Chitosan and its applications

Abstract

The present invention relates to crosslinked carboxyalkyl chitosan forming a matrix, to compositions comprising the same, to methods for the preparation thereof and to various applications thereof, in particular in the therapeutic, rheumatological, ophthalmic, cosmetic, plastic surgery, open surgery, dermatological, gynecological or aesthetic fields. The present invention in particular relates to a matrix comprising at least one carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units and glucosamine units substituted with carboxyalkyl groups, the degree of acetylation of said carboxyalkyl chitosan being comprised between 40% and 80%, expressed in number of moles of N-acetyl groups relative to the number of moles of total glucosamine units, said carboxyalkyl chitosan being crosslinked by covalent bonds between carboxyalkyl chitosan chains.

Description

TECHNICAL FIELD

[0001] The present invention relates to cross-linked carboxyalkyl chitosan forming a matrix, to compositions comprising the cross-linked carboxyalkyl chitosan, to methods for their preparation and to various applications thereof, in particular in the therapeutic, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, open surgery, dermatology, gynecology or cosmetic fields. BACKGROUND

[0002] Chitosan derivatives are known, notably 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 the patent applications of Kiomed Pharma filed as PCT / EP2018 / 080763 and PCT / EP2018 / 080767 and their family, the content of which is incorporated by reference in the present invention.

[0003] According to the present inventors, it would be advantageous to be able to modulate the biomechanical behavior of carboxyalkyl chitosan compositions, or even to be able to increase the persistence or the effect of the treatment by the presence of carboxyalkyl chitosans. However, it would not be obvious to the person skilled in the art to provide such a composition with improved biomechanical properties, in particular when it is necessary to prepare a hydrogel. Among the biopolymer compositions of the prior art (notably hydrogels), one of the technical problems of the biopolymer-based compositions known to the person skilled in the art is that some compositions are not in the form of cohesive hydrogels, i.e. the hydrogels spontaneously disintegrate into different parts in the presence of an aqueous medium, thereby forming particles, fragments. This is also referred to as a fragmented or fragmented hydrogel.

[0004] It is recognized that such non-cohesive hydrogels present a risk of long-term inflammatory nodule formation or granulomatous reactions when the product is implanted in human or animal tissues, which is considered undesirable for many medical applications (Bergerey-Galley, Aesth Surf J 24, 33, 2004). It is therefore important, in terms of health safety for the subject or patient, to be able to avoid the formation of significant fragments and to obtain a composition in the form of a cohesive hydrogel.

[0005] Moreover, in certain cases, it is desirable to avoid such aggregates to improve the aesthetic (visual and / or tactile) appearance of the tissue filled with such a composition, which is properly biointegrated into the tissue thereby allowing a homogeneous filling.

[0006] Thus, for many applications, a cohesive hydrogel is preferred, e.g. which remains in one piece when an aqueous medium is added thereto. This is also referred to as a "homogenous" hydrogel. Furthermore, for most applications, a hydrogel is preferred which is referred to as "smooth" hydrogel, as it has no or hardly any lumps in its visual appearance.

[0007] In addition to the cohesiveness, the composition according to the present application, in particular the hydrogel, should be suitable for use in humans or animals, in particular with respect to safety, immunocompatibility, bioabsorbability, biomechanical properties and duration or activity time. However, not all compositions in the prior art exhibit these properties satisfactorily and thus do not comply with the present application.

[0008] Various methods are known to implement carboxyalkyl chitosan in the form of a hydrogel. In particular, Rufato et al. (Intechopen 81811, 2018), Upadhyaya et al. (J Controlled Release, 2014), and Fonseca-Santos et al. (Mater Sci Engineering C 77, 1349, 2017) have identified several chitosan-based hydrogels comprising carboxyalkyl chitosan for medical or pharmaceutical use. However, none of these hydrogels is what the present inventors were looking for, as they do not meet the expectations, in particular at the same time with respect to cohesiveness, safety, immunocompatibility, biomechanical properties, bioabsorbability and / or duration or activity time. In addition to the compositions of Comed Pharma according to the above-mentioned applications PCT / EP2018 / 080763 and PCT / EP2018 / 080767, none of the carboxyalkyl chitosans used to prepare the known hydrogels according to the prior art exhibits good immunocompatibility according to the present inventors. Not any chitosan can be used to form a hydrogel which can be used in humans or animals.

[0009] The chitosan-based hydrogels known so far are prepared by combining chitosan or one of its derivatives with other polymers, such as alginate, isopropyl acrylamide, polyurethane, polyacrylonitrile, gelatin, polyethylene glycol (PEG), polyvinyl alcohol (PVA). However, these polymers are either non-bioabsorbable or immunoreactive, which does not meet the objectives of the present application.

[0010] For example, Huang et al. (RCS Adv 2016 DOI: 10.1039 / C5RA26160K) prepared a glycol chitosan and hyaluronic acid hydrogel, however, since this glycol chitosan is immunoreactive, it is not suitable for use in humans. Song et al. (Sci Rep 6, 37600, 2016) prepared a carboxymethyl chitosan and oxidized hyaluronic acid based hydrogel by Schiff base reaction between the amino groups of carboxymethyl chitosan and the aldehyde groups of hyaluronic acid. However, in the experience of the present inventors, the carboxymethyl chitosan used does not have the molecular structure required to meet the purposes of the present invention. In particular, according to the in vitro and in vivo tests carried out, the described hydrogel is very rapidly resorbed. Therefore, this hydrogel needs to be improved, especially in terms of its durability, in order to be used for a wide range of indications.

[0011] Furthermore, the products of the past tend to be not versatile enough to meet the needs of different indications, especially different therapeutic indications. Therefore, there is a need to provide a product versatile enough in terms of properties, especially biomechanical properties, to easily adapt it to different applications.

[0012] For example, in regenerative medicine or surgery, it is often sought to repair altered tissues or fluids and / or prevent tissue alteration, fill tissues, or even separate tissues to avoid adhesions. The cause of the alteration of the tissues can be natural aging, external attacks (trauma, UV radiation, surgery...), pathologies such as inflammation, autoimmune pathologies, etc. But most of the alterations of the tissues involve oxidative stress, sometimes called oxidative stress, which is characterized by a high content of free radicals capable of damaging tissues or cells. Reducing the number of free radicals can allow the tissues to prevent / delay their aging and reduce their harmful consequences. There are several ways to reduce the number of free radicals in the tissues, for example by administering antioxidant substances such as vitamins C, B, E and / or ubiquinone. Alternatively, compositions capable of scavenging free radicals can be used, thus reducing their content and proliferation in the tissues.

[0013] As listed in the review by Ngo et al. (Adv Food Nutrition Res 73, 15, 2014), chitosan and some of its derivatives exhibit the ability to scavenge oxidative free radicals, as described for various formulations for biomedical use. For example, carboxymethyl chitosan with different structures and molecular weights were investigated for their ability to scavenge different types of free radicals using in vitro measurement methods, as described in particular 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).

[0014] However, it is difficult to provide a composition in therapeutic form to exploit the beneficial effects of chitosan, in particular its ability to scavenge free radicals, so as to both reduce the impact of oxidative stress on the tissues and better modulate the biomechanical behaviour of the product, even increasing the persistence or effectiveness of the treatment through the presence of this exogenous polymer.

[0015] The prior art therefore does not obviously enable the person skilled in the art to provide a composition that is satisfactory to overcome the problems set out in the present application. SUMMARY

[0016] One aim of the present application is to solve the technical problem of providing a chitosan derivative or a composition comprising it suitable for use on humans or animals, in particular in the therapeutic, surgical and cosmetic fields.

[0017] One aim of the present application is to solve the technical problem of providing a chitosan derivative or a composition comprising it in therapeutic form to exploit the beneficial effects of chitosan, in particular its ability to scavenge free radicals, so as to both reduce the impact of oxidative stress on the tissues and better modulate the biomechanical behaviour and increase the persistence or effectiveness of the treatment through the presence of this exogenous polymer.

[0018] In particular, one aim of the present application is to solve the technical problem of providing a composition, in particular in the form of a bioabsorbable hydrogel, suitable for use in contact with human or animal tissues, in particular in the context of regenerative medicine or anti-aging medicine, for example in the therapeutic, rheumatology, orthopedics, gynecology, ophthalmology, aesthetic medicine, plastic surgery, open surgery, dermatology or cosmetic fields, acceptable in terms of biomechanical properties, in situ persistence or active time, good health safety, in particular without immune and / or foreign body reactions in the short and long term, and with beneficial effects.

[0019] One object of the present invention is to solve the technical problem of providing a composition having good biomechanical properties, in particular adjustable biomechanical properties according to its intended indication.

[0020] One object of the present invention is to solve the technical problem of providing a product based on a chitosan derivative, to enable the preparation of a range of products having variable biomechanical properties adapted to each intended indication.

[0021] One object of the present invention is to solve the technical problem of providing a composition which provides, preferably simultaneously, cohesion, safety (including immunocompatibility), biomechanical properties, sufficient bioresorption for administration in humans or animals, and preferably has an appropriate duration or activity time.

[0022] 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 it, having a level acceptable for humans or animals suffering from an intended indication. DETAILED DESCRIPTION

[0023] To solve the technical problem raised in the present invention, the inventors sought to develop a chitosan having both good antioxidant properties and good mechanical properties for intended applications in humans or animals, referred to as biomechanical properties.

[0024] The inventors are aware from their own experience of the advantages of substituted chitosans, and in particular carboxyalkyl chitosans. In particular, the company Comed Pharmaceuticals has filed patent applications in PCT / EP2018 / 080763 and PCT / EP2018 / 080767. They sought to apply this teaching to solve the technical problem raised in the present invention.

[0025] The inventors noted that carboxyalkyl chitosan hydrogels formed by ionic (i.e. non-covalent) cross-linking do not retain their biomechanical properties long enough after implantation for some intended applications; in particular, this technique does not enable the duration or activity time to be widely adjusted. Furthermore, carboxyalkyl chitosan hydrogels formed by enzyme-catalysed cross-linking pose a risk of enzymatic immunoreactivity due to their protein nature, and complicate the final purification of the resulting cross-linked product.

[0026] Patent application CN 107325306 (Imeik Technology Development) describes the preparation of a gel based on carboxymethyl chitosan of crustacean origin by cross-linking with BDDE in several successive cross-linking steps (multiple cross-linking). However, this method cannot provide a hydrogel that meets the criteria of the present invention, in particular because the hydrogel obtained is not cohesive, since it is formed from particles of cross-linked chitosan derivatives dispersed in a solution of carboxymethyl chitosan, the whole being cross-linked again to form a gel. The cross-linking operation is repeated several times ("multiple cross-linking"). This product is likely to form granulomas, thus negatively affecting the immunocompatibility after contact with the human or animal body, which is precisely what the present invention seeks to avoid. The present invention further advantageously enables a greater diversity of indications, especially when a hydrogel is required that is cohesive (i.e. remains in one piece without breaking, for example when in contact with water) and / or has a "smooth" appearance. According to CN 107325306, the carboxymethyl chitosan used has a low DA (degree of deacetylation of 60-99%, preferably 80-95%, i.e. in practice a degree of acetylation (DA) well below 40%). Czechowska-Biskup et al. (DOI: 10.15259.PCACD.21.03) also describe carboxymethyl chitosan hydrogels with a low degree of acetylation. However, these hydrogels are not cohesive and do not meet the objectives of the present invention.

[0027] The inventors have found that the cross-linked carboxyalkyl chitosan matrix according to the present invention or the composition comprising it, in particular the hydrogel, is able to solve at least one, preferably all, of the technical problems set out in the present invention.

[0028] The present invention thus relates to a matrix comprising at least one carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units and glucosamine units substituted with a carboxyalkyl group, expressed in terms of the number of moles of N-acetyl relative to the number of moles of total glucosamine units, the degree of acetylation of said carboxyalkyl chitosan being between 40% and 80%, said carboxyalkyl chitosan being cross-linked by covalent bonds between the chains of carboxyalkyl chitosan.

[0029] In practice, it has been found that a cross-linked carboxyalkyl chitosan with a DA less than 40% is not possible to obtain a hydrogel with the required cohesiveness, since it breaks up into separate fragments during wetting, which is undesirable for many applications.

[0030] 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”:

[0031] 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 immunocompatibility, 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 application is characterized by a starting carboxyalkyl chitosan which is cross-linked to form the matrix according to the application.

[0037] According to a first aspect, a carboxyalkyl chitosan of fungal origin is used, the carboxyalkyl chitosan of fungal origin having carboxyalkyl-substituted glucosamine units, N-acetylglucosamine units and glucosamine units, expressed in moles of substituents relative to the moles of total units, said carboxyalkyl chitosan preferably having a degree of substitution of carboxyalkyl groups greater than 20%.

[0038] This is also referred to as chitosan derivative or substituted chitosan.

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

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

[0041] The chitosan used in the present application is advantageously of fungal origin and is preferably derived from mycelium of fungi of the Ascomycete type, in particular Aspergillus piger and / or Basidiomycete fungi, and in particular Lentinula edodes (Shiitake) and / or Agaricus bisporus (White mushroom). Preferably, the chitosan is derived from Agaricus bisporus. The chitosan is preferably of high purity, i.e. contains few impurities from its fungal origin or from the preparation process, with a microbiological level compatible with its use as an implant or a pharmaceutical composition. One method of preparing chitosan is the method described in patent WO 03 / 068824 (EP 1483299; US 7 556946).

[0042] Typically, chitin is suspended in an aqueous medium in the presence of sodium hydroxide, which medium is then heated at high temperature for a variable time according to the desired molecular weight. The chitosan is then dissolved in an acidic medium for purification, precipitated in a basic medium, washed and dried.

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

[0044] The chitosan is advantageously purified and then preferably dried. After purification, the process of the application can comprise a step of drying the carboxyalkyl chitosan, which can then optionally be ground into a powder. The carboxyalkyl chitosan can be dried, for example, by evaporation of water, for example by spray-drying (atomization), fluidized bed processes, or by drying under heating, either under vacuum or at atmospheric pressure, or by lyophilization.

[0045] The carboxyalkyl chitosan can be dissolved in an aqueous solution, for example in water of pharmaceutical grade suitable for injection or implantation into the body, in particular into the human body.

[0046] Such carboxyalkyl chitosan is then cross-linked to prepare the matrix according to the application.

[0047] The DA and DS of the cross-linked carboxyalkyl chitosan can be expressed as a function of the DA and DS of the non-cross-linked carboxyalkyl chitosan, since the DA and DS are essentially unchanged after cross-linking. However, if the cross-linking agent provides N-acetyl groups or carboxyalkyl groups, these extraneous groups are not counted in the DA and DS of the cross-linked carboxyalkyl chitosan compared to the starting non-cross-linked carboxyalkyl chitosan. The values of DA and DS are known to the person skilled in the art, as described below. Thus, DA and DS refer both before and after cross-linking.

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

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

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

[0052] The degree of acetylation of the carboxyalkyl chitosan can be determined by solid-state carbon-13 nuclear magnetic resonance, solid-state carbon-13 nuclear magnetic resonance or liquid-state proton nuclear magnetic resonance. The carboxyalkyl chitosan advantageously has a controlled degree of acetylation. The term "chitosan having a controlled degree of acetylation" refers to a product whose degree of acetylation (i.e. the proportion of N-acetyl-glucosamine units) can be adjusted in a controlled manner, in particular by acetylation.

[0053] Preferably, the carboxyalkyl chitosan is reacetylated.

[0054] According to one alternative embodiment, the process for preparing a carboxyalkyl chitosan according to the application comprises preparing chitosan of fungal origin, reacetylating the chitosan and carboxyalkylating the reacetylated chitosan. Thus, the present application relates to a reacetylated carboxyalkyl chitosan. In particular, the present application relates to an anionic carboxyalkyl chitosan.

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

[0056] According to one alternative embodiment, the process for preparing a carboxyalkyl chitosan according to the application comprises preparing chitosan, carboxyalkylating the chitosan and then reacetylating the carboxyalkylated chitosan. Advantageously, this process allows precise control of the degree of acetylation of the final carboxyalkyl chitosan, in order in particular to obtain a high degree of acetylation, for example a degree of acetylation of more than 40%. Thus, the present application relates to a reacetylated then carboxyalkylated chitosan or to a reacetylated carboxyalkyl chitosan.

[0057] According to one alternative embodiment, the process for preparing a carboxyalkyl chitosan according to the application comprises preparing chitosan of fungal origin, reacetylating the chitosan and carboxyalkylating the reacetylated chitosan. Thus, the present application relates to a reacetylated carboxyalkyl chitosan. In particular, the present application relates to an anionic carboxyalkyl chitosan.

[0058] According to one alternative embodiment, the process for preparing a carboxyalkyl chitosan according to the application comprises preparing chitosan of fungal origin, reacetylating the chitosan and carboxyalkylating the reacetylated chitosan. Thus, the present application relates to a reacetylated carboxyalkyl chitosan. In particular, the present application relates to an anionic carboxyalkyl chitosan.

[0059] According to one alternative embodiment, the carboxyalkyl chitosan has an average molecular weight of 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 herein, the average molecular weight is the viscosity average molecular weight (Mv) calculated from the intrinsic viscosity. This representation is the customary representation for the person skilled in the art. The intrinsic viscosity (η) is measured by capillary viscosimetry according to the method in the European Pharmacopoeia 2.2.9 monograph, using a capillary viscosimeter of the Ubbelohde type. The flow time of the solution is measured using an automatic I-Visc viscosimeter (Lauda) through a suitable capillary (Lauda, for example Ubbelohde 510 01 capillary with a diameter of 0.53 mm). The Mark-Houwink equation (η = K*Mv α ) is then applied to calculate the average viscosity mass of the carboxyalkyl chitosan, wherein:

[0066] Mv is the viscosity average molecular weight of the carboxyalkyl chitosan,

[0067] η is the intrinsic viscosity of the carboxyalkyl chitosan,

[0068] The values of the constants K and a are respectively 0.0686 and 0.7638 as previously determined for (unsubstituted) chitosan by size exclusion chromatography with a MALLS detector.

[0069] Thus, the intrinsic viscosity of the carboxyalkyl chitosan can generally be expressed as:

[0070] Chitosan can be hydrolyzed to reduce its molecular weight.

[0071] Generally, in the carboxyalkyl chitosan which is not cross-linked, the glucosamine units are D-glucosamine units (D-glucosamine units, N-acetyl-D-glucosamine units, and at least one of substituted D-glucosamine units and N-acetyl-D-glucosamine units).

[0072] According to an alternative embodiment, the substituted chitosan has only substitution of D-glucosamine units.

[0073] According to another alternative embodiment, the substituted chitosan has substitutions of both D-glucosamine and N-acetyl-D-glucosamine units, wherein the carboxyalkyl groups are covalently bound according to one alternative embodiment to only the amino groups of the chitosan, or according to another alternative embodiment to both the amino and hydroxyl groups of the chitosan.

[0074] The substitution is typically only partial and not all units need to be substituted.

[0075] According to one embodiment, the degree of substitution of the D-glucosamine units is in the range of 30% to 250% expressed in moles of D-glucosamine units relative to the moles of total units of the substituted chitosan (substituted or unsubstituted D-glucosamine units and N-acetyl-D-glucosamine units).

[0076] According to one embodiment, the carboxyalkyl chitosan has a degree of substitution of the carboxyalkyl groups of more than 20%, such as more than 50%, such as less than 200% expressed in moles of substituents relative to the moles of total units.

[0077] According to one embodiment, the degree of substitution of the carboxyalkyl groups is more than 50% expressed in moles of substituents relative to the moles of total units.

[0078] According to one embodiment, the degree of substitution of the D-glucosamine units is in the range of 50% to 200%, and preferably more than 70% expressed in moles of D-glucosamine units relative to the moles of total units of the substituted chitosan (substituted or unsubstituted D-glucosamine units and N-acetyl-D-glucosamine units).

[0079] According to one embodiment, the degree of substitution of the carboxyalkyl groups is less than 80% expressed in moles of substituents relative to the moles of total units.

[0080] Typically, the substitution is achieved by covalent bonding.

[0081] According to one alternative embodiment, the carboxyalkyl chitosan is an N,O- carboxyalkyl chitosan. The proportion of units substituted with carboxyalkyl groups at the O-position (03or 06of the D-glucosamine units and / or N-acetyl-D-glucosamine units) and / or at the N-position (of the D-glucosamine units) can vary. Thus, the degree of substitution can be more than 100%.

[0082] Advantageously, the degree of substitution (DS) and the degree of acetylation (DA) of carboxyalkyl chitosan are measured by solid-state carbon-13 NMR using a Bruker spectrometer (Avance III HD 400 MHz) equipped with a PH MAS VTN 400SB BL4 NP / H probe. For example, the spectrum is recorded at room temperature, with a relaxation time between 1 and 8 seconds, with a number of scans between 64 and 512. The areas of the carbon signals are determined after deconvolution. The carbons considered are: “CH3acetyl” (methyl carbon of the acetyl group of the substituted or unsubstituted N-acetyl-glucosamine units), “Cx” (carbon in position x of the glucosamine and N-acetyl-glucosamine units, x ranging from 1 to 6) and “C=0” (carbonyl carbon of the carbonyl group of the carboxyalkyl substituent and of the acetyl group of the substituted or unsubstituted N-acetyl-glucosamine units). In order to determine the DS of a given carboxyalkyl chitosan, the carbon 13 NMR spectrum of the precursor chitosan of this carboxyalkyl chitosan should also be recorded. From the spectrum of the precursor chitosan, the “CSU ratio” is calculated, i.e. the ratio of the signal area of the “CH3acetyl” group (methyl carbon of the acetyl group of the N-acetyl-glucosamine units) to the signal area of the “C=0” (carbonyl carbon of the acetyl group of the N-acetyl-D-glucosamine units). The DA of the carboxyalkyl chitosan is calculated according to formula 1 and the DS according to formula 2, where I represents the signal area of the considered carbon. X [mathematical formula 1]

[0083] Formula 1:

[0084] [mathematical formula 1]

[0085] [mathematical formula 1] [mathematical formula 1]

[0086] Formula 2: [mathematical formula 2]

[0087] [mathematical formula 2] [mathematical formula 2]

[0088] [mathematical formula 2] [mathematical formula 2] [mathematical formula 2]

[0089] Other known methods can be used to determine the DA and the DS of carboxyalkyl chitosan, for example by proton NMR in aqueous medium using a magnetic resonance spectrometer, for example according to the method described by Liu et al. (Carb Polym 137, 600, 2016), for example, with the addition of a concentrated solution of deuterated hydrochloric acid to hydrolyze the carboxyalkyl chitosan beforehand before analysis.

[0090] If another NMR method is more advantageous for reliably estimating the DA and / or the DS, such a method is suitable for use. The above-mentioned method should be adapted by the skilled person with respect to the sample preparation and the signals to be integrated, in particular with respect to the resolution, robustness and proton position of the signals used for calculating the degree of substitution.

[0091] The degree of carboxyalkylation of chitosan can advantageously range from 20 to 250 %, preferably from 50 to 200 %, and for example from 70 to 170 %, expressed in moles of carboxyalkyl groups with respect to the moles of total units.

[0092] According to an alternative embodiment, the degree of carboxyalkylation of chitosan can advantageously range from 40 to 130 %, for example from 70 to 130 %, expressed in moles of carboxyalkyl groups with respect to the moles of total units.

[0093] The degree of substitution of chitosan is generally related to the mass ratio of reactants to chitosan at the beginning of the reaction. Examples of carboxyalkylation agents include acyl chlorides (or salts thereof, for example sodium monochloroacetate), such as those bearing one or more carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl, etc. groups.

[0094] According to an alternative embodiment, the present application relates to a carboxyalkyl chitosan wherein the alkyl part of the carboxyalkyl group is linear or branched C1-C5.

[0095] According to an embodiment, the present application relates to a carboxymethyl chitosan.

[0096] According to this alternative embodiment, the substituted chitosan is an N- carboxyalkyl chitosan.

[0097] According to this embodiment, the substituted chitosan is an O-carboxyalkylated chitosan.

[0098] According to this alternative embodiment, the substituted chitosan is an N- carboxyalkylated and O-carboxyalkylated chitosan.

[0099] According to a second aspect, the present application relates to a chitosan derivative having glucosamine units, N-acetyl-glucosamine units and glucosamine units substituted with carboxyalkyl groups, said carboxyalkyl chitosan having 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, such chitosan derivative is able to limit the immune response of a subject to which it is administered, usually by instillation, injection or implantation, to the chitosan derivative or to a composition comprising it.

[0100] Advantageously, the zeta potential measured at pH 7.5 is less than or equal to -18 mV.

[0101] Advantageously, the carboxyalkyl chitosan has a zeta potential less than or equal to -22 mV, preferably less than or equal to -24 mV, measured at pH 7.5.

[0102] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0103] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0104] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0105] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0106] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0107] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0108] According to a 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0109] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0110] 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 %, the molecular weight preferably representing the molecular weight before substitution.

[0111] According to a specific alternative embodiment, the substituted chitosan has a degree of substitution of 20 % to 80 %, preferably 40 % to 60 %, and has a degree of acetylation of 40 % to 80 %, preferably 50 % to 75 %.

[0112] According to a specific alternative embodiment, the substituted chitosan has a degree of substitution of 50% to 200%, preferably 70% to 200%, and an acetylation degree of 40% to 80%, preferably 50% to 75%.

[0113] According to another specific alternative embodiment, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and an acetylation degree of 40% to 80%, the molecular weight preferably indicating the molecular weight before substitution.

[0114] According to a specific alternative embodiment, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and an acetylation degree of 40% to 60%, preferably 50% to 60%.

[0115] According to a specific alternative embodiment, the substituted chitosan has a degree of substitution of 90% to 200%, preferably 90% to 150%, and an acetylation degree of 50% to 75%.

[0116] 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 an acetylation degree of 50% to 75%, the molecular weight preferably indicating the molecular weight before substitution.

[0117] By substituting chitosan, it is possible to prepare solutions of carboxyalkyl chitosan that are soluble in aqueous solutions whose pH varies in a wide range, whereas unsubstituted chitosan is soluble only at a pH lower than 5.5-6.5. Therefore, due to the presence of the carboxyalkyl groups that modify its solubility profile, carboxyalkyl chitosan shows the ability to dissolve at different pHs, and in particular at physiological pH or at the pH of the physiological fluids altered in pathologies, such as inflammatory pathologies.

[0118] By "soluble in water" it is meant that the carboxyalkyl chitosan, when placed in an aqueous solution, does not present any visible turbidity to the naked eye. More specifically, the solubility (i.e. the absence of turbidity) of a carboxyalkyl chitosan solution having a concentration of, for example, 1% (m / m) in water or in a buffer (e.g. phosphate buffer) can be confirmed by an optical density of less than 0.5, preferably less than 0.2, measured at a wavelength of 500 nm by UV-visible spectroscopy, with respect to a reference cell containing only the aqueous solvent used for the sample under measurement, in the absence of substituted chitosan. Another method is the visual inspection according to the European Pharmacopoeia monograph 2.9.20. When the chitosan is not sufficiently substituted, the composition is not soluble at room temperature in a satisfactory pH range (e.g. pH 5.5 to pH 8.5).

[0119] According to one embodiment, the carboxyalkyl chitosan is sterile.

[0120] By "crosslinking by covalent bonds between the carboxyalkyl chitosan chains" is especially understood that the chitosan backbone (also called chitosan backbone chain) is covalently bound to one or more main chitosan chains. Advantageously, a three-dimensional network of chitosan molecules is thereby obtained. The present application is not limited to a particular method of covalent crosslinking, but a method using a chemical molecule (also called crosslinker) as crosslinker is preferred.

[0121] According to the present application, the carboxyalkyl chitosan is crosslinked.

[0122] According to one alternative embodiment, the crosslinking is formed by a crosslinker forming said covalent bonds.

[0123] Thus, several chitosan chains can be crosslinked, for example by reaction with one or more crosslinkers, for example selected from crosslinkers for crosslinking polysaccharides, such as 1,4-butanediol diglycidyl ether, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxy pentane, 1-chloro-2,3-epithiopropane, 1-bromo-2,3-epithiopropane, 1-bromo-3,4-epithiobutane, 1-bromo-4,5-epithiopentane, 2,3-dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanethiol, 2,4-dibromobutane- thiol, 2,5-dibromopentane-thiol, epichlorohydrin, 1-chloro-2,3-epithiopropane, dimethylaminopropyl carbodiimide, gallic acid, epigallocatechin gallate, curcumin, tannic acid, genipin or even a diisocyanate compound, such as hexamethylene diisocyanate or toluene diisocyanate, or even a diethylene sulfone.

[0124] Genipin is a naturally occurring crosslinker for crosslinking polysaccharides, in particular carboxymethyl chitosan (Yang et al. Acta Pharmacol Sin, 31, 1625, 2020). Genipin imparts a dark blue to black color to the hydrogel, which can be an advantage in some indications.

[0125] Preferably, the crosslinker is of the polyepoxide type, for example a bifunctional agent. The use of 1,4-butanediol diglycidyl ether (BDDE) or ethylene glycol diglycidyl ether (EGDE) as crosslinker is preferred, as they have already been used for the preparation of biomaterials for application to humans, in particular hyaluronic acid hydrogels for intradermal, intra-articular or intraocular administration. According to one alternative embodiment, the crosslinker is a diethylene sulfone.

[0126] Advantageously, the composition of the application can also comprise a biopolymer other than crosslinked carboxyalkyl chitosan. According to an advantageous alternative, the biopolymer is an oxidized or non-oxidized, crosslinked or non-crosslinked polysaccharide by covalent bonds, such as a glycosaminoglycan, and in particular a hyaluronic acid, such as hyaluronic acid or sodium hyaluronate.

[0127] The advantage of combining or crosslinking the crosslinked carboxyalkyl chitosan with some other polymers is to increase their biological and physicochemical properties, or even to create a synergistic effect.

[0128] According to an alternative embodiment, the matrix according to the application comprises crosslinked carboxyalkyl chitosan and hyaluronic acid, chondroitin sulfate and / or carboxymethylcellulose. To date, no hydrogel combining crosslinked carboxyalkyl chitosan, as defined in the application, and hyaluronic acid has been described. One of the objectives of the application is to combine these two polymers in order to be able to combine the well-recognized moisturizing properties of hyaluronic acid and the protective properties against oxidative stress of chitosan.

[0129] According to an alternative embodiment, the matrix comprises at least one hyaluronic acid.

[0130] Advantageously, the matrix according to the application comprises crosslinked carboxymethyl chitosan alone or crosslinked carboxymethyl chitosan combined with crosslinked or non-crosslinked hyaluronic acid. This makes it possible to adjust the desired properties.

[0131] The matrix comprises at least one carboxymethyl chitosan and a hyaluronic acid.

[0132] According to an alternative embodiment, the hyaluronic acid has a bio- average molecular weight of less than 5 million, and preferably greater than 1 million, preferably greater than 2 million, as determined by the capillary viscosimetry method. The molecular weight of hyaluronic acid is sometimes expressed by its density, since they are correlated by a linear relationship. Hyaluronic acid can have a density of up to 4.25 m 3 / kg, and can for example be designated as having a low density (e.g., about 1 to 2 m 3 / kg) hyaluronic acid or a high density (e.g., about 2 to 4 m 3 / kg) hyaluronic acid.

[0133] According to an alternative embodiment, the hyaluronic acid is obtained by fermentation (e.g., with streptococcus). According to another alternative embodiment, it is produced by extraction from rooster peaks.

[0134] According to an alternative embodiment, the matrix comprises at least one hyaluronic acid crosslinked by covalent bonds.

[0135] Thus, the crosslinked hyaluronic acid comprises covalent bonds between the different hyaluronic acid chains.

[0136] Different types of hyaluronic acid, such as hyaluronic acid of different molecular weight or different hyaluronic acid salts, can be cross-linked with each other.

[0137] The present application also relates to a process for preparing cross-linked carboxyalkyl chitosan.

[0138] According to an alternative embodiment, the process for preparing the matrix according to the application comprises:

[0139] contacting the carboxyalkyl chitosan with at least one cross-linking agent, the contacting preferably being carried out in an aqueous basic phase;

[0140] cross-linking the carboxyalkyl chitosan with the cross-linking agent; and

[0141] obtaining a matrix comprising cross-linked carboxyalkyl chitosan.

[0142] According to an alternative embodiment, the carboxyalkyl chitosan is cross-linked in an aqueous basic phase, for example in the presence of a sodium hydroxide (NaOH) solution.

[0143] Advantageously, the concentration of carboxyalkyl chitosan initially present in the aqueous phase is in the range 1-30% (w / v), preferably in the range 5-20% (w / v), expressed in weight of carboxyalkyl chitosan relative to the volume of the aqueous basic phase.

[0144] Advantageously, the mass ratio between the cross-linking agent and the polymer is in the range 0.1-30%, expressed in weight of cross-linking agent relative to the weight of polymer.

[0145] Preferably, the mass ratio between the cross-linking agent and the polymer is in the range 0.5-20%, expressed in weight of cross-linking agent relative to the weight of polymer, in particular when BDDE is used.

[0146] Generally, the reaction is carried out under heating, for example at 25-60°C, for example 50°C, for a time of, for example, 30 minutes to 48 hours, for example 1 hour to 5 hours. Generally, the cross-linking is stopped by neutralization and dilution, for example by adding an acid, for example by adding acetic acid or hydrochloric acid.

[0147] Advantageously, the reaction residues are removed by dialysis using a phosphate buffer.

[0148] A hydrogel comprising the matrix according to the application is thus obtained.

[0149] On the other hand, carboxyalkyl chitosan is an exogenous molecule which is more resistant to degradation than hyaluronic acid after implantation / injection / instillation into the body.

[0150] Thus, the present application relates to a matrix comprising a three-dimensional network based on these two polymers having different molecular weights. Thus, advantageously, a range of biomechanical properties, in situ product duration and treatment duration are provided, while preserving the ability of carboxyalkyl chitosan to scavenge free radicals.

[0151] The present application relates to a matrix comprising at least one hyaluronic acid co-crosslinked with carboxyalkyl chitosan through covalent bonds.

[0152] According to an alternative embodiment, the method of preparing a matrix comprising carboxyalkyl chitosan (preferably carboxyalkyl chitosan as defined according to the present application) co-crosslinked with another biopolymer (preferably hyaluronic acid), said method comprising:

[0153] contacting a mixture of carboxyalkyl chitosan and the other biopolymer (preferably hyaluronic acid) with at least one crosslinking agent, the contacting preferably being carried out in an alkaline phase;

[0154] crosslinking the carboxyalkyl chitosan and the other biopolymer (preferably hyaluronic acid) with the crosslinking agent;

[0155] obtaining a co-crosslinked matrix of carboxyalkyl chitosan and the other biopolymer (preferably hyaluronic acid).

[0156] According to an alternative embodiment, the matrix according to the present application is sterile.

[0157] It is advantageous to provide a hydrogel from the matrix according to the present application.

[0158] Thus, the present application relates to a hydrogel, and advantageously to a cohesive hydrogel.

[0159] Thus, the present application relates to a crosslinked carboxyalkyl chitosan hydrogel, wherein the carboxyalkyl chitosan has a high degree of acetylation (DA) (greater than 40%) and preferably also a high degree of substitution (DS) (greater than 20%, preferably greater than 50% and typically less than 200%).

[0160] The present application relates to a composition comprising at least one matrix according to the definition of the present application.

[0161] According to a preferred alternative embodiment, the matrix according to the present application is formulated in an aqueous medium to form a composition in the form of a hydrogel.

[0162] Advantageously, the concentration of the polymer (carboxyalkyl chitosan, with or without the other biopolymer such as hyaluronic acid) is less than 10%, for example less than or equal to 5%, in mass of polymer relative to the total mass of the composition (in particular of the hydrogel) (m / m).

[0163] 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%, in mass of polymer relative to the total mass of the composition (in particular the total mass of the hydrogel) (m / m).

[0164] The mass ratio (m / m) [carboxyalkyl chitosan / hyaluronic acid] is for example between 5% and 95%, for example between 10% and 90%, and also for example between 30% and 70%. The mass ratio (m / m) [hyaluronic acid / carboxyalkyl chitosan] is for example between 5% and 95%, for example between 10% and 90%, and further for example between 30% and 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).

[0165] The aqueous medium can be water, an aqueous solution, the pH and the tonicity of which are adjusted for example using an acid / base buffer system with the addition of salts and / or optionally polyols (sorbitol, mannitol, glycerol).

[0166] According to an alternative embodiment, the matrix according to the application is formulated in a hydrolipidic medium, so as to form a single or multiple, direct or inverse, emulsion.

[0167] According to one embodiment, the composition of the matrix has a tonicity of between 100 mosm / kg and 700 mosm / kg, preferably between 120 mosm / kg and 500 mosm / kg.

[0168] Advantageously, the composition of the matrix has a tonicity of between 250 mosm / kg and 400 mosm / kg, preferably between 270 mosm / kg and 330 mosm / kg.

[0169] According to an alternative embodiment, the composition of the matrix has a tonicity suitable for the joint.

[0170] According to an alternative embodiment, the composition of the matrix has a tonicity compatible with the ocular or intraocular surface.

[0171] According to an alternative embodiment, the composition of the matrix has a tonicity compatible with the dermis or mucous membranes.

[0172] According to an alternative embodiment, the tonicity of the composition of the matrix is preferably between 100 mosm / kg and 400 mosm / kg, more particularly between 120 mosm / kg and 380 mosm / kg.

[0173] According to an alternative embodiment, the composition according to the application is sterile.

[0174] Advantageously, the composition according to the application is contained in an injection, implantation or instillation device, such as a syringe or a vial.

[0175] Advantageously, the injection device, such as a syringe, can for example then be subjected to steam sterilization. This device, such as a syringe, can then preferably be packaged in a sterilized or sterile manner. It can also be a bag, a flapula or a vial for instilling the composition according to the application, these bags, flapulas or vials being aseptically filled after sterilization of the preparation or sterilized directly after filling.

[0176] According to an alternative embodiment, the composition according to the application, in particular the hydrogel according to the application, is sterilized by filtration and / or steam sterilization before filling an injection device, an implantation device or an instillation device, such as a syringe or a vial.

[0177] The person skilled in the art knows the techniques for sterilizing a hydrogel to obtain a sterile hydrogel as desired. They have several types of equipment for heat or steam sterilization and can use several types of cycles for removing the microbial load.

[0178] The application more particularly relates to an injectable composition comprising a matrix according to the application, preferably in the form of a hydrogel.

[0179] The application also relates to a pharmaceutical composition comprising at least one matrix according to the application, preferably in the form of a hydrogel.

[0180] According to an alternative embodiment, the composition according to the application is used as an injectable, implantable or instillable pharmaceutical composition, or as an injectable or implantable or instillable medical device.

[0181] The application further encompasses a composition according to the application in dry form, in particular in lyophilized form. The lyophilized product can in particular be (re)dispersed and preferably dissolved before use.

[0182] The application more particularly relates to the use of a composition according to the application for therapeutic treatment, for example comprising the injection of said composition by subcutaneous, intradermal, intraocular, or intra-articular, intramucosal, intramuscular route, for example to repair, regenerate or fill at least one body tissue / fluid in need of repair or filling.

[0183] It is advantageous to use chitosan having a sufficient purity for the intended application.

[0184] It is advantageous to use hyaluronic acid having a sufficient purity for the intended application.

[0185] The sought biomechanical properties of the composition according to the application can vary in nature according to the indication, for example according to the tissue in which the hydrogel is to be integrated, the mechanism of action or the effect intended to ensure a benefit for the patient, and the duration of the effect, vary in nature.

[0186] Advantageously, the properties of the composition according to the application, in particular of the hydrogel according to the application, are adapted according to the indication. To adapt these properties, in particular by the mass ratio of crosslinking agent / polymer, and / or the nature and / or the 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 for example varied.

[0187] In particular, the application relates to a highly elastic hydrogel (especially when it is necessary to ensure a sustained increase in volume at the skin, subcutaneous or periosteal level (for augmentation or reshaping)) or a viscoelastic gel, especially so as to be able to both dampen and have a lubricating effect at the joint. The application relates to a lubricating hydrogel especially when it is necessary to reduce the friction between two biological surfaces, for example the two cartilage surfaces in a joint, or the ocular surface and the eyelid in the eye. The composition of the application can have a variable level of elasticity adapted according to the indication and is characterized by an elastic modulus measured by rheometry.

[0188] Preferably, the matrix has antioxidant power by scavenging free radicals, especially with a normalized antioxidant power greater than 0.30, preferably greater than 0.50, even more preferably greater than 0.80, for example greater than 0.90.

[0189] The application relates to an injectable composition characterized in that it comprises at least one matrix as defined according to the application.

[0190] The application relates to a pharmaceutical composition characterized in that it comprises at least one matrix as defined according to the application.

[0191] According to an alternative embodiment, the composition according to the application is used as an injectable, implantable or instillable, or topically administrable pharmaceutical composition, or an injectable or implantable or instillable, or topically administrable medical device, for example for a therapeutic treatment method (for example comprising the local instillation or administration or injection of said composition by subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular, intraosseous route), for example to repair or fill at least one body tissue in need of repair or filling.

[0192] According to an alternative embodiment, the composition according to the application is used in a method for treating, repairing or filling at least one body fluid or body tissue in need of repair or filling, for example, the body tissue is chosen from a tissue belonging to the group consisting of vocal cords, muscles, ligaments, tendons, mucous membranes, sexual organs, bones, joints, eyes, dermis or any combination thereof, in particular dermis, cartilage, synovial membrane, skin wounds, even ocular surface.

[0193] The application relates to the use of the composition according to the application in a method for treating osteoarthritis or repairing a cartilage defect, for example by injection into a biological fluid, such as synovial fluid, or implantation into cartilage after mixing with a biological fluid, such as blood. Biological fluid means a fluid of body origin, which has or has not been subjected to a treatment modifying its composition.

[0194] The application relates to a medical device, such as a medical implant, characterized in that it comprises or consists of a composition according to the definition of the application.

[0195] The application particularly relates to the use of the composition according to the application for therapeutic, surgical or cosmetic treatment, in particular treatment in rheumatology, ophthalmology, gynaecology, aesthetic medicine, plastic surgery, open surgery, orthopaedic surgery, gynaecology to prevent postoperative tissue adhesions, and in dermatology.

[0196] The application also relates to the use of the composition according to the application for the therapeutic treatment of dry eye, corneal lesions or ocular or articular inflammation.

[0197] The application further relates to the use of the composition according to the application by instillation on the ocular surface to prevent or combat corneal lesions or dry eye, in particular for the purpose of lubricating or regenerating the ocular surface.

[0198] The application thus also relates to an eye drop composition comprising a carboxyalkyl chitosan according to the definition of the application.

[0199] According to an alternative embodiment, the subject suffers from an inflammatory condition, such as osteoarthritis, arthritis, dry eye.

[0200] The application more particularly relates to the use of the composition according to the application for treating arthrosis, arthritis or repairing a cartilage defect, for example by injection into the synovial cavity or by implantation at the site of the cartilage defect.

[0201] The application more particularly relates to a medical device, such as a medical implant, characterized in that it comprises or consists of a composition according to the application.

[0202] According to one preferred alternative embodiment, the present application thus relates to a medical device comprising a chamber containing a composition according to the present application in dry form, in particular lyophilized form, and optionally one or more other chambers containing one or more active products, additives or excipients.

[0203] The composition according to the present application can also comprise one or more active agents for the desired indication, and / or one or more additives or excipients for modulating the properties of the composition according to the present application.

[0204] The present application also relates to the use of the composition according to the present application in a therapeutic treatment method.

[0205] The present application also relates to the use of the composition according to the present application in a method for treating joint disease or repairing cartilage defects, for example by injection into the synovial bursa or implantation into cartilage / bone after mixing with blood.

[0206] The present application also relates to the use of the composition according to the present application in a cosmetic treatment or cosmetic care method by dermal filling ("dermal filling") or lip filling. This relates in particular to, for example, subcutaneous, intradermal, intramucosal or intramuscular injection of the composition according to the present application.

[0207] The present application also relates to the use of the composition according to the present application in a method for surface treatment of the skin (by multiple intradermal injections) or other tissues according to conventional mesotherapy known to the person skilled in the art. Such compositions are generally used in dermatology as a treatment for cosmetic purposes. The aim of this method is, for example, to plump up the skin to make its appearance wrinkle-free (treatment of wrinkles and / or fine lines). This treatment can be used for a subject who wishes to restore a youthful appearance to his / her skin.

[0208] The present application also relates to the use of the composition according to the present application in a treatment method, wherein the composition is a viscosupplement. Here, for example, intra-articular injection of the composition of the present application, in particular to limit friction on the surface of the articular cartilage.

[0209] The present application also relates to the use of the composition according to the present application as a cell carrier for one or more cell types and / or one or more active agents. These can be active agents from a pharmaceutical or biological point of view. The composition of the present application can indeed be compatible with the presence of cells, preferably living cells. Examples of living cells of interest include: chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), dermal fibroblasts (skin), tendon cells (tendon), myofibroblasts (muscle), mesenchymal stem cells, red blood cells (blood) and keratinocytes (skin). The composition of the present application can also be used as a therapeutic carrier for the targeted delivery and / or controlled release of at least one therapeutic agent.

[0210] According to an alternative embodiment, blood, or plasma, or platelet lysate, or platelet rich plasma, or any biological fluid is added with the composition of the application, for example to improve the performance of the product.

[0211] According to an alternative embodiment, the composition according to the application is formulated in a solid form (for example, a membrane or a porous foam) which swells / wets once implanted (for example, a lacrimal plug, a dressing).

[0212] According to an alternative embodiment, the composition is formulated in the form of a nebulizable composition (a spray).

[0213] The application also relates to the composition according to the application for use in a method for treating or cosmetically caring for one or more tissues or organs affected by high temperatures, such as in the case of burns.

[0214] The application also relates to the composition according to the application for use in a method for treating cartilage repair (for example, by implanting on a cartilage defect to promote its regeneration).

[0215] The application also relates to the composition according to the application for use in a method for prophylactically treating post-surgical tissue adhesions, the product being applied on the tissues at the end of the surgery (for example gynecological, abdominal, visceral, orthopedic, etc.).

[0216] The application relates to a physiological composition which is administered topically by injection or implantation to come into contact with one or more living tissues subjected to oxidative stress, for example:

[0217] - intra-articular injection (by synovial fluid supplementation, cartilage lubrication, shock absorption at the joint, synovial membrane regeneration) to treat osteoarthritis; intra-articular implant to promote repair of cartilage defects;

[0218] - intra-osseous implant to promote bone repair (osteoinduction / osteoconduction);

[0219] - subcutaneous and / or intradermal injection for filling or regenerating the skin or hair follicles to increase volume in the case of fat atrophy;

[0220] - ocular instillation to relieve ocular surface symptoms or prevent changes, for example to treat dry eye and comeal pathologies, and to administer active ingredients;

[0221] - intraocular injection, for example, as an adjuvant to cataract surgery, for optimizing the effects of glaucoma surgery or vitreous supplementation, to regenerate anterior or posterior ocular tissues, and to administer active ingredients intraocularly;

[0222] - administration on internal tissues and organs (membranes) to prevent post-surgical adhesions;

[0223] - to be applied on wounds, cracks, tears, cavities... of tissues and organs such as skin, bone, cartilage, cornea, tendons, meniscus... to promote their repair or regeneration;

[0224] - to be injected into the mucosa of the vulva for the treatment of vulvodynia.

[0225] The application also relates to a composition according to the application forming an artificial synovial fluid.

[0226] By seeking for example to improve its lubricating ability to reduce friction at the joint and / or its shock-absorbing properties (determinable by the elastic modulus G'), the composition according to the application can mimic healthy synovial fluid or improve healthy or defective synovial fluid, while being easy to inject, for example to fill a syringe, or to inject 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.

[0227] Advantageously, for intra-articular injection, the composition according to the application is easy to inject at room temperature through a fine needle, for example a 21 gauge needle. "Easy" injection preferably means that the force exerted on such a syringe to make the composition according to the application flow through a 21 gauge needle is less than 50 Newtons at a speed of 10 mm / min, preferably less than 20 Newtons.

[0228] Advantageously, for intradermal injection, the composition according to the application is easy to inject at room temperature through a fine needle, for example a 25 gauge or smaller needle. "Easy" injection preferably means that the force exerted on such a syringe to make the composition according to the application flow through a 27 gauge needle is less than 30 Newtons to be ejected into the air at a speed of 10 mm / min, preferably less than 20 Newtons.

[0229] The application also relates to a composition comprising a carboxyalkyl chitosan according to the application as an artificial tear.

[0230] Generally, the osmolality and the pH of the composition are in a range that is suitable and generally close to the osmolality and the pH of the tissue in contact with the composition according to the application.

[0231] Advantageously, the composition according to the application is sterile. Very advantageously, the composition according to the application is sterilized by heat, preferably in an autoclave.

[0232] According to one embodiment, the matrix has a lubricating ability with a low coefficient of friction (COF) (for example less than 20, and for example less than 10).

[0233] According to one alternative embodiment, the composition of the application is transparent or translucent.

[0234] "Translucent" means that the object can be recognized when the composition thereof is placed between the eye of the observer and the object. "Transparent" means that the alphanumerical characters can be recognized when the composition is placed between the eye of the observer and the characters observed. Generally, such evaluation is performed with a composition thickness of about 1 cm. The method for visual inspection according to European Pharmacopoeia monograph 2.9.20 can also be used. The optical density of the composition can also be measured, for example by UV-visible spectroscopy at 500 nm, and it is ensured that the optical density is less than 0.5, preferably less than 0.2, relative to the reference solvent.

[0235] According to an alternative embodiment, the composition of the application is not opalescent or only slightly opalescent.

[0236] "Opalescent" means that the solution produces a visible light diffraction by the naked eye, for example by visual inspection according to a method such as European Pharmacopoeia monograph 2.9.20 and by comparison with reference solutions of different opalescence levels of the European Pharmacopoeia. According to an alternative embodiment, the composition of the application is colorless, i.e. in particular, the naked eye observer does not assign a specific color to the composition. According to an alternative embodiment, the opalescence is lower than the maximum allowed for the intended application.

[0237] The application particularly relates to a preferably sterile preparation or package comprising one or more infusion or injection devices pre-filled with a composition according to the application, in particular in the form of a hydrogel. These are generally devices for infusing a product in the form of drops or pre-filled syringes.

[0238] The composition of the application can advantageously be stored, preferably in a preparation or package suitable for its indications, and preferably for several months.

[0239] Advantageously, the composition of the application can be sterilized. The application thus relates to a sterilized crosslinked carboxyalkyl chitosan. This crosslinked carboxyalkyl chitosan is thus sterile, in particular for applications where it is required.

[0240] According to an alternative embodiment, the composition of the application is steam sterilized according to methods known to the person skilled in the art and / or recommended by the European Pharmacopoeia.

[0241] According to another alternative embodiment, the composition can be sterilized by filtration using a filter for this purpose, for example a filter with a pore size of less than or equal to 0.2 pm.

[0242] Advantageously, according to a preferred embodiment, the loss of intrinsic viscosity of the crosslinked carboxyalkyl chitosan is less than 40% after steam sterilization.

[0243] The application also encompasses a method for therapeutic treatment comprising the injection of a composition according to the application.

[0244] The application also encompasses the use of a composition according to the application for the manufacture of a pharmaceutical composition, in particular for therapeutic treatment, for example as more particularly defined in the application.

[0245] The application also encompasses a method for cosmetic (in other words, non-therapeutic) treatment comprising the injection of a composition according to the application. This is, for example, to fill wrinkles or to fill one or more damaged visible tissue areas (for example resulting from an accident or a surgical operation) for cosmetic purposes.

[0246] A tissue is a group of similar cells of the same origin gathered in a functional unit, i.e. they all contribute to the same function. Among these tissues can be mentioned: dermal tissues (for example epithelial tissues), connective tissues, muscular tissues and nervous tissues.

[0247] "Composition according to the application" or equivalent terms mean a composition as defined in the application, independently or in any combination thereof, comprising the composition according to any alternative embodiment, specific or particular embodiment, including the composition according to the preferred features.

[0248] Further objects, features and advantages of the application will become apparent to the person skilled in the art from reading the explanatory description, which refers to examples, which are in no way intended to limit the scope of the application, but only to illustrate it.

[0249] These examples are an integral part of the application and any feature that is novel with respect to any prior art, in terms of its function and its generality, is an integral part of the application, according to the whole of the description, including the examples.

[0250] Thus, each example has a general scope.

[0251] On the other hand, in the examples, all percentages are by mass, unless otherwise stated, and the temperatures are expressed in degrees Celsius, and the pressures are atmospheric, unless otherwise stated.

[0252] Examples

[0253] Method for measuring zeta potential

[0254] The formulation to be analyzed is diluted in phosphate buffer to obtain a final concentration of 0.05% of the polymer, then it is slowly stirred until homogenization. The solution is then divided into several portions and the pH of each portion is 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 portion is measured with the "Nano-Z" device (Zeta-Sizer series, Malvern Instruments).

[0255] Method for measuring solubility range of chitosan polymers

[0256] The solubility range is determined by preparing a solution of the polymer to be tested at a concentration of 1% and pH 9, by dividing it into several portions and adjusting the pH of each portion to different pH values ranging from 9 to 1. For each portion, the solubility of the polymer is checked according to the visual inspection method of the European Pharmacopoeia monograph 2.9.20, i.e. it does not form turbidity. The pH range in which the polymer is soluble or insoluble is recorded.

[0257] Biomechanical profile determination by rheometry

[0258] The biomechanical profile of the samples is characterized using a DHR-2 hybrid rheometer (TA Instruments) equipped with a 20 mm flat geometry spaced 700 pm from the Peltier, at a temperature of 37°C, a frequency of 3.98 rad / s, a deformation amplitude ranging from 0.1% to 10%. Each measurement is performed three times and then the average of the elastic modulus (G'), the viscosity (G") and tan delta (G" / G') of the three measurements is calculated.

[0259] Lubricating ability

[0260] The lubricating ability is characterized by the coefficient of friction (COF) between two surfaces. The measurement of the coefficient of friction is carried out according to the following method, the parameters of which are chosen according to the product and the indication expected.

[0261] Method for viscosity extender

[0262] Two discs (16.15 mm in diameter) based on the polyacrylate biomaterial used to manufacture hydrophobic intraocular lenses (as described in patent EP 1830898) were pre-wetted by immersion in water at 60°C for about 2 hours, then fixed on the upper and lower geometries of a DHR-2 rheometer (TA Instruments). An approximate volume of 100 μί of the sample to be tested was placed on the lower disc, then the upper geometry was lowered until the two discs were in contact, with a normal force of 5 Newtons applied. The measurement of the coefficient of friction was carried out at 25°C, under constant normal force (5 N), oscillation frequency of 1.256 rad / s, deformation angle of about 0.05 rad, for a duration of 150 seconds, according to a protocol adapted from that described by Waller et al. (see: J 47 Rheumatol 39, 7, 1473, 2012). The option "Respect zero start of oscillation movement" was activated. At each measurement point, the torque value was recorded, then the coefficient of friction (COF) was calculated according to the following formula: COF = Torque / (1 / 3 x disc diameter x normal force). The measurement was repeated 5 times for each formulation. The value of the coefficient of friction was reported by extrapolation 5 of the intercept (COF0) at the start of each COF versus time curve.

[0263] Method for artificial tears

[0264] Two discs (16.15 mm in diameter) based on the polyacrylate biomaterial used to manufacture hydrophobic intraocular lenses (as described in patent EP 1830898) were pre-wetted by immersion in water at 60°C for about 2 hours, then fixed on the upper and lower geometries of a DHR-2 rheometer (TA Instruments). An approximate volume of 100 μί of the sample to be tested was placed on the lower disc, then the upper geometry was lowered until the two discs were in contact, with a normal force of 5 Newtons applied. The measurement of the coefficient of friction was carried out at 25°C, under constant normal force (5 N), oscillation frequency of 1.256 rad / s, deformation angle of about 0.05 rad, for a duration of 150 seconds, according to a protocol adapted from that described by Waller et al. (see: J 47 Rheumatol 39, 7, 1473, 2012). The option "Respect zero start of oscillation movement" was activated. At each measurement point, the torque value was recorded, then the coefficient of friction (COF) was calculated according to the following formula: COF = Torque / (1 / 3 x disc diameter x normal force). The measurement was repeated 5 times for each formulation. The value of the coefficient of friction was reported by extrapolation 5 of the intercept (COF0) at the start of each COF versus time curve.

[0265] Ejection force via needle

[0266] Measurements were performed using a MultiTest 2.5-i compression tester (Mecmesin) equipped with a 100 N compression cell. A suitable needle was fitted to a syringe containing the sample. The syringe was positioned on the tester and the piston of the syringe was pushed at a constant speed (e.g. 10 mm / min or 80 mm / min) and the force required to eject was measured. The maximum force that the rig could withstand was approximately 70 Newtons.

[0267] In vitro antioxidant capacity (ABTS test)

[0268] To measure the antioxidant activity of the carboxyalkyl chitosan formulations and compare them to commercialised products, an in vitro 'ABTS' test was performed. This test consists in determining the ability of a substance to capture 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+1) cationic radical, which is a chromophore that absorbs maximally at a wavelength of 734 nm in the form of a cationic radical. A protocol adapted from the method described by Valyova et al. (Int J Applied Res Nat Prod, 5, 19, 2012) was used to measure the absorbance, using Nunclon 96 polystyrene microplates (Thermo Fisher Scientific) and an Infinite M200 microplate reader (Tecan Life Sciences).

[0269] Each test series was performed in 4 steps.

[0270] 1) 1 g of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was diluted in a homogeneous solution of K2S2O8 (2.45 mM in MilliQ water) to obtain a concentration of 7 mM of ABTS. This mixture was protected from light and stirred at room temperature for 24 hours, which was the time required to generate an appropriate amount of ABTS+1 radical cations. The working solution of ABTS+1 was finally obtained by taking 600 μL of the stirred mixture and diluting this amount in MilliQ water to a concentration of 415 μM.

[0271] 2) A calibration curve of radical scavenging capacity is established by comparison with a reference antioxidant molecule (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) Trolox. Trolox solutions at concentrations of 30, 60, 90, 120, 150, 180 and 210 μM are obtained by dilution in MilliQ water of a stock solution of 15 mg Trolox in 5 mL of 100% methanol. The absorbance is measured at wavelength 734 nm, 1 hour after mixing 50 μL of ABTS·1 working solution and 50 μL of each Trolox solution. The relationship between absorbance and Trolox concentration in the linear region is read. The minimum absorbance value in the linear region corresponds to the detection limit.

[0272] 3) The product under test is either characterized as such at its initial concentration, or diluted in MilliQ water (depending on the product under test, so that the absorbance of the mixture with the ABTS·1 solution is above the detection limit). 50 μL of working solution and 50 μL of test product solution are mixed. After 1 hour of incubation at room temperature, the absorbance is measured at wavelength 734 nm. If the absorbance value is within the detection range of the instrument, the value is retained and the Trolox equivalent is calculated by means of the calibration curve, expressed as "trolox equivalent antioxidant capacity" TEAC.

[0273] 4) The antioxidant capacity is expressed from one series to another in a normalized manner using a positive control, which is ascorbic acid (vitamin C) in a solution at 0.02 mg / mL (20 μg / mL). First, the TEAC of ascorbic acid solutions at 0.005-0.05 mg / ml is measured. It is verified that the absorbance of the 0.02 mg / mL ascorbic acid solution is in the linear region. Finally, the normalized antioxidant capacity of the product under test is expressed by the ratio TEAC (product) / TEAC (ascorbic acid at 0.02 mg / mL).

[0274] Example 1

[0275] Using the reaction parameters in Table 1a given by way of example, carboxymethyl chitosan is prepared via carboxymethylation and acetylation reactions according to the following method. In addition, other reaction parameters can also be used to adjust the molecular structure of the carboxymethyl chitosan.

[0276] Step 1 : Carboxymethylation of chitosan.

[0277] Thirty grams of chitosan of origin Agaricus bisporus was dispersed in 600 ml of isopropanol, 41 ml of water and 163 ml of 50% sodium hydroxide (m / v). One hundred thirty-five grams of alkylating agent monochloroacetic acid (MCA) was dissolved in 135 ml of isopropanol and added to the chitosan suspension. The reaction was continued for 23 hours at 35°C. 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 CC4, Table 1b).

[0278] Step 2: Acetylation of carboxymethyl chitosan.

[0279] Twenty-one grams of mass of CC4 was distributed in 570 ml of water and the pH of the solution was adjusted to pH > 7. A volume of 10 ml of acetic anhydride was added and the solution was stirred for 30 minutes at 25°C. The pH of the solution was adjusted to pH > 7 and then 10 ml of anhydride was added. After homogenization (stirring for about 30 minutes at room temperature), the pH was adjusted to about pH 7.5. The polymer was recovered by precipitation in ethanol and then purified by several cycles of dissolution in water and precipitation. After drying in a ventilated oven, carboxymethyl chitosan was collected (reference CC3, Table 1b).

[0280] Carboxymethyl chitosans used to prepare the matrices of examples 2-11 are described in Table 1b. CC1 to CC6 are carboxymethyl chitosans of origin fungal chitosan and were prepared according to the method described above.

[0281] CC7 is a commercial carboxymethyl chitosan of origin crustacean provided by the company Kraeber (product code 5313009900, Ehretschossen, Germany).

[0282] [Table 1a]

[0283]

[0284]

[0285] [Table 1b]

[0286]

[0287] a: measured by solid-state carbon-13 NMR (formula 2); b: measured by potentiometric titration; c: measured by capillary viscosimetry; d: acetyl groups signal not detectable by carbon-13 NMR (low DA).

[0288] Example 2 - Matrix of carboxymethyl chitosan

[0289] Synthesis trials by covalent cross-linking were performed using the cross-linking agent 1,4-butanediol diglycidyl ether (CAS 245-79-8, BDDE) to provide matrices of carboxymethyl chitosan. Several carboxymethyl chitosans of fungal origin were used, produced by Kymed Pharmaceuticals according to the method of Example 1. Their characteristics are given in Table 1. BDDE (96%, specific gravity 1.049) was supplied by Alfa Aesar (Thermo Fisher, Candler, Germany).

[0290] Example 2a

[0291] A cross-linked matrix was prepared from carboxymethyl chitosan CC3 after adjusting the reaction parameters (Table 2a, reference M1 -A). The degree of acetylation of CC3 was 55% and the degree of carboxymethylation was 87% as measured by carbon-13 NMR (formula 2). After dialysis, the hydrogel formed from the matrix was transferred into a 3 ml glass syringe which had been steam sterilized in short cycles (conditions "A2") in a SYSTEC-DX-65 autoclave. The final polymer concentration of the resulting sterilized hydrogel (M1 -A) was determined by mass balance. The cohesive properties of the hydrogel were analyzed by the water test and the viscoelastic level of the hydrogel was determined by rheometry (scale 1 -4). The higher the score, the greater the viscoelasticity of the matrix forming the hydrogel. It was concluded that, after adjusting the reaction parameters, a matrix of BDDE cross-linked carboxyalkyl chitosan could be obtained which formed a cohesive hydrogel according to the water test. The elasticity score of this hydrogel was 1. It could be injected through an intradermal needle (27G 13 mm).

[0292] These same reaction parameters were then applied to two carboxymethyl chitosans of different molecular structure and with a degree of acetylation lower than 40% (Table 1 b): CC4 of fungal origin (Kymed Pharmaceuticals) and CC7 of crustacean origin (Kraeber).

[0293] [Table 2a]

[0294]

[0295] *A2: short cycles (SYSTEC DX-65 autoclave); ** ejection force less than 30 Newtons at a ejection speed of 10 mm / min.

[0296] The matrices M1 -B and M1 -C (Table 2a) obtained respectively under the same conditions as the matrix M1 -A did not form cohesive hydrogels according to the water test. In contrast, the matrix M1 -A was able to form a cohesive hydrogel, thus fulfilling this object of the application.

[0297] Example 2b

[0298] Attempts were made to modulate the biomechanical properties of cross-linked carboxyalkyl chitosan-based hydrogels, in particular their viscoelasticity (measured on a scale of 0 to 4). To this end, matrices were prepared from CC1, CC5 and CC6 (Table lb) having a DA greater than 40% by varying the molecular weight (expressed in intrinsic viscosity) of the carboxyalkyl chitosan and the parameters of the cross-linking reaction. The cross-linking agent (BDDE), the medium, the temperature and the duration of the reaction, as well as the neutralization and purification conditions were identical to those of matrix M1-A.

[0299] [Table 2b]

[0300]

[0301] *A2: short cycle; A1 : long cycle

[0302] It appears that the biomechanical properties of cross-linked carboxyalkyl chitosan-based hydrogels, in particular the viscoelasticity, can be modified by varying the reaction parameters (in particular the initial concentration of carboxyalkyl chitosan or the ratio cross-linking agent / carboxyalkyl chitosan, here BDDE / carboxymethyl chitosan) and the molecular weight of the carboxyalkyl chitosan.

[0303] Example 3 - matrices of co-crosslinked carboxymethyl chitosan and hyaluronic acid

[0304] Matrices were obtained by cross-linking a mixture of carboxymethyl chitosan of fungal origin and having a DA greater than 40% (Table lb) and hyaluronic acid ("co-crosslinking") using BDDE. Hyaluronic acids (HA) having an average viscosity molecular weight of 2.2 or 2.3 million (HA1 type) and 4.3 million (HA2 type) were used (Table 3a).

[0305] [Table 3]

[0306]

[0307] *Supplier reported value

[0308] The reagents of the cross-linking reaction (BDDE), the medium, the temperature and the duration, as well as the neutralization and purification conditions were identical to those of matrix M1-A of Example 2. The hydrogels formed from the matrices were sterilized by autoclaving as described in Example 2, according to cycle A1 or A2. Several hydrogels are described by way of illustration since other combinations and / or parameters can also give rise to cohesive hydrogels. All these hydrogels can be injected easily through an intradermic needle having a gauge of 27 and a length of 13 mm.

[0309] Example 3a

[0310] An attempt was made to demonstrate that carboxyalkyl chitosan (CC) could be co-crosslinked with hyaluronic acid (HA) to form cohesive hydrogels. To this end, a matrix was prepared from a mixture of CC and HA at a CC / HA mass ratio of 75:25 (Table 3a). The reference numbers for CC are consistent with the previous examples. Furthermore, by adjusting the parameters of the crosslinking reaction, an attempt was made to adjust the level of elasticity from 1 to 3 (on a scale of 0 to 4).

[0311] [Table 3a]

[0312]

[0313] Conditions of Example 2

[0314] It was observed that the hydrogel M2-B of CC co-crosslinked with 25% HA was more elastic than the hydrogel M1-A of CC alone in Example 2, at the same BDDE / polymer ratio (18%) and the same final polymer concentration of 23 mg / ml. It can also be concluded that by varying the molecular weight of HA, the percentage of crosslinking agent (here BDDE), it is possible to vary the viscoelasticity of the hydrogels of co-crosslinked carboxyalkyl chitosan and HA.

[0315] Example 3b

[0316] An attempt was made to obtain cohesive hydrogels from carboxyalkyl chitosan and HA co-crosslinked in different proportions.

[0317] [Table 3b]

[0318]

[0319] It was shown that it is possible to obtain cohesive hydrogels of carboxyalkyl chitosan and HA co-crosslinked in different proportions, and that their level of elasticity depends on the ratio of carboxyalkyl chitosan / HA.

[0320] Example 4 - Matrix of crosslinked carboxymethyl chitosan in combination with hyaluronic acid

[0321] In this example, an attempt was made to evaluate the possibility of forming cohesive hydrogels from a matrix of crosslinked carboxyalkyl chitosan in combination with HA. According to the method of Example 1, carboxyalkyl chitosan was first crosslinked with BDDE, then a solution of HA (type HA1) was added thereto. The resulting hydrogel was then sterilized via autoclaving through cycle A2. (Table 4).

[0322] [Table 4]

[0323]

[0324] It was easy to incorporate HA into the hydrogels based on the cross-linked carboxyalkyl chitosan matrix. The resulting hydrogels were cohesive according to the water test, and had a viscoelastic score of 3, while being easily injectable through a 27 gauge intradermal needle.

[0325] Example 5 - Biomechanical properties of the hydrogels

[0326] In this example, the biomechanical properties of some representative CC hydrogels from Examples 2 to 4 were characterized by rheometry (Table 5). The hydrogels were cohesive, injectable through a 27G needle, and had an elasticity level of 1 to 3. They were compared to three commercial cross-linked HA-based products used for intradermal injection for cosmetic purposes (Table 5, references B1 to B3): B1 was a viscous solution (tan delta > 1) and B2 and B3 were cohesive gels (tan delta < 1) according to the water test.

[0327] [Table 5]

[0328]

[0329]

[0330] It was confirmed that the cross-linked carboxyalkyl chitosan-based hydrogels according to the application had biomechanical properties, in particular elastic modulus (G'), comparable to commercial cross-linked HA-based products used for cosmetic medical intradermal injection.

[0331] Example 6 - Ability to scavenge ABTS°1 free radicals (in vitro)

[0332] An attempt was made to validate the matrix of cross-linked carboxyalkyl chitosan (CC) using a standard in vitro test called "ABTS" in which the free radical ABTS°1 is formed and calibrated with the antioxidant substance "Trolox". Each test product was diluted to obtain a total concentration of polymer Cp (CC, HA or CC and HA) of 8 mg / mL, 4 mg / mL and 1 mg / mL. The results were checked to ensure that they were within the detection area of the test, then the ability to scavenge the free radical ABTS°1 was expressed in Trolox equivalent. The antioxidant capacity of a solution of 20 μg / mL ascorbic acid was also measured (positive control). The antioxidant capacity of each product tested was normalized according to the following formula: Normalized antioxidant capacity = TEAC (product) / TEAC (ascorbic acid 20 μg / mL).

[0333] For comparison, a solution of non-crosslinked carboxyalkyl chitosan polymer (CC2) and a commercial product based on a non-crosslinked HA solution (reference B6) were tested. Also characterized were four commercial products for intradermal injection for cosmetic purposes: references B1 to B3 (based on crosslinked HA only, see Table 5 of Example 5) and B4 (based on a hydrogel combining crosslinked HA with a complex of several small molecules including an antioxidant molecule).

[0334] Table 6 reports the results obtained for all products at the same total polymer concentration (Cp) of 4 mg / mL.

[0335] [Table 6]

[0336]

[0337] It was observed that all CC-based compositions, whether 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 thus can be used as antioxidants. At the same polymer concentration, only the commercial HA-based products (B6, B1, B2 and B3) did not exhibit this ability.

[0338] Surprisingly, the hydrogels M1-E (CC) and M2-A (CC / HA 75:25) exhibited the highest antioxidant capacity among all the tested products, including compared to the solution of non-crosslinked CC S1. Both hydrogels had an antioxidant capacity similar to that of ascorbic acid at 20 pg / ml.

[0339] Among the commercial HA-based products, only B4 was able to significantly scavenge the free radical ABTS°1, but its capacity to scavenge the free radical ABTS°1 was only half that of M1-E and M2-A. In fact, B4 is a crosslinked hyaluronic acid combined with a complex of several small molecules, among which an antioxidant agent, which is the cause of the observed effect. However, since these substances are water-soluble small molecules, it is likely that after intradermal injection they will rapidly diffuse out of the B4 hydrogel, which will then lose its antioxidant capacity.

[0340] Example 7 - Ability of hydrogels to reduce oxidative stress in in vitro dermal cell cultures

[0341] Two hydrogels based on cross-linked CC (reference M1-E, see Example 2) and co-cross-linked CC / HA (M2-A, see Example 3) were evaluated in standard in vitro tests for their ability to protect human dermal cells from damage induced by "ROS" (reactive oxygen species) free radicals, which are the free radical species encountered in skin tissues under oxidative stress. They were compared with a non-cross-linked carboxyalkyl chitosan solution and a commercial product based on cross-linked hyaluronic acid for intradermal injection for cosmetic purposes (reference B3, see Example 5).

[0342] Human dermal fibroblasts (NHDF) with about 40% in vitro proliferative potential were cultured in monolayer in DMEM (Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum, penicillin and streptomycin at 37°C in a 5% CO2 atmosphere. The cultures were transferred into DMEM without fetal bovine serum and then divided into wells. The products to be tested were diluted in DMEM to a total concentration of polymer of 0.6 and 0.2 mg / mL and then added to the wells (3 wells for each product to be tested). After 72 hours of contact with the products to be tested, a probe of 2'-7'-dichloro-dihydrofluorescein diacetate that fluoresces under the action of free radicals was added for 30 minutes. The cultures in each well were then rinsed with HBSS to remove the product to be tested, the cells were returned to HBSS and all the wells were then irradiated with UVA at 12.5 J / cm2for 20 minutes to generate ROS. 2

[0343] Untreated, unirradiated cultures were used as a reference. Untreated and irradiated cultures were used as a negative control, ascorbic acid-treated (50 μg / mL) and irradiated cultures were used as a positive control. At the end of the UVA irradiation, the fluorescence intensity, which is directly proportional to the ROS content (excitation wavelength 485 nm, emission wavelength 520 nm), was measured and then the relative ROS content with respect to the unirradiated reference was calculated (Table 7). The reduction in ROS content with respect to the untreated and unirradiated control was then calculated, which characterizes the ability of the product to reduce oxidative stress.

[0344] [Table 7]

[0345]

[0346] * Total concentration of polymer (CC, CC / HA or HA) used for cell treatment

[0347] ​Under the in vitro culture conditions of the present experiment, it can be concluded that CC-based compositions, whether cross-linked (M1-E) or not (S2), have a good ability to reduce the ROS content, i.e. to reduce the oxidative stress that can alter the cells and the dermal tissue. This ability is at the same level as that of ascorbic acid (50 μg / mL, Vitamin C) and much higher than that of the commercial cross-linked HA product. The composition M2-A, containing 75% of CC, cross-linked CC / HA, also has a good ability to reduce the oxidative stress.

[0348] Example 8 - Fluid hydrogels based on carboxyalkyl chitosan matrix for ocular administration

[0349] In this example, it was attempted to obtain a cross-linked CC hydrogel whose viscosity makes it easy to instill in the form of well-defined droplets, while having a good lubricating ability suitable for artificial tear indications for ocular surface treatment.

[0350] To this end, cohesive cross-linked CC hydrogels (M8-B, Table 8a) were prepared by targeting the dynamic viscosity in the range of 1 to 60 mPa.s (at a shear rate of 10 s -1 Their instillability was verified and their lubricating ability between two polyacrylate surfaces, expressed in coefficient of friction, was measured according to the artificial tear method.

[0351] The properties of this hydrogel were compared with those of two commercial products based on non-cross-linked HA for ocular surface treatment (reference numbers B7 and B8, Table 8b). Their lubricating ability was measured in the same test series as M8-B.

[0352] [Table 8a]

[0353]

[0354] [Table 8b]

[0355]

[0356] It was concluded that it is possible to obtain a cohesive, fluid and instillable cross-linked CC hydrogel having a lubricating ability comparable to that of commercial products for ocular surface treatment.

[0357] Example 9 - Local effects (short-term) after intradermal implantation in rabbits

[0358] Three CC-based hydrogels were evaluated by intradermal administration in rabbits: Ml-A (crosslinked CC, see Example 1), M2-A and M2-B (co-crosslinked CC / HA, see Example 2). These formulations were filled in 1 mL glass syringes (Hypak, BD Medical) and sterilized. The endotoxin content, determined according to the method D-monograph EP 2.6.14 of the European Pharmacopoeia, was satisfactory. Two commercial products based on crosslinked hyaluronic acid for intradermal injection for cosmetic purposes (Bl and B2, see Example 5) were also evaluated.

[0359] According to a protocol meeting the ISO 10993-10 standard for the evaluation of primary irritation induced by intradermal implants, the formulations were administered to rabbits by intradermal injection with a needle of diameter 27G in a volume of 200 μL. Twelve injections per product were performed on six rabbits. The local effects on all injection sites were observed daily, in particular the level of erythema.

[0360] Table 9 reports the average level of erythema (score graded from 0 to 4) 7 days after injection. It is also noted whether papules are visible (score graded from 0 to 4) at 7 days. Macroscopic analysis or microscopic analysis (skin histology) of the injection sites of the animals euthanized 7 days after injection were performed to evaluate the presence of the product.

[0361] [Table 9]

[0362]

[0363] Intradermal injection of the hydrogels was associated with the appearance of a mild local effect, characterized by a maximum score of 1 for the average of erythema at 7 days (graded from 0 to 4). This corresponds to a mild level of erythema, comparable to that observed for the two commercial products. Moreover, the presence of the product in the dermis was demonstrated after euthanasia of the animals and histological analysis at day 7.

[0364] Example 10 - Hydrogels for articular viscosupplementation

[0365] In this example, the viscoelasticity and lubricating ability of two hydrogels based on crosslinked CC (Ml-E) and co-crosslinked CC / HA (M2-B) were evaluated and compared to those of two commercial products based on crosslinked HA (B9 and B10, see composition in Table 10) used for the treatment of osteoarthritis by articular viscosupplementation. The lubricating properties of the hydrogels were determined according to the method for viscosupplementation by their ability to reduce the coefficient of friction between two polyacrylate polymer discs mounted on a rheometer.

[0366] [Table 10]

[0367]

[0368]

[0369] * Standard deviation high, indicating a high friction between the two surfaces (low lubricating power of the product under test); ** Total concentration of polymers

[0370] It was observed that the elastic modulus G' of the crosslinked CC and of the co-crosslinked CC / HA hydrogels was in the same range as that of B9, while the elastic modulus of B10 was higher. It was observed that both CC and CC / HA hydrogels exhibited a significant lubricating power (characterized by a low coefficient of friction between the two surfaces), comparable to that of the crosslinked HA viscosity supplement B10, and superior to that of the crosslinked HA viscosity supplement B11.

[0371] In examples 11 to 14, the polymers CC and HA used were those described in Table 11a and Table 11b.

[0372] [Table 11a]

[0373]

[0374] a: value evaluated according to the DA of the starting chitosan; b: value evaluated according to the DS of the CC after acetylation measured by carbon-13 NMR; c: measured by solid-state carbon-13 NMR (formula 2).

[0375] [Table 11b]

[0376]

[0377] Example 11 - Co-crosslinking test of HA with CC having an acetylization degree less than 40%

[0378] An attempt was made to verify whether it was possible to obtain a cohesive hydrogel by co-crosslinking CC and HA starting from CC having a DA less than 40% (CC8, Table 11a) 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 M2-I) are reported in Table 11c and compared with those of the hydrogel of Example 3 (according to the application) with reference M2-A.

[0379] It was observed that, as determined by the tan delta value (tan delta, measured by rheometry), with CC8, co-crosslinking and autoclave sterilization did not lead to a gel. In fact, the M2-I formulation included a tan delta value of 1.6, higher than 1, indicating the behavior of a viscous solution rather than a gel. On the contrary, according to the application, the tan delta value of the hydrogel M2-A was 0.4, i.e. less than 1, indicating the behavior of a gel.

[0380] [Table 11c]

[0381]

[0382]

[0383] As the obtained preparation is not a gel, the water test is not applicable.

[0384] Example 12 - Hydrogel for volume restoration or filling of large skin depressions

[0385] This example illustrates the use of a cross-linked CC-based hydrogel for restoring facial volume or filling large skin depressions by subcutaneous injection or injection into the deep dermis. For both indications, a hydrogel with a viscoelasticity level of 4 is sought, i.e. a hydrogel with an elastic modulus G' of about 150 Pa or more, while the hydrogel is cohesive according to the water test and easy to inject through a needle of 27 gauge and 13 mm in length. As a reference, two commercial products B11 and B12 (Table 12) are used in these indications, wherein B11 and B12 are cohesive hydrogels based on cross-linked hyaluronic acid with an elastic level of 4.

[0386] The hydrogel M2-J is obtained by co-crosslinking CC5 and HA1 type of HA (CC / HA ratio of 25:75) overnight at room temperature with 13% of BDDE. It has an elastic modulus of 295 Pa (corresponding to the desired elastic level 4), while remaining cohesive and easy to inject, in line with the expectations for the intended indications (Table 12).

[0387] [Table 12]

[0388]

[0389] Example 13 - Volume retention after 1 month of intradermal injection of co-crosslinked CC / HA hydrogel

[0390] According to the reaction conditions of Example 12, a hydrogel is prepared by co-crosslinking CC9 (see Table 11a) and HA2, with a CC / HA mass ratio of 40:60. The obtained hydrogel (reference M2-K) is filled in 1 mL glass syringes (Hypak, BD Medical) and sterilized in the same way as in Example 9. Its final concentration of polymer is 23 mg / mL and it is cohesive, injectable through a 27G needle, with a viscoelasticity level of 3.

[0391] According to a protocol similar to Example 9, the same volume of hydrogel M2-K and commercial product B12 (see Table 12, viscoelasticity level 4) was injected intradermally into rabbits by means of a 27 gauge needle. The local reactions were evaluated periodically over a period of 26 days after injection, then the volume of the papules visible on the skin surface formed by the injected product was evaluated by scoring on a scale of 0 to 4. The volume of the papules is indicative of the presence of the product and its ability to locally increase the volume of the skin tissue.

[0392] The injection of the two products did not cause any significant local reaction during the follow-up period. Immediately after injection of the two products, papules with an average volume score of 3 ± 0 (on 20 injection sites evaluated) were formed. Over the next few days, the papules faded slightly but were still practically present. 26 days after injection, the papules were still present, with an average score of the volume of the papules equal to 2.0 ± 0.0 for M2-L and equal to 2.4 ± 0.5 for B12 (20 sites evaluated), which is consistent with their relative elasticity level. At this time point, the difference between the volume scores provided by the hydrogel M2-K and B12 was not significant.

[0393] Thus, it was confirmed that the hydrogel M2-K was practically still present in the dermis at least 26 days after intradermal injection in rabbits and maintained a significant volumetric increase effect around its injection site, as expected for the indication of filling skin depressions.

[0394] Example 14 - Preservation of co-crosslinked CC / HA hydrogels

[0395] The feasibility of preserving the co-crosslinked CC / HA hydrogels was evaluated by submitting them to accelerated aging conditions in an oven at 40°C and monitoring their biomechanical properties. As long as the hydrogel remains cohesive and easy to inject according to the water test and is considered acceptable from a biomechanical point of view, it comprises a gel-like behavior (tan delta value lower than 1) and its viscoelasticity level remains unchanged with respect to the initial level at t0 and complies with the expected indication.

[0396] To obtain a viscoelasticity level of 2, a hydrogel with reference M2-L was prepared by co-crosslinking CC9 (see Table 11a) and HA2 in a 70:30 CC / HA ratio according to the reaction conditions of Example 12. This is a product packaged in 1 mL glass syringes (Hypak, BD Medical) and sterilized in the same way as in Example 9. The syringes were placed in an oven at 40°C for 6 months. Table 13 gives the properties measured at 3 months of storage time.

[0397] [Table 13]

[0398] Thus, it was confirmed that the hydrogel M2-K was practically still present in the dermis at least 26 days after intradermal injection in rabbits and maintained a significant volumetric increase effect around its injection site, as expected for the indication of filling skin depressions.

[0399] After 3 months of accelerated aging at 40°C, the product M2-L remains a hydrogel (since tan δ < 1) and maintains its cohesiveness, ease of injectability and level of viscoelasticity 2. Thus, by extrapolation, this co-crosslinked CC / HA hydrogel should be able to maintain acceptable properties for the intended indications for at least 12 months at room temperature.

Claims

1. A hydrogel matrix comprising at least one carboxyl chitosan, wherein the carboxyl chitosan has 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.

2. A hydrogel matrix, said hydrogel matrix comprising at least one carboxyl chitosan, said carboxyl chitosan having glucosamine units, N-acetylglucosamine units, and carboxyl-substituted glucosamine units, wherein, The hydrogel matrix is ​​sterile and cohesive. 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 through covalent bonds between carboxyalkyl chitosan chains.

3. A hydrogel matrix, said hydrogel matrix comprising at least one carboxyl chitosan, said carboxyl chitosan having glucosamine units, N-acetylglucosamine units, and carboxyl-substituted glucosamine units, wherein, The hydrogel matrix is ​​cohesive, injectable, implantable, or drip-injectable and suitable for contact with human or animal tissues. 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. The hydrogel matrix is ​​suitable for flow through a 21-gauge needle when injected with an injection force of less than 50 Newtons at a speed of 10 mm / min; or when injected with an injection force of less than 30 Newtons at a speed of 10 mm / min, the hydrogel matrix is ​​suitable for flow through a 27-gauge needle.

4. A hydrogel matrix comprising at least one carboxyl chitosan, wherein the carboxyl chitosan has glucosamine units, N-acetylglucosamine units, and carboxyl-substituted glucosamine units, wherein, The hydrogel matrix is ​​cohesive and has a coefficient of friction of less than 20. 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.

5. The hydrogel matrix according to claim 3, characterized in that, The hydrogel matrix is ​​sterilized by steam.

6. The hydrogel matrix according to claim 3, characterized in that, The hydrogel matrix has a coefficient of friction of less than 10.

7. The hydrogel matrix according to any one of claims 1-4, characterized in that, The degree of substitution of the carboxyl groups in the carboxyl chitosan is less than 200%, expressed as the number of moles of substituents relative to the total number of units.

8. The hydrogel matrix according to any one of claims 1-4, characterized in that, The chitosan is derived from basidiomycetes. Basidiomycete Fungi or derived from ascomycetes Ascomycete The mycelium of a certain type of fungus.

9. The hydrogel matrix according to any one of claims 1-4, characterized in that, The chitosan is derived from Aspergillus niger. Aspergillus piger Basidiophyte (Shiitake mushroom) Lentinula edodes Or button mushroom Agaricus bisporus .

10. The hydrogel matrix according to any one of claims 1-4, characterized in that, The carboxyalkyl chitosan is reacetylated.

11. The hydrogel matrix according to any one of claims 1 or 3-4, characterized in that, The hydrogel matrix is ​​sterile.

12. The hydrogel matrix according to claim 1, characterized in that, The hydrogel matrix forms a cohesive hydrogel.

13. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix includes at least one hyaluronic acid.

14. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix includes at least one type of hyaluronic acid obtained through fermentation.

15. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix includes at least one hyaluronic acid cross-linked by covalent bonds.

16. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix includes at least one hyaluronic acid that is co-crosslinked with carboxyl chitosan via covalent bonds.

17. The hydrogel matrix according to any one of claims 1-4, characterized in that, Crosslinking is formed by a crosslinking agent that forms the covalent bonds.

18. The hydrogel matrix according to claim 17, characterized in that, The cross-linking agent is selected from cross-linking agents used for cross-linking polysaccharides.

19. The hydrogel matrix according to claim 18, characterized in that, 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 divinyl sulfone.

20. The hydrogel matrix according to claim 19, characterized in that, The diisocyanate compound is selected from hexamethylene diisocyanate and toluene diisocyanate.

21. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix forms a hydrogel.

22. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix has antioxidant capabilities by scavenging free radicals.

23. The hydrogel matrix according to any one of claims 1-4, characterized in that, The hydrogel matrix has a normalized antioxidant capacity greater than 0.

30.

24. A composition, characterized in that, The composition comprises at least one hydrogel matrix according to any one of claims 1-23.

25. An injectable composition, characterized in that, The injectable composition comprises at least one hydrogel matrix according to any one of claims 1-23.

26. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one hydrogel matrix according to any one of claims 1-23.

27. The composition according to claim 24 or 26, characterized in that, The composition may be used as an injectable, implantable, infusion-promoted, or locally administered pharmaceutical composition, or as an injectable, implantable, infusion-promoted, or locally administered medical device.

28. Use of the composition according to any one of claims 24 to 27 in the preparation of a medicament for therapeutic treatment, wherein the therapeutic treatment comprises local instillation or administration or injection of the composition via subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular routes.

29. The use according to claim 28, characterized in that, The treatment is used to repair or fill at least one body tissue that needs repair or filling.

30. The use according to claim 28, for treating osteoarthritis or repairing cartilage defects.

31. A medical device, characterized in that, The medical device comprises or is composed of any one of claims 24 to 27.

32. A method for preparing a hydrogel matrix according to any one of claims 1 to 23, the method comprising: Contact the carboxyl chitosan with at least one crosslinking agent; The carboxyl chitosan is crosslinked with the crosslinking agent; A hydrogel matrix comprising the crosslinked carboxyl chitosan was obtained.

33. A method for preparing a hydrogel matrix, the hydrogel matrix comprising carboxyalkyl chitosan co-crosslinked with other biopolymers, wherein the carboxyalkyl chitosan is the carboxyalkyl chitosan according to any one of claims 1-23, the method comprising: Contact a mixture of carboxyalkyl chitosan and other biopolymers with at least one crosslinking agent; Crosslinking the carboxyl chitosan and other biopolymers; A co-crosslinked hydrogel matrix of the carboxyl chitosan and other biopolymers was obtained.

34. The method according to claim 33, wherein, The biopolymer is hyaluronic acid.

35. The method according to claim 33, wherein, The hydrogel matrix is ​​sterilized by steam sterilization.

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