New molecules and their use in the treatment of pathologies related to oxidative stress

Abstract

Pharmaceutical compositions are described, comprising (CAR-HNE), i.e. a molecule obtained from carnosine and 4-hydroxy-2-nonenal, for the prevention and / or treatment of ophthalmic diseases resulting from an imbalance of the redox equilibria in ocular cells and tissues. A chemical conjugate HA-CAR-HNE obtained from hyaluronic acid (HA), carnosine (CAR), and hydroxynonenal (HNE), the synthesis process, and its use as such or in pharmaceutical compositions for the prevention and / or treatment of diseases related to oxidative stress, whose pathophysiology is linked to an imbalance of redox equilibria in cells and tissues, are also described.

Description

[0001] "NEW MOLECULES AND THEIR USE IN THE TREATMENT OF PATHOLOGIES RELATED TO OXIDATIVE STRESS"

[0002] ****

[0003] SUBJECT OF THE INVENTION

[0004] The present invention relates to new molecules and their use for the treatment of pathologies related to oxidative stress. More specifically, the present invention relates to the use of the molecule (CAR-HNE) obtained by the reaction of carnosine (CAR) with 4-hydroxy-2-nonenal (HNE), the chemical conjugate HA-CAR-HNE obtained from hyaluronic acid (HA), carnosine (CAR) and 4-hydroxy-2-nonenal (HNE), the relative pharmaceutical compositions and the relative use as a medicine in the prevention and / or treatment of pathologies related to oxidative stress, i.e., pathologies whose physiopathology is linked to an imbalance of the oxidative-reductive (redox) equilibria in cells and tissues, in particular in the prevention and / or treatment of ophthalmic pathologies due to increased oxidative stress in ocular cells and tissues.

[0005] STATE OF THE ART

[0006] Reactive oxygen species (ROS) are formed as a natural byproduct of aerobic cellular metabolism. Mitochondrial respiration is the primary cause of ROS formation, but these compounds can also be produced by a variety of enzymes such as NADPH oxidase, xanthine oxidase, nitric oxide synthase, and peroxisome constituents. ROS can also be produced as a consequence of exposure to ionizing or UV radiation, or from the metabolism of numerous drugs and xenobiotics. In the endoplasmic reticulum, ROS can be released during protein folding and the formation of disulfide bonds. (Juan et al., IntJMol Set 2021, 22:4642).

[0007] ROS are highly reactive chemical compounds, which are formed by the propensity of molecular oxygen O2 to accept electrons, thus generating unstable molecules such as superoxide anion (*O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH“) and singlet oxygen. C

[0008]

[0009] O2). Physiological levels of ROS are intrinsic to normal cell functionality and are essential for maintaining homeostasis and cell signalling. When ROS are produced in excess, on the other hand, or when cellular defense systems are unable to metabolize them, oxidative stress occurs, with concomitant damage to the cell structures. In general, the harmful effects of ROS on the cell can include: DNA and RNA damage, lipid peroxidation, and protein oxidation. The damage caused by ROS to DNA, lipids and enzymes present in the cellular cytosol is irreversible, as the cellular components involved are oxidized and modified in such a way that they are no longer able to exert their original function (Juan et al., IntJMol Sci 2021, 22(9):4642).

[0010] An important biomarker of oxidative stress is lipid peroxidation, whose occurrence increases rapidly and proportionally with an increase in the intracellular presence of free radicals. Lipid peroxidation can be described in general terms as a complex process in which oxidizing molecules, such as free radicals, attack lipids containing carbon-carbon double bonds, especially polyunsaturated fatty acids (PUFAs), such as membrane phospholipids, triggering a chain reaction that leads to the formation of a large number of oxidation products. Lipid hydroperoxides are the main primary products resulting from this process; they are unstable compounds, however, that tend to decompose into secondary products. Among the numerous aldehydes that can be formed as primary products, malondialdehyde (MDA), propionaldehyde, hexanal, and 4- hydroxy-2-nonenal (HNE) can be mentioned. MDA appears to be the most mutagenic product of lipid peroxidation, whereas HNE is considered the most toxic (Ayala et al., Oxid Med Cell Longev 2014, 2014:360438; Csala et al., Biochim Biophys Acta 2015, 1852:826-838).

[0011] OH

[0012] 4-hydroxy-2-nonenal

[0013]

[0014] HNE (4-hydroxy-trans-2,3-nonenal, also known as 4-hydroxynonenal or 4- hydroxy-2-nonenal, PubChem CID 5283344) is the major a,P-unsaturated hydroxyalkenal formed as a peroxidation product of co-6 PUFAs, such as arachidonic acid and linoleic acid. The high toxicity of HNE is mainly due to its capacity of reacting rapidly with a wide spectrum of molecules present in the cell, including proteins, lipids and nucleic acids; HNE, however, can also exert physiologically beneficial effects on the cell depending on its intracellular concentration, exerting a hermetic action. HNE is, in fact, capable of promoting cell survival and proliferation when present in low concentrations (<2 pM), whereas it is toxic at higher concentrations (>10 pM), to the point of causing cell death at concentrations higher than 100 pM (Ayala et al., OxidMed Cell Longev 2014, 2014:360438; Shoeb et al., CurrMedChem 2014, 21:230-237).

[0015] HNE can cause cell death both through apoptosis, primarily via the extrinsic pathway, and through necrosis. Different cell types can show a varying sensitivity to the same exposure times and concentrations to HNE, and can activate different cell death mechanisms in response. It has been demonstrated, for example, that cells belonging to the Hep G2 cell line, deriving from human hepatocellular carcinoma, mainly activate apoptosis (detected by flow cytometry measurements, analysis of Caspase-3 activation and proteolytic cleavage of PARP) for HNE concentrations within the range of 5-40 pM, whereas they preferentially undergo necrosis when treated with HNE concentrations within the range of 80 and 100 pM (Ayala et al., OxidMed Cell Longev 2014, 2014:360438; Chaudhary, et al., Biochemistry 2010, 49: 6263-6275). Similarly, Caspase-3 protein levels and activity have proved to have increased in SH-SY5Y human neuroblastoma-derived cells treated with HNE at concentrations equal to or greater than 5 pM (Abarikwu, et al., Basic Clin Pharmacol Toxicol 2012, 110:441-448).

[0016] HNE also exerts important direct and indirect effects on proinflammatory signalling by acting on various intracellular proteins, including the transcription factor NF-KB, a known positive regulator of inflammation. It has been demonstrated, for example, that HNE in high concentrations (40 pM) is capable of binding to IKB, a cytoplasmic inhibitor of NF-KB, preventing its activity and thus promoting the nuclear translocation of NF-KB and the expression of its target genes, which include numerous pro-inflammatory cytokines (G^gotek and Skrzydlewska, Chem Phys Lipids 2019, 221:46-52). HNE has proved to stimulate the inflammatory response in cells deriving from the retinal pigment epithelium (RPE), pigmented cells located in the outermost layer of the retina and having primarily trophic functions towards retinal photoreceptor cells. In the presence of HNE at a concentration of 10 pM, in fact, RPE cells show an increased production of proinflammatory cytokines. IL-6, IL-ip and TNF-a (Yang et al., Exp Eye Res 2019, 188:107792).

[0017] The numerous and diverse effects of HNE derive from its capacity of interacting with most intracellular signalling pathways, mainly due to the formation of covalent bonds with key proteins for their regulation. The aldehyde group in Ci position can react with primary amines to form Schiff bases, i.e. imines bearing a hydrocarbon group on the nitrogen atom ('Schiff bases' in IUPAC Compendium of Chemical Terminology, 3rd ed. International Union of Pure and Applied Chemistry; 2006. Online version 3.0.1, 2019; Csala et al., Biochim Biophys Acta 2015, 1852:826-838). The double bond between the C2 and C3 atoms of HNE is responsible for the reaction with thiol or amine compounds, via Michael addition. Michael addition involves the reaction between a donor (enolate or other nucleophile) and an acceptor (usually an a,P-unsaturated carbonyl compound), resulting in the formation of a so-called Michael adduct through the formation of a carbon-carbon bond between the two reacting molecules, corresponding to the P-carbon of the acceptor. Whereas the formation of Schiff bases is relatively slow and reversible, Michael adducts are stable. Cysteine, lysine, and histidine residues in proteins are the primary targets of Michael addition (Csala et al., Biochim Biophys Acta 2015, 1852:826-838).

[0018] Many pathologies are related to an alteration of the intracellular redox equilibria and the increased presence of intracellular ROS can represent both the primary cause and a secondary contributor to the progression of diseases of different origins. For some diseases, this aspect has not yet been clarified, such as obesity. A large amount of evidence demonstrates that obesity is characterized by chronic oxidative stress, but it is not yet fully understood whether this condition is a trigger or a consequence of obesity itself (Savini Isabella, Valeria Gasperi, and Maria Valeria Catani, "Oxidative stress and obesity" Obesity: A Practical Guide [book] 2016: 65-86).

[0019] ROS formation is the primary etiological agent of radiation-induced lung injury, Paraquat poisoning and atherosclerosis. In radiation-induced lung injury, radiation exposure is known to induce the formation of ROS, which oxidize intracellular macromolecules and promote a strong inflammatory response, characterized by immune cell infiltration into the lung, cell death, and, in the long term, fibrosis related to the continued production of cytokines. Similarly, Paraquat causes damage by inducing the production of ROS in type II alveolar cells, as well as in the liver and kidneys. Long-term exposure to Paraquat has also been associated with the onset of Parkinson's disease. Oxidative stress is directly responsible for the conversion of LDL cholesterol to its oxidized form (OxLDL) which plays a key role in the formation of atherosclerotic plaques (Forman HJ and Zhang H, Nat Rev Drug Discov 2021, 20:689-709; Hajam et al., Cells 2022, 11:552).

[0020] It should also be noted that oxidative stress can also arise as a side effect of therapeutic strategies. This is the case, for example, with radiotherapy, i.e., targeted exposure to ionizing radiation frequently used for treating oncological diseases, which can cause, as a side effect, the development of radiation-related fibrosis (radiation-induced tissue fibrosis, RIF). This condition is characterized by the abnormal activation of myofibroblasts and the excessive accumulation of extracellular matrix, and can manifest in multiple organs, such as the lungs, skin, liver, and kidneys. Oxidative stress, resulting from oxidative damage directly resulting from exposure to ionizing radiation, is one of the main causes of abnormal myofibroblast activation. For this reason, antioxidant and antiinflammatory therapies represent the main strategies for combating RIF (Yu et al., J Transl Med 2023, 21:708).

[0021] In many pathologies, oxidative stress occurs secondary to the initiation of the pathogenic mechanism by other factors. For example, oxidative stress can be caused by the increased production of ROS by NADPH oxidase in the inflammatory reaction following initial tissue injury, or by xanthine oxidase in the case of ischemia-reperfusion events. Oxidative stress can also modulate various intracellular signalling pathways and influence essential biological processes, such as, for example, promoting inflammation, inducing apoptosis, deregulating autophagy, and compromising the mitochondrial functionality. These events accelerate the progression and exacerbate the symptoms of numerous diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), hypertension, type 2 diabetes mellitus, Alzheimer's disease, cancer, and systemic inflammatory response syndrome (SIRS) and the above-mentioned ischemia-reperfusion injury (Forman HJ and Zhang H, Nat Rev Drug Discov 2021, 20:689-709; Hajam et al., Cells 2022, 11:552).

[0022] Ocular structures are particularly sensitive to increased oxidative stress, due to both the intrinsic vulnerability of their tissues and also their external location, which leads to direct exposure to harmful chemical and physical agents, including atmospheric oxygen and UV radiation. In ophthalmology, the separation of the eye into two distinct anatomical portions: the anterior segment and the posterior segment, is of considerable importance, both from a diagnostic and surgical perspective. The anterior segment includes the cornea, iris, and pupil, the lens with the structures that support it, and the ciliary body. These structures define two cavities, filled with aqueous humor and are also considered part of the anterior segment of the eye: the anterior chamber, bounded 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. The posterior segment of the eye, on the other hand, comprises the posterior two-thirds of the eyeball, the vitreous cavity and the retina (Gray H. & Lewis W. H., 1918; Anatomy of the human body, 20th ed. Philadelphia: Lea & Febiger).

[0023] The retina is the component of the eye that is sensitive to light, it adheres internally to the posterior third of the eyeball and is made up of different cell types organized for forming ten overlapping layers that follow each other from the outside inwards in the following order:

[0024] • retinal pigment epithelium layer, composed of a single layer of epithelial cells in contact with the inner side of the eyeball, having trophic functions;

[0025] • photoreceptor layer, or the rod and cone layer, which represents the specialized structure for detecting light stimuli that enter the eyeball through the pupil; • external limiting membrane, which separates the portion of photoreceptor cells present in the photoreceptor layer from the cell bodies, including the nuclei, of the same cells;

[0026] • outer granule layer or outer nuclear layer, consisting of the cell bodies of the photoreceptor cells, i.e. cones and rods;

[0027] • external plexiform layer, the region that contains the synapses between the terminals of the photoreceptor cells and the dendrites of the bipolar and horizontal cells, i.e. neuronal-type cells whose function is to transmit signals from the photoreceptors to the retinal ganglion cells;

[0028] • granule or inner nuclear layer, containing the nuclei and cell bodies of bipolar and horizontal cells, and of amacrine cells, which also contribute to the vertical transmission of signals along the retinal layers;

[0029] • internal plexiform layer, where the synapses are found between the axons of the bipolar cells and the dendrites of the amacrine and ganglion cells, which are the neurons responsible for collecting all the visual information coming from the retina to transmit it to the brain through the optic nerve, a structure composed by definition of the axonal extensions of these cells; • ganglion cell layer, which contains the nuclei of retinal ganglion cells and some amacrine cells;

[0030] • nerve fiber layer, in which the axons of the ganglion cells run horizontally, which then converge in the optic nerve;

[0031] • internal limiting membrane, i.e. the basement membrane that is in contact with the vitreous humor and is produced by Muller glial cells (Mahabadi and Khalili, “Neuroanatomy, Retina”, StatPearls [Internet] 2023, PMID: 31424894).

[0032] Among the main disorders of the anterior segment of the eye whose pathophysiology can be related to oxidative stress are dry eye, keratoconus, conjunctivitis, uveitis, and cataracts. Numerous conditions characterized by retinal dysfunction have also been linked to alterations in the endogenous systems that protect against oxidative stress, including both widespread diseases (age-related macular degeneration, glaucoma) and rare hereditary vision disorders (retinitis pigmentosa, Leber hereditary optic neuropathy or LHON). (Dalleau S et al., Cell Death Differ 2013, 20:1615-1630; Domenech EB and Marfany G, Antioxidants (Basel) 2020, 9:347; Dammak A et al., Biomedicines 2023, 11 :292).

[0033] Leber hereditary optic neuropathy (LHON) is a clear example of oxidative stress-related retinopathy. This rare disease is characterized by the degeneration of retinal ganglion cells (RGCs) due to mutations in the mitochondrial genes encoding NADH dehydrogenase, an enzyme essential for the production of the cell's energy molecule, adenosine triphosphate (ATP). These mutations cause a decrease in ATP synthesis and a concomitant significant increase in intracellular ROS levels, which in turn trigger RGC apoptosis. Treatment of this and other posterior segment ocular diseases caused by oxidative stress is hampered both by the poor stability of all the antioxidant therapeutic molecules currently in use and by their reduced permanence within the eyeball, due to the high turnover rate and typical washout of the vitreous humor (Domenech EB and Marfany G, Antioxidants (Basel) 2020, 9:347).

[0034] In order to extend life expectancy and improve its quality, it is therefore recommended to monitor oxidative stress, improving systems for detecting its increase, and supporting the natural antioxidant mechanisms of the cell. At the same time, numerous therapeutic strategies for the above-mentioned diseases are being studied, based on reducing oxidative stress and / or modulating its related intracellular pathways (Dalleau S et al., Cell Death Differ 2013, 20:1615-1630; Forman HJ and Zhang H, Nat Rev Drug Discov 2021, 20:689-709).

[0035] The main endogenous defenses of the cell against ROS are represented by superoxide dismutase (SOD) enzymes, including Zn-Cu SOD or SOD1 in the cytosol and intermembrane space of mitochondria and Mn-SOD or SOD2 in the mitochondrial matrix, which catalyze the dissociation of the superoxide anion (O2*“) into molecular oxygen (O2) and hydrogen peroxide (H2O2), through redox reactions. The hydrogen peroxide thus obtained is in turn converted into water (H2O) by other enzymes, including catalase, glutathione peroxidase, and thioredoxin reductase, thus preventing damage to cell structures caused by ROS. In some vertebrate tissues, such as skeletal muscle and the brain, characterized by high levels of oxidative metabolism, high concentrations of small molecules with an antioxidant activity are present, including glutathione and other peptides (carnosine, homocarnosine, anserine, etc.), which have the specific function of strengthening the cell's protection from damage related to oxidative stress (Boldyrev et al., Physiol Rev 2013, 93:1803-1845; Juan et al., Int J Mol Sci 2021, 22:4642).

[0036] In particular, with respect to the anti-radical and anti-peroxidative properties of carnosine, preclinical studies have demonstrated its capacity of preventing, and partially treating, inflammatory disturbances, as demonstrated in tissues of the digestive tract, eyes, and skin. Furthermore, carnosine has shown inhibitory effects against copper-induced Cu(II) oxidation of human LDL (Bellia et al., Eur J Med Chem 2007, 43:373-380), and its scavenger activity against the hydroxyl radical has been specifically demonstrated (La Mendola et al., Helv Chim Acta, 2002, 85:1633-1643). It is also interesting to note that carnosine complexes with Cu(II) show synergistic activity against two toxic radical species, Ch’-and »OH (Bonomo et al., Dalton Trans 2003, 23:4406-4415). The scavenger activity of these carnosine complexes with Cu(II) is also present when they dimerize (Tamba and Torreggiani, Int J Radiat Biol 1999, 75:1177-1188).

[0037] Based on the literature discussed above, carnosine appears to be an excellent candidate for counteracting oxidative stress in the prevention and treatment of related diseases. The peptide nature of carnosine, however, imposes some limitations on its use, due to its degradability by specific peptidases called camosinases and other enzymes normally present in biological tissues. In order to overcome this limitation, the use of N-acetylcamosine has been proposed in topical ophthalmic formulations intended for the treatment of cataracts and glaucoma. N-acetylcarnosine is a small molecule, produced through processes known to persons skilled in the field and purified chromatographically using large quantities of solvents (WO9510294). A further attempt to reduce the degradability of carnosine was effected by derivatizing it with cyclodextrins, compounds widely used as drug carriers, through a chemical procedure that requires subsequent purification steps by means of chromatographic techniques (EPl 176154). Based on the same rationale, a conjugate of carnosine with trehalose was subsequently patented. The synthesis of the compound requires subsequent purification by cation exchange column chromatography (EP 1860116). The exact degradation profile of derivatized carnosine as described in WO9510294, EPl 176154, and EPl 860116 by enzymes present in biological tissues, however, is unknown.

[0038] Hyaluronic acid (HA) is a heteropolysaccharide composed of alternating residues of glucuronic acid (P-D-glucopyranuronic) and acetylglucosamine (2-acetylamino-2-deoxy-P-D-glucopyranose). It is a natural linear-chain polymer with a molecular weight ranging from 50,000 to 13 x 106Da, depending on the source from which it is obtained and the preparation methods used. It is naturally present in pericellular gels, in the ground substance of the connective tissue of vertebrate organisms (of which it is a major component), in the synovial fluid of joints, in the vitreous humor, and in the umbilical cord. HA provides mechanical support to cells in many tissues, such as skin, tendons, muscles, and cartilage, and is also capable of modulating many different processes related to cell physiology and biology, such as proliferation, migration, cell differentiation, and angiogenesis (Fallacara et al., Polymers 2018, 10:701).

[0039] Due to the properties described above, HA is used in the treatment of a broad spectrum of pathologies: from wound healing to counteracting degenerative processes affecting joint cartilage. The healing of various types of wounds and ulcers, even chronic, is significantly improved following the local application of HA-containing ointments or creams. HA is also suitable for incorporation into molecular scaffolds suitable for guiding tissue regeneration in tissue engineering (Fakhari and Berkland, Acta Biomater, 2013, 9:7081-92). HA is also used as a vitreous humor substitute in ophthalmology, as a dermal filler, and as a moisturizing agent in cosmetics. One of the most important biomedical uses of HA is undoubtedly intra-articular injection to combat cartilage degeneration associated with various diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA), which are characterized by inflammatory conditions affecting the joints (Fallacara et al., Polymers 2018, 10:701).

[0040] Furthermore, HA is used as a vehicle for various kinds of drugs, which are salified with it or formulated in simple association, as its specific properties of biocompatibility, biodegradability, non-immunogenicity, viscosity and hydratability make it particularly suitable as a delivery system for drugs and molecules both at a topical and systemic level. Although HA is suitable for these purposes in its natural form, its range of applications can be further expanded through its chemical modification, aimed at obtaining higher-performance products capable of meeting specific needs, such as extending their half-life. The presence of HA confers to its synthetic derivatives a series of positive properties typical of the native molecule, such as mucoadhesiveness, biodegradability, and biocompatibility (Fallacara et al., Polymers 2018, 10:701).

[0041] EP2648715 describes a compound comprising HA salified, or at least partially salified, with carnosine for the purpose of obtaining preparations that offer the biopharmacological properties of both compounds. The compound described is characterized in that the chemical bond between HA and carnosine is ionic, therefore weak and exclusively electrostatic in nature. It is well known that in aqueous solution, chemical species, or electrolytes, held together by an ionic bond dissolve, immediately releasing free carnosine and HA, which are both rapidly degraded by numerous enzymes normally present, including carnosinase, nonspecific peptidases, and hyaluronidase.

[0042] A conjugate between hyaluronic acid (HA) and carnosine has also been prepared in which the amino group of the peptide is engaged in an amide bond with the carboxyl of some of the glucuronic acid units present in the polysaccharide chain (EP3174555). This conjugate has proved to exhibit an interesting improved resistance to the action of human serum carnosinase (CN-1). It has also been described that HA conjugated to carnosine via an amide bond and with a high degree of derivatization is useful in the treatment of osteoarthritis (OA), rheumatoid arthritis (RA) and a series of pathologies directly resulting from them (EP3691691).

[0043] EP3922268 is also known, which describes a conjugate in which carnosine is covalently linked to hyaluronic acid (HA) through a peptidase-resistant amino bond, different from those described previously. The objective of the present invention is therefore to identify new molecules that overcome the drawbacks of the molecules known from the state of the art indicated above, that are particularly effective in preventing and / or treating diseases related to oxidative stress, as well as damage due to an imbalance of the redox equilibria in the body in general, and in particular to prevent and / or treat the same damage at an ophthalmic level.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates to pharmaceutical compositions comprising, or consisting of, a molecule obtained by the reaction of carnosine with 4-hydroxy-2-nonenal (CAR-HNE) and at least one pharmaceutically acceptable excipient, wherein the CAR-HNE molecule has the following structural formula (1):

[0046]

[0047] (1),

[0048] said compositions optionally comprising hyaluronic acid (HA) or its derivatives in simple association, for use as a medicinal product in the prevention and / or treatment of ophthalmic diseases related to oxidative stress, affecting both the anterior and posterior segment of the eye, such as dry eye, keratoconus, conjunctivitis, uveitis, cataract, age-related macular degeneration, glaucoma, retinitis pigmentosa, Leber hereditary optic neuropathy (LHON), preferably for use in the prevention and / or treatment of age-related macular degeneration and Leber hereditary optic neuropathy (LHON).

[0049] The functional groups present in the CAR-HNE molecule having formula (1) are susceptible to reacting at an intramolecular level, giving rise to the formation of isomers which in aqueous solution are in equilibrium with (1) and which do not differ in any way from (1) with regards to the chemical reactivity and biological activity. These isomers have the formulas (2), (3) and (4) indicated hereunder.

[0050]

[0051] Specifically:

[0052] • the molecule formed by reaction between CAR and HNE in open form can give the aldehyde tautomer (1) and the enol tautomer (2), in equilibrium with each other;

[0053] • the cyclic hemiacetal form (3) is obtained from the tautomer (1) by reaction of the aldehyde group with the alcohol group present in the portion of the molecule deriving from HNE, thus generating a five-membered 2-hydroxy-tetrahydrofuran ring;

[0054] • the cyclic imine (4) is formed by reaction between the aldehyde group of the tautomer (1) with the free amino group of the P-Ala residue and loss of a water molecule.

[0055] For the sake of simplicity, in this description, the Applicant will refer to the mixture of the chemical species (1), (2), (3) and (4) indicated above and present in the pharmaceutical compositions for use according to the present invention with the abbreviation CAR-HNE.

[0056] Pharmaceutical compositions for use according to the present invention, comprising or consisting of CAR-HNE and at least one pharmacologically acceptable excipient, can be advantageously formulated with excipients, stabilizers, viscosifiers, and / or preservatives as known to the person skilled in the field. Said pharmaceutical compositions can take various pharmaceutical forms, suitable for topical, oral, systemic, and injectable administration, including intravitreal administration, comprising solutions, ointments, gels, suspensions, and the like. Furthermore, said compositions can be impregnated or deposited on gauze, bandages, adhesive dressings, plasters, three-dimensional structures or various kinds of scaffolds, that can be used in the ophthalmic field, including ophthalmic surgery. The compositions comprising or consisting of CAR-HNE for use in accordance with the present invention are preferably in the pharmaceutical form of a solution suitable for injectable administration, intravitreously.

[0057] The Applicant has in fact surprisingly found that the pharmaceutical compositions for use according to the present invention comprising or consisting of CAR-HNE are extremely effective in preventing damage due to an imbalance in the redox equilibria that can occur at an ophthalmic level. In particular, the antiinflammatory and anti-apoptotic effect that CAR-HNE is capable of exerting on retinas subjected ex vivo to hypoxia, a condition known for triggering oxidative stress, is surprising and completely unexpected based on the scientific literature available to date. The Applicant has also surprisingly found that CAR-HNE is capable of extraordinarily counteracting the loss of functionality of retinas subjected in vivo to light damage, a condition characterized by a strong increase in oxidative stress.

[0058] As indicated above, the pharmaceutical compositions comprising or consisting of CAR-HNE for use according to the present invention can optionally comprise hyaluronic acid (HA) or its derivatives in simple association.

[0059] The HA that can be used in simple association with CAR-HNE for the purposes of the invention described herein can derive from any source, for example, from rooster combs (EP 138572) or from fermentation (from Streptococcus equi or zooepidemicus. EP0716688), and can be purified according to different techniques. (EP3491027, EP3655138). The preferred source of HA is fermentation by Streptococcus equi or zooepidemicus, subspecies equi 68222 mutant H-l (EP3491027). The weight average molecular weight of HA for the applications described herein can range from IxlO4to 3xl06Da, preferably from IxlO5Da to IxlO6Da, even more preferably within the range of 130-230 kDa or within the range of 500-750 kDa and mixtures thereof. For the sake of brevity, HA with a weight average molecular weight (MW) ranging from 130 to 230 kDa is generally referred to as “HA with a weight average MW of 200 kDa,” whereas HA with a weight average molecular weight (MW) ranging from 500 to 750 kDa is generally referred to as “HA with a weight average MW of 700 kDa.” Average molecular weight refers to the weight average MW calculated using the “intrinsic viscosity” method (Terbojevich et al., Carbohydr Res, 1986, 363-377).

[0060] The HA derivatives, which can be used in simple association with CAR-HNE for the purposes of the present invention, are listed hereunder:

[0061] - HYAFF®: HA esters with alcohols of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series, with an esterification percentage that can vary depending on the type and length of the alcohol used, preferably ranging from 50% to 75%, whereas the remaining percentage of non-esterified HA can be salified with organic and / or inorganic bases (EP216453), preferably the HA ester with benzyl alcohol having a derivatization percentage equal to 50%, called HYAFF®llp50;

[0062] - HYADD®: HA amides with amines of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series, with an amidation percentage ranging from 0.1% to 10%, whereas the remaining percentage of HA not subjected to amidation can be salified with organic and / or inorganic bases (EP 1095064), preferably the hexadecyl amide of hyaluronic acid having a derivatization percentage ranging from 0.1% to 5%, called HYADD®-4;

[0063] O-sulfated derivatives of HA up to the fourth degree of sulfation (EP702699);

[0064] ACP®: internal esters of HA with an esterification percentage not exceeding 20%, preferably ranging from 0.05% to 5% of esterification, whereas the remaining percentage of non-esterified HA can be salified with organic and / or inorganic bases (EP341745);

[0065] - Deacetylated HA: deriving from the deacetylation of the N-acetyl- glucosamine fraction with a deacetylation percentage preferably ranging from 0.1% to 30%, whereas all the carboxylic groups of HA can be salified with organic and / or inorganic bases (EP1313772);

[0066] - HYOXX®: percarboxylated derivatives of HA obtained from the oxidation of the primary hydroxyl of the N-acetyl-glucosamine fraction with a percarboxylation degree ranging from 0.1% to 100% and, preferably, from 25% to 75%. All the carboxylic groups of HA can be salified with organic and / or inorganic bases (EP1339753).

[0067] For the purposes of the present invention, the HA derivatives that can be used in simple association with CAR-HNE are preferably the HA ester with benzyl alcohol having a derivatization percentage equal to 50%, called HYAFF®1 lp50, or the hexadecyl amide of hyaluronic acid having a derivatization percentage that ranges from 0.1% to 5%, called HYADD®-4.

[0068] The pharmaceutical compositions for use according to the invention, comprising or consisting of said CAR-HNE and at least one pharmacologically acceptable excipient, are extraordinarily suitable for ophthalmic use as a medicinal product, in the prevention and / or treatment of ophthalmic diseases related to oxidative stress, affecting any ocular structure, including damage resulting from exposure to molecular oxygen and UV radiation. Said pharmaceutical compositions are described for use as a medicinal product in the treatment and / or prevention of any ocular disease related to oxidative stress, affecting both the anterior and posterior segments of the eye, including hereditary diseases, such as dry eye, keratoconus, conjunctivitis, uveitis, cataracts, age-related macular degeneration, glaucoma, retinitis pigmentosa, and Leber hereditary optic neuropathy (LHON). Pharmaceutical compositions comprising, or consisting of said CAR-HNE for use according to the present invention are preferably described in the treatment and / or prevention of age-related macular degeneration and Leber hereditary optic neuropathy (LHON).

[0069] The Applicant has in fact demonstrated that treatment with CAR-HNE is capable of exerting an extraordinary protective effect against oxidative stress on retinas cultured ex vivo subjected to hypoxia, drastically reducing inflammation and cell death by apoptosis. This effect is completely unexpected, as scientific literature has shown that HNE stimulates inflammation even in retina-derived cells (G^gotek and Skrzydlewska, Chem Phys Lipids 2019, 221:46-52; Yang et al., Exp Eye Res 2019, 188:107792) and is capable of inducing cell death by apoptosis at low concentrations, equal to or greater than 5 pM, in different cell types, including the neuronal line SH-SY5Y (Ayala et al., OxidMed Cell Longev 2014, 2014:360438; Chaudhary, et al., Biochemistry 2010, 49: 6263-6275; Abarikwu, et al., Basic Clin Pharmacol Toxicol 2012, 110:441-448). The Applicant has subsequently even more surprisingly clearly demonstrated that CAR-HNE is capable of protecting in vivo the functionality of retinas subjected to light damage, a stimulus known to cause intense oxidative stress. This evidence supports the efficacy of CAR-HNE for ophthalmic use, and preferably in the treatment and / or prevention of the above-mentioned diseases.

[0070] The present invention further relates to a chemical conjugate HA-CAR-HNE, obtained from hyaluronic acid (HA), carnosine (CAR) and 4-hydroxy-2-nonenal (HNE), the pharmaceutical, nutraceutical or cosmetic compositions comprising or consisting of said conjugate, and the use of both the conjugate and the pharmaceutical composition comprising it as a medicinal product in the prevention and / or treatment of all pathologies whose physiopathology is related to oxidative stress.

[0071] The present invention relates in fact to a chemical conjugate HA-CAR- HNE, obtained from hyaluronic acid (HA), carnosine (CAR) and 4-hydroxy-2-nonenal (HNE), having the following structural formula (5):

[0072]

[0073] The structure 5 is part of a polysaccharide chain wherein n refers to the conjugated disaccharide and in said polysaccharide chain n ranges from 1 to 4500, preferably from 25 to 750, depending on the weight average molecular weight of the HA used in the synthesis of HA-CAR and its derivatization degree.

[0074] The functional groups present in the HA-CAR-HNE conjugate having formula (5) are susceptible to reacting at an intramolecular level, giving rise to the formation of isomers which in aqueous solution are in equilibrium with (5) and which do not differ in any way with respect to the chemical reactivity and biological activity, whose formulas (6) and (7) are provided hereunder:

[0075] H3G^

[0076] "\ OH

[0077]

[0078] (5)

[0079]

[0080] (7)

[0081] More specifically:

[0082] the molecule formed by the reaction between CAR and HNE, in turn linked to HA in the HA-CAR-HNE conjugate, in open form can give the aldehyde tautomer (5) and the enol tautomer (6) in equilibrium with each other;

[0083] • the cyclic hemiacetal form (7) is obtained from the tautomer (5) by reaction of the aldehyde group with the alcohol group present in the portion of the molecule deriving from HNE, thus generating a five-membered 2-hydroxy-tetrahydrofuran ring.

[0084] For the sake of simplicity, in this description, the Applicant will refer to the mixture of the chemical species (5), (6) and (7) indicated above and present in the pharmaceutical compositions according to the present invention with the abbreviation HA-CAR-HNE. By way of example, the HA-CAR-HNE conjugate according to the invention can be obtained through a synthetic process comprising the following steps:

[0085] • the HA-CAR conjugate, obtained by reaction between HA and CAR according to the process described in EP3174555 and / or in EP3691691, is reacted with hydroxynonenal (HNE) in a solvent based on water and ethanol, preferably in a 1:1 ratio (H2O:EtOH, 1:1), at room temperature, for a time exceeding 100 hours;

[0086] • the above solvent is removed under reduced pressure;

[0087] • the residue is crushed or ground with EtOH and dried, to obtain the HA- CAR-HNE product having the appearance of a glassy solid.

[0088] The HA-CAR conjugate used for the synthesis of HA-CAR-HNE according to the invention preferably has a derivatization level, referring to the number of CAR residues per 100 total disaccharide units, linked through a peptide bond to the free carboxylic group of glucuronic acid, ranging from 5% to 60%, and even more preferably from 10% to 30%. The Applicant has verified that the synthesis process described above obtains the bonding of HNE with all the CAR residues present in the starting HA-CAR conjugate.

[0089] The HA that can be used for the synthesis of the HA-CAR-HNE conjugate between hyaluronic acid (HA), carnosine (CAR) and hydroxynonenal (HNE), according to the invention described herein, can derive from any source, for example, from rooster combs (EP 138572) or from fermentation (from Streptococcus equi or zooepidemicus. EP0716688), and can be purified according to different techniques. (EP3491027, EP3655138). The preferred source of HA is fermentation by Streptococcus equi or zooepidemicus, subspecies equi 68222 mutant H-l (EP3491027). The weight average molecular weight of HA for the applications described herein can range from IxlO4to 3xl06Da, preferably from IxlO5Da to IxlO6Da, even more preferably within the range of 130-230 kDa or within the range of 500-750 kDa and mixtures thereof. For the sake of brevity, HA with a weight average molecular weight (MW) ranging from 130 to 230 kDa is generally referred to as “HA with a weight average MW of 200 kDa” whereas HA with a weight average molecular weight (MW) ranging from 500 to 750 kDa is generally referred to as “HA with a weight average MW of 700 kDa”. Average molecular weight refers to the weight average MW calculated using the “intrinsic viscosity” method (Terbojevich et al., Carbohydr Res, 1986, 363-377).

[0090] The present invention further relates to pharmaceutical, nutraceutical, or cosmetic compositions comprising or consisting of said HA-CAR-HNE conjugate and at least one pharmaceutically acceptable excipient.

[0091] The invention also relates to the non-pharmaceutical cosmetic use of said HA-CAR-HNE conjugate or of the composition comprising or consisting of said HA-CAR-HNE conjugate for preventing and / or treating damage caused by an imbalance in the body's redox equilibria, preferably skin and other damage related to excessive solar radiation.

[0092] The present invention further relates to a medical device comprising the conjugate and / or the pharmaceutical or nutraceutical composition according to the present invention, optionally in the presence of at least one excipient, said medical device preferably being a gauze, bandage, adhesive dressing, plaster, three-dimensional structure or scaffold.

[0093] Said compositions can be advantageously formulated with excipients, stabilizers, viscosifiers, and / or preservatives, as known to skilled persons in the field. Furthermore, said pharmaceutical compositions can be in various pharmaceutical forms, suitable for topical, oral, systemic, and injectable administration, including intravitreal administration, comprising: solid compositions for oral use (e.g., tablets), solutions (e.g., drops, ophthalmic drops), ointments, gels, suspensions, and the like. Said compositions can further be impregnated or deposited on various types of supports, including gauze, bandages, adhesive dressings, plasters, and three-dimensional structures or various kinds of scaffolds, that can be used in any field, including surgery and the field of advanced dressings for the treatment of tissue lesions of any nature.

[0094] In view of the antioxidant properties, the HA-CAR-HNE conjugate and the pharmaceutical or nutraceutical compositions comprising or consisting of said HA-CAR-HNE conjugate are described and claimed for medical use, preferably for use in the prevention and / or treatment of pathological conditions related to an imbalance of intracellular redox equilibria, of which a non-exhaustive list is provided hereunder: radiation-induced lung injury, Paraquat poisoning, atherosclerosis, radiation-induced tissue fibrosis (RIF), tissue injury, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), hypertension, type 2 diabetes mellitus, Alzheimer's disease, cancer, systemic inflammatory response syndrome (SIRS), ischemia-reperfusion injury, obesity.

[0095] In consideration of the antioxidant properties, the HA-CAR-HNE conjugate and the pharmaceutical, nutraceutical or cosmetic compositions comprising, or consisting of said HA-CAR-HNE conjugate are described and claimed for use in the prevention and / or treatment of conditions related to an imbalance of the redox equilibria.

[0096] Due to their anti-inflammatory properties, the HA-CAR-HNE conjugate and the pharmaceutical or nutraceutical compositions comprising, or consisting of said HA-CAR-HNE conjugate are also described and claimed for use in the prevention and / or treatment of inflammatory conditions affecting the joints, resulting from degeneration of the same, in particular in the prevention and / or treatment of Osteoarthritis (OA) and in the treatment of Rheumatoid Arthritis (RA), as well as in the treatment of pathologies directly caused by RA or indirectly related / dep endent on RA.

[0097] The HA-CAR-HNE conjugate and pharmaceutical or nutraceutical compositions comprising or consisting of said HA-CAR-HNE conjugate are also exceptionally suitable for use as medicinal products, preferably in the prevention and / or treatment of ophthalmic diseases related to oxidative stress, affecting any ocular structure, including damage resulting from exposure to molecular oxygen and UV radiation. The HA-CAR-HNE conjugate and pharmaceutical compositions comprising or consisting of said HA-CAR-HNE conjugate are described and claimed for use as medicinal products in the treatment and / or prevention of any ocular disease related to oxidative stress, both affecting the anterior and posterior segments of the eye, including hereditary diseases, such as dry eye, keratoconus, conjunctivitis, uveitis, cataracts, age-related macular degeneration, glaucoma, retinitis pigmentosa, Leber hereditary optic neuropathy (LHON). The HA-CAR-HNE conjugate and pharmaceutical compositions comprising, or consisting of said HA-CAR-HNE conjugate according to the present invention are preferably described for use as medicinal products in the treatment and / or prevention of age-related macular degeneration and Leber hereditary optic neuropathy (LHON).

[0098] The Applicant has in fact found and demonstrated, as indicated hereunder in this description, that the HA-CAR-HNE conjugate is capable of exerting an extraordinary protective effect against oxidative stress in the retinas of animals subjected in vivo to light damage, a stimulus known to cause intense oxidative stress. In vivo treatment with HA-CAR-HNE has in fact proved to be capable of drastically reducing inflammation and cell death by apoptosis at the retinal level in the presence of oxidative stress, and these effects at a cellular level are accompanied by a clear protective effect on the retinal functionality. In particular, the data provided in this description demonstrate that HA-CAR-HNE is capable of exerting a protective effect on different cell types that form the retina, and in particular on rod cells, bipolar cells, Mullerian glial cells, and retinal ganglion cells (RGCs). Rod cells are one of the cell types involved in age-related macular degeneration, whereas retinal ganglion cells (RGCs) are the cell type primarily affected in Leber Hereditary Optic Neuropathy (LHON).

[0099] The presence of HA in the HA-CAR-HNE conjugate confers a number of beneficial properties for the uses described and claimed, comprising:

[0100] • an increased stability and resistance to degradation;

[0101] • an increased adhesiveness, which provides considerable resistance to washout in biological fluids in vivo, including the vitreous humor;

[0102] • in the case of topical application in vivo, a better healing effect and improved local-regional adhesiveness;

[0103] • an improved capacity of reaching the therapeutic target, thanks to the interaction with receptors known for interacting with HA expressed in physiological conditions, and sometimes differentially regulated in the presence of pathologies, at the level of numerous cell types, in different organs and tissues, for example it is known that the HA receptor CD44 is expressed in physiological conditions at the retinal level (Too LK et al., Acta Histochem 2017, 119:142-149), and in human skin, both in normal conditions and in skin tumours (Yasaka N et al., J Dermatol 1995, 22:88- 94).

[0104] At the same time, the data obtained by the Applicant and indicated hereunder reveal how the activity of the CAR-HNE molecule, intended as a protective effect against oxidative stress, is in no way compromised by conjugation with HA. The steric hindrance due to the considerable size of HA could in fact mask the CAR-HNE substituent, compromising its functionality in terms of anti-inflammatory and anti-apoptotic effects. Conversely, the data obtained demonstrate that the HA-CAR-HNE conjugate retains all the antiinflammatory and anti-apoptotic properties of CAR-HNE and is also capable of protecting tissue functionality in the presence of oxidative stress.

[0105] The above considerations, as a whole, support the efficacy of the HA-CAR-HNE conjugate in accordance with the present invention, for use in the prevention and / or treatment of all diseases whose pathophysiology is linked to oxidative stress.

[0106] Brief description of the figures.

[0107] A brief description is provided hereunder of the contents of figures 1-14, which are an integral part of this description.

[0108] FIGURE 1. Densitometry of protein levels detected by Western Blotting of the inflammatory biomarker IL-ip in retinal explants cultured under normoxic or hypoxic conditions, and treated or untreated with CAR-HNE at different doses (1, 10, 100 pM). For each condition, the Optical Density value is indicated, related to IL-ip normalized on the Optical Density detected for P-actin, expressed in Arbitrary Units (A.U.).

[0109] FIGURE 2. Densitometry of protein levels detected by Western Blotting of the inflammatory biomarker IL- 10 in retinal explants cultured under normoxic or hypoxic conditions, and treated or not treated with CAR-HNE at different doses (1, 10, 100 pM). For each condition, the Optical Density value is indicated, related to IL- 10 normalized on the Optical Density detected for P-actin, expressed in Arbitrary Units (A.U.).

[0110] FIGURE 3. Densitometry of protein levels detected by Western Blotting of the apoptosis biomarker active Caspase-3 in retinal explants cultured under normoxic or hypoxic conditions, and treated or not treated with CAR-HNE at different doses (1, 10, 100 pM). For each condition, the Optical Density value of active Caspase-3 is indicated, normalized on the Optical Density detected for P-actin, expressed in Arbitrary Units (A.U.).

[0111] FIGURE 4. Representative scotopic ERG tracings recorded at a light intensity of 10 cd-s / m2in control eyes (CTRL) or in eyes subjected to light damage, in the absence of treatment (DL) or treated with CAR-HNE (CAR-HNE+DL).

[0112] FIGURE 5. Average of the amplitudes detected for the a-wave by effecting scotopic ERG recorded at a light intensity of 10 cd-s / m2, in control eyes (CTRL) or subjected to light damage, in the absence of treatment (DL) or treated with CAR-HNE (CAR-HNE+DL). N=6 eyes per condition.

[0113] FIGURE 6. Average of the amplitudes detected for the b-wave by effecting scotopic ERG recorded at a light intensity of 10 cd-s / m2, in control eyes (CTRL) or subjected to light damage, in the absence of treatment (DL) or treated with CAR-HNE (CAR-HNE+DL). N=6 eyes per condition.

[0114] FIGURE 7. Average of the amplitudes detected for the a-wave by effecting scotopic ERG recorded at a light intensity of 10 cd-s / m2, in control eyes (CTRL) or subjected to light damage, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). N=6 eyes per condition.

[0115] FIGURE 8. Average of the amplitudes detected for the b-wave by effecting scotopic ERG recorded at a light intensity of 10 cd-s / m2, in control eyes (CTRL) or subjected to light damage, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). N=6 eyes per condition.

[0116] FIGURE 9. Representative ERG pattern (PERG) tracings recorded in control (CTRL) or light-damaged eyes, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL).

[0117] FIGURE 10. Average of the amplitudes detected for the Nl-Pl wave by effecting Pattern ERG (PERG) in control eyes (CTRL) or subjected to light damage, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). N=6 eyes per condition.

[0118] FIGURE 11. Average of the amplitudes detected for the P1-N2 wave by effecting Pattern ERG (PERG) in control eyes (CTRL) or subjected to light damage, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). N=6 eyes per condition.

[0119] FIGURE 12. Densitometry of protein levels detected by Western Blotting of the inflammatory biomarker IL-ip in protein extracts of retinas explanted from control (CTRL) or light-damaged animals, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). For each condition, the Optical Density value for IL-ip is indicated, normalized on the Optical Density detected for P-actin, expressed in Arbitrary Units (U. A.).

[0120] FIGURE 13. Densitometry of protein levels detected by Western Blotting of the inflammatory biomarker IL- 10 in protein extracts of retinas explanted from control (CTRL) or light-damaged animals, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). For each condition, the Optical Density value for IL- 10 is indicated, normalized on the Optical Density detected for P-actin, expressed in Arbitrary Units (U. A.).

[0121] FIGURE 14. Densitometry of protein levels detected by Western Blotting of the apoptosis biomarker active Caspase-3 in protein extracts of retinas explanted from control (CTRL) or light-damaged animals, in the absence of treatment (DL) or treated with HA-CAR-HNE (HA-CAR-HNE+DL). For each condition, the Optical Density value related to active Caspase-3 is indicated, normalized on the Optical Density detected for P-actin, expressed in Arbitrary Units (A.U.).

[0122] Some examples are provided for a better illustration of the objectives and advantages of the present invention, which, however, in no way limit the scope of the claims.

[0123] EXAMPLES

[0124] Unless otherwise indicated, commercially available reagents were used in all of the following examples. EXAMPLE 1. Synthesis of carnosine-hydroxynonenal (CAR-HNE).

[0125] 1.2 mg of HNE (generated from 1.9 mg of diethyl acetal in 500 pL of 1 mM HC1) was mixed with 1.8 mg of carnosine in 1.5 mL of phosphate buffer (10 mM, pH 7.4). After 24 hours of incubation at 37°C, the formation of the CAR-HNE product was detected by mass spectrometry and NMR analysis, which was freeze-dried under vacuum.

[0126] EXAMPLE 2. Ex vivo treatment of explanted rat retinas with carnosine-hydroxynonenal (CAR-HNE), under normoxic and hypoxic conditions.

[0127] Sprague Dawley rats (Envigo, San Pietro al Natisone, Italy) were sacrificed by a lethal injection of sodium pentobarbital. The eyes were enucleated and the retinas dissected in Modified Eagle Medium (MEM; Sigma-Aldrich, St. Louis, MO, USA), separated from other ocular tissues using microsurgical procedures. The retinas were transferred and stretched on Millicell-CM inserts (Merck Millipore, Darmstadt, Germany), after having undergone four radial cuts, placing the ganglion cell layer upwards. The retinas were cultured in 6-well plates with 1 ml of basal medium containing 50% MEM (Sigma-Aldrich), 25% of Hank's buffer salt solution (HBSS; Sigma-Aldrich), 25% of Dulbecco's Phosphate Buffered Saline (D-PBS; Sigma-Aldrich), 25 U / mL of penicillin (Sigma-Aldrich), 25 mg / mL of streptomycin (Sigma-Aldrich), 1 pg / mL of amphotericin B (Sigma-Aldrich), and 200 pM L-glutamine (Sigma-Aldrich). CAR-HNE, obtained as described in EXAMPLE 1, was diluted in basal medium to 1, 10, or 100 pM concentrations. The retinas were cultured at 37°C and 5% v / v of CO2. The culture medium was changed every day. The retinas from all of the experimental groups were pre-adapted at 21% v / v of O2 for the first 24 hours, then incubated at 21% (normoxic groups) or 1% (hypoxic groups) v / v O2 for a further 24 hours. In the treated normoxic and hypoxic groups, CAR-HNE was administered for the entire culture period. Hypoxic conditions were achieved using an incubator (Thermo Fisher Scientific, Waltham, MA) in which nitrogen was introduced to reduce the O2 levels. After 48 hours of culture, the retinas were used for subsequent analyses. EXAMPLE 3. Western Blotting analysis of explanted rat retinas treated ex vivo with Camosine-Hydroxynonenal (CAR-HNE), under normoxic and conditions.

[0128] The retinas treated as described in EXAMPLE 2, maintained under normoxic or hypoxic conditions, in the presence or absence of CAR-HNE treatment, were lysed with RIPA lysis buffer (Santa Cruz Biotechnology, Dallas, TX) to which a cocktail of phosphatase and protease inhibitors was added (1:100 dilution; Roche Applied Science, Indianapolis, IN). The concentration of protein extracts was assessed by Micro BCA protein assay (Thermo Fisher Scientific, Waltham, MA). Thirty micrograms of protein per sample were separated by SDS-PAGE using a 4-20% polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and subsequently transferred to nitrocellulose membranes. (Bio-Rad Laboratories). The membranes were blocked with 5% w / v of skimmed milk in a TBS / T buffer (Tris-buffered saline solution with 0.1% v / v of Tween 20 detergent) for 1 hour at room temperature. The membranes were subsequently incubated with a primary antibody solution, including either: anti-IL-ip (1:100 dilution; P420B, Invitrogen, Waltham MA, United States), anti-IL-10 (1:200 dilution; PA5-95524, Invitrogen), anti-active caspase-3 (1:1000 dilution; 9664S, Cell Signaling Technology, Danvers, MA, USA), or anti-P-actin (1:2500 dilution; A2228, Sigma-Aldrich, St. Louis, MO, United States) overnight at 4°C. The membranes were then incubated for 2 hours at room temperature with appropriate secondary antibodies conjugated with horseradish peroxidase (HRP), anti-mouse (1:5000 dilution; A9044, Sigma-Aldrich) or anti-rabbit (1:5000 dilution; 170-6515, Bio-Rad Laboratories, Inc). The membranes were finally developed using a Clarity Western ECL substrate (Bio-Rad Laboratories, Inc) and images were acquired using ChemiDoc XRS+(Bio-Rad Laboratories, Inc). The optical densities of the bands were calculated using Image Lab 3.0 software (Bio-Rad Laboratories, Inc.). The optical densities of the target bands were normalized for the optical densities of P-actin, used as a loading control. The results obtained are indicated in FIGURES 1, 2 and 3.

[0129] As shown in the graph in FIGURE 1, in the absence of treatment, oxidative stress caused by hypoxia causes a marked increase in the protein levels of the pro-inflammatory cytokine IL-ip in the retina compared to the control condition of normoxia, in the absence of treatment. Interestingly, treatment with CAR-HNE according to the present invention has no effect on retinas explanted under normoxia conditions, whereas under hypoxic conditions it determines a dosedependent reduction in the protein levels of the pro-inflammatory cytokine IL-ip, thus contributing to a beneficial reduction in retinal inflammation resulting from the oxidative stress.

[0130] The graph shown in FIGURE 2 reveals a drastic decrease in protein levels of the anti-inflammatory cytokine IL- 10 following oxidative stress caused by hypoxia compared to the normoxic control condition, in the absence of treatment. Further confirming the extraordinary anti-inflammatory effect that CAR-HNE exerts on the retina, FIGURE 2 also shows a dose-dependent increase in protein levels of the anti-inflammatory cytokine IL-10 in retinas subjected to hypoxia and treated with CAR-HNE until reaching IL-10 protein levels equal to those of the control retinas, kept under normoxic conditions and in the absence of treatment. Interestingly, CAR-HNE does not in any way perturb the inflammation mechanisms under physiological conditions, IL- 10 in fact remains at constant levels for all doses of CAR-HNE tested when the treatment was effected on retinas kept under hypoxic conditions.

[0131] As a whole, the evidence reported in FIGURES 1 and 2 demonstrate a surprising and completely unexpected anti-inflammatory effect of CAR-HNE when used according to the present invention, which is extremely effective in counteracting the inflammatory processes that are activated in the retina following oxidative stress caused by hypoxia.

[0132] In accordance with what is indicated in literature, FIGURE 3 reveals how oxidative stress caused by hypoxia causes a considerable increase in cell death by apoptosis, detected as an increase in the protein levels of active Caspase-3, compared to control retinas maintained in normoxia and in the absence of treatment. CAR-HNE, when used in accordance with the present invention, surprisingly does not produce any pro-apoptotic effect on retinas kept under physiological conditions, at any of the concentrations tested (1-10-100 pM), and actually exerts a significant anti-apoptotic effect on retinas subjected to oxidative stress caused by hypoxia. This anti-apoptotic effect is dose-dependent, and for the CAR-HNE dose of 100 pM, very low protein levels of active Caspase-3 are observed, equal to those observed for untreated control retinas maintained under normoxic conditions, demonstrating an extraordinary beneficial effect of CAR-HNE in accordance with the present invention.

[0133] EXAMPLE 4. In vivo model of light damage and treatment with CAR-HNE.

[0134] Sprague Dawley rats were purchased from Envigo (San Pietro al Natisone, Italy), housed in a controlled environment (23°C ± 1°C, 50% ± 5% humidity) with a 12-hour light / dark cycle, and fed with a standard diet (2018 Teklad Global Diets; Envigo, Italy). The animals were anesthetized with 30 mg / kg of sodium pentobarbital intraperitoneally. One eye per animal was subsequently treated with 5 pl of a solution containing 100 pM CAR-HNE (dissolved in a saline solution) administered via intravitreal injection using a Hamilton syringe with a 36-gauge needle, whereas the contralateral eye was untreated and served as a “stressed” control. The rats were then dark-adapted overnight and subjected to light damage (DL), as described hereunder, 24 hours after injection. Immediately before exposure to light, the animals' pupils were dilated with drops of 1% tropicamide (Allergan S.p.A., Rome, Italy). Each animal was housed separately in a small cage and placed in the center of a box (72 * 61 * 52 cm) equipped with LED bulbs. The light intensity in the chamber was measured using a digital luxmeter (Dr. Meter, Ahem Ave, Union City, CA, USA). The rats were exposed for 24 hours to 1000 lux of continuous white light emitted by six LED bulbs. The temperature of the chamber was maintained at 25 ± 1.5 °C using a ventilation cooling system consisting of a small fan. At the end of the damage, the rats were kept in dim light for 48 hours, and then subjected to scotopic ERG as described in the following Example 5.

[0135] EXAMPLE 5. Evaluation of the protective effect of CAR-HNE against light damage on retinal functionality using flash scotopic ERG.

[0136] Before being subjected to flash electroretinogram (ERG) under scotopic conditions, i.e., for a low illumination level, the rats treated and light-damaged as described in EXAMPLE 4, together with a group of non-stressed and untreated control animals, were adapted to total darkness for one night and subsequently anesthetized with an intraperitoneal injection of 30 mg / kg of sodium pentobarbital. Pupil dilation was induced by topical administration of 1% tropicamide (Allergan S.p.A., Rome, Italy). The animals' body temperature was maintained at 37°C using a heating pad. Electroretinographic responses were recorded using corneal electrodes inserted under the eyelids, avoiding visual field obstruction. Ocular drying was prevented by instilling saline solution. Each corneal electrode was referenced to a subcutaneous electrode at the level of the corresponding frontal region. The ground electrode was inserted subcutaneously at the base of the tail. Scotopic responses were induced by flashes of intensity 10 cd-s / m2emitted by a Ganzfeld stimulator (Retimax Advanced; CSO, Florence, Italy) and recorded simultaneously from both eyes, amplified at 1000 gain and filtered with a 0.2-500 Hz bandpass before being digitized at 5 Hz with a data acquisition program (Retimax Advanced; CSO, Florence, Italy). All ERG tracings were analyzed using the same Retimax Advanced software (CSO, Florence, Italy). In accordance with the guidelines of the International Society for Clinical Electrophysiology, the a-wave amplitude was measured from the pre-stimulus baseline to the negative peak of the a-wave, whereas the b-wave amplitude was measured from the negative peak of the a-wave to the positive peak of the b-wave. It should be noted that the amplitude of the a-wave indicates rod activity, whereas the amplitude of the b-wave reflects the activity of bipolar cells and Muller glial cells. Rods, along with cones, represent the photoreceptor cells of the retina, specialized nerve cells capable of converting light stimuli into variations in the membrane potential. Bipolar cells are one of the types of neuronal cells present in the retina that are responsible for collecting and transmitting stimuli from the photoreceptors to the brain, along with amacrine, horizontal, and retinal ganglion cells. Finally, Muller cells are non-neuronal glial cells with mainly trophic and structural functions (Mahabadi N, Al Khalili Y. Neuroanatomy, Retina. [Updated 2023 Aug 8], In: StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK545310 / ). The results obtained are illustrated in FIGURES 4, 5 and 6. FIGURE 4 shows one of the ERG tracings considered representative for each experimental condition (CTRL, untreated control; DL, light damage; CAR-HNE+DL, light damage and treatment with CAR-FINE). For illustrative purposes, the amplitude of the a-wave and that of the b-wave are indicated in the tracing relating to the CTRL condition. In the representative images shown, it is evident that these waves have an extremely reduced amplitude following light damage (DL), demonstrating a significant loss in the functionality of the retinal cells. When light damage is associated with treatment with CAR-HNE (CAR-HNE+DL), in accordance with the present invention, the a and b waves appear surprisingly similar to the CTRL condition, indicating a significant protective effect of CAR-HNE on retinas subjected to light damage. This observation is confirmed by the quantification of the amplitudes of the a-wave in 6 biological replicates per condition: FIGURE 5 shows the average amplitude of the a-wave for each of the three conditions tested and it is evident how the reduction of this parameter following light damage (DL) is effectively counteracted by treatment with CAR-HNE in the CAR-HNE+DL condition. Similarly, FIGURE 6 shows the average amplitude of the b-wave for each of the three conditions tested, from which it emerges that CAR-HNE in the CAR-HNE+DL condition is extraordinarily effective in counteracting the reduction in the amplitude of the b-wave that is observed following light damage (DL) compared to the control condition. (CTRL).

[0137] These findings represent a clear demonstration that CAR-HNE exerts a surprising protective activity in vivo against the functionality of retinas subjected to light damage, a stimulus known to cause intense oxidative stress.

[0138] EXAMPLE 6. Preparation of the Hyaluronic Acid-Camosine-Hydroxynonenal Conjugate (HA-CAR-HNE).

[0139] The hyaluronic acid-camosine conjugate (HA-CAR), having a derivatization degree equal to 24.5%, was prepared as described in Example 3 of patent EP3691691, starting from HA having a weight average molecular weight equal to 200 kDa and CAR protected at the carboxyl group by the formation of a methyl ester, reducing the concentration of N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC HC1) to obtain the desired derivatization degree, as known to persons skilled in the field. 50.0 mg of HA-CAR thus obtained were dissolved in 700 pL of H2O, a solution of 5.86 mg of hydroxynonenal (HNE) dissolved in 700 pL of EtOH was then added to this solution and the reaction was allowed to proceed under magnetic stirring at room temperature for 100 hours and 32 minutes. After removing the solvent under reduced pressure, in order to concentrate and dehydrate the desired product, the residue was triturated with EtOH (700 pL x 6) and dried at 40°C for 48 hours to obtain 50.0 mg of HA-CAR-HNE, which proved to have the appearance of a glassy solid.

[0140] EXAMPLE 7. In vivo model of light damage and treatment with the conjugate HA-CAR-HNE.

[0141] Sprague Dawley rats were purchased from Envigo (San Pietro al Natisone, Italy), housed in a controlled environment (23°C ± 1°C, 50% ± 5% humidity) with a 12-hour light / dark cycle, and fed with a standard diet (2018 Teklad Global Diets; Envigo, Italy). The animals were anesthetized with 30 mg / kg of sodium pentobarbital intraperitoneally. One eye per animal was subsequently treated with 5 pl of a solution containing the HA-CAR-HNE conjugate (prepared as per EXAMPLE 6), in a concentration sufficient for providing a quantity of conjugate equivalent to 100 pM CAR-HNE, dissolved in a saline solution, administered by intravitreal injection, using a Hamilton syringe with a 36-gauge needle. The contralateral eye was not treated and was used as a “stressed” control. The rats were then dark-adapted overnight and subjected to light injury (DL), as described hereunder, 24 hours after injection. Immediately before exposure to light, the animals' pupils were dilated with drops of 1% tropicamide (Allergan S.p.A., Rome, Italy). Each animal was housed separately in a small cage and placed in the center of a box (72 * 61 * 52 cm) equipped with LED bulbs. The light intensity in the chamber was measured using a digital luxmeter (Dr. Meter, Ahem Ave, Union City, CA, USA). The rats were exposed for 24 hours to 1000 lux of continuous white light emitted by six LED bulbs. The temperature of the chamber was maintained at 25 ± 1.5 °C using a ventilation cooling system consisting of a small fan. At the end of the damage, the rats were kept in dim light for 48 hours, and then subjected to ERG as described in the following EXAMPLES 8 and 9.

[0142] EXAMPLE 8. Evaluation of the protective effect of HA-CAR-HNE against light damage on retinal functionality using flash scotopic ERG.

[0143] The rats treated and subjected to light damage as described in EXAMPLE 7, together with a group of unstressed and untreated control animals, were subjected to flash scotopic ERG as described in the previous EXAMPLE 5.

[0144] The results obtained are illustrated in FIGURES 7 and 8.

[0145] FIGURE 7 shows the average amplitude of the a-wave, an index of the rod activity, for each of the three conditions tested (CTRL, untreated control; DL, light damage; HA-CAR-HNE+DL, light damage and treatment with the HA-CAR-HNE conjugate), in 6 biological replicates per condition. It is evident that light damage causes a significant reduction in the amplitude of this wave, demonstrating a significant loss of functionality of the rods present in the retinas of the group of animals subjected to light damage in the absence of pharmacological treatment (DL) compared to the unstressed and untreated control (CTRL). The conjugate object of the present invention, HA-CAR-HNE, proves to be extraordinarily effective in counteracting this loss of functionality; the animals subjected to light damage and treatment with HA-CAR-HNE + DL, in fact, have an average a-wave amplitude equal to more than double that of the DL group, and close to that of the CTRL group. Similarly, FIGURE 8 indicates the average amplitude of the b-wave, which however reflects the activity of bipolar cells and Muller glial cells, for each of the three conditions tested, in 6 biological replicates per condition. Also in this case, HA-CAR-HNE in the HA-CAR-HNE+DL condition proves to be extraordinarily effective in counteracting the reduction in the amplitude of the b-wave, which is observed following light damage (DL) compared to the control condition (CTRL). Taken together, these findings demonstrate that HA-CAR-HNE extraordinarily counteracts the damage caused by a stimulus capable of increasing oxidative stress at the retinal level, helping to preserve its functionality.

[0146] EXAMPLE 9. Evaluation of the protective effect of HA-CAR-HNE against light damage on retinal ganglion cell functionality by Pattern ERG (PERG).

[0147] In order to evaluate the efficacy of HA-CAR-HNE on retinal ganglion cell functionality the rats in EXAMPLE 8 were subsequently subjected to pattern electroretinogram (PERG). This test involves measuring the retinal bioelectrical response to a visual stimulus, which consists of a structured pattern reproduced on a television monitor, featuring black and white bars or checks that alternate in a rhythmic manner over time. The resulting pattern reflects the activity of the innermost retinal layers, i.e., the retinal ganglion cells (RGCs).

[0148] For the PERG recordings indicated herein, a 19" diode display was used, aligned approximately 20 cm from the animal's corneal surface, to provide pattern-reversing stimuli consisting of black and white horizontal bars (spatial frequency: 0.05 cycles / degree; temporal frequency: 1 Hz; contrast: 98%) with an average luminance of 50 cd / m2. After averaging a total of 200 consecutive responses to reduce noise contamination, the PERG waveforms were analyzed by detecting the early negative inflection point (Nl), the positive peak (Pl), and the late negative sulcus (N2) to measure the Nl-Pl and P1-N2 amplitudes, which represent the functionality indices of retinal ganglion cells (RGCs). Representative images of the PERG tracings obtained for each experimental condition are shown in FIGURE 9. It is evident that in the tracing relating to the light damage condition in the absence of treatment (DL), the amplitudes of both the Nl-Pl and P1-N2 components are significantly reduced compared to the untreated and unstressed controls (CTRL). It is also evident that the administration of HA-CAR-HNE in conjunction with light damage (HA-CAR-HNE+DL) determines a significant protection of the amplitude of both the Nl-Pl and P1-N2 components of the PERG response. This evidence is confirmed by the quantification of the amplitudes of the Nl-Pl and P1-N2 components in 6 biological replicates for each condition, indicated in FIGURE 10 and 11 respectively. It is evident, in fact, on observing FIGURE 10, that the Nl-Pl component has an average amplitude almost halved under conditions of light damage (DL) compared to the control (CTRL), and a significant increase is observed compared to DL in the presence of a treatment with HA-CAR-HNE (HA-CAR-HNE+DL). Similarly, FIGURE 11 shows that the average amplitude of the P1-N2 component following light damage (DL) is less than half that observed for the control condition (CTRL), and settles at a value close to the control value when light damage is added to treatment with HA-CAR-HNE (HA-CAR-HNE+DL). These results clearly and unequivocally demonstrate that the HA-CAR-HNE conjugate of the present invention counteracts the damage caused by a stimulus that induces oxidative stress at the retinal level, preserving the functionality of the retina and in particular of the retinal ganglion cells (RGCs), known to play a key role in the collection of visual stimuli and their transmission to the brain. RGCs are also the cellular component of the retina most affected in Leber hereditary optic neuropathy (LHON).

[0149] EXAMPLE 10. Western Blotting analysis on retinas taken from rats subjected in vivo to light damage and treatment with the conjugate HA-CAR-HNE.

[0150] Following the functional analyses described in EXAMPLES 8 and 9 above, the animals were sacrificed by lethal injection of sodium pentobarbital. The eyes were enucleated and the retinas were isolated, from which protein extracts were then prepared. Western blotting experiments were conducted following the same protocol described in EXAMPLE 3. The results obtained are indicated in FIGURES 12, 13 and 14.

[0151] The graph indicated in FIGURE 12 reveals that oxidative stress following light damage (DL) is capable of causing a marked increase in the protein levels of the pro-inflammatory cytokine IL-ip in the retina, which are doubled compared to the unstressed and untreated control condition (CTRL). The animals subjected to light damage and treatment with the HA-CAR-HNE conjugate (HA-CAR-HNE+DL) show a reduction in the protein levels of IL-ip compared to the DL condition, supporting a surprising anti-inflammatory effect of the conjugate HA-CAR-HNE.

[0152] FIGURE 13 shows a drastic decrease in protein levels of the antiinflammatory cytokine IL- 10 following oxidative stress caused by light damage (DL) compared to the unstressed and untreated control condition (CTRL). As further confirmation of the extraordinary anti-inflammatory effect that the HA- CAR-HNE conjugate exerts on the retina, FIGURE 13 shows an increase in protein levels of the anti-inflammatory cytokine IL- 10 in retinas taken from animals subjected to light damage and treatment with HA-CAR-HNE (HA-CAR-HNE+DL).

[0153] Taken together, the evidence indicated in FIGURES 12 and 13 demonstrates a surprising and completely unexpected anti-inflammatory effect of the HA-CAR-HNE conjugate, which is extremely effective in counteracting the inflammatory processes that are activated in the retina following oxidative stress caused by exposure to a strong and prolonged light stimulus.

[0154] Finally, FIGURE 14 reveals how oxidative stress caused by light causes a significant increase in cell death by apoptosis, detected as an increase in the protein levels of active Caspase-3, compared to the untreated and non-stressed control condition (CTRL). The HA-CAR-HNE conjugate according to the present invention surprisingly exerts a significant anti-apoptotic effect on retinas taken from animals subjected to treatment and light damage. (HA-CAR-HNE+DL).

[0155] As a whole, the above evidence demonstrates that the HA-CAR-HNE conjugate according to the present invention is capable of counteracting oxidative stress caused by exposure to an intense, prolonged light stimulus, exerting an antiinflammatory and anti-apoptotic effect that results in protection of the retina from loss of functionality. The fact that HA-CAR-HNE is capable of protecting the functionality of different cell types present in the retina, including rod cells, bipolar cells, Mullerian glial cells, and retinal ganglion cells (RGCs), is particularly interesting and noteworthy.

Claims

CLAIMS1. A pharmaceutical composition comprising, or consisting of, a molecule obtained by the reaction of carnosine with 4-hydroxy-2-nonenal (CAR-HNE) and at least one pharmaceutically acceptable excipient, wherein the CAR-HNE molecule has the following structural formula (1):M(1),optionally comprising hyaluronic acid (HA) or its derivatives in simple association, for use as a medicinal product in the prevention and / or treatment of ophthalmic diseases related to oxidative stress, affecting both the anterior and posterior segment of the eye, preferably in the prevention and / or treatment of diseases such as dry eye, keratoconus, conjunctivitis, uveitis, cataracts, age-related macular degeneration, glaucoma, retinitis pigmentosa, Leber hereditary optic neuropathy (LHON).

2. The pharmaceutical composition for use according to claim 1 wherein the pathology is age-related macular degeneration and Leber hereditary optic neuropathy (LHON).

3. A HA-CAR-HNE chemical conjugate, obtained by reaction of hyaluronic acid (HA), carnosine (CAR) and 4-hydroxy-2-nonenal (HNE), having the following structural formula (5):(5)4. A pharmaceutical, nutraceutical or cosmetic composition, comprising, or consisting of the conjugate according to claim 3, and at least one pharmaceutically acceptable excipient.

5. A conjugate according to claim 3 or pharmaceutical or nutraceutical composition according to claim 4 for use as a medicinal product, preferably for use as a medicinal product in the prevention and / or treatment of pathological conditions related to oxidative stress, preferably related to an imbalance of intracellular redox equilibris, more preferably in the prevention and / or treatment of radiation-induced lung injury, Paraquat poisoning, atherosclerosis, radiation-induced tissue fibrosis (RIF), tissue injury, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), hypertension, type 2 diabetes mellitus, Alzheimer's disease, cancer, systemic inflammatory response syndrome (SIRS), ischemia-reperfusion injury, obesity.

6. A conjugate according to claim 3 or pharmaceutical or nutraceutical composition according to claim 4 for use as a medicinal product in the prevention and / or treatment of inflammatory conditions affecting the joints, resulting from degeneration of the same, preferably in the prevention and / or treatment of Osteoarthritis (OA) and in the treatment of Rheumatoid Arthritis (RA), in the treatment of pathologies directly caused by RA or indirectly related / dependent on RA.

7. A conjugate according to claim 3 or pharmaceutical or nutraceutical composition according to claim 4 for use as a medicinal product in the preventionand / or treatment of ophthalmic diseases, preferably related to oxidative stress affecting any ocular structure, including damage resulting from exposure to molecular oxygen and UV radiation.

8. A conjugate according to claim 3 or pharmaceutical or nutraceutical composition according to claim 4 for use as a medicinal product in the treatment and / or prevention of any ophthalmic disease related to oxidative stress, affecting both the anterior and posterior segment of the eye, including hereditary diseases, such as dry eye, keratoconus, conjunctivitis, uveitis, cataract, age-related macular degeneration, glaucoma, retinitis pigmentosa, Leber hereditary optic neuropathy (LHON).

9. A conjugate according to claim 3 or pharmaceutical or nutraceutical composition according to claim 4 for use as a medicinal product in the treatment and / or prevention of age-related macular degeneration and Leber hereditary optic neuropathy (LHON).

10. A pharmaceutical composition for use according to claims 1 or 2, a conjugate according to claim 3 or pharmaceutical, nutraceutical or cosmetic composition according to claim 4, formulated with excipients, stabilizers, viscosifiers and / or preservatives, in a pharmaceutical form selected from solutions, ointments, gels, suspensions, for topical, oral, systemic and injectable administration, preferably in the form of a solution for intravitreal injection.

11. A pharmaceutical composition for use according to claims 1 or 2, a conjugate according to claim 3 or pharmaceutical, nutraceutical or cosmetic composition according to claim 4, formulated with excipients, stabilizers, viscosifiers and / or preservatives, by impregnation or deposition on gauze, bandages, adhesive dressings, plasters, three-dimensional structures or scaffolds.

12. A conjugate according to claim 3 or pharmaceutical or nutraceutical composition according to claim 4, formulated with excipients, stabilizers, viscosifiers and / or preservatives, by impregnation or deposition on gauze, bandages, adhesive dressings, plasters, three-dimensional structures or scaffolds for medical use, including in the surgical field and for advanced dressings for the treatment of tissue lesions of any nature.

13. Non-pharmaceutical cosmetic use of the HA-CAR-HNE conjugate according to claim 3 or of the composition comprising or consisting of said HA-CAR-HNE conjugate according to claim 4, for preventing and / or treating damage due to an imbalance of the oxidation-reduction equilibria in the body, preferably skin and non-skin damage, linked to excessive solar radiation.

14. A medical device comprising the conjugate according to claim 3 and / or the pharmaceutical or nutraceutical composition according to claim 4, optionally in the presence of at least one excipient, said medical device preferably being a gauze, bandage, adhesive dressing, plaster, three-dimensional structure or scaffold.

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