, the present invention also relates to the combination of the combination and the suspension according to any of the embodiments described her ly for the above uses, preferably for simultaneous, separate or sequential use in medicine, more preferably as an ophthalmic dye.

[0100] Surprisingly, injecting REmark M into the vitreous cavity makes it possible to clearly stain, specifically stain blue, the ILM, allowing it to be distinguished from the underlying white (unstained) retina, and thus allowing selective removal of that membrane. Furthermore, thanks to the components of the solution, REmark M can be injected directly into the eye even when the latter is filled with a sterile ocular irrigation solution such as, for example, BSS® or BSS® Plus marketed by Alcon. Under these conditions, REmark M precipitates immediately and forms a cohesive sphere in the fundus, selectively staining the desired tissue, i.e. the ILM, without spreading throughout the posterior segment. The riboflavin-based aqueous suspension that is the subject of the invention is also particularly advantageous in that the particulate matter present in the REmark V suspension, upon intraocular injection, deposits on the posterior hyaloid and ERM, facilitating their identification and, consequently, their removal. REmark V is also recommended for staining the vitreous, especially in cases of vitreousschisis.

[0101] For example, in multi-step surgical procedures, the REmark V solution can be used in a first step to selectively stain the epiretinal membrane, for example in procedures to remove this membrane by vitrectomy or in procedures to peel the hyaloid membrane, and subsequently REmark M can be used to stain the internal limiting membrane in procedures such as, for example, peeling of this membrane.

[0102] The present invention therefore also relates to the use, preferably in vitro or ex vivo, of the composition, suspension or kit of the invention as a histological stain, preferably of collagen-and / or gelatin-based tissues.

[0103] The present invention also relates to the composition, suspension, combination and kit of parts of the invention for use in a method of in vivo or in vitro diagnosis of an ophthalmic pathology, such as those mentioned above. The diagnostic (or surgical) methods mentioned aboveinclude at least one step of contact between the composition or suspension, as defined above, and at least one ocular tissue, or a fraction thereof, taken or isolated; such methods may further comprise one or more steps such as, for example, image recording, measurement of the staining intensity of the ocular tissue or fraction thereof, or comparison with healthy ocular tissue or fraction thereof.

[0104] The present invention further provides an in vitro or ex vivo method for staining ophthalmic membranes, preferably the internal limiting membrane and / or the epiretinal membrane ( ), which involves at least one contact step between said membranes and the composition or aqueous suspension that is the subject of the first and second objects of the invention, respectively; preferably, the duration of contact is between 10 and 120 seconds, more preferably, the duration of contact is 60 seconds; this in vitro or ex vivo method comprises at least one further step of removing the staining composition, for example by irrigation with BSS®.

[0105] Atthe sametime, the present invention provides a composition consistingof or comprising at least one extract of Arthrospira spp. or pigments derived therefrom, Trypan Blue and tocopherol or tocopherol derivatives, as per the first aspect of the invention, an aqueous suspension comprising basic riboflavin in an amount greater than 0.5% w / w or a kit comprising them for in vivo use as an ophthalmic dye in surgical and / or diagnostic procedures involving at least one step of contact between said composition and / or suspension with at least one ophthalmic membrane, preferably the internal limiting membrane and / or the epiretinal membrane, and one step of removal of said composition and / or suspension, for example by irrigation with BSS®. The duration of contact is preferably between 10 and 120 seconds, more preferably, the duration of contact is 60 seconds.

[0106] The present invention also provides a method of treatment, prophylaxis or diagnosis of an ophthalmic condition or a surgical method for treating an ophthalmic condition comprising at least one administration of the composition, suspension, combination or kit according to any of the embodiments described herein.

[0107] EXAMPLES

[0108] Example 1 - Method of preparing a colouring composition according to an embodiment of the present invention (REmark M composition)

[0109] An extract of A. platensis was solubilised in VE-TPGS in a 1:10 weight / volume ratio. The mixture obtained was kept under agitation for about 30 minutes and then filtered to obtain a clear solution. This solution was added at 1% to a second solution containing 0.3% hyaluronic acid,0.18% Trypan blue, 0.0001% riboflavin phosphate and water in the quantity necessary to reach a volume of 100%.

[0110] Example 2 - Intraoperative efficacy and clinical results of two dye compositions according to an embodiment of the present invention used in vitrectomy for idiopathic epiretinal membrane The intraoperative efficacy study was based on data obtained from a questionnaire comprising questions relating to the intraoperative performance of colouring compositions according t s according to the present invention, also in comparison with commercially available ophthalmic dyes (e.g., Membra Blue-Dual®). The dye compositions according to the present invention investigated here correspond to compositions according to preferred embodiments of the invention, in particular:

[0111] REmark V (yellowish colour): 2% basic riboflavin in aqueous suspension.

[0112] REmark M (intense blue colour): Arthrospira platensis + TPGS (weight / volume ratio 1:10 w / v between them) 1% w / w of the weight of the composition, Trypan Blue 0.18% v / v of the volume of the composition, hyaluronic acid 0.3% w / w of the weight of the composition, riboflavin phosphate 0.0001% w / w of the weight of the composition.

[0113] Data relating to human participants were collected and managed in accordance with ethical standards and the 1964 Declaration of Helsinki and its subsequent amendments. All patients signed a general informed consent form that was specifically designed and approved for participants in observational studies.

[0114] Postoperative clinical outcomes were also analysed retrospectively.

[0115] Study design

[0116] In this non-interventional, cross-sectional observational study, a customised questionnaire was administered to a single vitreoretinal surgeon, asking the following questions regarding the performance of the dye compositions used during surgery: (1) How would you rate the cohesiveness of the dye solution? (2) How would you rate the colouring effectiveness of the ERM? (3) How would you rate the colouring effectiveness of the ILM? In addition, the number of injections of the dye compositions required during surgery was also recorded. The survey was conducted at the Department of Ophthalmology of the Pellegrini Hospital over a period of 6 months, from September 2023 to March 2024, during which the ophthalmic operating theatre was supplied with the REmark V and REmark M dye compositions, which are the subject of the invention, and the MembraneBlue-Dual® solution. These dyes were used alternately in each operation. The questionnaire was submitted to the surgeon immediately after the operation. Patients were managed routinely in the ophthalmology clinic with standard visits scheduled 1 to3 days before the operation, then 7, 30 and 90 days after the operation, as for all patients operated on for ERM. Preoperative visits and those performed 7, 30 and 90 days after surgery, in addition to the physician's evaluation, also included structural spectral optical coherence tomography (SD-OCT).

[0117] Study participants

[0118] Compliance with the inclusion and exclusion criteria, detailed below, was verified in the preoperative phase.

[0119] Patient inclusion criteria: age >18 years and diagnosis of idiopathic ERM involving the fovea with and / or without vitreous schisis (diagnosed by biomicroscopy and SD-OCT).

[0120] Patient exclusion criteria: ERM secondary to any ocular disease (e.g., diabetic retinopathy, uveitis, retinal vein occlusion, retinal dystrophies), glaucoma, previous posterior segment surgery (including retinal laser photocoagulation), previous corneal surgery, previous surgery for complicated cataract surgery, and axial length < 22 mm or > 26 mm.

[0121] Surgical procedure

[0122] Transconjunctival pars plana vitrectomy without sutures using a 25-gauge three-port technique with ERM and ILM peeling was performed by a single vitreoretinal surgeon. ERM and ILM peeling was achieved using the pinch technique, first removing the posterior hyaloid attached to the optic disc and all parts of the ERM, then grasping the ILM near the macula with macular forceps and providing a round maculorhexis. ERM and ILM were removed by peeling in all patients using a dye solution (REmark V or REmark M). Fluid-air and (in selected cases) air-gas exchanges were performed in appropriate cases. Standard postoperative therapy consisted of topical netilmicin and dexamethasone 4 times a day for 4 weeks and 1% tropicamide twice a day for 1 week.

[0123] Questionnaire on the intraoperative performance of dye solutions

[0124] The surgeon was asked to answer the questions in the above questionnaire and to rate them on a scale of 1 to 3.

[0125] Scores for question (1): 1 (minimum) in case of complete clouding of the vitreous cavity at the time of injection of the dye composition, requiring complete rinsing with the cutter in aspiration mode; 3 (maximum) in case of no clouding and no need for aspiration; 2 (intermediate) in case only a brief aspiration of the composition was required.

[0126] Scores for questions 2 and 3: 1 (minimum) in the absence of ERM / ILM staining, requiring an additional injection of dye; 3 (maximum) in the absence of staining of the ERM / ILM, which could be grasped and easily detached; 2 (intermediate) if the staining of the ERM / ILM was noticeable but with an uneven pattern, requiring multiple evaluation of the tissues before peeling.Cohesion ILM staining ERM staining Number of injections MembraneBlu MembraneBlu MembraneBlu MembraneBlu REmark M REmark M REmark V REmark M REmark V

[0127] e-Dual® e-Dual® e-Dual® e-Dual® 2 2 2 3 3 3 1 2 1 2 3 2 3 3 3 1 1 1 3 3 2 3 3 2 1 1 1 2 3 2 3 3 2 1 1 1 3 3 2 3 3 3 1 2 2 3 3 2 3 3 2 1 1 1 2 3 2 3 3 2 2 1 1 3 3 2 3 3 3 2 2 2 2 3 2 2 2 2 1 1 1 2 3 2 3 3 2 2 1 2

[0128]

[0129] Table 1

[0130] As can be seen from the survey results shown in Table 1, the cohesion and staining efficacy of REmark M and REmark V on ERM and ILM are comparable to those obtained with MembraneBlue-Dual® (a staining solution containing Trypan Blue and Brilliant Blue G). This result would therefore confirm that REmark M and REmark V are a valid alternative to commercially available staining solutions.

[0131] Measurements

[0132] The electronic medical records and imaging data of the patients participating in the study were analysed retrospectively. Best-corrected visual acuity (BCVA) and central foveal thickness (CRT) from structural SD-OCT were collected both before surgery and approximately 1 and 3 months after surgery. The presence of adverse effects was also verified.

[0133] BCVA was converted to logarithm of minimum angle of resolution (logMAR) for calculation purposes. SD-OCT and BA images were recorded with the Spectralis OCT device (Heidelberg Engineering, Heidelberg, Germany). ERM stage was recorded according to the classification of Govetto and colleagues.

[0134] Statistical analysis

[0135] A Student's t-test (orT-test) was performed.

[0136] Results

[0137] Twenty patients who met the inclusion / exclusion criteria were included in the study: 10 in the REmark V + REmark M group (test group) and 10 in the MembraneBlue-Dual® group (controlgroup). The patients and their ocular characteristics were found to be equally matched in the two groups before surgery. In all 20 cases, the surgery was performed successfully, and the questionnaire was completed by a single surgeon.

[0138] The questionnaire results showed that the REmark V and REmark M dye compositions received comparable ILM staining cohesion and efficacy scores compared to MembraneBlue-Dual®; furthermore, the ERM staining score and the number of injections required were similar in both groups (Table 1).

[0139] Visual acuity improved significantly when comparing BCVA at 1 month and 3 months with BCVA at time 0 in both groups during the observation period, reaching similar values at both 1 month (P >0.01) and 3 months (P >0.01) after surgery. Central foveal thickness was reduced similarly after surgery in both groups, with no significant differences between groups at each time point (P >0.01 and P>0.01), respectively, at 1 and 3 months (Table 2).

[0140] No other clinically relevant complications, such as retinal detachment, increased intraocular pressure, endophthalmitis, or unexplained visual loss, were observed.

[0141] BCVA BCVA BCVA BCVA AGE Gender

[0142] (logMAR)to (logMAR)t7(logMAR)t30 (logMAR)t90

[0143] 61 M 0.5 0.4 0.4 0.25

[0144] 44 M 0.6 0.5 0.5 0.5

[0145] 56 M 0.7 0.6 0.5 0.55

[0146] 59 M 0.5 0.5 0.4 0.3

[0147] REmark M 47 F 0.4 0.2 0.2 0.2

[0148] 53 M 0.6 0.3 0.3 0.4

[0149] 73 F 0.4 0.2 0.2 0.15

[0150] 57 F 0.4 0.2 0.2 0.2

[0151] 62 F 0.6 0.3 0.2 0.2

[0152] 51 F 0.3 0.1 0.1 0.1

[0153] BCVA BCVA BCVA BCVA AGE Gender

[0154] (logMAR)to (logMAR)t7(logMAR)t3o (logMAR)t9o

[0155] 52 F 0.6 0.4 0.4 0.3

[0156] 59 F 0.4 0.2 0.2 0.2

[0157] 48 F 0.7 0.5 0.4 0.4

[0158] 66 M 0.5 0.3 0.2 0.2 MembraneBlu 64 M 0.5 0.4 0.4 0.4 e-Dual® 52 F 0.5 0.3 0.3 0.3

[0159] 37 F 0.4 0.3 0.2 0.2

[0160] 61 F 0.7 0.5 0.5 0.4

[0161] 56 M 0.5 0.4 0.3 0.2

[0162]

[0163] 55 M 0.4 0.2 0.2 0.2AGE Sex CTFto CTFt7CTFtso CTFt9o

[0164] 61 M 521 444 446 431

[0165] 44 M 518 432 412 410

[0166] 56 M 503 423 431 424

[0167] 59 M 498 412 410 405

[0168] 47 F 509 422 422 420 REmarkV 53 M 517 417 426 422

[0169] 73 F 512 419 416 416

[0170] 57 F 510 430 423 418

[0171] 62 F 486 409 415 419

[0172] 51 F 514 465 458 465

[0173] AGE Sex CTFto CTFt7 CTFt3o CTFtso

[0174] 52 F 521 467 461 450

[0175] 59 F 509 434 421 432

[0176] 48 F 516 437 438 433

[0177] 66 M 501 435 426 412

[0178] MembraneBlu 64 M 493 452 455 424

[0179] e-Dual® 52 F 511 473 456 451

[0180] 37 F 517 434 426 423

[0181] 61 F 482 443 426 433

[0182] 56 M 522 476 438 441

[0183]

[0184] 55 M 512 447 429 432

[0185] Table 2

[0186] Discussion

[0187] This study aimed to compare the performance of dye compositions according to an embodiment of the present invention. Their properties were evaluated by a single surgeon, verifying their staining capabilities on a homogeneous sample of idiopathic ERM. The customised questionnaire showed that the dyes under examination obtained good scores for cohesion and ILM staining efficacy. It can be hypothesised that these properties may be due to both the particular composition of colouring agents present in the compositions of the invention and the presence of the viscoelastic moisturising agent (thickener), or to both of these factors. The good cohesion resulting from this study can be used by the surgeon to direct a few drops of the dye composition precisely onto the area to be stained, without dispersion or blurring, resulting in easier and faster removal of the ocular membranes. In addition, the stability of each drop may be beneficial in macular hole surgery, preventing unintended subretinal migration of the dye through the hole. Functional and morphological results confirmed the safety profiles of each dye composition, with no significant differences between the two. Visual acuity and macular thickness showed similar trends of improvement. Although idiopathic ERMs are thought to be related to a generalsubclinical inflammatory state, clinically manifest inflammation resolves within 30 days, regardless of the calibre chosen. However, in the present study, no significant signs of intraocular inflammation were ever found.

[0188] At the end of the study, no major adverse events, such as endophthalmitis, retinal detachment or retinal vascular events, were recorded.

[0189] REmark V and REmark M are advantageously free of preservatives such as benzyl alcohol, which is present as a vehicle in other surgical dyes that are known to cause retinal damage.

[0190] In conclusion, the REmark V and REmark M dye compositions are suitable dyes with an equivalent safety and efficacy profile, both intraoperatively and postoperatively. The dye compositions of the invention therefore meet the need for faster, ready-to-use and efficient dyes, as well as offering a solution for operators who prefer a more cohesive and stable dye than those known in the art or commercially available, and therefore suitable for obtaining localised staining with rapid washing.

[0191] Example 3 - Two dye compositions for vitreoretinal surgery

[0192] Purpose of the study

[0193] The purpose of the study is to evaluate the staining capacity and safety of the dye compositions covered by the invention, in particular REmark V for staining the vitreous, posterior hyaloid and ERM, and REmark M for staining the internal limiting membrane (ILM) in pig eyes, for the evaluation of their use in vitreoretinal surgery.

[0194] Dyes and dye preparation

[0195] REmark V and REmark M were included in this investigation and evaluated at the following concentrations: REmark V, 3%, 2% and 0.5%; REmark M at 2%, 1% and 0.5% diluted in BSS® plus saline solution. For controls, balanced saline solution (BSS® plus; Alcon Laboratories, Inc., Fort Worth, TX) alone or containing 0.5% ICG was applied to one eye. A total of 10 pig eyes were treated.

[0196] The concentrations of the dye compositions were chosen to be higher than in the in vitro investigation in order to detect potential adverse effects and to better classify the staining characteristics. For both dye compositions, the evaluation began with the highest concentration. Lower concentrations were applied only if a satisfactory staining effect was observed with the previously injected concentration. The staining characteristics were evaluated by two examiners who also performed the surgery.

[0197] In vitro laboratory testsThe Matrigel basement membrane matrix (Corning 354234) was thawed overnight at 4 °C, dissolved 1:4 in cold phosphate-buffered saline (PBS), and 500 pL of this solution was added to each well of a 24-well plate. The plate was incubated at 37 °C for 1 hour to allow the matrix to gel. After gelation, the wells were gently washed with 500 pL of phosphate-buffered saline (PBS) to remove any unbound material. Three test solutions were tested: Alone 1 (Arthrospira platensis and riboflavin phosphate), Alone 2 (Arthrospira platensis, Trypan blue and riboflavin phosphate) and Alone 3 (Trypan blue and riboflavin phosphate). Each solution was added to the wells in triplicate and incubated for 24 hours at 37 °C with gentle agitation. After incubation, the residual solutions were carefully removed and the wells were washed three times with PBS to remove nonpermeated substances. To dissolve the matrigel and all compounds, 200 pL of 2% SDS solution was added to each well and incubated for 30 minutes at room temperature. The dissolved samples were then transferred to wells, and absorbance was measured using a spectrophotometer (Multiskan Go, Thermo Scientific, Waltham, MA, USA) at 615 nm for Trypan Blue and 607 nm for the A. platensis extract. Control wells included 2% SDS solution alone as a negative control and Alone 1-3 solutions without Matrigel as positive controls.

[0198] Animals

[0199] Ten eyes from five pigs (aged 4 months or older) were included in this study. To limit the number of animals, both eyes were treated with the dye compositions of the invention or BSS®. The animals were sedated by intramuscular injection of a mixed solution of ketamine (10 mg / kg; Imalgene 1000, Merial) and azaperone (2 mg / kg; Stenil, Jansen-Cilag). Anaesthesia was induced using an intravenous solution of sodium thiopental (10 mg / kg; Pentothal, Abbott). Anaesthesia was maintained after intubation with 2% isoflurane in 100% oxygen with respiratory assistance. Cefamendol (20 mg / kg; Pan-pharma) was administered at the start of surgery and every 2 hours until the end of surgery. At the end of the experiment, the animals (country of origin: Italy) were euthanised with an intravenous injection of pentobarbital (50 mg / kg; Do-lethal, Vetoquinol). All animals received veterinary care in accordance with the "Guide for the Care and Use of Laboratory Animals", and the procedures were approved by the institutional ethics committee.

[0200] Experimental procedures

[0201] Ophthalmic surgery consisted of standard pars plana vitrectomy (Fig. 4). To better visualise the vitreous base and hyaloid, various concentrations of REmark V were injected into the vitreous cavity. If necessary, posterior vitreous detachment was induced by aspiration with the vitrectomy probe at the optic nerve head, and the vitreous, identified by the yellowish colour intensity produced by the basic riboflavin crystal particles adhering thereto, was removed. This wasfollowed by fluid-air exchange. Subsequently, various concentrations of REmark M for ILM staining were injected into the globe. After a period of 1 minute, this staining composition was removed by irrigation with BSS®. The intensity of retinal surface staining was subjectively graded by the surgeon and an additional examiner who observed the surgical procedure. The evaluations are reported in Table 3.

[0202] Staining effect REMARK M REMARKV

[0203] Very good 3% 3%

[0204] Good 1-2% 1-2%

[0205]

[0206] Weak 0.5% 0.5%

[0207] Table 3 - Subjective assessment of the colouring effect on the retinal surface Histology

[0208] The enucleated eyes were sectioned horizontally. One half of the eye was placed in 4% paraformaldehyde and the other half was placed in a 1:1 mixture of 2.5% glutaraldehyde and 4% paraformaldehyde. Paraffin sections were obtained from the paraformaldehyde-fixed tissue and stained with haematoxylin / eosin to study the general appearance of the tissue.

[0209] From selected regions of glutaraldehyde / paraformaldehyde-fixed tissue (central retina near the optic nerve and peripheral retina nearthe equator), semi-thin sections were stained with toluidine blue for optical microscopic evaluation, and ultrathin sections were stained with lead citrate and uranyl acetate for electron microscopy. Optical microscopy of haematoxylin / eosin-stained sections of the posterior ocular segment showed regions of normal-appearing retina alongside regions of mechanical disruption of the internal limiting membrane due to the surgical procedure (Fig. 6). Free erythrocytes were frequent and were taken up as a result of intraoperative rupture of small retinal vessels. Morphological alterations were limited to areas of attempted ILM removal and were considered to be mechanical trauma. Regardless of the stain and concentration applied, no signs of necrosis, pathological swelling, or apoptosis were visible in any other section.

[0210] Semi-thin and ultra-thin sections of intact retinal regions revealed no morphological changes in any of the retinal layers.

[0211] Results

[0212] Discussion

[0213] The dye compositions of the invention have certain advantages, such as high absorption coefficients, high extinction coefficients, high solubility in BSS® and good adsorption properties towards the target tissue. In addition, the selected dyes show high photochemical stability.Both REmark V and REmark M proved to be promising candidates as new ophthalmic dyes, particularly applicable to vitrectomy, as they revealed good staining characteristics of the posterior segments, even at low concentrations. No toxic effects were observed in a cell culture model using retinal pigment epithelial cells, and no histological abnormalities occurred after shortterm application in the animals used.

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Claims

CLAIMS1. A composition comprising:• Arthrospira spp. extract or pigments derived therefrom,• Trypan Blue; and• tocopherol or its derivatives.

2. The composition according to claim 1 further comprising riboflavin, preferably riboflavin phosphate.

3. The composition according to claim 1 or 2 further comprising a viscoelastic moisturising ingredient, preferably selected from hyaluronic acid, carboxymethylcellulose, hydroxypropylmethylcellulose, glycerine, povidone, polyethylene glycol, sorbitol, xanthan gum, agarose, sodium chondroitin sulphate and mixtures thereof; more preferably hyaluronic acid.

4. The composition according to any of the preceding claims, wherein the weight / volume ratio between the Arthrospira spp. extract and tocopherol is between 20:0.001 and 0.001:20, preferably between 10:0.01 and 0.01:15; more preferably between 1:0.1 and 0.1:10; more preferably 1:10.

5. The composition according to any of the preceding claims, in which composition the amount of Trypan Blue is in the range of 0.001-5% v / v, preferably in the range of 0.01-2.5%, v / v, more preferably in the range of 0.1-1% v / v, even more preferably 0.18-0.25% v / v of the total volume of the composition.

5. The composition according to any of claims 1 to 5 for use as an ophthalmic dye in vitreous surgery; preferably for use in surgical procedures for the treatment of idiopathic, infectious or traumatic vitreitis, haemorrhagic vitreous opacities, exudative vitreous opacities, endophthalmitis, proliferative diabetic retinopathy, idiopathic retinal detachment, tractional retinal detachment, macular hole, tractional maculopathies, macular puckers and vitreoschisis.

7. The composition for use according to claim 6, for use in combination with an aqueous suspension comprising basic riboflavin in an amount of not less than 0.5% w / v of the total volume of the suspension, preferably 2% w / v of the total volume of the suspension.

8. The composition for use according to claim 6 or 7, for use in the selective staining of the internal limiting membrane (ILM) and / or the epiretinal membrane (ERM).

9. Kit of parts comprising a container containing the composition according to any of claims 1 to 8 or a kit of parts comprising a first container containing at least one extract of Arthrospiraspp. or pigments derived therefrom, a second container containing Trypan Blue, a third container containing tocopherol or tocopherol derivatives and, optionally, further containers containing riboflavin and / or a viscoelastic moisturising ingredient, preferably wherein said container is a syringe, a pre-filled syringe, an ampoule or a vial.

10. In vitro or ex vivo use of a composition according to any of claims 1 to 5 as a dye, preferably as a histological dye, more preferably as a dye for collagen- or gelatin-based tissues.

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