Chitosan and application thereof

Abstract

The present invention relates to matrix-forming cross-linked carboxyalkyl chitosan, to compositions comprising same, to a process for the preparation thereof and to various uses thereof, in particular in therapeutic, rheumatology, ophthalmology, cosmetic medicine, plastic surgery, open surgery, dermatology, gynecology or cosmetic fields. In particular, the invention relates to a substrate comprising at least one carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units and glucosamine units substituted with a carboxyalkyl group, expressed in the number of moles of N-acetyl groups relative to the number of moles of total glucosamine units, the acetylation degree of the carboxyalkyl chitosan is 40%-80%, and the carboxyalkyl chitosan is crosslinked through covalent bonds between carboxyalkyl chitosan chains.

Description

[0001] This application is a divisional application of the invention patent application filed on May 20, 2020, with application number 202080049342.6 and invention title "Chitosan and its Application". Technical Field

[0002] This invention relates to cross-linked carboxyl chitosan forming a matrix, compositions comprising the cross-linked carboxyl chitosan, methods for preparing the same, and various applications thereof, particularly in the fields of therapeutics, rheumatology, ophthalmology, cosmetic medicine, plastic surgery, open surgery, dermatology, gynecology, or cosmetic surgery. Background Technology

[0003] Chitosan derivatives are known, particularly in the applications and corresponding patents of Kiomed Pharma, disclosed in WO 2016 / 016463 and WO 2016 / 016464. Also known from Kiomed Pharma are advantageous chitosan derivatives, such as carboxyalkyl chitosans described in Kiomed Pharma's patent applications and their families filed PCT / EP2018 / 080763 and PCT / EP2018 / 080767, the contents of which are incorporated herein by reference.

[0004] According to the inventors, it is advantageous to be able to modulate the biomechanical behavior of carboxyl chitosan compositions, or even to increase the durability or efficacy of treatment through the presence of carboxyl chitosan. However, providing such compositions with improved biomechanical properties (especially when it is necessary to prepare hydrogels) is not obvious to those skilled in the art. One of the technical problems with prior art biopolymer compositions (especially hydrogels) known to those skilled in the art is that some compositions are not in the form of cohesive hydrogels; that is, the hydrogel spontaneously decomposes into different parts in the presence of an aqueous medium, thereby forming particles or fragments. This is also referred to as fragmented gels or fragmented hydrogels.

[0005] It has been recognized that when products are implanted into human or animal tissues, such non-cohesive hydrogels carry the risk of long-term inflammatory nodule formation or granulomatous reactions, which is considered undesirable for many medical applications (Bergerey-Galley, Aesth Surf J 24, 33, 2004). Therefore, it is important to obtain compositions in a cohesive hydrogel form that avoids significant fragmentation in terms of the health and safety of subjects or patients.

[0006] Moreover, in some cases, for several reasons, it is desirable to avoid such aggregates in order to improve the aesthetic (visual and / or tactile) appearance of tissues filled with such a composition (which is properly biointegrated into the tissue to allow for homogeneous filling).

[0007] Therefore, for many applications, cohesive hydrogels are preferred, for example, because they remain as a single, monolithic mass when an aqueous medium is added to them. This is also known as a "homogeneous" hydrogel. Furthermore, for most applications, hydrogels, often referred to as "smooth" hydrogels, are preferred because they have little or no visual clumping.

[0008] In addition to cohesiveness, the compositions according to the invention, particularly hydrogels, should be suitable for use in humans or animals, especially in terms of safety, immunocompatibility, bioabsorbability, biomechanical properties, and persistence or duration of activity. However, not all compositions in the prior art satisfactorily exhibit these properties and therefore do not conform to the present invention.

[0009] Various methods for realizing carboxyl chitosan in the form of hydrogels are known. In particular, Rufato et al. (Intechopen 81811, 2018), Upadhyaya et al. (J Controlled Release, 2014), and Fonseca-Santos et al. (Mater Sci Engineering C77, 1349, 2017) have identified several chitosan-based hydrogels containing carboxyl chitosan for medical or pharmaceutical applications. However, none of these hydrogels is what the inventors are looking for, as they do not meet expectations, particularly in terms of cohesiveness, safety, immunocompatibility, biomechanical properties, bioabsorbability, and / or durability or duration of activity. Apart from the compositions of Comed Pharmaceuticals Inc. according to the aforementioned applications PCT / EP2018 / 080763 and PCT / EP2018 / 080767, none of the carboxyl chitosans used in the prior art for preparing known hydrogels exhibits good immunocompatibility, according to the inventors. Not all chitosans can be used to form hydrogels that can be used in humans or animals.

[0010] Currently known chitosan-based hydrogels are prepared by combining chitosan or one of its derivatives with other polymers, such as alginate / ester, isopropylacrylamide, polyurethane, polyacrylonitrile, gelatin, polyethylene glycol (PEG), and polyvinyl alcohol (PVA). However, these polymers are either non-bioresorbable or immunoreactive, which does not meet the objectives of this invention.

[0011] For example, Huang et al. (RCS Adv 2016 DOI:10.1039 / C5RA26160K) prepared a glycol chitosan and hyaluronic acid hydrogel; however, because this glycol chitosan is immunoreactive, it is not suitable for humans. Song et al. (Sci Rep 6, 37600, 2016) prepared a hydrogel based on carboxymethyl chitosan and oxidized hyaluronic acid via a Schiff base reaction between the amino group of carboxymethyl chitosan and the aldehyde of hyaluronic acid. However, in the inventors' experience, the carboxymethyl chitosan used does not have the molecular structure required to satisfy the purposes of this invention. In particular, according to in vitro and in vivo tests, the described hydrogel dissolves very rapidly. Therefore, this hydrogel needs improvement, especially in its durability, for use in a wide range of indications.

[0012] Furthermore, previous products were often not very versatile and could not meet the needs of different indications, especially different therapeutic indications. Therefore, there is a need to provide a product that is sufficiently versatile in terms of performance, especially biomechanical performance, so as to easily adapt it to different applications.

[0013] For example, in regenerative medicine or surgery, the aim is often to repair altered tissue or fluid and / or prevent tissue alteration, fill tissue, or even separate tissue to avoid adhesions. Tissue alterations can be caused by natural aging, external aggression (trauma, UV radiation, surgery, etc.), or pathology (such as inflammation, autoimmune pathology, etc.). However, most tissue alterations involve oxidative stress, sometimes called oxidative stress, characterized by high levels of free radicals that can damage tissues or cells. Reducing the number of free radicals can enable tissues to prevent / delay their aging and reduce their harmful consequences. Several methods exist to reduce the number of free radicals in tissues, such as by administering antioxidants like vitamins C, B, E, and / or ubiquinone. Alternatively, compositions capable of scavenging free radicals can be used, thereby reducing their concentration and proliferation in tissues.

[0014] As listed in the review by Ngo et al. (Adv Food Nutrition Res 73, 15, 2014), and as described for various formulations for biomedical use, chitosan and some of its derivatives exhibit the ability to scavenge oxidative free radicals. For example, particularly as described by Ujang et al. (The Development, Characterization and Application of Water Soluble Chitosan; in Biotechnology of Biopolymers, InTech, 2011. ISBN: 978-953-307-179-4), the ability of carboxymethyl chitosan with different structures and molecular weights to scavenge different types of free radicals was studied using in vitro measurement methods.

[0015] However, it is difficult to provide compositions in therapeutic form to take advantage of the beneficial effects of chitosan, especially its ability to scavenge free radicals, so as to both reduce the effects of oxidative stress on tissues and better modulate the biomechanical behavior of the product, or even increase the durability or efficacy of the treatment through the presence of this exogenous polymer.

[0016] Therefore, the prior art does not readily enable those skilled in the art to provide satisfactory compositions to overcome the problems presented in this invention. Summary of the Invention

[0017] One object of the present invention is to solve the technical problem of providing chitosan derivatives or compositions comprising them suitable for human or animal use (particularly in therapeutic, surgical and cosmetic fields).

[0018] One object of the present invention is to solve the technical problem of providing a therapeutic form of chitosan derivative or a composition comprising it to take advantage of the beneficial effects of chitosan, particularly its ability to scavenge free radicals, thereby reducing the effects of oxidative stress on tissues, better modulating biomechanical behavior, and increasing the durability or efficacy of treatment through the presence of such exogenous polymers.

[0019] In particular, one object of the present invention is to solve the following technical problem: to provide a composition, especially in the form of a bioabsorbable hydrogel, which is particularly suitable for use in contact with human or animal tissues in the context of regenerative medicine or anti-aging medicine (e.g., in the fields of therapeutics, rheumatology, orthopedic surgery, gynecology, ophthalmology, cosmetic medicine, plastic surgery, open surgery, dermatology or plastic surgery), and is acceptable in terms of biomechanical properties, in situ persistence or active time, good health safety (especially no immune response and / or foreign body reaction in the short and long term), and has beneficial effects.

[0020] One object of the present invention is to solve the technical problem of providing compositions having good biomechanical properties (in particular, biomechanical properties that can be adjusted according to their indications).

[0021] One object of the present invention is to solve the technical problem of providing a product based on chitosan derivatives, so as to enable the preparation of a series of products having variable biomechanical properties adapted to each intended indication.

[0022] One object of the present invention is to solve the technical problem of providing a composition that provides, preferably simultaneously, cohesiveness, safety (including immunocompatibility), biomechanical properties, bioreabsorption sufficient for administration in humans or animals, and preferably has suitable persistence or duration of activity.

[0023] One object of the present invention is to solve the technical problem raised in the present invention by providing, in particular, a chitosan derivative or a composition comprising the chitosan derivative or a composition comprising the chitosan derivative having an acceptable grade for humans or animals suffering from the intended indication. Detailed Implementation

[0024] In order to solve the technical problem proposed in this invention, the inventors seek to develop a chitosan that has both good antioxidant properties and good mechanical properties (referred to as biomechanical properties) for intended use in humans or animals.

[0025] The inventors have learned from their own experience the advantages of substituted chitosans, especially carboxyalkyl chitosans. In particular, Comed Pharmaceuticals has filed patent applications under PCT / EP2018 / 080763 and PCT / EP2018 / 080767. They sought to apply these teachings to solve the technical problems presented in this invention.

[0026] The inventors have noted that carboxyl chitosan hydrogels formed by ionic (i.e., non-covalent) crosslinking do not maintain their biomechanical properties sufficiently for some intended applications after implantation; in particular, this technique does not allow for extensive tuning of persistence or activity time. Furthermore, carboxyl chitosan hydrogels formed by enzymatic crosslinking are at risk of enzymatic immunoreactivity due to their protein properties, and complicate the final purification of the resulting crosslinked products.

[0027] Patent application CN 107325306 (Imeik Technology Development) describes the preparation of a crustacean-derived carboxymethyl chitosan-based gel via crosslinking using BDDE in several successive crosslinking steps (multiple crosslinking). However, this method cannot provide a hydrogel that meets the criteria of this invention, particularly because the resulting hydrogel is not cohesive. This is because the hydrogel is formed from particles of crosslinked chitosan derivatives dispersed in a carboxymethyl chitosan solution, which are then crosslinked again to form a gel. The crosslinking operation is repeated several times (“multiple crosslinking”). Such a product is likely to form granulomas, thus negatively impacting immunocompatibility upon contact with human or animal bodies, which is precisely what this invention seeks to avoid. This invention further advantageously enables greater diversity of indications, especially when a cohesive (i.e., to remain as a single block without crumbling, for example, upon contact with water) and / or “smooth” appearance of the hydrogel is required. According to CN107325306, the carboxymethyl chitosan used has low DA (degree of deacetylation of 60-99%, preferably 80-95%, i.e., practically a degree of acetylation (DA) much lower than 40%). Czechowska-Biskup et al. (DOI: 10.15259.PCACD.21.03) also described carboxymethyl chitosan hydrogels with low acetylation. However, these hydrogels are not cohesive and do not meet the objectives of this invention.

[0028] The inventors have discovered that the cross-linked carboxyl chitosan matrix or composition comprising it (especially hydrogels) according to the present invention can solve at least one, preferably all, of the technical problems raised in the present invention.

[0029] Therefore, the present invention relates to a matrix comprising at least one carboxyl chitosan having glucosamine units, N-acetylglucosamine units and carboxyl-substituted glucosamine units, expressed as the number of N-acetylated moles relative to the total number of glucosamine units, wherein the degree of acetylation of the carboxyl chitosan is 40%-80%, and the carboxyl chitosan is cross-linked by covalent bonds between carboxyl chitosan chains.

[0030] In fact, it has been found that cross-linked carboxyl chitosan with a DA of less than 40% cannot produce hydrogels with the desired cohesiveness because it breaks into separate fragments during wetting, which is undesirable for many applications.

[0031] According to the present invention, a cohesive hydrogel is understood to be a hydrogel that maintains its cohesiveness by adjusting methods conventionally used to characterize hydrogels for intradermal use, such as those described by Micheels et al. (J Clin Aesth Dermatol 10, 29, 2017 and J Drugs Dermatol 15, 1092, 2016), according to the following cohesiveness test known as the “water test”: Place 1 g of the hydrogel to be tested in the center of a 5 cm diameter glass Petri dish. Add 1 ml of distilled water to the periphery of the dish. Gently agitate the Petri dish until the water covers the hydrogel and then return it to a horizontal position. Immediately after the substrate comes into contact with the water, preferably 15 to 25 seconds after contact, and preferably at least 30 seconds after contact, observe whether the hydrogel remains intact (i.e., forms a single unit when it is cohesive) or spontaneously separates into distinct parts or forms visible particles when it is non-cohesive.

[0032] Furthermore, it has been advantageously found that the matrix according to the invention is capable of scavenging free radicals. Maintaining this chitosan property is far from obvious to those skilled in the art. While it is known that the molecular structure (DS) and molecular weight of carboxyl chitosans affect their ability to scavenge free radicals, conflicting results have been disclosed. Therefore, it is unclear whether cross-linked carboxyl chitosans would exhibit the ability to scavenge free radicals.

[0033] Furthermore, the hydrogel according to the invention exhibits such antioxidant activity while also possessing appropriate cohesiveness, biomechanical properties, durability, and safety.

[0034] Furthermore, it is unclear in the prior art whether the cross-linked carboxyl chitosan formulated into a hydrogel is cohesive, preferably smooth (i.e., without obvious, visible, or tactilely perceptible fragments), and possesses suitable safety, particularly in terms of immunocompatibility, biomechanical properties, and durability. The present invention can provide such a matrix or composition, particularly in the form of a hydrogel. Regarding an immunocompatible cross-linked matrix, i.e., a non-immunoreactive cross-linked matrix that substantially does not activate an immune response, it should be prepared from at least one or more non-immunoreactive polymers. The non-immunoreactive nature of the polymer is verified using specific and standardized tests, such as human whole blood tests (in vitro) and subcutaneous injection into mouse air sacs.

[0035] Acceptably, the hydrogel formed from the matrix according to the invention is not perfectly smooth and has, for example, visible or palpable clumps, provided that it is cohesive according to the above water test.

[0036] The matrix according to the invention is characterized by starting with carboxyl chitosan, which is crosslinked to form the matrix according to the invention.

[0037] According to the first aspect, a fungal-derived carboxyl chitosan is used, which has carboxyl-substituted glucosamine units, N-acetylglucosamine units and glucosamine units, expressed as the number of moles of substituents relative to the total number of units, wherein the carboxyl chitosan preferably has a degree of substitution of more than 20% of carboxyl groups.

[0038] This is also known as chitosan derivatives or substituted chitosan.

[0039] Carboxyalkyl chitosans are prepared by substitution of chitosan. Typically, carboxyalkyl chitosans are prepared according to patent applications filed by Commodity Pharmaceuticals under PCT / EP2018 / 080763 and its families (especially FR 17 61314 and EP 18799772.1) and PCT / EP2018 / 080767 and its families (especially FR 1761323 and EP 18799773.9), which are specifically incorporated herein by reference to illustrate the preparation of carboxyalkyl chitosans.

[0040] Chitosan, for example, is the chitosan represented by CAS number 9012-76-4.

[0041] The chitosan used in this invention is advantageously derived from fungi, and preferably from ascomycetes. Ascomycete Mycelium of certain types of fungi, especially Aspergillus niger Aspergillus piger and / or basidiomycetes Basidiomycete Fungal mycelium, especially shiitake mushrooms Lentinula edodes (Shitak mushroom) and / or button mushroom Agaricus bisporus （ White mushroom). Preferably, the chitosan is derived from button mushrooms. Agaricus bisporus Chitosan is preferably of high purity, meaning it contains very few impurities from its fungal origin or from the preparation process, and has a microbiological grade commensurate with its use as an implant or pharmaceutical composition. One method for preparing chitosan is described in patent WO 03 / 068824 (EP 1483299; US 7 556946).

[0042] Typically, chitosan is suspended in an aqueous medium in the presence of sodium hydroxide, and then the medium is heated at a high temperature for a variable time, depending on the desired molecular weight. Chitosan is then dissolved in an acidic medium for purification, precipitated in an alkaline medium, washed, and dried.

[0043] Preferably, the chitosan has a sufficient purity grade for pharmaceutical use.

[0044] Chitosan is advantageously purified and then preferably dried. After purification, the method of the present invention may include the step of drying carboxyl chitosan, which may then optionally be ground into powder. Carboxyl chitosan can be dried, for example, by evaporating water, such as by spray drying (atomization), fluidized bed methods, or by heating under vacuum or atmospheric pressure, or by freeze-drying.

[0045] Carboxyalkyl chitosan can be dissolved in aqueous solutions, such as pharmaceutical-grade water suitable for injection or implantation, particularly in the human body.

[0046] The carboxyalkyl chitosan is then crosslinked to prepare the matrix according to the invention.

[0047] The DA and DS of cross-linked carboxyl chitosan can be expressed as functions of the DA and DS of uncross-linked carboxyl chitosan because DA and DS remain essentially unchanged after cross-linking. However, if the cross-linking agent provides N-acetyl or carboxyl groups, these foreign groups are not included in the DA and DS of the cross-linked carboxyl chitosan compared to the initial uncross-linked carboxyl chitosan. As described below, the values ​​of DA and DS are known to those skilled in the art. Therefore, DA and DS refer to both before and after cross-linking.

[0048] The degree of acetylation (DA) of chitosan is determined by potentiometric titration, as described, for example, in patent applications WO2017009335 and WO2017009346. Alternatively, DA can be determined by other known methods for chitosan, such as liquid-phase proton nuclear magnetic resonance, solid-phase carbon-13 nuclear magnetic resonance, and infrared spectroscopy.

[0049] Advantageously, the degree of acetylation of the carboxyalkyl chitosan is between 40% and 80%, expressed as the molar number of N-acetylglucosamine units relative to the total number of units. The degree of acetylation is expressed as the number of N-acetyl groups (of D-glucosamine units) relative to the number of total glucosamine units (N-acetyl-D-glucosamine, substituted N-acetyl-D-glucosamine, D-glucosamine, and substituted D-glucosamine) present in the chitosan.

[0050] Advantageously, the degree of acetylation of carboxyalkyl chitosan, expressed as the number of N-acetyl groups relative to the total number of glucosamine units, is between 40% and 80%.

[0051] According to one alternative embodiment, the degree of acetylation ranges from 40% to 50%. According to one alternative embodiment, the degree of acetylation ranges from 50% to 60%. According to one alternative embodiment, the degree of acetylation ranges from 60% to 75%.

[0052] The degree of acetylation of carboxyl chitosan can be determined by solid-phase carbon-13 NMR, solid-phase carbon-13 NMR, or liquid-phase proton NMR. This carboxyl chitosan advantageously possesses a controlled degree of acetylation. The term "chitosan with a controlled degree of acetylation" refers to a product whose degree of acetylation (i.e., the ratio of N-acetyl-glucosamine units) can be adjusted in a controlled manner, particularly through an acetylation reaction.

[0053] Preferably, the carboxyl chitosan is reacetylated.

[0054] According to an alternative embodiment, a method for preparing the carboxyalkyl chitosan according to the invention includes preparing a fungal-derived chitosan, reacetylifying the chitosan, and carboxylating the reacetylated chitosan. Therefore, the present invention relates to a reacetylated carboxyalkyl chitosan. In particular, the present invention relates to an anionic carboxyalkyl chitosan.

[0055] According to one embodiment, chitosan can be dissolved in an aqueous, preferably slightly acidified medium (e.g., pH 6). Acetic anhydride may be added to the chitosan solution in one or more steps. An alkaline reagent, such as sodium bicarbonate and / or urea, is then added. An alkylating agent, such as sodium monochloroacetate (i.e., the sodium salt of chloroacetic acid) or chloroacetic acid, is then added. The substituted chitosan is then purified, recovered, and dried.

[0056] According to an alternative embodiment, a method for preparing carboxyalkyl chitosan according to the invention includes preparing chitosan, carboxylating the chitosan, and subsequently reacetylifying the carboxylated chitosan. Advantageously, this method allows for precise control of the degree of acetylation of the final carboxyalkyl chitosan, to obtain, in particular, a high degree of acetylation, for example, more than 40%. Therefore, the present invention relates to a reacetylated and then carboxylated chitosan or a reacetylated carboxyalkyl chitosan.

[0057] According to an alternative embodiment, a method for preparing carboxyalkyl chitosan according to the invention comprises preparing a fungal-derived chitin, carboxylating the chitin, and optionally reacetylating the carboxylated chitin to obtain the carboxyalkyl chitosan according to the invention.

[0058] According to an alternative embodiment, a method for preparing carboxyalkylated chitosan according to the invention comprises preparing chitin of fungal origin, deacetylifying the chitin, carboxylating the chitin, and optionally reacetylifying the carboxylated chitin to obtain the carboxyalkylated chitosan of the invention.

[0059] According to an alternative embodiment, the average molecular weight of carboxyalkyl chitosan is less than 400,000.

[0060] According to one embodiment, the average molecular weight is between 20,000 and 60,000.

[0061] According to another embodiment, the average molecular weight is between 60,000 and 120,000.

[0062] According to another embodiment, the average molecular weight is between 100,000 and 400,000.

[0063] According to another embodiment, the average molecular weight is between 120,000 and 400,000.

[0064] According to another embodiment, the average molecular weight is between 180,000 and 400,000.

[0065] Preferably, in this document, the average molecular weight is the viscosity-average molecular weight (Mv) calculated based on the intrinsic viscosity. This representation is customary for those skilled in the art. The intrinsic viscosity (η) is measured by capillary viscometry using a capillary viscometer of the Ubbelohde type, according to the method described in Monograph 2.2.9 of the European Pharmacopoeia. The flow time of the solution is measured using an automated I-Visc viscometer (Lauda) through a suitable capillary (Lauda, ​​e.g., a Ubbelohde 510 01 capillary with a diameter of 0.53 mm). The Mark-Houwink equation (η = ...) is then applied. K * Mv α To calculate the average viscosity mass of carboxyl chitosan, where: Mv is the viscosity-average molecular weight of carboxyalkyl chitosan. η is the intrinsic viscosity of carboxyalkyl chitosan. As previously determined using size exclusion chromatography with a MALLS detector for (unsubstituted) chitosan, the constants K and α were 0.0686 and 0.7638, respectively.

[0066] Therefore, the intrinsic viscosity of carboxyalkyl chitosan can usually be used to represent it.

[0067] Chitosan can be hydrolyzed to reduce its molecular weight.

[0068] Typically, in uncrosslinked carboxyalkyl chitosan, the glucosamine unit is a D-glucosamine unit (at least one of D-glucosamine unit, N-acetyl-D-glucosamine unit, and substituted D-glucosamine unit and N-acetyl-D-glucosamine unit).

[0069] According to an alternative embodiment, the substituted chitosan has only the substitution of D-glucosamine units.

[0070] According to another alternative embodiment, the substituted chitosan has substitutions for both D-glucosamine and N-acetyl-D-glucosamine units, wherein, according to an alternative embodiment with only the amino group of chitosan, or according to another alternative embodiment with both amino and hydroxyl groups of chitosan, the carboxyl groups are covalently bonded.

[0071] Replacement is usually only partial; not all units need to be replaced.

[0072] According to one embodiment, the molar number of D-glucosamine units is expressed relative to the molar number of total units of the substituted chitosan (substituted or unsubstituted D-glucosamine units and N-acetyl-D-glucosamine units), and the degree of substitution of the D-glucosamine units is 30%-250%.

[0073] According to one embodiment, the carboxyalkyl chitosan has a degree of substitution of carboxyalkyl groups greater than 20%, for example greater than 50%, and for example less than 200%, expressed as the number of moles of substituents relative to the total number of units.

[0074] According to one embodiment, the degree of substitution of the carboxyl group is greater than 50%, expressed as the number of moles of substituents relative to the total number of units.

[0075] According to one embodiment, the molar number of D-glucosamine units is expressed relative to the molar number of total units of the substituted chitosan (substituted or unsubstituted D-glucosamine units and N-acetyl-D-glucosamine units), and the degree of substitution of the D-glucosamine units is in the range of 50%-200%, and more preferably higher than 70%.

[0076] According to one embodiment, the degree of substitution of the carboxyl group is less than 80%, expressed as the number of moles of substituents relative to the total number of units.

[0077] Typically, substitution is achieved through covalent bonding.

[0078] According to an alternative embodiment, the carboxyl chitosan is an N,O-carboxyl chitosan. The proportion of units substituted with carboxyl groups at the O-position (O3 or O6 of the glucosamine unit and / or N-acetyl-glucosamine unit) and / or at the N-position (of the glucosamine unit) can vary. Therefore, the degree of substitution can be greater than 100%.

[0079] Advantageously, the degree of substitution (DS) and degree of acetylation (DA) of carboxyl chitosan were measured by solid-phase carbon-13 NMR using a Bruker spectrometer (AvanceIII HD 400MHz) equipped with a PH MAS VTN 400SB BL4 NP / H probe. For example, spectra were recorded at room temperature, relaxation times between 1 and 8 seconds, and scan numbers between 64 and 512. The area of ​​the carbon signal was determined after deconvolution. The carbons considered were: “CH3acetyl” (the methyl carbon of the acetyl group of the substituted or unsubstituted N-acetyl-glucosamine unit), “C…”, and “C…”. X "(The x-position carbon of glucosamine and N-acetyl-glucosamine units, where x ranges from 1 to 6)" and "C=O" (the carbonyl carbon of the carboxyl substituent and the C=O carbonyl carbon of the acetyl group of the substituted or unsubstituted N-acetyl-glucosamine unit). To determine the DS of a given carboxyl chitosan, the carbon-13 NMR spectrum of the precursor chitosan of that carboxyl chitosan should also be recorded. Based on the spectrum of the precursor chitosan, the "CSU ratio" is calculated, which is the ratio of the signal area of ​​the "CH3acetyl" group (the methyl carbon of the acetyl group of the N-acetyl-glucosamine unit) to the signal area of ​​the "C=O" (the carbonyl carbon of the acetyl group of the N-acetyl-D-glucosamine unit). The DA of the carboxyl chitosan is calculated according to Equation 1, and the DS is calculated according to Equation 2, where I represents the signal area of ​​the carbon considered.

[0080] Formula 1: [Mathematical Expression 1]

[0081] Formula 2: [Mathematical Expression 2]

[0082] Other known methods can be used to determine the DA and DS of carboxyl chitosan, such as by proton NMR in an aqueous medium using magnetic resonance spectroscopy, for example, according to the method described by Liu et al. (Carb Polym 137, 600, 2016), for example, by adding a concentrated solution of deuterated hydrochloric acid to pre-hydrolyze the carboxyl chitosan before analysis.

[0083] If an alternative NMR method is more advantageous for reliably estimating DA and / or DS, then this method is suitable. The above method should be adjusted by a technician relative to sample preparation and the signal to be integrated, particularly regarding the resolution, robustness, and proton position of the signal used to calculate the degree of substitution.

[0084] The degree of carboxylation of chitosan can advantageously be in the range of 20-250%, preferably in the range of 50-200%, and for example in the range of 70-170%, expressed as the number of moles of carboxyl groups relative to the total number of units.

[0085] According to an alternative embodiment, the degree of carboxylation of chitosan can advantageously be in the range of 40-130%, for example, in the range of 70-130%, expressed as the number of moles of carboxyl groups relative to the total number of units.

[0086] The degree of substitution of chitosan is generally related to the mass ratio of reactants to chitosan at the start of the reaction. Examples of carboxylalkylating agents include acyl chlorides (or their salts, such as sodium monochloroacetate), such as those with one or more carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl, or other similar groups.

[0087] According to an alternative embodiment, the present invention relates to a carboxyl chitosan, wherein the alkyl portion of the carboxyl group is a straight-chain or branched C1-C5 group.

[0088] According to one embodiment, the present invention relates to carboxymethyl chitosan.

[0089] According to this alternative embodiment, the substituted chitosan is N-carboxyalkyl chitosan.

[0090] According to this embodiment, the substituted chitosan is O-carboxyalkylated chitosan.

[0091] According to this alternative embodiment, the substituted chitosan is N-carboxyalkylated and O-carboxyalkylated chitosan.

[0092] According to a second aspect, the present invention relates to a chitosan derivative having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with carboxyl groups, wherein the carboxyl chitosan has a zeta potential less than or equal to -10 mV, preferably less than or equal to -15 mV, measured at pH 7.5. In particular, this chitosan derivative is capable of limiting the immune response in subjects who are typically administered the chitosan derivative or compositions comprising it via infusion, injection, or implantation.

[0093] Advantageously, the zeta potential measured at pH 7.5 is less than or equal to -18 mV.

[0094] Advantageously, the carboxyl chitosan has a zeta potential of less than or equal to -22 mV, preferably less than or equal to -24 mV, as measured at pH 7.5.

[0095] According to a specific alternative embodiment, the substituted chitosan preferably has an average molecular weight of 150,000 to 220,000 and a degree of substitution of 50% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0096] According to another specific alternative embodiment, the substituted chitosan preferably has an average molecular weight of 120,000 to 150,000 and a degree of substitution of 70% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0097] According to a specific alternative embodiment, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000 and a degree of substitution of 70% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0098] According to another specific alternative embodiment, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution of 50% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0099] According to another specific alternative embodiment, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution of 50% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0100] According to another specific alternative embodiment, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution of 70% to 200%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0101] According to another specific alternative embodiment, the substituted chitosan preferably has an average molecular weight of 120,000 to 150,000 and a degree of substitution of 20% to 50%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0102] According to another specific alternative embodiment, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution of 20% to 50%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0103] According to another specific alternative embodiment, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution of 20% to 50%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0104] According to a specific alternative implementation, the substituted chitosan has a degree of substitution of 20% to 80%, preferably 40% to 60%, and a degree of acetylation of 40% to 80%, preferably 50% to 75%.

[0105] According to a specific alternative implementation, the substituted chitosan has a degree of substitution of 50% to 200%, preferably 70% to 200%, and a degree of acetylation of 40% to 80%, preferably 50% to 75%.

[0106] According to another specific alternative embodiment, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 40% to 80%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0107] According to a specific alternative implementation, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 40% to 60%, preferably 50% to 60%.

[0108] According to a specific alternative embodiment, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 50% to 75%.

[0109] According to a specific alternative embodiment, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000, a degree of substitution of 90% to 200%, preferably 90% to 150%, and a degree of acetylation of 50% to 75%, wherein the molecular weight preferably represents the molecular weight before substitution.

[0110] By substituting chitosan, solutions of carboxyl-based chitosans can be prepared that are soluble in aqueous solutions with a wide pH range, whereas unsubstituted chitosans are only soluble at pH values ​​below 5.5–6.5. Therefore, due to the altered solubility profile caused by the presence of carboxyl groups, carboxyl-based chitosans exhibit the ability to dissolve at different pH values, particularly at physiological pH values ​​or at pH values ​​altered by pathological conditions (e.g., inflammatory pathology).

[0111] The term "water-soluble" means that when carboxyl chitosan is placed in an aqueous solution, no visible turbidity is observed. More specifically, the solubility (i.e., absence of turbidity) of a carboxyl chitosan solution at a concentration of, for example, 1% (m / m) in water or a buffer solution (e.g., phosphate buffer) can be confirmed by an optical density of less than 0.5, preferably less than 0.2, where the optical density is measured at a wavelength of 500 nm by UV-Vis spectroscopy relative to a reference cell containing only the aqueous solvent used for the measurement sample and without substituted chitosan. Another method is visual inspection according to Monograph 2.9.20 of the European Pharmacopoeia. When the chitosan is not sufficiently substituted, the composition is insoluble at room temperature within a satisfactory pH range (e.g., pH 5.5 to pH 8.5).

[0112] According to one implementation, the carboxyl chitosan is sterile.

[0113] The term "crosslinking via covalent bonds between carboxyl chitosan chains" is specifically understood to mean that the chitosan backbone (also called the chitosan skeletal chain) is covalently bonded to one or more main chitosan chains. Advantageously, this yields a three-dimensional network of chitosan molecules. The invention is not limited to a specific covalent crosslinking method, but methods using chemical molecules (also called crosslinking agents) as crosslinking agents are preferred.

[0114] According to the present invention, carboxyl chitosan is cross-linked.

[0115] According to an alternative embodiment, crosslinking is formed by a crosslinking agent that forms the covalent bonds.

[0116] Therefore, several chitosan chains can be cross-linked, for example, by reacting with one or more cross-linking agents, such as those selected for cross-linking polysaccharides, including 1,4-butanediol diglycidyl ether, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxypentane, 1-chloro-2,3-cyclothiopropane, 1-bromo-2,3-cyclothiopropane, 1-bromo-3,4-cyclothiobutane, 1-bromo-4,5-cyclothiopentane, and 2,3-dibromopropanol. 2,4-Dibromobutanol, 2,5-Dibromopentanol, 2,3-Dibromopropanethiol, 2,4-Dibromobutanethiol, 2,5-Dibromopentane-thiol epichlorohydrin, 2,3-Dibromopropanol, 1-chloro-2,3-cyclothiopropane, dimethylaminopropylcarbodiimide, gallic acid, epigallocatechin gallate, curcumin, tannic acid, genipin, or even diisocyanate compounds, such as hexamethylene diisocyanate or toluene diisocyanate, or even divinyl sulfone.

[0117] Genipin is a naturally occurring cross-linking agent used to cross-link polysaccharides, particularly carboxymethyl chitosan (Yang et al. Acta Pharmacol Sin, 31, 1625, 2020). Genipin gives hydrogels a deep blue to black color, which may be an advantage in some indications.

[0118] Preferably, the crosslinking agent is a polyepoxide type, such as a bifunctional reagent. 1,4-Butanediol diglycidyl ether (BDDE) or ethylene glycol diglycidyl ether (EGDE) is preferred as the crosslinking agent because they have been used to prepare biomaterials for human use, particularly hyaluronic acid hydrogels for intradermal, intra-articular, or intraocular administration. According to an alternative embodiment, the crosslinking agent is diethylene sulfone.

[0119] Advantageously, the compositions of the present invention may also include biopolymers other than cross-linked carboxyalkyl chitosan. According to an advantageous alternative, the biopolymer is an oxidized or unoxidized, covalently cross-linked or uncross-linked polysaccharide, such as glycosaminoglycans, and in particular hyaluronic acid, such as hyaluronic acid or sodium hyaluronate.

[0120] The advantage of combining or crosslinking crosslinked carboxyl chitosan with other polymers is that it increases their biological and physicochemical properties, or even produces synergistic effects.

[0121] According to an alternative embodiment, the matrix according to the invention comprises cross-linked carboxyl chitosan and hyaluronic acid, chondroitin sulfate, and / or carboxymethyl cellulose. To date, there are no hydrogels combining cross-linked carboxyl chitosan (as defined in this invention) with hyaluronic acid. One object of the invention is to combine these two polymers to combine, for example, the well-known moisturizing properties of hyaluronic acid with the protective properties against oxidative stress of chitosan.

[0122] According to an alternative embodiment, the matrix comprises at least one hyaluronic acid.

[0123] Advantageously, the matrix according to the invention comprises cross-linked carboxymethyl chitosan alone or cross-linked carboxymethyl chitosan combined with cross-linked or uncross-linked hyaluronic acid. This allows for the adjustment of desired properties.

[0124] The matrix includes at least one carboxymethyl chitosan and hyaluronic acid.

[0125] According to an alternative embodiment, such as as determined by capillary viscosity, the bioaverage molecular weight of hyaluronic acid is less than 5 million, and preferably greater than 1 million, more preferably greater than 2 million. The molecular weight of hyaluronic acid is sometimes expressed by its density, as they are correlated by a linear relationship. Hyaluronic acid can have a molecular weight as high as 4.25 m... 3 The density is specified as per kg, and can be, for example, specified as having a low density (e.g., about 1 to 2 m³). 3 / kg) hyaluronic acid or high-density (e.g., about 2 to 4 m) 3 / kg) hyaluronic acid.

[0126] According to one alternative embodiment, hyaluronic acid is obtained through fermentation (e.g., using Streptococcus). According to another alternative embodiment, it is produced by extraction from rooster peaks.

[0127] According to an alternative embodiment, the matrix comprises at least one hyaluronic acid cross-linked by covalent bonds.

[0128] Therefore, cross-linked hyaluronic acid includes covalent bonds between different hyaluronic acid chains.

[0129] Different types of hyaluronic acid (such as hyaluronic acid of different molecular weights or different hyaluronic acid salts) can cross-link with each other.

[0130] This invention also relates to a method for preparing cross-linked carboxyl chitosan.

[0131] According to an alternative embodiment, a method for preparing the matrix according to the present invention includes: Carboxyalkyl chitosan is brought into contact with at least one crosslinking agent, preferably in an alkaline aqueous phase; Crosslinking carboxyl chitosan with a crosslinking agent; and A matrix comprising cross-linked carboxyl chitosan was obtained.

[0132] According to an alternative embodiment, the carboxyl chitosan is crosslinked in an alkaline aqueous phase (e.g., in the presence of a sodium hydroxide (NaOH) solution).

[0133] Advantageously, the concentration of carboxyl chitosan initially present in the aqueous phase is in the range of 1%-30% (w / v), preferably in the range of 5%-20% (w / v), based on the weight of carboxyl chitosan relative to the volume of the alkaline aqueous phase.

[0134] Advantageously, the weight of the crosslinking agent is expressed relative to the weight of the polymer, with a mass ratio of 0.1% to 30% between the crosslinking agent and the polymer.

[0135] Preferably, the weight of the crosslinking agent is expressed relative to the weight of the polymer, and in particular, when BDDE is used, the mass ratio between the crosslinking agent and the polymer is 0.5%-20%.

[0136] Typically, the reaction is carried out under heat (e.g., at 25-60°C, such as 50°C) for a duration of, for example, 30 minutes to 48 hours, or, for example, 1 hour to 5 hours. Crosslinking is usually stopped by neutralization and dilution, for example, by adding an acid, such as acetic acid or hydrochloric acid.

[0137] Advantageously, phosphate buffer is used to remove reaction residues by dialysis.

[0138] This results in a hydrogel comprising the matrix of the present invention.

[0139] On the other hand, carboxyalkyl chitosan is an exogenous molecule that is more resistant to degradation than hyaluronic acid after implantation / injection / infusion into the body.

[0140] Therefore, the present invention relates to a matrix comprising a three-dimensional network based on two polymers with different molecular weights. Advantageously, this provides a range of biomechanical properties, in-situ product duration, and therapeutic duration, while retaining the free radical scavenging ability of carboxyalkyl chitosan.

[0141] The present invention relates to a matrix comprising at least one hyaluronic acid, which is co-crosslinked with carboxyl chitosan via covalent bonds.

[0142] According to an alternative embodiment, a method for preparing a matrix comprising a carboxyl chitosan (preferably a carboxyl chitosan as defined according to the invention) co-crosslinked with another biopolymer (preferably hyaluronic acid) includes: A mixture of carboxyl chitosan and other biopolymers (preferably hyaluronic acid) is contacted with at least one crosslinking agent, preferably in an alkaline phase; Carboxyalkyl chitosan and other biopolymers (preferably hyaluronic acid) are cross-linked with a cross-linking agent. A co-crosslinked matrix of carboxyl chitosan and other biopolymers (preferably hyaluronic acid) is obtained.

[0143] According to an alternative embodiment, the substrate according to the invention is sterile.

[0144] It is advantageous to provide a hydrogel derived from the matrix according to the invention.

[0145] Therefore, the present invention relates to a hydrogel, and advantageously forms a cohesive hydrogel.

[0146] Therefore, the present invention relates to cross-linked carboxyl chitosan hydrogels, wherein the carboxyl chitosan has a high degree of acetylation (DA) (greater than 40%) and preferably also has a high degree of substitution (DS) (greater than 20%, preferably greater than 50%, and typically less than 200%).

[0147] The present invention relates to a composition comprising at least one matrix as defined in the present invention.

[0148] According to a preferred alternative embodiment, a composition according to the matrix of the invention is formulated in an aqueous medium to form a hydrogel.

[0149] Advantageously, the concentration of the polymer (carboxyalkyl chitosan with or without other biopolymers such as hyaluronic acid) is less than 10%, for example less than or equal to 5%, based on the mass of the polymer relative to the total mass of the composition (especially the total mass of the hydrogel) (m / m).

[0150] According to an alternative embodiment, the concentration of the polymer (carboxyalkyl chitosan with or without other biopolymers such as hyaluronic acid) is less than 4%, for example less than or equal to 3%, based on the mass of the polymer relative to the total mass of the composition (particularly the total mass of the hydrogel) (m / m).

[0151] The mass ratio (m / m) [carboxyalkyl chitosan / hyaluronic acid] is, for example, 5%-95%, for example, 10%-90%, and further for example, 30%-70%. The mass ratio (m / m) [hyaluronic acid / carboxyalkyl chitosan] is, for example, 5%-95%, for example, 10%-90%, and further for example, 30%-70%. According to an alternative embodiment, the mass ratio (m / m) [carboxyalkyl chitosan / hyaluronic acid] is 1:1 (i.e., 50% chitosan and 50% hyaluronic acid).

[0152] The aqueous medium can be water or an aqueous solution, the pH of which and the permeability are adjusted, for example, using an acid / base buffer system with added salt and / or optional polyols (sorbitol, mannitol, glycerol).

[0153] According to an alternative embodiment, the matrix according to the invention is formulated in a hydrolipidic medium to form a single or multiple, direct or inverse emulsion.

[0154] According to one embodiment, the permeability of the matrix composition is 100 mosm / kg-700 mosm / kg, preferably 120 mosm / kg-500 mosm / kg.

[0155] Advantageously, the composition of the matrix has a permeability of 250 mosm / kg-400 mosm / kg, preferably 270 mosm / kg-330 mosm / kg.

[0156] According to an alternative embodiment, the matrix composition has a permeability suitable for joints.

[0157] According to an alternative embodiment, the matrix composition has permeability compatible with the surface of the eye or the inner eye.

[0158] According to an alternative embodiment, the matrix composition has permeability compatible with dermis or mucous membranes.

[0159] According to an alternative embodiment, the composition of the matrix preferably has a permeability between 100 mosm / kg and 400 mosm / kg, more specifically between 120 mosm / kg and 380 mosm / kg.

[0160] According to an alternative embodiment, the composition according to the invention is sterile.

[0161] Advantageously, the composition according to the invention is contained in an injection, implantation or infusion device (e.g., a syringe or vial).

[0162] Advantageously, the injection device (such as a syringe) can then undergo steam sterilization, for example. Such a device (such as a syringe) can then preferably be packaged in a sterile or aseptic manner. It can also be a bag, ampoule, or vial for instilling the composition according to the invention, which is aseptically filled after sterilization of the formulation, or sterilized directly after filling.

[0163] According to an alternative embodiment, the composition according to the invention, particularly the hydrogel according to the invention, is sterilized by filtration and / or steam sterilization before being filled into an injection device, implantation device, or infusion device (such as a syringe or vial).

[0164] Those skilled in the art know the techniques for sterilizing hydrogels to obtain the desired sterile hydrogel. They have several types of equipment for heating or steam sterilization and can use several types of cycles to remove microbial loads.

[0165] More specifically, the present invention relates to injectable compositions comprising a matrix (preferably in the form of a hydrogel) according to the invention.

[0166] The present invention also relates to a pharmaceutical composition comprising at least one matrix according to the invention (preferably in the form of a hydrogel).

[0167] According to an alternative embodiment, the composition according to the invention is used as an injectable, implantable, or infusion-compatible pharmaceutical composition, or an injectable, implantable, or infusion-compatible medical device.

[0168] The invention further covers compositions according to the invention in dried form, especially lyophilized form. Lyophilized products can be (re)dispersed, and preferably dissolved, before use.

[0169] More specifically, this invention relates to the use of the compositions according to the invention in therapeutic treatments, such therapeutic treatments comprising, for example, injection of the compositions via subcutaneous, intradermal, intraocular, or intra-articular, intramucosal, or intramuscular routes, for example, to repair, regenerate, or fill at least one body tissue / fluid requiring repair or filling.

[0170] For the intended application, it is advantageous to use chitosan with sufficient purity.

[0171] It is advantageous to use hyaluronic acid with sufficient purity to meet the intended application.

[0172] The biomechanical properties sought by the compositions according to the invention may vary in nature depending on the indication, such as the tissue in which the hydrogel will be integrated, the mechanism of action, or the intended effect to ensure a beneficial effect on the patient, and the duration of the effect may also vary in nature.

[0173] Advantageously, the properties of the compositions according to the invention, particularly the properties of the hydrogels according to the invention, are adjusted according to the indications. To adjust these properties, in particular by the mass ratio of crosslinking agent to polymer, and / or the nature and / or amount of ions, and / or the initial molecular weight of the polymer, the final concentration of the polymer (carboxyalkyl chitosan and / or other biopolymers (such as hyaluronic acid)) and / or the crosslinking rate are varied.

[0174] In particular, this invention relates to highly elastic hydrogels (especially when it is necessary to ensure a continuous increase in volume at the skin, subcutaneous tissue, or periosteum (for bulging or remodeling)) or viscoelastic gels, particularly to provide both shock absorption and lubrication at joints. This invention relates to lubricating hydrogels, especially when it is necessary to reduce friction between two biological surfaces (e.g., two cartilage surfaces in a joint, or the ocular surface and eyelids in the eye). The compositions of this invention can have a variable level of elasticity adjustable according to the indication, and are characterized by measuring the elastic modulus by rheological methods.

[0175] Preferably, the matrix has antioxidant capacity by scavenging free radicals, especially having a normalized antioxidant capacity greater than 0.30, preferably greater than 0.50, and even more preferably greater than 0.80, for example greater than 0.90.

[0176] The present invention relates to an injectable composition, characterized in that it comprises at least one matrix as defined in the present invention.

[0177] This invention relates to a pharmaceutical composition characterized by comprising at least one matrix as defined in this invention.

[0178] According to an alternative embodiment, the composition according to the invention is used as an injectable, implantable, instillable, or locally administered pharmaceutical composition, or an injectable, implantable, instillable, or locally administered medical device, for example for therapeutic treatments (e.g., including local instillation, application, or injection of the composition via subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular, intraosseous routes), for example to repair or fill at least one body tissue requiring repair or filling.

[0179] According to an alternative embodiment, in a method of using the composition according to the invention to treat, repair or fill at least one body fluid or body tissue that needs repair or filling, for example, the body tissue is selected from tissues belonging to vocal cords, muscles, ligaments, tendons, mucous membranes, sexual organs, bones, joints, eyes, dermis or any combination thereof, particularly dermis, cartilage, synovium, skin wounds, and even the ocular surface.

[0180] This invention relates to methods of using compositions according to the invention for treating osteoarthritis or repairing cartilage defects, such as by injection into a biofluid (e.g., synovial fluid) or by implantation into cartilage after mixing with a biofluid (e.g., blood). A biofluid refers to a fluid of bodily origin, which may or may not have undergone treatment to alter its composition.

[0181] The present invention relates to medical devices (e.g., medical implants), characterized in that they comprise or consist of compositions defined according to the present invention.

[0182] This invention particularly relates to the use of compositions according to the invention for therapeutic, surgical, or cosmetic treatments, especially in rheumatology, ophthalmology, gynecology, cosmetic medicine, plastic surgery, open surgery, orthopedic surgery, gynecological treatments to prevent postoperative tissue adhesions, and in dermatology.

[0183] The present invention also relates to the use of compositions according to the invention for the therapeutic treatment of dry eye, corneal damage, or inflammation of the eye or joints.

[0184] The present invention further relates to the application of compositions according to the invention by instillation onto the ocular surface to prevent or combat corneal damage or dry eye (particularly for the purpose of lubricating or regenerating the ocular surface).

[0185] Therefore, the present invention also relates to eye drop compositions comprising carboxyalkyl chitosan as defined in the present invention.

[0186] According to an alternative implementation, the subject suffers from an inflammatory condition (e.g., osteoarthritis, arthritis, dry eye).

[0187] The present invention relates more particularly to the use of compositions according to the invention for treating arthropathy, arthritis or repairing cartilage defects, for example by injection into the synovial cavity or by implantation at the site of the cartilage defect.

[0188] The present invention relates more particularly to a medical device, such as a medical implant, characterized in that it comprises or is composed of a composition according to the invention.

[0189] According to a preferred alternative embodiment, the present invention therefore relates to a medical device comprising a chamber for receiving a composition according to the invention in a dried form (especially a lyophilized form), and optionally comprising one or more other chambers for receiving one or more active products, additives or excipients.

[0190] The compositions according to the invention may further include one or more active agents for the desired indication, and / or one or more additives or excipients for modulating the properties of the compositions according to the invention.

[0191] The present invention also relates to the use of compositions according to the invention in therapeutic treatment methods.

[0192] The present invention also relates to methods of using the compositions according to the invention for treating arthropathy or repairing cartilage defects, for example by injection into the synovial bursa or by implantation into cartilage / bone after mixing with blood.

[0193] The present invention also relates to the use of the compositions according to the invention in cosmetic treatments or cosmetic care methods via dermal fillers (“dermal fillers”) or lip fillers. This particularly relates to, for example, subcutaneous, intradermal, intramucosal, or intramuscular injection of the compositions according to the invention.

[0194] This invention also relates to compositions according to the invention for use in methods of superficial treatment of the skin (by multiple intradermal injections) or other tissues according to conventional mesotherapy known to those skilled in the art. Such compositions are commonly used in dermatology as treatments for cosmetic purposes. The aim of this method is, for example, to plump up the skin to make it appear wrinkle-free (treating wrinkles and / or fine lines). This treatment can be used on subjects who wish to restore the appearance of their skin.

[0195] The present invention also relates to the use of compositions according to the invention in therapeutic applications, wherein the composition is a viscous supplement. Here, for example, intra-articular injection of the compositions of the invention is particularly effective in limiting friction on the surface of articular cartilage.

[0196] This invention also relates to the use of compositions according to the invention as cell carriers for one or more cell types and / or one or more active agents. These can be active agents from a pharmaceutical or biological perspective. The compositions of the invention are indeed compatible with the presence of cells, preferably living cells. Examples of living cells of interest include: chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligaments), skin fibroblasts (skin), tendon cells (tendons), myofibroblasts (muscles), mesenchymal stem cells, erythrocytes (blood), and keratinocytes (skin). The compositions of the invention can also serve as therapeutic carriers for targeted delivery and / or controlled release delivery of at least one therapeutic agent.

[0197] According to an alternative embodiment, blood, or plasma, or platelet lysate, or platelet-rich plasma, or any biological fluid may be added together with the composition of the present invention, for example, to improve the performance of the product.

[0198] According to an alternative embodiment, the composition according to the invention is formulated in a solid form (e.g., a membrane or porous foam) that expands / wets once implanted (e.g., a lacrimal duct plug, a dressing).

[0199] According to an alternative embodiment, the composition is formulated into an atomizable composition (spray).

[0200] The present invention also relates to a method of treating or cosmetically caring for one or more tissues or organs affected by excessively high temperatures (such as in the case of burns) using compositions according to the invention.

[0201] The present invention also relates to a method of using the composition according to the invention for treating cartilage repair (e.g., by implantation on cartilage defects to promote their regeneration).

[0202] The present invention also relates to a method of using the composition according to the invention for the preventive treatment of postoperative tissue adhesions, wherein the product is applied to the tissue at the end of surgery (e.g., gynecological, abdominal, visceral, orthopedic, etc.).

[0203] This invention relates to a physiological composition administered topically by injection or implantation to contact one or more living tissues subjected to oxidative stress, for example: - Intra-articular injections (through synovial fluid replenishment, cartilage lubrication, joint shock absorption, and synovial regeneration) to treat osteoarthritis; Intra-articular implants to promote cartilage defect repair; - Intraosseous implants to promote bone repair (bone induction / bone conduction); - Subcutaneous and / or intradermal injections for filling or regenerating skin or hair follicles to increase volume in cases of fat atrophy; - Eye drops to relieve ocular surface symptoms or prevent changes, such as treating dry eye and corneal diseases, and to administer active ingredients; - Intraocular injection, for example, as an adjuvant in cataract surgery, to optimize the effects of glaucoma surgery or vitreous replenishment to promote the regeneration of anterior or posterior ocular tissues, and intraocular application of active ingredients; - Apply to internal tissues and organs (membranes) to prevent postoperative adhesions; - Apply to wounds, cracks, tears, cavities, etc. of tissues and organs such as skin, bones, cartilage, cornea, tendons, meniscus, etc., to promote their repair or regeneration; - Injected into the vulvar mucosa for the treatment of vulvar pain.

[0204] The present invention also relates to compositions according to the invention for forming artificial synovial fluid.

[0205] By seeking to improve, for example, its lubrication capacity to reduce friction at joints and / or its damping properties (which can be determined by the elastic modulus G'), the compositions according to the invention can mimic or improve healthy or defective synovial fluid, while being easy to inject, for example, into a filler syringe, or injected into the human or animal body. As an indication, the elastic modulus G' of healthy synovial fluid is between 40 and 100 Pa, and its loss modulus G" is between 1 and 10 Pa.

[0206] Advantageously, for intra-articular injection, the composition according to the invention is readily injected at room temperature through a fine needle (e.g., a 21-gauge needle). “Preferably” means that the force applied to such a syringe is less than 50 Newtons (at a speed of 10 mm / min) to allow the composition according to the invention to flow through a 21-gauge needle, preferably less than 20 Newtons.

[0207] Advantageously, for intradermal injection, the composition according to the invention is readily injected at room temperature using a fine needle (e.g., a 25-gauge or smaller diameter needle). “Preferably” means that the composition according to the invention is propelled through a 27-gauge needle by a force of less than 30 Newtons (at a speed of 10 mm / min) applied to such a syringe and directed into the air. Preferably, the force is less than 20 Newtons.

[0208] The present invention also relates to compositions comprising carboxyl chitosan according to the invention as artificial tears.

[0209] Typically, the range of permeability and pH of the composition is suitable and generally close to the permeability and pH of the tissue in contact with the composition according to the invention.

[0210] Advantageously, the compositions according to the invention are sterile. Most advantageously, the compositions according to the invention are sterilized by heating, preferably in an autoclave.

[0211] According to one embodiment, in tests conducted according to an embodiment of the invention, the matrix has a low coefficient of friction (COF) (e.g., less than 20, and, for example, less than 10) lubrication capability.

[0212] According to an alternative embodiment, the composition of the present invention is transparent or translucent.

[0213] "Semi-transparent" means that the object can be identified when the composition is placed between the observer's eye and the object. "Transparent" means that alphanumeric characters can be identified when the composition is placed between the observer's eye and the observed characters. Generally, this assessment is performed when the composition thickness is approximately 1 cm. The method for visual inspection as described in European Pharmacopoeia Monograph 2.9.20 can also be used. The optical density of the composition can also be measured, for example by UV-Vis spectroscopy at 500 nm, ensuring that the optical density relative to the reference solvent is less than 0.5, preferably less than 0.2.

[0214] According to an alternative embodiment, the composition of the present invention is not milky white or only slightly milky white.

[0215] "Milk white" means, for example, that the solution produces visible light diffraction by visual inspection according to methods such as European Pharmacopoeia Monograph 2.9.20 and by comparison with reference solutions of different opalescence levels in the European Pharmacopoeia. According to an alternative embodiment, the compositions of the present invention are colorless, i.e., in particular, no particular color is assigned to the composition by the naked eye. According to an alternative embodiment, the opalescence is below the maximum permissible for the intended application.

[0216] This invention particularly relates to articles or packaging that are preferably sterile, comprising one or more infusion or injection devices pre-filled with compositions (particularly compositions in hydrogel form) according to the invention. These are typically devices for administering products in the form of drops or pre-filled syringes.

[0217] The compositions of the present invention can be advantageously stored, preferably in articles or packaging suitable for their indications, and preferably stored for several months.

[0218] Advantageously, the compositions of the present invention can be sterilized. Therefore, the present invention relates to a sterilized cross-linked carboxyl chitosan. Thus, this cross-linked carboxyl chitosan is sterile, particularly for applications requiring it.

[0219] According to an alternative embodiment, the compositions of the present invention are steam sterilized according to methods known to those skilled in the art and / or recommended by the European Pharmacopoeia.

[0220] According to another alternative embodiment, the composition can be sterilized by filtration using a filter designed for this purpose (e.g., a filter with a pore size of less than or equal to 0.2 μm).

[0221] Advantageously, according to a preferred embodiment, the loss of intrinsic viscosity of cross-linked carboxyalkyl chitosan is less than 40% after steam sterilization.

[0222] The present invention also covers methods for therapeutic treatment, comprising injecting a composition according to the invention.

[0223] The present invention also covers the use of the compositions according to the invention in the preparation of pharmaceutical compositions (particularly for therapeutic treatment, such as those defined more specifically in the present invention).

[0224] The present invention also covers a method for cosmetic (in other words, non-therapeutic) treatment comprising injecting a composition according to the invention. This is, for example, filling wrinkles or filling one or more areas of damaged visible tissue (e.g., due to an accident or surgery) for cosmetic purposes.

[0225] Tissues are groups of similar cells of the same origin that aggregate in functional units, meaning they all contribute to the same function. Among these tissues are: dermal tissue (e.g., epithelial tissue), connective tissue, muscle tissue, and nerve tissue.

[0226] "Compositions according to the invention" or equivalent terms refer to compositions as defined in this invention, including compositions according to any alternative, specific, or particular embodiment, either independently or in any combination thereof, including compositions according to preferred features.

[0227] Upon reading the explanatory description, those skilled in the art will more clearly understand the further objects, features, and advantages of the invention, which refers to embodiments for illustrative purposes only and in no way intended to limit the scope of the invention.

[0228] These embodiments are integral parts of the present invention, and any feature that is novel relative to any prior art, according to the entire specification (including the embodiments), is integral to the present invention in terms of its functionality and versatility.

[0229] Therefore, each embodiment has a general scope.

[0230] On the other hand, in the embodiments, unless otherwise stated, all percentages are in mass, and unless otherwise stated, temperature is in degrees Celsius, and unless otherwise stated, pressure is in atmospheres.

[0231] Example Methods for measuring ζ potential The formulation to be analyzed was diluted in phosphate buffer to obtain a final polymer concentration of 0.05%, and then slowly stirred until homogenized. The solution was then aliquoted, and the pH of each aliquot was adjusted to the desired value (pH 4 to pH 8) by adding 0.1 N sodium hydroxide or 0.1 N hydrochloric acid. The zeta potential of each aliquot was measured using a Nano-Z device (Zeta-Sizer series, Malvern Instruments).

[0232] Methods for measuring the solubility range of chitosan polymers The solubility range was determined by preparing a 1% solution of the polymer at pH 9, dividing it into several portions, and adjusting the pH of each portion to different values ​​within the range of 9 to 1. For each portion, the solubility of the polymer was checked according to the visual inspection method of European Pharmacopoeia Monograph 2.9.20, i.e., whether it formed turbidity. The pH range in which the polymer was soluble or insoluble was recorded.

[0233] Biomechanical profile determined by rheometry The biomechanical profile of the samples was characterized using a DHR-2 hybrid rheometer (TA instrument) equipped with a 20 mm planar geometry spaced 700 μm apart from the Peltier, at a temperature of 37 °C, a frequency of 3.98 rad / s, and a deformation range of 0.1% to 10%. Each measurement was performed three times, and the average value of the elastic modulus (G'), viscosity (G"), and tan δ (G") of the three measurements was calculated.

[0234] Lubrication ability Lubrication capability is characterized by the coefficient of friction (COF) between two surfaces. The coefficient of friction is measured according to the following method, with the parameters selected based on the intended product and indication.

[0235] Methods for use with viscosity supplements Two discs (16.15 mm in diameter) of polyacrylate biomaterial used in the manufacture of hydrophobic intraocular lenses (as described in patent EP 1830898) were pre-wetted by immersion in water at 60°C for approximately 2 hours and then fixed onto the upper and lower geometries of a DHR-2 rheometer (TA instrument). Approximately 100 μL of the sample to be tested was placed on the lower disc, and the upper geometry was then lowered until the two discs made contact, with a normal force of 5 N applied. The coefficient of friction was measured at 25°C for 150 seconds, following a scheme adapted from that described by Waller et al. (see: J 47 Rheumatol 39, 7, 1473, 2012), under a constant normal force (5 N), an oscillation frequency of 1.256 rad / s, and a deformation angle of approximately 0.05 radians. The option "Observe the zero starting point of the oscillating motion" was activated. At each measurement point, the torque value was recorded, and the coefficient of friction (COF) was calculated using the following formula: COF = Torque / (1 / 3 × disk diameter × normal force). For each formulation, the measurements were repeated five times. The value of the coefficient of friction was reported by extrapolating by 5 from the intercept (COF0) at the beginning of each COF relative to the time curve.

[0236] Methods for artificial tears Two discs (16.15 mm in diameter) of polyacrylate biomaterial used in the manufacture of hydrophobic intraocular lenses (as described in patent EP 1830898) were pre-wetted by immersion in water at 60°C for approximately 2 hours and then fixed onto the upper and lower geometries of a DHR-2 rheometer (TA instrument). Approximately 100 μL of the sample to be tested was placed on the lower disc, and the upper geometry was then lowered until contact was made between the two discs, with a normal force of 5 N applied. The coefficient of friction was measured at 25°C for 150 seconds, following a scheme adapted from that described by Waller et al. (see: J 47 Rheumatol 39, 7, 1473, 2012), under a constant normal force (5 N), an oscillation frequency of 1.256 rad / s, and a deformation angle of approximately 0.05 radians. The option "Observe the zero starting point of the oscillating motion" was activated. At each measurement point, the torque value was recorded, and the coefficient of friction (COF) was calculated using the following formula: COF = Torque / (1 / 3 × disk diameter × normal force). For each formulation, the measurements were repeated five times. The value of the coefficient of friction was reported by extrapolating the intercept (COF0) at the beginning of each COF-time curve by 5.

[0237] via the ejection force of the needle Measurements were performed using the MultiTest 2.5-i compression tester (Mecmesin) equipped with a 100N compression unit. A suitable needle was fitted to the syringe containing the sample. The syringe was positioned on the tester, and the piston was pushed at a constant speed (e.g., 10 mm / min or 80 mm / min), measuring the force required for ejection. The maximum force that this equipment can withstand is approximately 70 Newtons.

[0238] In vitro antioxidant capacity (ABTS test) To measure the antioxidant activity of carboxyl chitosan formulations and compare them with commercial products, an in vitro 'ABTS' assay was performed. This assay determines the ability of a substance to capture the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•1) cationic radical, a chromophore that exhibits maximum absorption at a wavelength of 734 nm in cationic radical form. Absorbance was measured using a protocol adapted from the method described by Valyova et al. (Int J Applied ResNat Prod, 5, 19, 2012) using a Nunclon 96 polystyrene microplate (Thermo Fisher Scientific) and an Infinite M200 microplate reader (Tecan Life Sciences).

[0239] Each test series is conducted in four steps.

[0240] 1) Dilute 1 g of 2,2'-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in a homogeneous solution of K₂S₂O₈ (2.45 mM in MilliQ water) to obtain a concentration of 7 mM ABTS. Protect the mixture from light and stir at room temperature for 24 hours, allowing sufficient time to generate the appropriate amount of ABTS•1 radical cations. The final working solution of ABTS•1 is obtained by taking 600 μL of the stirred mixture and diluting it in MilliQ water to a concentration of 415 μM.

[0241] 2) A calibration curve for free radical scavenging ability was established by comparison with the reference antioxidant molecule (6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-carboxylic acid) Trolox. Trolox solutions with concentrations of 30, 60, 90, 120, 150, 180, and 210 µM were obtained by diluting 15 mg of Trolox in 5 mL of 100% methanol with MilliQ water. After mixing 50 μL of each Trolox solution with 50 μL of the working solution in 1 hour, absorbance was measured at 734 nm. The relationship between absorbance and Trolox concentration in the linear region was read. The minimum absorbance value in the linear region corresponds to the detection limit.

[0242] 3) The test product is either characterized as is at its initial concentration or diluted in MilliQ water (depending on the test product, to ensure that the absorbance of the mixture containing the ABTS•1 solution is above the detection limit). Mix 50 μL of the working solution and 50 μL of the test product solution. After incubating at room temperature for 1 hour, measure the absorbance at a wavelength of 734 nm. If the absorbance value is within the instrument's detection range, retain the value and calculate the Trolox equivalent using the calibration curve, expressed as "trolox equivalent antioxidant capacity" (TEAC).

[0243] 4) A positive control was used to standardize the antioxidant capacity from one series to another. This positive control was ascorbic acid (vitamin C) in a solution of 0.02 mg / mL (20 μg / mL). First, the TEAC of ascorbic acid solutions in the range of 0.005–0.05 mg / mL was measured. The absorbance of the 0.02 mg / mL ascorbic acid solution was validated to be in the linear region. Finally, the standardized antioxidant capacity of the tested product was expressed as the ratio of TEAC (product) to TEAC (0.02 mg / mL ascorbic acid).

[0244] Example 1 Carboxymethyl chitosan was prepared via carboxymethylation and acetylation reactions using the reaction parameters given in Table 1a as examples. Furthermore, other reaction parameters can be used to adjust the molecular structure of carboxymethyl chitosan.

[0245] Step 1: Carboxymethylation of chitosan.

[0246] 30 g of chitosan from Agaricus bisporus was dispersed in 600 ml isopropanol, 41 ml water, and 163 ml 50% sodium hydroxide (m / v). 135 g of alkylating agent monochloroacetic acid (MCA) was dissolved in 135 ml isopropanol and added to the chitosan suspension. The reaction was continued at 35 °C for 23 hours. The polymer was recovered by precipitation in ethanol, and then purified by several cycles of dissolution in water and precipitation in ethanol. After drying in a ventilated oven, carboxymethyl chitosan was collected (reference number CC4, Table 1b).

[0247] Step 2: Acetylation of carboxymethyl chitosan.

[0248] 21 g of CC4 was dispensed into 570 ml of water, and the pH of the solution was adjusted to >7. 10 ml of acetic anhydride was added, and the solution was stirred at 25°C for 30 minutes. The pH of the solution was adjusted to >7, and then 10 ml of acetic anhydride was added. After homogenization (stirring at room temperature for approximately 30 minutes), the pH was adjusted to approximately pH 7.5. The polymer was recovered by precipitation in ethanol, and then purified by several cycles of dissolution and precipitation in water. After drying in a ventilated oven, carboxymethyl chitosan (reference number CC3, Table 1b) was collected.

[0249] Table 1b describes the carboxymethyl chitosans used to prepare the matrices of Examples 2-11. CC1 to CC6 are carboxymethyl chitosans derived from fungal chitosans and were prepared according to the methods described above.

[0250] CC7 is a commercially available carboxymethyl chitosan derived from crustaceans, supplied by Kraeber (product code 5313009900, Ellebec, Germany).

[0251] [Table 1a]

[0252] [Table 1b]

[0253] a: Measurement by solid-phase carbon-13 NMR (Equation 2); b: Measurement by potentiometric titration; c: Measurement by capillary viscosity method; d: Acetyl group signal cannot be detected by carbon-13 NMR (low DA).

[0254] Example 2 — Carboxymethyl chitosan matrix Synthetic experiments were conducted using a crosslinking agent, 1,4-butanediol diglycidyl ether (CAS 245-79-8, BDDE), via covalent crosslinking to provide a matrix for carboxymethyl chitosan. Agaricus bisporus, manufactured by Colmed Pharmaceuticals, was used according to the method of Example 1. Agaricus bisporus Several carboxymethyl chitosans from various sources are described. Their characteristics are shown in Table 1. BDDE (96%, specific gravity 1.049) was supplied by Alfa Aesar (Thermo Fisher Scientific, Kandel, Germany).

[0255] Example 2a After adjusting the reaction parameters, a cross-linked matrix (Table 2a, reference number M1-A) was prepared from carboxymethyl chitosan CC3. The degree of acetylation of CC3 was 55% and the degree of carboxymethylation was 87%, as measured by carbon-13 NMR (Equation 2). After dialysis, the hydrogel formed from the matrix was transferred to a 3 ml glass syringe, which was sterilized by short-cycle steam in a SYSTEC-DX-65 autoclave (condition "A2"). The final polymer concentration of the sterilized hydrogel (M1-A) was determined by mass balance. The cohesive properties of the hydrogel were analyzed by water testing, and the viscoelasticity level (grades 1-4) of the hydrogel was determined by rheological assay. The higher the fraction, the greater the viscoelasticity of the matrix forming the hydrogel. It was concluded that, after adjusting the reaction parameters, a BDDE-crosslinked carboxyalkyl chitosan matrix could be obtained, and this matrix formed a cohesive hydrogel according to the water test. The elasticity fraction of this hydrogel was 1. It could be injected via an intradermal needle (27G 13 mm).

[0256] These same reaction parameters were then applied to two carboxymethyl chitosans with different molecular structures and degrees of acetylation below 40% (Table 1b): CC4 (Colmed Pharmaceuticals) from fungi and CC7 (Kraeber) from crustaceans.

[0257] [Table 2a]

[0258] *A2: Short cycle (SYSTEC DX-65 autoclave); **When the ejection speed is 10mm / min, the ejection force is less than 30 Newtons.

[0259] According to water tests, matrices M1-B and M1-C (Table 2a), obtained under the same conditions as matrix M1-A, did not form cohesive hydrogels. In contrast, matrix M1-A was able to form cohesive hydrogels, thus satisfying this objective of the present invention.

[0260] Example 2b Attempts were made to modulate the biomechanical properties of crosslinked carboxyl chitosan-based hydrogels, particularly their viscoelasticity (measured on a scale of 0 to 4). To this end, matrices were prepared from CC1, CC5, and CC6 (Table 1b) with DA greater than 40% by varying the molecular weight of the carboxyl chitosan (expressed as intrinsic viscosity) and the parameters of the crosslinking reaction. The crosslinking agent (BDDE), medium, temperature, reaction time, and neutralization and purification conditions were identical to those for matrix M1-A.

[0261] [Table 2b]

[0262] *A2: Short loop; A1: Long loop This indicates that the biomechanical properties, especially viscoelasticity, of crosslinked carboxyl chitosan-based hydrogels can be altered by changing reaction parameters (particularly the initial concentration of carboxyl chitosan or the ratio of crosslinking agent to carboxyl chitosan, in this case BDDE / carboxymethyl chitosan) and the molecular weight of carboxyl chitosan.

[0263] Example 3 — A matrix of co-crosslinked carboxymethyl chitosan and hyaluronic acid The matrix was obtained by using a mixture of carboxymethyl chitosan and hyaluronic acid of BDDE crosslinking fungal origin with a DA greater than 40% (Table 1b) (“co-crosslinking”). Hyaluronic acid (HA) with an average viscosity molecular weight of 2.2 or 2.3 million (HA1 type) and 4.3 million (HA2 type) was used (Table 3a).

[0264] [Table 3]

[0265] *Values ​​reported by the supplier The reagents (BDDE), media, temperature, and duration of the crosslinking reaction, as well as the neutralization and purification conditions, were the same as those for matrix M1-A in Example 2. The hydrogels formed from the matrix were sterilized in an autoclave according to cycle A1 or A2, as described in Example 2. Since other combinations and / or parameters may also produce cohesive hydrogels, several hydrogels are described by way of example. All these hydrogels can be easily injected using an intradermal needle of size 27 and a length of 13 mm.

[0266] Example 3a An attempt was made to demonstrate that carboxyalkyl chitosan (CC) can be co-crosslinked with hyaluronic acid (HA) to form a cohesive hydrogel. For this purpose, a matrix was prepared from a mixture of CC and HA at a CC / HA mass ratio of 75:25 (Table 3a). The reference number for CC is consistent with that in the previous examples. Furthermore, by adjusting the parameters of the crosslinking reaction, an attempt was made to adjust the elasticity level from 1 to 3 (based on a scale of 0 to 4).

[0267] [Table 3a]

[0268] *Conditions of Example 2 It was observed that, at the same BDDE / polymer ratio (18%) and the same final polymer concentration of 23 mg / m³, the hydrogel M2-B co-crosslinked with 25% HA was more elastic than the hydrogel M1-A containing only CC in Example 2. It was also concluded that the viscoelasticity of the co-crosslinked carboxyalkyl chitosan and HA hydrogels can be altered by changing the molecular weight of HA and the percentage of the crosslinking agent (in this case, BDDE).

[0269] Example 3b An attempt was made to obtain cohesive hydrogels by co-crosslinking carboxyl chitosan and HA in different proportions.

[0270] [Table 3b]

[0271] This indicates that co-crosslinked endogenous hydrogels of carboxyalkyl chitosan and HA in different ratios can be obtained, and their elasticity level depends on the carboxyalkyl chitosan / HA ratio.

[0272] Example 4 — A matrix of cross-linked carboxymethyl chitosan combined with hyaluronic acid In this embodiment, the possibility of forming an intracellular hydrogel from a matrix of cross-linked carboxyl chitosan combined with HA was attempted to be evaluated. Following the method of Example 1, BDDE cross-linked carboxyl chitosan was first used, and then an HA (HA1 type) solution was added thereto. The resulting hydrogel was then sterilized by autoclaving via cycle A2 (Table 4).

[0273] [Table 4]

[0274] HA can be readily incorporated into hydrogels based on cross-linked carboxyl chitosan matrices. Water tests showed that the resulting hydrogels were cohesive with a viscoelastic score of 3 and were easily injected via a 27-gauge intradermal needle.

[0275] Example 5 — Biomechanical properties of hydrogels In this embodiment, the biomechanical properties of some representative CC hydrogels from Examples 2 to 4 were characterized by rheological assays (Table 5). The hydrogels were cohesive, injectable via a 27G needle, and exhibited elasticity levels of 1 to 3. They were compared with three commercially available cross-linked hyaluronic acid-based products used for intradermal injection for cosmetic purposes (Table 5, reference numbers B1 to B3): B1 was a viscous solution (tan δ > 1) according to water tests, while B2 and B3 were cohesive gels (tan δ < 1).

[0276] [Table 5]

[0277] The cross-linked carboxyl chitosan-based hydrogels according to the present invention have been shown to have biomechanical properties, particularly elastic modulus (G'), comparable to those of commercially available cross-linked HA-based products used for intradermal injection in cosmetic medicine.

[0278] Example 6 — Ability to scavenge ABTS°1 free radicals (in vitro) An attempt was made to verify the ability of a cross-linked carboxyalkyl chitosan (CC) matrix to scavenge oxidative free radicals, where free radicals ABTS°1 are formed, using a standard in vitro test known as “ABTS”, calibrated with the antioxidant “Trolox”. Each test product was diluted to obtain total concentrations of polymer Cp (CC, HA, or CC and HA) of 8 mg / mL, 4 mg / mL, and 1 mg / mL. The results were verified to ensure they were within the detection range of the test, and the ability to scavenge free radicals ABTS°1 was then expressed as Trolox equivalents. The antioxidant capacity of a 20 µg / mL ascorbic acid solution (positive control) was also measured. The antioxidant capacity of each test product was normalized using the following formula: Normalized antioxidant capacity = TEAC (product) / TEAC (ascorbic acid 20 μg / mL).

[0279] For comparison, a non-crosslinked carboxyalkyl chitosan polymer (CC2) in solution and a commercially available product based on a non-crosslinked HA solution (reference number B6) were tested. Four commercially available products for cosmetic intradermal injection were also characterized: reference numbers B1 to B3 (based solely on crosslinked HA, see Table 5 of Example 5) and B4 (based on a hydrogel composed of crosslinked HA combined with a complex of several small molecules, including antioxidant molecules).

[0280] Table 6 reports the results obtained for all products at the same total polymer concentration (Cp) of 4 mg / mL.

[0281] [Table 6]

[0282] It was observed that all CC-based compositions, whether in solutions of non-crosslinked CC (S1) or hydrogels of crosslinked CC (M1-E and M2-A), were able to significantly scavenge the free radical ABTS°1, and therefore could be used as antioxidants. At the same polymer concentration, only the commercially available HA products (B6, B1, B2, and B3) did not exhibit this ability.

[0283] Surprisingly, hydrogels M1-E (CC) and M2-A (CC / HA 75:25) exhibited the highest antioxidant capacity among all tested products, including solution S1 with uncrosslinked CC. Both hydrogels showed antioxidant capacity similar to that of 20 µg / ml ascorbic acid.

[0284] Among commercially available HA-based products, only B4 can significantly scavenge the free radical ABTS°1, but its ability to scavenge ABTS°1 is only half that of M1-E and M2-A. In fact, B4 is a cross-linked hyaluronic acid complexed with several small molecules, including antioxidants, which are responsible for the observed effects. However, because these substances are water-soluble small molecules, they are likely to rapidly diffuse out of the B4 hydrogel after intradermal injection, and the hydrogel will then lose its antioxidant capacity.

[0285] Example 7 — The ability of hydrogels to reduce oxidative stress in in vitro dermal cell culture The ability of two hydrogels, based on cross-linked CC (reference number M1-E, see Example 2) and co-cross-linked CC / HA (M2-A, see Example 3), to protect human dermal cells from damage caused by ROS (reactive oxygen species), which are free radical species encountered in skin tissue under oxidative stress, was evaluated in standard in vitro tests. They were compared with a non-cross-linked carboxyalkyl chitosan solution and a commercially available product based on cross-linked hyaluronic acid for cosmetic intradermal injection (reference number B3, see Example 5).

[0286] Human dermal fibroblasts (NHDF) with approximately 40% in vitro proliferative potential were cultured as a monolayer in Dulbecco's Modified Eagle Medium (DMEM) at 37°C and a 5% CO2 atmosphere, containing 10% fetal bovine serum, penicillin, and streptomycin. Cultures were transferred to DMEM without fetal bovine serum and aliquoted into wells. Test products were diluted in DMEM to total polymer concentrations of 0.6 and 0.2 mg / mL and added to wells (each test product was added to 3 wells). After 72 hours of contact with the test products, a 2'-7'-dichloro-dihydrofluorescein diacetate probe, which fluoresces under free radical susceptibility, was added for 30 minutes. The culture in each well was then rinsed with HBSS to remove the test products, and the cells were returned to HBSS and then treated with 12.5 J / cm² water. 2 All pores were irradiated with UVA for 20 minutes to generate ROS.

[0287] Untreated, unirradiated cultures were used as references. Untreated and irradiated cultures served as negative controls, while ascorbic acid-treated (50 μg / mL) and irradiated cultures served as positive controls. At the end of UVA irradiation, fluorescence intensity (excitation wavelength 485 nm, emission wavelength 520 nm), proportional to ROS content, was measured, and the relative ROS content compared to the unirradiated reference was calculated (Table 7). The reduction in ROS content relative to the untreated and unirradiated controls was then calculated, characterizing the product's ability to reduce oxidative stress.

[0288] [Table 7]

[0289] *Total concentration of polymers (CC, CC / HA, or HA) used for cell treatment Under the in vitro culture conditions of this experiment, it can be concluded that CC-based compositions, whether cross-linked (M1-E) or non-cross-linked (S2), exhibit excellent ability to reduce ROS levels, i.e., to reduce oxidative stress that may alter cells and dermal tissue. This ability is comparable to that of ascorbic acid (50 μg / mL, vitamin C) and significantly higher than that of commercially available cross-linked HA products. The co-cross-linked CC / HA composition M2-A, containing 75% CC, also demonstrates good ability to reduce oxidative stress.

[0290] Example 8 — A fluid hydrogel based on a carboxyl chitosan matrix for ocular drug delivery In this embodiment, an attempt is made to obtain a cross-linked CC hydrogel with a viscosity that makes it easy to drip in the form of well-defined droplets, while having good lubrication properties suitable for artificial tear indications for ocular surface treatment.

[0291] Therefore, the target dynamic viscosity is set in the range of 1 to 60 mPa·s (within 10 s). -1 Cohesive crosslinked CC hydrogels (M8-B, Table 8a) were prepared at a shear rate of [missing value]. Their drip permeability was verified, and their lubrication ability between two polyacrylate surfaces was measured using methods similar to artificial tears, expressed as the coefficient of friction.

[0292] The properties of this hydrogel were compared with those of two commercially available non-crosslinked HA-based products (reference numbers B7 and B8, Table 8b) for ocular surface treatment. Their lubrication capabilities were measured in the same test series as M8-B.

[0293] [Table 8a]

[0294] [Table 8b]

[0295] The conclusion is that cohesive, fluid, and drip-readable cross-linked CC hydrogels can be obtained, which have lubrication capabilities comparable to commercial products used for ocular surface treatment.

[0296] Example 9 — Local effects (short-term) after intradermal implantation in rabbits Three CC-based hydrogels—M1-A (crosslinked CC, see Example 1), M2-A, and M2-B (co-crosslinked CC / HA, see Example 2)—were evaluated for intradermal administration to rabbits. These formulations were packaged into 1 mL glass syringes (Hypak, BD Medical) and sterilized. Endotoxin levels, determined according to European Pharmacopoeia Method D-Monograph EP 2.6.14, were satisfactory. Two commercially available products (B1 and B2, see Example 5) based on crosslinked hyaluronic acid for cosmetic intradermal injection were also evaluated.

[0297] Following the protocol of ISO 10993-10, which meets the criteria for evaluating primary irritation induced by intradermal implants, rabbits were administered 200 µL of the formulation via intradermal injection using a 27G needle. A total of twelve injections of each product were performed on six rabbits. Local effects at all injection sites, particularly erythema levels, were observed daily.

[0298] Table 9 reports the average erythema level (scoring 0 to 4) 7 days post-injection. Also note the presence of papules (scoring 0 to 4) at day 7. Macroscopic or microscopic analysis (skin histology) of the injection site in animals euthanized 7 days post-injection is used to assess the presence of the product.

[0299] [Table 9]

[0300] Intradermal injection of the hydrogel was associated with the appearance of mild local effects, characterized by a maximum average erythema score of 1 (grades 0 to 4) at day 7. This corresponds to a level of mild erythema, comparable to that observed with both commercial products. Furthermore, the presence of the product in the dermis was demonstrated after euthanasia of animals on day 7 and histological analysis.

[0301] Example 10 — Hydrogel for Adhesive Joint Supplementation In this embodiment, the viscoelasticity and lubrication of two hydrogels based on cross-linked CC (M1-E) and co-cross-linked CC / HA (M2-B) were evaluated and compared with the viscoelasticity and lubrication of two commercially available products (B9 and B10, see composition in Table 10) based on cross-linked HA used for the treatment of osteoarthritis via viscous supplementation at joints. The lubrication properties of the hydrogels were determined by their ability to reduce the coefficient of friction between two polyacrylate polymer disks mounted on a rheometer, according to the method used for viscous supplementation.

[0302] [Table 10]

[0303] *A high standard deviation indicates high friction between the two surfaces (low lubrication capability of the tested product);** Total polymer concentration It was observed that the elastic modulus G' of crosslinked CC and co-crosslinked CC / HA hydrogels was in the same range as that of B9, while the elastic modulus of B10 was higher. It was also observed that both CC and CC / HA hydrogels exhibited significant lubrication capabilities (characterized by a low coefficient of friction between the two surfaces), comparable to the lubrication capabilities of crosslinked HA viscosity supplement B10, and superior to those of crosslinked HA viscosity supplement B11.

[0304] In Examples 11 to 14, the polymers CC and HA used are those described in Tables 11a and 11b.

[0305] [Table 11a]

[0306] a: Value evaluated based on the DA of the starting chitosan; b: Value evaluated based on the DS of the acetylated CC measured by carbon-13 NMR; c: Measured by solid-phase carbon-13 NMR (Equation 2).

[0307] [Table 11b]

[0308] Example 11 — Co-crosslinking test of HA with CC with acetylation degree less than 40% An attempt was made to verify whether it was possible to obtain a cohesive hydrogel by co-crosslinking CC and HA, starting with CC (CC8, Table 11a) having less than 40% DA and HA of type HA1 (Table 11b), using the same conditions as in Table 3a of Example 3. The conditions and properties of the resulting formulation (reference number M2-I) are recorded in Table 11c and compared with the conditions and properties of the hydrogel of reference number M2-A of Example 3 (according to the invention).

[0309] It was observed that, as determined by the tan δ (measured by rheological assay), no gel was obtained using CC8 via co-crosslinking and autoclaving. In fact, the M2-I formulation has a tan δ value greater than 1, 1.6, indicating behavior of a viscous solution rather than a gel. Conversely, according to the invention, the hydrogel M2-A has a tan δ value of 0.4, i.e., less than 1, indicating gel behavior.

[0310] [Table 11c]

[0311] *Because the obtained formulation is not a gel, the water test is not applicable.

[0312] Example 12—A hydrogel for volume restoration or filling large skin depressions This embodiment illustrates the use of cross-linked hyaluronic acid (CC)-based hydrogels, administered subcutaneously or injected deep into the dermis, for restoring facial volume or filling large skin depressions. For both indications, a hydrogel with a viscoelasticity level of 4 is sought, meaning the hydrogel has an elastic modulus G' of approximately 150 Pa or higher, while also being cohesive according to water testing and easily injectable through a 27-gauge needle of 13 mm length. Two commercially available products, B11 and B12 (Table 12), are used as references for these indications; B11 and B12 are cohesive hydrogels based on cross-linked hyaluronic acid with an elasticity level of 4.

[0313] Hydrogel M2-J was obtained by co-crosslinking CC5 and HA1 type HA (CC / HA ratio of 25:75) overnight at room temperature with 13% BDDE. It has an elastic modulus of 295 Pa (corresponding to the desired elasticity level 4) while maintaining cohesion and being easy to inject, which meets the expectations for the intended indication (Table 12).

[0314] [Table 12]

[0315] Example 13—Volume retention of intradermal co-crosslinked CC / HA hydrogel after 1 month Hydrogels were prepared by co-crosslinking CC9 (see Table 11a) and HA2 under the reaction conditions of Example 12, wherein the CC / HA mass ratio was 40:60. The resulting hydrogels (reference number M2-K) were loaded into 1 mL glass syringes (Hypak, BD Medical) and sterilized in the same manner as in Example 9. The final polymer concentration was 23 mg / mL, and it was cohesive, injectable through a 27G needle, with a viscoelasticity level of 3.

[0316] Following a protocol similar to Example 9, equal volumes of hydrogel M2-K and commercially available product B12 (see Table 12, viscoelasticity level 4) were intradermally injected into rabbits using a 27-gauge needle. Local responses were assessed periodically over 26 days post-injection, and the volume of papules visible on the skin surface formed by the injected product was evaluated using a scoring system ranging from 0 to 4. Papule volume indicates the presence of the product and its ability to locally increase skin tissue volume.

[0317] Neither product caused any significant local reactions during the follow-up period. Immediately after injection, papules with a mean volume score of 3 ± 0 (out of 20 injection sites evaluated) formed. Over the next few days, the papules slightly subsided but remained. At 26 days post-injection, the papules persisted, with a mean volume score of 2.0 ± 0.0 for M2-L and 2.4 ± 0.5 for B12 (out of 20 sites evaluated), consistent with their relative elasticity levels. At this time point, there was no significant difference in volume scores between the hydrogels M2-K and B12.

[0318] Therefore, it was confirmed that the hydrogel M2-K remained in the dermis for at least 26 days after intradermal injection in rabbits, maintaining a significant increase in volume around the injection site, as expected for the indication of filling skin depressions.

[0319] Example 14—Preservation of Co-crosslinked CC / HA Hydrogels The feasibility of preserving the co-crosslinked CC / HA hydrogel was assessed by placing it under accelerated aging conditions in a 40°C oven and monitoring its biomechanical properties. The hydrogel was considered biomechanically acceptable as long as it remained cohesive and easily injectable according to water tests, exhibited gel-like behavior (tan δ value less than 1), and its viscoelasticity remained constant relative to its initial level at t0, thus meeting the intended indication.

[0320] To obtain a viscoelasticity level of 2, a hydrogel with reference number M2-L was prepared by co-crosslinking CC9 (see Table 11a) and HA2 in a CC / HA ratio of 70:30 according to the reaction conditions of Example 12. This was a sterilized product packaged in a 1 mL glass syringe (Hypak, BD Medical) in the same manner as in Example 9. The syringe was placed in an oven at 40°C for 6 months. Table 13 shows the properties measured during a 3-month storage period.

[0321] [Table 13]

[0322] After 3 months of accelerated aging at 40°C, product M2-L remained a hydrogel (because tan δ < 1) and maintained its cohesiveness, injectability, and viscoelasticity level of 2. Therefore, by extrapolation, it is estimated that this co-crosslinked CC / HA hydrogel should be able to maintain properties acceptable for the intended indication for at least 12 months at room temperature.

Claims

1. Use of a carboxyalkyl chitosan in the preparation of compositions for injection, implantation, or infusion in cosmetic care methods for human or animal bodies, said carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units, and carboxyalkyl-substituted glucosamine units, wherein, The carboxyalkyl chitosan is N,O-carboxyalkyl chitosan, expressed as the number of moles of substituents relative to the total number of units. The carboxyalkyl chitosan has a degree of carboxyalkyl substitution greater than 70%, expressed as the number of moles of N-acetyl groups relative to the total number of glucosamine units. The carboxyalkyl chitosan has a degree of acetylation greater than 40% and less than or equal to 80%. The carboxyalkyl chitosan is cross-linked by covalent bonds between carboxyalkyl chitosan chains.

2. The use according to claim 1, wherein, The method includes injecting the composition.

3. The use according to claim 2, wherein, The method includes filling wrinkles or filling one or more areas of damaged visible tissue.

4. The use according to claim 1, wherein, The composition is a sterile, cohesive hydrogel matrix.

5. The use according to claim 1, wherein, The composition is a hydrogel matrix, injected with an injection force of less than 50 Newtons at a speed of 10 mm / min, the hydrogel matrix being suitable for flow through a 21-gauge needle; or injected with an injection force of less than 30 Newtons at a speed of 10 mm / min, the hydrogel matrix being suitable for flow through a 27-gauge needle.

6. The use according to claim 1, wherein, The composition is a hydrogel matrix that is cohesive and has a coefficient of friction of less than 20.

7. The use according to claim 1, wherein, The method is used in cosmetic medicine, plastic surgery, dermatology, or cosmetic treatments.

8. The use according to claim 1, wherein, The carboxyl chitosan has a zeta potential of less than or equal to -20 mV, measured at pH 7.

5.

9. The use according to claim 1, wherein, It is expressed as the number of moles of substituent carboxyl groups relative to the total number of units, and the degree of substitution of carboxyl groups is less than 200%.

10. The use according to claim 1, wherein, Chitosan is derived from basidiomycetes Basidiomycete Fungi or derived from ascomycetes Ascomycete The mycelium of a certain type of fungus.

11. The use according to claim 1, wherein, The carboxyalkyl chitosan is water-soluble at pH values ​​below 3.5 and at physiological pH values.

12. The use according to claim 1, wherein, The carboxyalkyl chitosan is water-soluble in a pH range of 3.1-9.

13. The use according to claim 1, wherein, The carboxyalkyl chitosan is sterile.

14. The use according to claim 1, wherein, The method includes applying an aqueous solution comprising the carboxyalkyl chitosan.

15. The use according to any one of claims 1 to 4, wherein, The composition includes at least one hyaluronic acid.

16. The use according to any one of claims 1 to 4, wherein, The composition includes at least one type of hyaluronic acid obtained through fermentation.

17. The use according to any one of claims 1 to 4, wherein, The composition includes at least one hyaluronic acid cross-linked by covalent bonds.

18. The use according to any one of claims 1 to 4, wherein, The composition includes at least one hyaluronic acid that is co-crosslinked with carboxyl chitosan via covalent bonds.

19. The use according to any one of claims 1 to 4, wherein, Crosslinking is formed by a crosslinking agent that forms the covalent bonds.

20. The use according to claim 19, wherein, The cross-linking agent is selected from those used for cross-linking polysaccharides.

21. The use according to claim 20, wherein, The crosslinking agent is selected from the group consisting of: 1,4-butanediol diglycidyl ether, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxypentane, 1-chloro-2,3-cyclothiopropane, 1-bromo-2,3-cyclothiopropane, 1-bromo-3,4-cyclothiobutane, 1-bromo-4,5-cyclothiopentane, 2,3-dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanethiol, 2,4-dibromobutanethiol, 2,5-dibromopentane-thiol, epichlorohydrin, dimethylaminopropylcarbodiimide, gallic acid, epigallocatechin gallate, curcumin, tannic acid, genipin, diisocyanate compounds, and diethylene sulfone.

22. The use according to claim 21, wherein, The diisocyanate compound is selected from hexamethylene diisocyanate and toluene diisocyanate.

23. The use according to claim 1, wherein, The aqueous solution also includes reducing sugars.

24. The use according to claim 1, wherein, The aqueous solution is included in the medical device.

25. The use according to claim 1, wherein, The method is used to plump up the skin or improve its appearance.

26. The use according to claim 1, wherein, The method is used to treat wrinkles and / or fine lines on the skin surface.

27. The use according to claim 1, wherein, The method is used to revitalize the skin of the subject.

28. The use according to claim 1, wherein, The carboxyalkyl chitosan is contained in an intradermal implant or the intradermal implant is composed of the carboxyalkyl chitosan.

29. The use according to claim 1, wherein, The composition is an aqueous preparation used for cosmetic treatment via subcutaneous or intradermal injection or implantation as a dermal filler.

30. The use according to claim 1, wherein, The composition is steam sterilized.

31. The use according to claim 1, wherein, The carboxyl chitosan is derived from Aspergillus niger. Aspergillus piger Basidiophyte (Shiitake mushroom) Basidiomycete Lentinula edodes Or button mushroom Agaricus bisporus It is made from chitosan.

32. The use according to claim 1, wherein, The carboxyalkyl chitosan is reacetylated.

33. Use of a carboxyl chitosan in the preparation of a composition for the treatment of human or animal eyes, said carboxyl chitosan having glucosamine units, N-acetylglucosamine units, and carboxyl-substituted glucosamine units, wherein, The carboxyalkyl chitosan is N,O-carboxyalkyl chitosan, expressed as the number of moles of substituents relative to the total number of units. The carboxyalkyl chitosan has a degree of carboxyalkyl substitution greater than 70%, expressed as the number of moles of N-acetyl groups relative to the total number of glucosamine units. The carboxyalkyl chitosan has a degree of acetylation greater than 40% and less than or equal to 80%. The carboxyalkyl chitosan is cross-linked by covalent bonds between carboxyalkyl chitosan chains.

34. The use according to claim 33, wherein, The treatment includes local instillation, application, or injection of the composition via subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular routes.

35. An eye composition, wherein, The ocular composition comprises an aqueous solution, gel, or solid insert, film, or dressing, including carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units, and carboxyalkyl-substituted glucosamine units, wherein the carboxyalkyl chitosan is N,O-carboxyalkyl chitosan, expressed as the number of moles of substituents relative to the total number of units, the carboxyalkyl chitosan having a degree of carboxyalkyl substitution greater than 70%, expressed as the number of moles of N-acetyl groups relative to the total number of glucosamine units, the carboxyalkyl chitosan having a degree of acetylation greater than 40% and less than or equal to 80%, and the carboxyalkyl chitosan being cross-linked by covalent bonds between carboxyalkyl chitosan chains.

36. The ocular composition according to claim 35, wherein, The aqueous solution also includes one or more pharmaceutical or biological active agents.

37. The ocular composition according to claim 35, wherein, The aqueous solution also includes hyaluronic acid or sodium hyaluronate that are covalently cross-linked or uncross-linked.

38. The ocular composition according to claim 35, wherein, The aqueous solution also includes reducing sugars.

39. The ocular composition according to claim 35, wherein, The aqueous solution also includes glycerol, sorbitol, trehalose, or combinations thereof.

40. The ocular composition according to claim 35, wherein, The ocular composition is selected from eye drop formulations and lubricating eye drop formulations.

41. The ocular composition according to claim 35, wherein, The viscosity of the ocular composition is 10-30 mPa·s under conditions of eye movement.

42. The ocular composition according to claim 35, wherein, The viscosity of the ocular composition is less than 10 mPa·s under conditions of eye movement.

43. The ocular composition according to claim 35, wherein, The ocular composition is formulated in a dry form as a film, lens, foam, or insert.

44. The ocular composition according to claim 35, wherein, The eye composition is formulated as a solid or gel.

45. An article characterized in that, The article comprises one or more instillation or application devices loaded with the ocular composition of claim 35.

46. ​​The article of claim 45, wherein, The device is an eye drop dispensing system or a solid / gel system.

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