Ophthalmic pharmaceutical compositions and relative use

Abstract

Ophthalmic viscoelastic pharmaceutical compositions, of the cohesive type, having a medium viscosity at rest, stained but without a staining capacity, comprising or consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF, and their use in surgery of the anterior segment of the eye, preferably in cataract surgery, are described.

Description

[0001] “OPHTHALMIC PHARMACEUTICAL COMPOSITIONS AND RELATIVE USE"

[0002] ****

[0003] OBJECT OF THE INVENTION

[0004] The present invention describes an innovative ophthalmic viscoelastic pharmaceutical composition of the cohesive type, having a medium viscosity at rest, stained but without a staining capacity, comprising or consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF, and its use in surgery of the anterior segment of the eye, preferably in cataract surgery.

[0005] STATE OF THE ART

[0006] The wall of the human eye is composed, from the outside to the inside, of three concentric tunics: fibrous tunic, vascular tunic or uvea, and nervous tunic. The fibrous tunic primarily exerts a structural and protective function, and is composed of the sclera and cornea. The sclera is the opaque white portion made up of collagen and elastic fibers that externally covers about five-sixths of the eyeball in the posterior part, whereas the cornea is the transparent multilayered structure that covers a portion of about one-sixth positioned anteriorly.

[0007] The trophic and vascular functions are exerted by the vascular tunic or uvea, which includes the iris, ciliary body and choroid. The iris is the anterior segment of the uvea, characterized by a thin ring-like structure, whose characteristic pigmentation defines the colour of the eyes and whose central hole is called the pupil. The ciliary body extends from the iris to the choroid and includes the ciliary muscle, which is primarily responsible for focusing. The ciliary muscle, by contracting and relaxing, is in fact capable of determining a variation in the curvature, and therefore in the dioptric power, of the natural lens of the eye connected to it, the crystalline lens. The function of supporting and maintaining the position of the crystalline lens however is effected by a system of fibers stretched between the crystalline lens itself and the ciliary body, called the ciliary zonule or suspension apparatus of the crystalline lens. The choroid, on the other hand, is a structure extremely rich in blood vessels and capillaries, which occupies the posterior portion of the uvea, constituting a thin layer between the sclera and the retina. The border area between the ciliary body and the choroid is called ora serrata.

[0008] Finally, the retina or nervous tunic is the component of the eye sensitive to light, and is composed of several layers of neurons interconnected by synapses and a pigmented epithelium that provides a trophic and functional support. The retina adheres internally to the posterior third of the eyeball, and its positioning is essential for guaranteeing its function, allowing it to collect light stimuli that penetrate the eyeball through the pupil. (Gray H. & Lewis W. H., 1918; Anatomy of the human body, 20th ed. Philadelphia: Lea & Febiger).

[0009] In ophthalmology, the separation of the eye into two distinct portions, the anterior segment and the posterior segment, is of considerable importance, both from a diagnostic and surgical point of view.

[0010] The posterior segment of the eye includes the posterior two-thirds of the eyeball and the cavity between the posterior surface of the lens and the retina, known as the vitreous chamber. This chamber is filled with a transparent gel called vitreous humor, mainly composed of water, sugars, salts, hyaluronic acid and collagen. The vitreous humor mainly plays a structural role, maintaining the spherical shape of the eye, but it is also involved in regulating the intraocular pressure and, consequently, keeping the retina in place. (Alovisi et al., Journal of Ophthalmology 2017 , 2017:3172138).

[0011] The anterior segment includes the cornea, the iris and the pupil, the lens with the structures that support it, and the ciliary body. These structures define two cavities: the anterior chamber, delimited by the posterior surface of the cornea and the anterior surface of the iris, and the posterior chamber, between the iris and the structures that connect the lens to the ciliary body. These cavities, which communicate through the pupil, are occupied by the aqueous humor, a liquid that is formed by secretion from the ciliary body and is mainly composed of water, amino acids and electrolytes. This liquid acts as a refractive medium, provides nutrients to the cornea and the lens and helps maintain the intraocular pressure.

[0012] The anterior segment is the portion of the eye most exposed to traumatic lesions, but many pathologies are also known that can affect it, such as allergies or infections affecting the cornea, dry eye syndrome, and keratoconus, i.e. a degenerative pathology of the cornea that causes a progressive deformation.

[0013] One of the most well-known diseases affecting the anterior segment of the eye is cataract, a clouding of the lens that can cause various vision problems including blurry or double vision, halos around lights, faded colours, problems with bright lights, and difficulty in seeing close up. Cataracts are estimated to affect 94 million people worldwide. (Cicinelli MV et al., Lancet 2023, 401 :377-389).

[0014] The crystalline lens is a transparent biconvex structure in which the following can be identified, from the outside towards the inside: a relatively thick basement membrane, mainly composed of type IV collagen, called capsule, in which the zonular fibers that allow accommodation in conjunction with the ciliary body are inserted; a single layer of epithelial cells, which form the epithelium of the lens; a parenchyma composed of lenticular fibers, in which an internal and central portion (nucleus) and a more superficial one (cortical layer) can be distinguished.

[0015] The pathogenesis of cataracts is a complex and multifactorial process, not yet fully understood, which involves the denaturation and coagulation of proteins present in the lenticular fibers present in the nucleus and / or cortical layer, resulting in loss of transparency of the lens and, finally, the formation of cataracts. The process can start as a consequence of chronic diseases or trauma, or following the intake of certain drugs, but more often it is related to aging. Exposure to UV radiation, smoking and exposure to corticosteroids also represent important risk factors. (Nizami AA et al., StatPearls [Internet] 2023, PMID: 30969521).

[0016] There are no pharmacological treatments for curing, stopping or slowing down the progressive worsening of cataracts and, when the patient's quality of life reaches a critical level of worsening, it is necessary to intervene surgically by replacing the opacified crystalline lens with an artificial intraocular lens (Intra Ocular Lens, IOL). Over the years, various surgical techniques have been developed and applied for the treatment of cataracts, with progressive improvements that have allowed the current success rate ranging from 90% to 95% to be reached.

[0017] Intracapsular Cataract Extraction (ICCE) is characterized by the total removal of the opacified lens, through breakage of the ciliary zonules. The main side-effects associated with ICCE result from the need for making a large incision on the cornea and the risk that, once the lens has been extracted, the vitreous humor prolapses towards the anterior structures of the eye causing retinal detachment, macular edema or corneal decompensation. Despite these possible side-effects, ICCE remained the primary approach to cataract treatment in the United States until the 1970s and is still practised in developing countries today. Extracapsular cataract extraction (ECCE), on the other hand, involves removing only part of the anterior portion of the capsule, leaving the ciliary zonules and the rest of the capsule intact, and extracting the cataract by acting directly on the nucleus and / or cortex of the lens. In its documented surgical applications dating back to the eighteenth century, this procedure required a large corneal incision (10 mm) and had a series of considerable complications, including healing difficulties, persistence of lens debris, opacification of the posterior portion of the capsule, and infections. The introduction of progressive technical improvements in conventional ECCE has led to modern techniques of cataract surgery.

[0018] In 1967, the American ophthalmologist Charles Kelman introduced the surgical technique called phacoemulsification (from the Greek, (|)aK6< , “lens”, and emulsification), in which an ultrasonic needle emulsifies and aspirates the crystalline lens through a very small incision (3-4 mm) on the cornea, making the procedure minimally invasive and revolutionizing cataract surgery. This technique is continuously improving and, also due to the high prevalence of the pathology, it represents one of the most commonly performed surgical procedures in the world.

[0019] Phacoemulsification is currently characterized by the following steps: in the pre-operative stage, the patient is given drugs to dilate the pupil and topical anesthetics; in the operating room, the patient's eye is disinfected and exposed, the surgical microscope is positioned, and the incisions are effected on the cornea to make the lens accessible; a circular opening is then made on the anterior part of the lens (capsulorhexis); a phacoemulsifier probe is inserted into this opening, an instrument capable of emitting high-frequency ultrasounds, which emulsifies the contents of the lens, allowing its removal; what remains after phacoemulsification is the lens capsule, deprived of the anterior portion removed with the capsulorhexis, which serves as a housing for the artificial intraocular lens (IOL).

[0020] The operation does not require stitches as the incision on the cornea is effected in such a way that it is able to heal itself. Techniques based on the use of laser can be adopted for effecting an initial segmentation of the crystalline lens content, so as to accelerate the subsequent phacoemulsification phase, and effect the capsulorhexis with greater precision and reproducibility. In some cases, the flaking of the internal layers of the lens, useful for reducing complications by making the subsequent phacoemulsification more rapid and effective, can also be obtained through the hydrodissection procedure, which consists of the injection of a fluid directed in such a way as to surgically separate the tissues less traumatically with respect to incision with a scalpel. In cataract surgery, materials with viscous and elastic properties, called ophthalmic viscosurgical devices (OVDs), are also used, which allow the volume and shape of the eye structures to be maintained, also protecting the intraocular tissues avoiding possible complications related for example to discontinuous capsulorhexis, loss of vitreous humor or damage to the corneal epithelium. These measures and the reduced invasiveness of the procedure allow for a generally rapid post-operative recovery for the patient. (Moshirfar M et al., StatPearls [Internet] 2023, PMID: 32644679; Davis G, Mo Med 2016, 113:58-62).

[0021] Ophthalmic viscosurgical devices (OVDs) are transparent solutions that play a fundamental role in all ophthalmic surgical procedures involving the anterior chamber and in particular in cataract surgery, where they serve to keep the anterior chamber pervious during the incision of the cornea and capsule (capsulorhexis) and during the insertion of the intraocular lens (IOL), as the aqueous humor exits from the anterior chamber, in order to protect the corneal endothelium from the turbulence caused by the aspiration phase of the crystalline lens, to prevent the prolapse of the iris into the anterior chamber and to trap the nuclear fragments of the crystalline lens, residual from the operation. Among the main desirable characteristics for OVDs the following can be mentioned: ease of extrusion and positioning; ease of removal by aspiration, or the possibility of not removing it completely or partially after use; transparency and the capacity of not interfering with the use of surgical instruments and with the phases of the intervention, e.g. insertion of the artificial lens; the possibility of being visualized to identify it and to stain the ocular structures; non-toxicity, sterility and the absence of adverse and undesired effects, including for example interference with the intraocular pressure.

[0022] OVDs are generally classified into cohesive and dispersive based on their chemi cal -physical characteristics, and more specifically with reference to the cohesion-dispersion coefficient. (Cohesion-Dispersion Index, CDI) (Poyer JF, et al., J Cataract Refract Surg 1998; 24: 1130-5).

[0023] Dispersive OVDs (CDI < 30%asp / mmHg) are characterized by a high dispersivity, which makes them suitable for coating and adhering to intraocular structures. For this reason, they are suitable for protecting ocular structures from damage that ultrasounds can cause during the phacoemulsification phase of cataract surgery. As they persist at the injection site, they are also useful for dividing spaces within the eye. Examples of dispersive OVDs include: Viscoat (Alcon) and OcuCoat (Bausch&Lomb).

[0024] Cohesive OVDs (CDI > 30%asp / mmHg) have a higher viscosity, and are therefore useful for preserving the anterior chamber, maintaining capsule depth during capsulorhexis, moving and manipulating the iris or other tissues, and keeping the empty capsular bag open for IOL insertion. Examples of cohesive OVDs include: ProVisc (Alcon), Healon Pro (Johnson& Johnson) and Am Vise (Bausch&Lomb).

[0025] Another important parameter for defining the characteristics and surgical behaviour of OVDs is the (dynamic) viscosity at rest (zero-shear viscosity, r]0), i.e. the viscosity in the absence of or for extremely low values of shear stress (shear rate). Cohesive OVDs (CDI > 30%asp / mmHg) generally have a relatively high viscosity at rest (rO > 100,000 mPa s), whereas dispersive OVDs (CDI < 30%asp / mmHg) are more often characterized by viscosity values at rest lower than 100,000 mPa s.

[0026] To date, no cohesive OVDs characterized by a low or medium viscosity at rest have been described. (Arshinoff SA and Jafari M, J Cataract Refract Surg 2005, 31 :2167-71; Arshinoff SA, 2005; Ophthalmic Viscosurgical Devices. In: Kohnen, T., Koch, D.D. (eds) Cataract and Refractive Surgery. Essentials in Ophthalmology. Springer, Berlin, Heidelberg; Borkenstein AF, et al., Ophthalmol Ther 2021, 10:831-843).

[0027] Hyaluronic acid (HA) is historically the first polymer to be used as an OVD, and is currently present in many commercialized OVDs, including for example Viscoat (Alcon) and Am Vise (Bausch&Lomb). It is a linear chain polymer consisting of a variable number of repeated disaccharide units, each composed of residues of glucuronic acid (p-D-glucopyranuronic acid) and acetyl-glucosamine (2-acetylamino-2-deoxy-P-D-glucopyranose), bound together by a glycosidic bond between the anomeric carbon atom of glucuronic acid and the C-3 of acetyl- glucosamine. HA is a major constituent of the extracellular matrix (ECM) of vertebrate tissues, with its presence being particularly relevant in the synovial fluid, vitreous humor and hyaline cartilage, but it is also abundant in skin, tendons and muscles. HA plays a key role in providing mechanical support to tissues, as well as supporting a wide variety of cellular processes. It is also known that HA, through its membrane receptor CD44, modulates many different processes related to cell physiology and biology such as, for example, proliferation, migration, cell differentiation and angiogenesis, and that it also exerts other functions such as tissue hydration and joint lubrication. It has also been demonstrated that HA is crucial in the tissue repair process both from a structural point of view (in the organization of the extracellular matrix and in the regulation of its hydration), and as a stimulating substance of a vast series of processes in which it intervenes directly and indirectly (clot formation, phagocytic activity, fibroblast proliferation, neovascularization, re- epithelialization, etc.) (Weigel P. et al., J Theoretical Biol, 1986, 119:219-234; Abatangelo G. et al., J Surg Res, 1983, 35:410-416; Goa K. et al., Drugs, 1994, 47:536-566).

[0028] Compositions containing vital dyes, i.e. molecules capable of staining living tissues or cells, are also used in ocular surgery. Their main function is to stain ophthalmic structures, making them more easily recognizable and identifiable by the surgeon. Various ophthalmic surgical procedures today can involve the use of vital dyes, including, for example, interventions for the treatment of strabismus, cataracts and retinal detachment, or for performing a corneal transplant (keratoplasty) or the removal of the vitreous (vitrectomy).

[0029] Among the most widely-used vital dyes in ophthalmic surgery, Trypan Blue, an azo dye, can be mentioned, i.e. characterized by the presence of double bonds between nitrogen atoms having the structure -N=N- This dye is widely used both in ophthalmic surgery of the posterior segment, as it is capable of selectively staining the internal limiting membrane of the retina (ILM, the basement membrane that acts as a border between the retinal epithelium and the vitreous humor) and the epiretinal membrane (ERM, a thin layer of scar tissue that can be formed on the retina, generating a visual defect), and the anterior segment, where it is capable of revealing the crystalline capsule and Descemet' s membrane, a basement membrane positioned between the stroma and the corneal endothelium.

[0030] Trypan Blue, however, should be used with caution, as it has been associated with a risk of developing cancer (National Center for Biotechnology Information; 2023. PubChem Compound Summary for CID 6296, Trypan blue).

[0031] Another vital dye that has a high affinity for ILM and ERM is Brilliant Blue G, also known as Coomassie Blue G, Acid Blue 90, C. I. 42655, and Brilliant or Coomassie Blue G-250, a triphenylmethane-based blue dye. Although generally considered as being safe and non-toxic, some side-effects associated with staining of the macula, i.e. the central area of the retina, with Brilliant Blue G, have been described in literature (National Center for Biotechnology Information; 2023. PubChem Compound Summary for CID 6324599, Brilliant Blue G; Soni A et al., Semin Ophthalmol 2022, 37: 117-122).

[0032] AU2015202819 describes staining compositions, and their use as staining compositions for staining ocular tissues or components of ocular tissues, including the retinal membrane in the posterior segment and the lens capsule in the anterior segment. These compositions comprise a combination of a first and a second dye, and a compound capable of increasing the density of the composition without reducing the staining capacity of the dye, selected from the group consisting of soluble polymers, in particular polyethylene glycol, and small inert molecules. WO201 1122947 describes a staining composition based on a combination of two or more vital dyes and a compound capable of increasing the density and viscosity of the composition, which can be polyethylene glycol (PEG) or an iodinated organic compound. This composition is used for staining ophthalmic membranes in retinal surgery or for the purpose of performing capsulorhexis in cataract surgery. EPl 819366 describes a derivative of Brilliant Blue G to be used as a dye for ophthalmic membranes, and in particular for the internal limiting membrane (ILM) or the anterior capsule, in order to facilitate their surgical removal. EP2274019 describes a biocompatible aqueous preparation for the selective staining of the internal limiting membrane (ILM) or epiretinal membranes (ERM) in the human or animal eye, comprising or consisting of at least one dye selected from the groups based on triphenylmethane, azo, cyanine and / or natural dyes or mixtures thereof. EP3630202 describes and claims staining compositions, methods for their preparation, and their use for staining ocular tissues during ophthalmic surgery, comprising hyaluronic acid in a low concentration (< 0.8%) and a combination of the two dyes Trypan Blue and Chicago Sky Blue. WO 9958160 describes the use of a dye selected from Trypan Blue, Trypan Red and Brilliant Crysyl Blue for staining the lens capsule in order to facilitate the exertion of capsulorhexis.

[0033] Staining cells and tissues involves the diffusion of vital dyes outside their original composition, into the patient's fluids and tissues. On the one hand, this may cause the composition, initially stained and easily identifiable, to lose its colouring, becoming completely colourless, and on the other hand, it may allow the dyes to give undesirable side-effects to the patient. In order to avoid possible toxic effects of vital dyes and still allow the surgeon to distinguish the cavities filled by ophthalmic viscosurgical devices (OVDs), as well as to visualize the OVD itself by distinguishing it from the surrounding tissues, it is more convenient to use a stained OVD that does not have any staining properties towards cells and tissues.

[0034] WO86 / 02548 describes a composition for ophthalmic use, containing an aqueous solution in gel form of a high-molecular-weight polymer to which a polymeric dye is added. It is stated that, in order to avoid diffusion problems and to keep the dye within the composition, this dye must be polymeric and have a molecular weight higher than 10,000 Da, preferably higher than 30,000 Da, even more preferably to have the highest possible molecular weight; this high-molecular- weight dye however will inevitably contribute to altering the original viscosity of the ophthalmic composition containing it, making this composition excessively viscous and, therefore, difficult to extrude. The objective of the present invention is to identify an ophthalmic pharmaceutical composition, belonging to the category of ophthalmic viscoelastic devices (OVDs), capable of overcoming the drawbacks of the prior art specified above.

[0035] The present invention relates to an innovative ophthalmic pharmaceutical composition belonging to the category of ophthalmic viscoelastic devices (OVDs) of the cohesive type, stained but without a staining capacity, comprising or consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF, wherein said dye is present in a concentration ranging from 0.001 mg / ml to 0.1 mg / ml, and its use in surgery of the anterior segment of the eye, preferably in cataract surgery.

[0036] The pharmaceutical composition object of the present invention is presented in the form of a gel, easily identifiable by its blue colour, which however maintains its transparency, allowing any object behind it to be visualized, including biological structures.

[0037] The Applicant has in fact surprisingly discovered and subsequently demonstrated that, by adopting non-polymeric dyes with a molecular weight lower than 1,000 Da, there is no diffusion of dye from the composition object of the present invention to the aqueous solvent in which it is immersed, and that this composition does not possess any staining activity.

[0038] Furthermore, the composition according to the invention has the advantage of being well tolerated both in vitro, where it does not cause any reduction in the cell viability, and in vivo, where it does not cause side-effects and does not permanently alter the intraocular pressure.

[0039] The composition described herein is therefore suitable for use in ophthalmic surgery and in particular in cataract surgery, as it has all the characteristics and advantages desirable for this class of products: the viscoelastic characteristics do not compromise its good extrudability; it can be easily removed, but no side-effects are observed even if a part remains in place after use, as demonstrated hereunder, thus making the procedure simpler and faster for the surgeon, who does not need to spend time removing all visible residues dispersed in the patient's tissue; it is stained and non-staining, but transparent, allowing the biological structures behind it to be visualized, and it is perfectly compatible with ophthalmic surgical procedures, and in particular with the phacoemulsification procedure; it is perfectly stable, sterilizable through different methods, non-toxic, and does not produce any undesired effects or side-effects neither does it interfere with the intraocular pressure, as demonstrated hereunder.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to an ophthalmic pharmaceutical composition in gel form, belonging to the category of ophthalmic viscoelastic devices (OVDs: Ophthalmic Viscosurgical Devices) of the cohesive type, stained but without a staining capacity, comprising or consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF with one or more pharmacologically acceptable excipients, wherein said dye is present in a concentration ranging from 0.001 mg / ml to 0.1 mg / ml.

[0042] The present invention further relates to an ophthalmic pharmaceutical composition in gel form, belonging to the category of ophthalmic viscoelastic devices (OVDs: Ophthalmic Viscosurgical Devices) of the cohesive type, stained but without a staining capacity, comprising or consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF with one or more pharmacologically acceptable excipients, for use in surgery of the anterior segment of the eye, preferably in cataract surgery.

[0043] Said gel is intended as a network of polymers that is soaked by a fluid for its entire volume (IUPAC. Compendium of Chemical Terminology, 2nded. (the "Gold Book'"). This composition is therefore neither a viscous solution nor a colloidal gel.

[0044] Said pharmaceutical composition according to the present invention is characterized in that it is: stained but without a staining capacity; the dye contained in the above-mentioned stained composition does not diffuse outside the composition itself.

[0045] The hyaluronic acid (HA) used in the pharmaceutical composition of the present invention can derive from any source, for example, from rooster combs (as described in EP 138572), from fermentation (from Streptococcus equi or zooepidemicus . or biotechnologically (from Bacillus, as described in EP2614088, EP2614087), and can be purified according to various techniques (as described in EP3491027, EP3655138). HA from fermentation is preferred and even more preferred HA from fermentation from Streptococcus equi or zooepidemicus, more preferably from Streptococcus equi sub species 68222, mutant H-l (EP0716688).

[0046] According to a preferred aspect, the weight average molecular weight of the HA used in the preparation of the composition of the invention for the applications described herein is within the range of IxlO6Da to 3xl06Da, preferably from 1.6xl06Da to 2.6xl06Da..

[0047] Molecular weight (MW) refers to the weight average molecular weight calculated with the “intrinsic viscosity” method, applying a specific increase in the refractive index (dn / dc) equal to 0.155 for the instrumental calculation (Terbojevich et al., Carbohydr Res 1986, 363-377; Guarise et al., J Meeh Behav Biomed Mater 2023, 143: 105908).

[0048] According to a preferred aspect of the present invention, the hyaluronic acid is present in the composition in a percentage ranging from 1% w / v to 3% w / v, preferably from 1.5% w / v to 2.5% w / v, more preferably from 1.8% w / v to 2.2% w / v, and even more preferably equal to 2% w / v, said percentages being expressed by weight with respect to the total volume of the composition.

[0049] The hyaluronic acid is therefore present in a concentration ranging from 10 mg / ml to 30 mg / ml, preferably from 15 mg / ml to 25 mg / ml, more preferably from 18 mg / ml to 22 mg / ml, even more preferably it is equal to 20 mg / ml. The present invention in its preferred embodiment also relates to an ophthalmic pharmaceutical composition in gel form, belonging to the category of ophthalmic viscoelastic devices (OVDs) of the cohesive type, stained but without a staining capacity, consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF with one or more pharmacologically acceptable excipients, wherein said dye is present in a concentration ranging from 0.001 mg / ml to 0.1 mg / ml, and its use in surgery of the anterior segment of the eye, preferably in cataract surgery.

[0050] The dye used for the purposes of the invention is selected from Brilliant Blue G and Brilliant Blue FCF. It should be pointed out that the dye that can be used in accordance with the present invention is not polymeric, it has a molecular weight lower than 1,000 Da, and does not belong to the family of azo dyes, as it does not have double bonds between nitrogen atoms of the -N=N- structure. Brilliant Blue G, also known as Coomassie Blue G, Acid Blue 90, C. I. 42655 and Brilliant or Coomassie Blue G-250, is a blue dye based on triphenylmethane with a molecular weight of 854 Da (National Center for Biotechnology Information; 2023. PubChem Compound Summary for CID 6324599, Brilliant Blue G).

[0051] The chemical formulas of the two dyes are provided hereunder

[0052] Brilliant Blue G Brilliant Blue FCF Brilliant Blue G should not be confused with the dye Coomassie Brilliant Blue R-250 (PubChem CID 61365), which lacks two methyl groups compared to Brilliant Blue G, and which is not among the dyes that can be used for the purposes of the invention. It should also not be confused with the dye Brilliant Cresyl Blue (PubChem CID 20841696), which cannot be used for the purposes of the present invention, which belongs to the family of oxazines, heterocyclic compounds containing one oxygen atom and one nitrogen atom.

[0053] According to a preferred embodiment, the dye Brilliant Blue G can be used in the composition of the present invention in a concentration ranging from 0.001 mg / ml to 0.1 mg / ml, preferably from 0.016 mg / ml to 0.050 mg / ml, more preferably from 0.02 mg / ml to 0.04 mg / ml.

[0054] Brilliant Blue FCF, known by a number of alternative names including E133, erioglaucine, C.I. 42090, and acid blue 9, is a blue dye with a molecular weight of 792.9 Da classified as a triarylmethane dye, which is widely used in the food industry, as well as in pharmaceuticals, dietary supplements, and cosmetics (National Center for Biotechnology Information; 2023. PubChem Compound Summary for CID 19700, Brilliant Blue FCF).

[0055] Brilliant Blue FCF dye is used in a concentration ranging from 0.001 mg / ml to 0.1 mg / ml, preferably from 0.016 mg / ml to 0.050 mg / ml, more preferably from 0.02 mg / ml to 0.04 mg / ml.

[0056] The ophthalmic pharmaceutical composition in gel form belonging to the category of ophthalmic viscoelastic devices of the cohesive type object of the present invention, comprises or consists of hyaluronic acid and a dye, selected from Brilliant Blue G and Brilliant Blue FCF, appropriately formulated, wherein the above-mentioned dye is surprisingly in no way capable of being diffused outside the composition and the composition is completely devoid of any staining capacity. The Applicant has in fact discovered and subsequently demonstrated how the dye contained in the pharmaceutical composition according to the present invention is not capable of being diffused outside the composition itself and is therefore not capable of staining the surrounding and adjacent tissues.

[0057] It has also been possible to demonstrate that live cells kept in contact with the composition object of the present invention are in no way stained. The composition according to the present invention retains the dye within its interior, preventing its diffusion and being stained itself. As it is stained, the composition can be easily distinguished from the tissues surrounding the injection site, but is nevertheless transparent, allowing any object behind it to be seen, including biological structures.

[0058] According to recent guidelines, ophthalmic pharmaceutical compositions in gel form, belonging to the category of ophthalmic viscoelastic devices (OVDs) of the cohesive type, are characterized by a cohesion-dispersion coefficient (Cohesion-Dispersion Index, CDI) greater than or equal to 30%asp / mmHg.

[0059] The cohesive OVDs described to date have a high (dynamic) viscosity at rest (zero-shear viscosity, qO) (qO > 100,000 mPa.s), whereas no cohesive OVDs are known with a low (103-104mPa.s) or medium (104-105mPa.s) viscosity at rest (Arshinoff SA and Jafari M, J Cataract Refract Surg 2005, 31 :2167-71 ; Arshinoff SA, 2005; Ophthalmic Viscosurgical Devices. In: Kohnen, T., Koch, D.D. (eds) Cataract and Refractive Surgery. Essentials in Ophthalmology. Springer, Berlin, Heidelberg; Borkenstein AF, et al., Ophthalmol Ther 2021, 10:831-843).

[0060] According to a preferred aspect of the present invention, the ophthalmic pharmaceutical composition in gel form, belonging to the category of OVDs of the cohesive type, has a Cohesion-Dispersion Index (CDI), measured according to the protocol described by Poyer and colleagues (Poyer JF, et al., J Cataract Refract Surg 1998; 24: 1130-5), which ranges from 30 to 45 %asp / mmHg, preferably equal to 35 %asp / mmHg.

[0061] According to a further preferred aspect of the present invention, the ophthalmic pharmaceutical composition in gel form belonging to the category of OVDs of the cohesive type has an average (dynamic) viscosity at rest that ranges from 50,000 mPa.s to 200,000 mPa.s (50,000 mPa.s < qO < 200,000 mPa.s), preferably from 80,000 mPa.s to 120,000 mPa.s (80,000 mPa.s < qO < 120,000 mPa.s), at a temperature of 25°C, and furthermore has a dynamic viscosity (q) at 25 °C, for a shear stress (shear-rate) of 10 s-1, which ranges from 12,000 mPa.s to 22,000 mPa.s (12,000 mPa.s < q < 22,000 mPa.s), preferably 15,000 mPa.s to 19,000 mPa.s (15,000 mPa.s < q < 19,000 mPa.s).

[0062] Said composition, object of the present invention, is therefore an OVD of the cohesive type having a medium viscosity: it is therefore a completely innovative OVD in gel form, as to date no cohesive-type OVDs with such viscosity characteristics are known in the state of the art.

[0063] The pharmaceutical composition according to the present invention preferably has a physiological pH, preferably ranging from 6.8 to 7.6, and an osmolality ranging from 250 mOsm / kg to 350 mOsm / kg.

[0064] The excipients present in the composition according to the present invention are preferably selected from preservatives, water and various mineral salts, wherein said preservative can be selected from all known preservatives suitable for the purpose, for example the preservative can be selected from the following list, which has an exemplary and non-limiting value: sodium thiosulfate, sorbitol, mannitol. According to a further preferred aspect, the composition according to the present invention can comprise sodium thiosulfate as a preservative, in a percentage ranging from 0.1 w / v to 2% w / v, or sorbitol in a percentage ranging from 1 w / v to 5% w / v, or mannitol in a percentage ranging from 1 w / v to 5% w / v, said percentages being expressed by weight with respect to the total volume of the composition, the composition according to this preferred form of the invention therefore comprises sodium thiosulfate in a concentration ranging from 1 mg / ml to 20 mg / ml, sorbitol in a concentration ranging from 10 mg / ml to 50 mg / ml or mannitol in a concentration ranging from 10 mg / ml to 50 mg / ml.

[0065] The composition according to the present invention can be sterilized according to any of the known methods, comprising: moist heat (steam, autoclave sterilization), dry heat, radiation. Said composition is preferably subjected to autoclave sterilization at a temperature equal to or greater than 121 °C for a time equal to or greater than 15 minutes, in accordance with the indications of the European Pharmacopoeia (Ph. Eur. 5.1.1; EMA Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container, 6 March 2019).

[0066] The ophthalmic pharmaceutical composition in gel form, belonging to the category of cohesive-type ophthalmic viscoelastic devices OVDs according to the present invention, is particularly advantageous for use in surgery of the anterior segment of the eye, which includes the cornea, the iris and the pupil, the crystalline lens with the structures that support it, the ciliary body and the cavities formed by these structures, i.e. the anterior chamber, delimited by the posterior surface of the cornea and the anterior surface of the iris, and the posterior chamber, included between the iris and the formations that bind the crystalline lens to the ciliary body. Corneal transplantation (also known as keratoplasty), iris or pupil reconstruction, cataract surgery are some examples of anterior segment surgery of the eye in which the cohesive-type OVD object of the invention can be used.

[0067] The composition according to the present invention is preferably used in cataract surgery, more preferably in surgery aimed at treating cataracts by intracapsular, extracapsular or phacoemulsion cataract extraction.

[0068] Cataract surgery refers to any of the known surgical procedures suitable for the treatment of this disease, comprising intracapsular cataract extraction (ICCE) and extracapsular cataract extraction (ECCE), using conventional or modern methods, and phacoemulsification, including situations in which it is associated with procedures based on the use of laser or hydrodissection or any other technique that can improve its safety and efficacy.

[0069] The OVD of the cohesive type in gel form according to the present invention can be used in all phases of surgical interventions aimed at the treatment of cataracts, and is particularly suitable for this purpose as it has all the desirable characteristics for this class of products: the viscoelastic characteristics do not compromise its good extrudability; it can be easily removed, but no side-effects are observed even if a part remains in place after use, (as demonstrated hereunder) thus making the procedure simpler and faster for the surgeon who does not necessarily have to spend time in removing all visible residues dispersed in the patient's tissue; it is stained and non-staining but transparent, allowing all the biological structures behind it to be visualized, and is perfectly compatible with ophthalmic surgical procedures, and in particular with the phacoemulsification procedure; it is perfectly stable, sterilizable by different methods, non-toxic, and does not produce any undesired effects or side-effects neither does it interfere with the intraocular pressure, as demonstrated hereunder.

[0070] According to a further preferred aspect, the pharmaceutical composition object of the present invention is used during operations aimed at treating cataracts to keep the anterior chamber open during the incision of the cornea and the capsule (capsulorhexis) and during the insertion of the intraocular lens (IOL), to protect the corneal endothelium from the turbulence caused by the aspiration phase of the crystalline lens, to prevent the prolapse of the iris on the anterior chamber or to trap the nuclear fragments of the crystalline lens, residual from the operation.

[0071] EXAMPLE 1. Composition of HA 2% with Brilliant Blue G (0.016 mg / ml) and sodium thiosulfate 0.5%.

[0072] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 1.8 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS, containing Na2S2Ch as preservative (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; Na2S2Ch 5 mg / ml; pH 7.4) and 0.4 ml of BBG-sol (final dye concentration 0.016 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0073] EXAMPLE 2. Composition of 2% HA with Brilliant Blue G (0.032 mg / ml) and 0.5% sodium thiosulfate.

[0074] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 2.5 mg / ml; NaHPC OA mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.2 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml PBS, containing Na2S2Ch as preservative (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; Na2S20s 5 mg / ml; pH 7.4) and 0.8 ml of BBG-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0075] EXAMPLE 3. Composition of 2% HA with Brilliant Blue G (0.048 mg / ml) and 0.5% sodium thiosulfate.

[0076] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.3 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml PBS, containing Na2S2Ch as preservative (NaCl 2.5 mg / ml; NaHPCh OA mg / ml; Na2S2Ch 5 mg / ml; pH 7.4) and 1.2 ml of BBG-sol (final dye concentration 0.048 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0077] The Cohesion-Dispersion Index (CDI) of the product obtained was found to be 34%asp / mmHg.

[0078] EXAMPLE 4. Composition of 1.5% HA with Brilliant Blue G (0.032 mg / ml) and 0.5% sodium thiosulfate.

[0079] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.5 MDa) was then prepared by adding 1.5 g of HA (Fidia) to 100 ml PBS, containing Na2S2Ch as preservative (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; NaiSiCh 5 mg / ml; pH 7.4) and 0.8 ml of BBG-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0080] EXAMPLE 5. Composition of 2.5% HA with Brilliant Blue G (0.032 mg / ml) and 0.5% sodium thiosulfate.

[0081] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.6 MDa) was then prepared by adding 2.5 g of HA (Fidia) to 100 ml PBS, containing NaiSiCh as preservative (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; NaiSiCh 5 mg / ml; pH 7.4) and 0.8 ml of BBG-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0082] EXAMPLE 6. Composition of 2% HA with Brilliant Blue G (0.016 mg / ml) and 3% sorbitol.

[0083] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.6 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS, containing sorbitol as a preservative (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; sorbitol 30 mg / ml; pH 7.4) and 0.4 ml of BBG-sol (final dye concentration 0.016 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0084] EXAMPLE 7. Composition of 2% HA with Brilliant Blue G (0.032 mg / ml) and 3% sorbitol. An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.6 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS, containing sorbitol as a preservative (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; sorbitol 30 mg / ml; pH 7.4) and 0.8 ml of BBG-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0085] The Cohesion-Dispersion Index (CDI) of the resulting product was found to be 37%asp / mmHg.

[0086] EXAMPLE 8. Composition of 2% HA with Brilliant Blue G (0.048 mg / ml) and 3% sorbitol.

[0087] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 1.5 mg / ml; NaHPCU OA mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.4 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS containing sorbitol as preservative (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; sorbitol 30 mg / ml; pH 7.4) and 1.2 ml of BBG-sol (final dye concentration 0.048 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0088] EXAMPLE 9. Composition of 2% HA with Brilliant Blue G (0.016 mg / ml) and 3% mannitol.

[0089] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.4 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS, containing mannitol as a preservative (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; mannitol 30 mg / ml; pH 7.4) and 0.4 ml of BBG-sol (final dye concentration 0.016 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0090] EXAMPLE 10. Composition of 2% HA with Brilliant Blue G (0.032 mg / ml) and 3% mannitol.

[0091] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 1.5 mg / ml; NaHPCU 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 2.5 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS, containing mannitol as a preservative (NaCl 1.5 mg / ml; NaHPCU 0.4 mg / ml; mannitol 30 mg / ml; pH 7.4) and 0.8 ml of BBG-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0092] EXAMPLE 11. Composition of 2% HA with Brilliant Blue G (0.048 mg / ml) and 3% mannitol.

[0093] An 0.4% stock solution of Brilliant Blue G (BBG) in PBS (NaCl 1.5 mg / ml; NaHPCU 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBG to 5 ml of PBS (BBG-sol). A solution of 20 mg / ml of HA (MW 1.8 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS containing mannitol as a preservative (NaCl 1.5 mg / ml; NaHPCU 0.4 mg / ml; mannitol 30 mg / ml; pH 7.4) and 1.2 ml of BBG-sol (final dye concentration 0.048 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0094] The Cohesion-Dispersion Index (CDI) of the resulting product was found to be 35%asp / mmHg.

[0095] EXAMPLE 12. Composition of 2% HA with Brilliant Blue FCF (0.032 mg / ml) and 0.5% sodium thiosulfate.

[0096] An 0.4% stock solution of Brilliant Blue FCF (BBFCF) in PBS (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBFCF to 5 ml of PBS (BBFCF-sol). A solution of 20 mg / ml of HA (MW 2.2 MDa) was then prepared by adding 2 g HA (Fidia) to 100 ml PBS, containing sodium thiosulfate as preservative (NaCl 2.5 mg / ml; NaHPCh 0.4 mg / ml; Na2S2Ch 5 mg / ml; pH 7.4) and 0.8 ml of BBFCF-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave..

[0097] EXAMPLE 13. Composition of HA 2% with Brilliant Blue FCF (0.032 mg / ml) and 3% sorbitol.

[0098] An 0.4% stock solution of Brilliant Blue FCF (BBFCF) in PBS (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBFCF to 5 ml of PBS (BBFCF-sol). A solution of 20 mg / ml of HA (MW 2.4 MDa) was then prepared by adding 2 g of HA (Fidia) to 100 ml of PBS, containing sorbitol as preservative (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; sorbitol 30 mg / ml; pH 7.4) and 0.8 ml of BBFCF-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave.

[0099] The Cohesion-Dispersion Index (CDI) of the resulting product was found to be 36%asp / mmHg.

[0100] EXAMPLE 14. Composition of 2% HA with Brilliant Blue FCF (0.032 mg / ml) and 3% mannitol.

[0101] An 0.4% stock solution of Brilliant Blue FCF (BBFCF) in PBS (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; pH 7.4) was prepared by adding 20 mg of BBFCF to 5 ml of PBS (BBFCF-sol). A solution of 20 mg / ml of HA (MW 2.4 MDa) was then prepared by adding 2 g HA (Fidia) to 100 ml PBS, containing mannitol as preservative (NaCl 1.5 mg / ml; NaHPCh 0.4 mg / ml; mannitol 30 mg / ml; pH 7.4) and 0.8 ml of BBFCF-sol (final dye concentration 0.032 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes and sterilized in an autoclave..

[0102] EXAMPLE 15. Composition of 2% HA with Brilliant Blue G (0.03 mg / ml) and 0.5% sodium thiosulfate.

[0103] A 3.66% stock solution of BBG in ultrapure water was prepared by adding 36.6 mg of BBG to 1 ml of ultrapure water (BBG-wsol). A solution of 20 mg / ml of HA (MW 2.6 MDa) was prepared by adding 2 g HA (Fidia) to 100 ml PBS containing Na2S2Ch as preservative (NaCl 2.8 mg / ml; Na2HPO4*12H2O 0.6 mg / ml; NaH2PO4*2H2O 0.05 mg / ml; Na2S2Ch 5 mg / ml; pH 7.3±0.05) and a small quantity of BBG-wsol (0.08 ml; final dye concentration 0.03 mg / ml). The resulting composition was mixed under orbital shaking for 24 hours at room temperature (25°C), then stored at 2-8°C for a further 24 hours. The resulting gel was filtered at 100 pm, divided into 2 ml glass syringes, and sterilized by autoclaving. The product thus obtained was subsequently subjected to a series of analyses, to detect the chemi cal -physical parameters of interest indicated hereunder.

[0104] On visual examination, the product proved to be stained blue but transparent, allowing any object behind it to be clearly distinguished, including biological structures.

[0105] The pH was measured on an undiluted sample of the product using a HACH pH meter, SensION+ PH31, and was found to be 6.96.

[0106] The Cohesion-Dispersion Index (CD I) was found to be 35%asp / mmHg.

[0107] The viscous properties were analyzed using an Anton Paar MCR 92 Rheometer, equipped with a cone-plate measuring device with a diameter of 50 mm and an angle of 1°. The analysis was carried out at a temperature of 25°C, with a gap of 0.103 mm. The dynamic viscosity was measured by increasing the shear stress (shear rate) from 0.001 s'1to 1000 s'1with a 33-point logarithmic ramp; the zero-shear viscosity is obtained using the Carreau model described in literature (Palacio-Pastrana C, et al., Med Devices (Auckl) 2022, 15:293-305). The dynamic viscosity at a temperature of 25°C and for a shear stress equal to 10 s-1 was found to be equal to 18,248 mPa- s, whereas the zero-shear viscosity was found to be equal to 87,000 mPa s.

[0108] EXAMPLE 16. Evaluation of the staining capacity and cell viability in vitro.

[0109] The staining capacity and cell viability following direct contact with the composition of the present invention were evaluated on the cell line of BALB / 3T3 murine fibroblast. For this purpose, a sample solution was prepared by combining the product of Example 15 and the cell culture medium DMEM + 10% FBS, in a 1 : 1 ratio. The BALB / 3T3 cells were grown in monolayer in plastic multiwell microplates. The culture medium was removed and, after a brief washing with PBS, replaced with fresh culture medium (CTRL) or with 5 mM SDS dissolved in culture medium (CTRL TOX) or finally with the sample solution described above. Cells placed in different wells were left in contact with said sample solution for a time range of 15, 30 or 60 minutes (TEST 15, TEST 30 and TEST 60, respectively).

[0110] At the end of the experiment, in order to evaluate the staining capacity of the sample solution described above, the cells were analyzed by observation under a microscope, in bright field, with 100X magnification (XDS-3 microscope, Optika Italy). For the purposes of this analysis, the experimental conditions CTRL, TEST 15, TEST 30 and TEST 60 were considered and for each of them 3 different wells were prepared and analyzed (N=3).

[0111] Result: no cell staining was detected for any of the conditions considered.

[0112] The cell viability was tested by performing the Alamar blue (resazurin) assay according to a known protocol (Kumar P et al., Cold Spring Harb Protoc 2018, 2018(6)). Resazurin is a molecule capable of permeating cell membranes. Once inside the cells, resazurin is reduced to resorufin by the activity of cellular redox enzymes, which catalyze the transfer of electrons from NADPH, FADH2, FMNH2, NADH, and cytochromes. This redox reaction causes a change in the colour of the molecule, which changes from indigo blue to a bright fluorescent red, and subsequently diffuses out of the cell into the culture medium, where it can be easily detected with colorimetric or fluorimetric methods. Live cells convert resazurin to resorufin continuously, making the method suitable for the quantitative measurement of cell viability.

[0113] For the purposes of cell viability analysis, the different contact intervals with the composition object of the present invention were evaluated (TEST 15, TEST 30 and TEST 60), the untreated control (CTRL) and a further control treated with 5 mM SDS dissolved in the culture medium (CTRL TOX). The expected effect of this last experimental condition is a reduction in cell viability. For each condition, 6 different wells were prepared and analyzed (N = 6), the fluorescence of the samples was detected using a TEC AN INFINITE 200 PRO plate reader (excitation wavelength (kex) = 530 nm, emission wavelength (kern) = 590 nm).

[0114] The results obtained are shown in Figure 1. The correct functioning of the test is confirmed by the low viability value detected for the CTRL TOX control. Direct contact of the cells with the object of the invention described herein does not cause any reduction in the cell viability for any of the exposure times considered, all the experimental conditions (TEST 15, TEST 30 and TEST 60) in fact show viability values completely similar to those of the untreated control (CTRL).

[0115] In short, the data obtained demonstrate that the composition according to the invention surprisingly retains the dye without releasing it and without staining the cells with which it comes into contact in any way, and at the same time, despite the direct contact between said composition and the cells, no toxicity is observed. The composition object of the invention is therefore safe and allows the surgeon’s work to be simplified and accelerated as it is not necessary to spend time in removing all of the residues possibly present in the patient's eye at the end of the surgical procedure.

[0116] EXAMPLE 17. Dye diffusion test in aqueous medium.

[0117] The experimental procedure described hereunder was effected in order to verify whether the dye included in the composition object of the invention is capable of being diffused outside of the same.

[0118] 1 ml of the product of Example 15 (i.e. HA 2%+Brilliant Blue G 0.03 mg / ml prepared according to Example 15), was immersed in 10 ml of physiological solution (NaCl 0.9%). The physiological solution was completely aspirated after 60 minutes of contact, transferred to a container of transparent inert material and analyzed by means of a spectrophotometer at a wavelength X=610 nm: Sample A. In order to have a negative reference control, the absorbance of an aliquot of physiological solution that had never been in contact with any other substance was also measured (Sample CTRL). The data obtained are indicated in the following table:

[0119] In parallel, again 1 ml of the product of Example 15 was left in contact with 10 ml of physiological solution (NaCl 0.9%) for 10 hours, at the end of which the physiological solution was completely aspirated, transferred to a container of transparent inert material and analyzed by means of a spectrophotometer again at a wavelength X=610 nm: Sample B. The absorbance results are the same as those obtained in the case of the 60 minutes of contact indicated above for Sample A.

[0120] Finally, 1 ml of the product of Example 12 (i.e. HA 2% + Brilliant FCF 0.032 mg / ml prepared according to Example 12) was left in contact with 10 ml of physiological solution (NaCl 0.9%) for 10 hours at the end of which said physiological solution was completely aspirated, transferred to a container of transparent inert material and analyzed by means of a spectrophotometer at a wavelength X = 610 nm: Sample C. Also in this case, no diffusion of dye from the composition to the physiological solution was revealed, which was found to be completely devoid of any staining, similar to the control sample CTRL.

[0121] In short, no diffusion of dye was revealed from the ophthalmic pharmaceutical compositions (according to the present invention) to the physiological solution in which said compositions were immersed, in fact both in the case of the product of Example 15 and in the case of the product of Example 12, regardless of the duration of contact, the colour of each composition remained unchanged whereas the physiological solutions in which the samples were immersed as previously described, were completely transparent and free of any shade of colour, similar to the control sample CTRL which had never come into contact with any substance, which therefore had an absorbance value of almost zero as it was transparent.

[0122] This evidence demonstrates that the composition according to the present invention does not allow the diffusion of the dye, whether it be Brilliant Blue G or Brilliant Blue FCF, to the surrounding structures and therefore the dye cannot exert any potentially toxic effect or perform a staining activity with respect to the surrounding structures.

[0123] EXAMPLE 18. In vivo tolerability assessment.

[0124] The in vivo tolerability of the composition object of the present invention was evaluated through a protocol specifically designed for this purpose. The study, approved by the Animal Welfare Body (OPBA, protocol no. 133 / 2019-PR, 14 February 2019) of the University of Pisa, was conducted at the Department of Biology of the same University. A total of 4 male Sprague Dawley rats (8 eyes), 8 weeks old and weighing between 250 and 300 g, were included in the study. Each rat was lightly sedated with ether, and treated with topical lidocaine on the eye. A defined volume of the composition of the invention prepared according to Example 15, was subsequently injected into the anterior chamber of the eye of each rat using syringes with a 30G needle. The rats were divided into two groups, the first receiving a volume of the composition equal to 1 pL (GROUP 1) whereas the second equal to 2 pL (GROUP 2). After the injection, the composition of the invention was left in place and the rats were monitored until complete recovery from the anesthesia. The intraocular pressure (IOP) was measured experimentally (Tonolab instrument; Icare, Finland) before the injection, and at defined intervals in the following 24 hours (0.25, 0.5, 1, 2, 3, 6, 8 and 24 hours after the injection). During the experiment, the possible onset of inflammatory reactions or any other adverse reaction related to the injection of the composition object of the invention was monitored.

[0125] The results obtained are indicated in the graphs of FIGURES 2 and 3.

[0126] The data obtained for GROUP 1 (Figure 2) show that the injection with 1 pl of the composition object of the invention causes an initial increase in the IOP, which reaches a peak of 25 mmHg, but within 8 hours a return to IOP values completely similar to the preoperative values is observed, which can be considered physiological (< 21 mmHg; Tranos PG et al., J Cataract Refract Surg2003 , 29:508- 12). These values are stably maintained 24 hours after the injection. Similarly, in the case of the animals that received 2 pl of the composition of the invention (GROUP 2), an initial increase in the IOP with a peak of 28 mmHg was observed, followed by a return to preoperative and physiological IOP values (< 21 mmHg) within 8 hours, which were stably maintained 24 hours after the injection (Figure 3). During the entire experiment, no inflammatory reaction or any other adverse reaction related to the injection of the composition of the invention was observed.

[0127] It is known that many ophthalmic surgical procedures can be followed by a transient increase in the IOP, even in the absence of intra- or post-operative complications and concomitant ocular pathologies of the patient. It is also known that particularly high peaks of IOP can cause damage to the eye, whereas a chronic increase in the IOP is the main risk factor for the development of glaucoma, and therefore the detection of this parameter after ophthalmic surgical interventions is particularly important. Literature indicates however that, in the absence of other symptoms such as comeal edema or discomfort for the patient, postoperative IOP peaks < 40 mmHg followed by a spontaneous return to physiological values (< 21 mmHg) within 1 week of surgery do not require any treatment as they have no consequences. (Tranos PG et al., J Cataract Refract Surg 2003, 29:508-12; Tranos P et al., Eye (Lond) 2004, 18:673-9).

[0128] In light of the above information, the evidence obtained demonstrates that the composition of the invention is well tolerated and does not cause any adverse reactions, either in terms of inflammation or increased IOP, even in the case of prolonged contact (24 hours) with the eye. These data, together with the results indicated in Example 16, help to demonstrate that should residues of the composition according to the invention remain in the patient's eye after surgery, no adverse effect is expected, thus making the procedures in which it is used simpler and faster for the surgeon, who does not need to spend time in removing all visible residues dispersed in the patient's tissue.

Claims

CLAIMS1. An ophthalmic pharmaceutical composition in gel form, belonging to the category of ophthalmic viscoelastic devices OVDs of the cohesive type, stained but without a staining capacity, comprising or consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF, with one or more pharmacologically acceptable excipients, preferably consisting of hyaluronic acid and a dye selected from Brilliant Blue G and Brilliant Blue FCF, with one or more pharmacologically acceptable excipients, wherein said dye is present in a concentration ranging from 0.001 mg / ml to 0.1 mg / ml.

2. The pharmaceutical composition according to claim 1, characterized in that said dye does not diffuse outside the composition.

3. The pharmaceutical composition according to one of claims 1-2, characterized in that the weight average molecular weight of the hyaluronic acid used in the preparation of the composition is within the range of IxlO6Da to 3xl06Da, preferably from 1.6xl06Da to 2.6 xlO6Da.

4. The pharmaceutical composition according to one or more of the previous claims, characterized in that hyaluronic acid is present in the composition in a percentage ranging from 1% w / v to 3% w / v, preferably from 1.5% w / v to 2.5% w / v, more preferably from 1.8% w / v to 2.2% w / v, and is even more preferably equal to 2% w / v, said percentages being expressed by weight with respect to the total volume of the composition.

5. The pharmaceutical composition according to one or more of the previous claims, characterized in that said dye is present in a concentration ranging from 0.016 mg / ml to 0.050 mg / ml, more preferably from 0.02 mg / ml to 0.04 mg / ml.

6. The pharmaceutical composition according to one or more of the previous claims, characterized by a dispersion-cohesion coefficient CDI ranging from 30 to 45%asp / mmHg, preferably equal to 35%asp / mmHG.

7. The pharmaceutical composition according to one or more of the previous claims, characterized in that it has a medium viscosity at rest at 25°C which ranges from 50,000 mPa.s to 200,000 mPa.s, preferably from 80,000 to 120,000 mPa.s.

8. The pharmaceutical composition according to one or more of the previousclaims, characterized in that it has a dynamic viscosity (r|) at 25 °C, for a shear stress equal to 10 s'1, which ranges from 12,000 mPa.s to 22,000 mPa.s, preferably from 15,000 mPa.s to 19,000 mPa.s.

9. The pharmaceutical composition according to one or more of the previous claims, characterized in that it has a physiological pH, preferably a pH that ranges from 6.8 to 7.6 and an osmolarity that ranges from 250 mOsm / kg to 350 mOsm / kg.

10. An ophthalmic pharmaceutical composition in gel form according to one or more of the previous claims 1-9, characterized in that said one or more pharmaceutically acceptable excipients are selected from preservatives, water and various mineral salts, preferably said preservatives are selected from mannitol, sorbitol or sodium thiosulfate.

11. An ophthalmic pharmaceutical composition in gel form according to one or more of the previous claims 1-10 for use in surgery of the anterior segment of the eye, preferably in cataract surgery.

12. An ophthalmic pharmaceutical composition in gel form for use according to claim 11, in surgery of the anterior segment of the eye, preferably in corneal transplantation, iris or pupil reconstruction or in cataract surgery, preferably in cataract surgery by intracapsular, extracapsular or phacoemulsion cataract extraction.

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