Chitosan and use thereof

Abstract

To provide a chitosan derivative or a composition containing the same, suitable for use in humans or animals, especially in the fields of treatment, surgery and beauty.SOLUTION: The inventors set out to develop a chitosan having both good antioxidant properties and good mechanical properties (called biomechanical properties) for the intended applications in humans or animals. The present invention relates to a crosslinked carboxyalkyl chitosan forming a matrix, to a composition comprising it, to a process for preparing it and in particular to therapeutic, rheumatological, ophthalmological, cosmetological, plastic surgery, internalsurgery, dermatological, gynaecological or cosmetic. In particular, the present invention relates to a matrix comprising a carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkyl chitosan has a degree of acetylation, expressed as moles of N-acetyl groups relative to moles of total glucosamine units, in the range of greater than 40% up to 80%, and wherein the carboxyalkyl chitosan is crosslinked by covalent bonds between the carboxyalkyl chitosan chains.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to matrix-forming crosslinked carboxyalkylchitosans, compositions containing them, methods for their preparation, and in particular to therapeutic, rheumatological, ophthalmological, aesthetic, plastic surgery, laparotomy, dermatological, gynecological or cosmetic fields. [Background technology]

[0002] Chitosan derivatives are known, in particular from the patent applications and corresponding patents of Kiomed Pharma published under WO 2016 / 016463 and WO 2016 / 016464. Advantageous chitosan derivatives, such as carboxyalkylchitosans, are also known from Kiomed Pharma, as described in the patent applications of Kiomed Pharma filed under PCT / EP2018 / 080763 and PCT / EP2018 / 080767 and in their families, the contents of which are incorporated herein by reference.

[0003] According to the present inventors, it would be advantageous to be able to tailor the biomechanical behavior of carboxyalkylchitosan compositions or increase the duration or effectiveness of treatments due to the presence of carboxyalkylchitosan. However, providing such compositions with improved biomechanical properties is not obvious to those skilled in the art, especially when it is desired to prepare hydrogels. One of the technical problems with biopolymer compositions, and particularly with state-of-the-art hydrogels, known to those skilled in the art is the fact that some compositions are not in the form of cohesive hydrogels. That is, the hydrogel spontaneously disintegrates into separate parts in the presence of an aqueous medium, thus forming particles or fragments. This is also known as a fragmented gel or 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 and are considered undesirable for many medical applications (Bergerey-Galley, Aesth Surf J 24, 33, 2004). Therefore, from the standpoint of subject or patient health safety, it is important to be able to avoid the formation of separate fragments and obtain the composition in the form of a cohesive hydrogel.

[0005] Furthermore, for several reasons, it may be desirable to avoid such aggregates in order to improve the aesthetic (visual and / or tactile) appearance of tissues filled with such compositions and properly biointegrated into the tissue, allowing for uniform filling.

[0006] Therefore, for many applications, a cohesive hydrogel is preferred, which remains in one mass when, for example, an aqueous medium is added to it. This is also called a "homogeneous" hydrogel. Furthermore, for most applications, a hydrogel is preferred, which is called a "smooth" hydrogel because of its visual appearance, with little or no lumps.

[0007] In addition to cohesiveness, the compositions, particularly hydrogels, according to the invention should be suitable for human or animal use, particularly in terms of safety, immunocompatibility, bioabsorbability, biomechanical properties, and duration or activity. However, not all compositions in the art adequately exhibit such properties and therefore would not be amenable to the present invention.

[0008] Various methods for converting carboxyl alkyl chitosan into hydrogel form are known. In particular, Rufato et al. (Intechopen 81811, 2018), Upadhyaya et al. (J Controlled Release 2014), and Fonseca-Santos et al. (Mater Sci Engineering C 77, 1349, 2017) identified several chitosan-based hydrogels containing carboxyl alkyl chitosan for medical or pharmaceutical applications. However, none of these hydrogels met the expectations of the present inventors, especially in terms of cohesion, safety, immunocompatibility, biomechanical properties, bioresorbability, and / or duration or activity time, particularly simultaneously. With the exception of the compositions of Kiomed Pharma according to the aforementioned patent applications PCT / EP2018 / 080763 and PCT / EP2018 / 080767, none of the carboxyalkylchitosans used to prepare known hydrogels according to the state of the art exhibit good immunocompatibility according to the inventors. Not all chitosans can be used to form hydrogels suitable for human or animal use.

[0009] Chitosan-based hydrogels known to date are prepared by combining chitosan or one of its derivatives with other polymers, such as alginate, isopropylacrylamide, polyurethane, polyacrylonitrile, gelatin, polyethylene glycol (PEG), and polyvinyl alcohol (PVA). However, these polymers are either non-bioabsorbable or immunoreactive and do not meet the objectives of the present invention.

[0010] For example, Huang et al. (RCS Adv 2016 D01:10.1039 / C5RA26160K) prepared a hydrogel of glycol chitosan and hyaluronan, but such glycol chitosan is immunoreactive and therefore unacceptable to humans. Song et al. (Sci Rep 6, 37600, 2016) prepared a hydrogel based on carboxymethyl chitosan and oxidized hyaluronan via a Schiff base reaction between the amine groups of carboxymethyl chitosan and the aldehydes of hyaluronan. However, in our experience, the carboxymethyl chitosan used does not have the molecular structure required to meet the objectives of this invention. In particular, the described hydrogel resorbs very rapidly, according to the in vitro and in vivo tests described. Therefore, such hydrogels need to be improved, especially in terms of their shelf life, for use in a wide range of indications.

[0011] Furthermore, previous products are often not versatile enough to meet the needs of various indications, particularly various therapeutic indications. Therefore, there is a need to provide products that are versatile enough in terms of properties, particularly biomechanical properties, to be easily adaptable to various uses.

[0012] For example, in regenerative medicine or surgery, the goal is generally to repair altered tissues or fluids and / or prevent tissue alterations, tissue filling, or tissue separation to avoid adhesions. The causes of tissue alterations can be natural aging, external aggression (trauma, UV radiation, surgery, etc.), inflammation, autoimmunity, and other pathological conditions. However, most tissue alterations involve oxidizing stress, sometimes called oxidative stress, which is characterized by a high content of free radical species that can damage tissues or cells. By reducing the amount of free radical species, tissues can prevent / delay aging and reduce its harmful consequences. There are several ways to reduce the amount of free radical species in tissues, for example, by administering antioxidants such as vitamins C, B, E, and / or ubiquinone. Alternatively, compositions capable of scavenging free radicals can be used, thereby reducing their content and proliferation in tissues.

[0013] Chitosan and some of its derivatives exhibit the ability to scavenge oxidative free radical species, as described in a review by Ngo et al. (Adv Food Nutrition Res 73, 15, 2014) and in many formulations for biomedical use. For example, carboxymethylchitosans of different structures and molecular weights have been studied for their ability to scavenge various types of free radicals using in vitro assays, 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 for applying the beneficial effects of chitosan, in particular its ability to scavenge free radicals, in the form of a treatment that allows both reducing the impact of oxidative stress on the tissue and better adjusting the biomechanical behavior of the product, or even increasing the duration or effectiveness of the treatment by the presence of this polymer of exogenous origin.

[0015] Thus, the state of the art does not obviously enable a person skilled in the art to provide a satisfactory composition to overcome the problem set out in the present invention. Summary of the Invention

[0016] Object of the invention One of the aims of the present invention is to solve the technical problem of providing chitosan derivatives or compositions containing them that are suitable for use in humans or animals, in particular in the therapeutic, surgical and cosmetic fields.

[0017] One of the aims of the present invention is to solve the technical problem consisting in providing a chitosan derivative or a composition comprising it, in order to apply the beneficial effects of chitosan, in particular its ability to scavenge free radicals, in the form of a therapy that allows both to reduce the impact of oxidative stress on tissues and to better regulate the biomechanical behavior and, through the presence of this polymer of exogenous origin, to increase the duration or effect of the therapy.

[0018] In particular, one of the objects of the present invention 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 tissue, acceptable in terms of biomechanical properties, in situ duration or duration of activity, good health safety (in particular absence of immune and / or foreign body reactions in the short and long term), and which provides beneficial effects, in particular in the context of regenerative or anti-aging medicine, e.g. therapy, rheumatology, orthopedics, gynecological-ophthalmology, aesthetic medicine, plastic surgery, laparotomy, dermatology or cosmetic fields.

[0019] One of the aims of the present invention is to solve the technical problem of providing a composition with good biomechanical properties, in particular biomechanical properties that can be adjusted according to the indication.

[0020] One of the aims of the present invention is to solve the technical problem of providing products based on chitosan derivatives that make it possible to prepare a range of products with different biomechanical properties adapted to each intended indication.

[0021] One of the aims of the present invention is to solve the technical problem of providing a composition that preferably simultaneously has cohesiveness, safety (including immunocompatibility), biomechanical properties, sufficient bioabsorbability for administration to humans or animals, and preferably an adequate duration or duration of activity.

[0022] One of the aims of the present invention is to solve the technical problem described in the present invention, in particular by providing chitosan derivatives or compositions containing them in a grade that is acceptable to humans or animals for the intended indications. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention In order to solve the technical problem described in this invention, the inventors have attempted to develop a chitosan that has both good antioxidant properties and good mechanical properties (referred to as biomechanical properties) for the intended use in humans or animals.

[0024] The present inventors have knowledge from their own experience about the advantages of substituted chitosans, especially carboxyalkyl chitosans. In particular, Kiomed Pharma has filed patent applications under PCT / EP2018 / 080763 and PCT / EP2018 / 080767. They have attempted to apply this teaching to solve the technical problem set forth in the present invention.

[0025] The present inventors have found that carboxyalkylchitosan hydrogels formed by ionic (i.e., noncovalent) crosslinking do not retain their biomechanical properties long enough after implantation for some intended applications; in particular, this technique does not allow for wide tuning of duration or activity. Furthermore, carboxyalkylchitosan hydrogels formed by enzymatically catalyzed crosslinking are at risk of enzyme immunoreactivity due to their proteinaceous nature, making final purification of the resulting crosslinked product difficult.

[0026] Patent application CN107325306 (Imeik Technology Development) describes the preparation of a gel based on carboxymethylchitosan derived from crustaceans by crosslinking with BDDE in several successive crosslinking steps (multi-crosslinking). However, this method does not provide a hydrogel according to the present invention, particularly because the resulting hydrogel is not cohesive, since it is formed by particles of crosslinked chitosan derivative dispersed in a solution of carboxymethylchitosan, and the whole is crosslinked again to form a gel. The crosslinking operation is repeated several times ("multi-crosslinking"). Such products are likely to form granulomas and therefore may adversely affect immune compatibility after contact with the human or animal body, which is exactly what the present invention seeks to avoid. The present invention further advantageously allows for a greater variety of indications, especially when a cohesive (i.e., one that remains in one mass without fragmenting upon contact with water, for example) and / or "smooth" appearance hydrogel is desired. According to CN107325306, the carboxymethylchitosan used has a low DA (deacetylation degree of 60-99%, preferably 80-95%; in practice, the acetylation degree (DA) is much lower than 40%). Carboxymethylchitosan hydrogels with low acetylation degrees have also been described by Czechowska-Biskup et al. (DO1: 10.15259.PCACD.21.03). However, these hydrogels are not cohesive and do not meet the objectives of the present invention.

[0027] The inventors have found that the crosslinked carboxyalkylchitosan matrix or composition according to the invention, and in particular the hydrogels comprising it, make it possible to solve at least one, and preferably all, of the technical problems described in the present invention.

[0028] The present invention therefore relates to a matrix comprising at least one carboxyalkylchitosan having glucosamine units, N-acetylglucosamine units, and glucosamine units substituted with carboxyalkyl groups, the carboxyalkylchitosan having a degree of acetylation, expressed as the number of moles of N-acetyl groups relative to the number of moles of total glucosamine units, ranging from greater than 40% up to 80%, and the carboxyalkylchitosan being crosslinked by covalent bonds between the carboxyalkylchitosan chains.

[0029] Indeed, it has been found that cross-linked carboxyalkyl chitosans with a DA of less than 40% fail to yield hydrogels with the desired cohesive properties in that they fragment into separate pieces during humidification, which is undesirable for many applications.

[0030] According to the present invention, a cohesive hydrogel is understood to be a hydrogel that retains its cohesiveness according to the following cohesion test, called the "water test", by adapting methods traditionally used to characterize hydrogels for intradermal use, such as those described by Michaels et al. (J Clin Aesth Dermatol 10, 29, 2017 and J Drugs Dermatol 15, 1092, 2016):

[0031] A 1 g block of the hydrogel to be tested is placed in the center of a 5 cm diameter Petri dish. 1 mL of distilled water is added around the perimeter of the dish. The Petri dish is gently rocked until the water covers the hydrogel, then returned to a horizontal position. The hydrogel is observed immediately after contact between the matrix and water, preferably after 15-25 seconds of contact, and preferably after at least 30 seconds of contact, to see whether it forms a single fragment and is cohesive, or whether it spontaneously separates into separate pieces or forms particles visible to the naked eye and is non-cohesive.

[0032] Furthermore, it has been advantageously found that the matrix according to the present invention can scavenge free radical species. This retention of chitosan properties was not obvious to those skilled in the art. It is known that the molecular structure (DS) and molecular weight of carboxyalkylchitosan affect its ability to scavenge free radicals, but conflicting results have been published. Therefore, it was not clear that crosslinked carboxyalkylchitosan exhibited free radical scavenging ability.

[0033] Moreover, the hydrogels according to the present invention exhibit such antioxidant activity while possessing suitable cohesion, biomechanical profile, longevity and safety.

[0034] Furthermore, it has not been shown that cross-linked carboxyalkyl chitosan formulated as a hydrogel is cohesive, preferably smooth, i.e., without distinct, visible, or perceptible fragments to the touch, and has adequate safety, particularly immunocompatibility, biomechanical profile, and duration. The present invention makes it possible to provide such a matrix or composition, particularly in the form of a hydrogel. To ensure that the cross-linked matrix is ​​immunocompatible, i.e., non-immunoreactive and does not substantially activate the immune response, it should be prepared from at least one non-immunoreactive polymer(s). Certain standardized tests are used to confirm that the polymer is non-immunoreactive, such as in vitro human whole blood tests and subcutaneous injection into an airbag in mice.

[0035] It can be appreciated that the hydrogel formed by the matrix according to the present invention is not completely smooth and may have visible or perceptible lumps to the touch, provided that it is cohesive, for example according to the water test described above.

[0036] The matrix according to the invention can be characterized by a starting carboxyalkylchitosan which is crosslinked to form the matrix according to the invention.

[0037] According to a first embodiment, a carboxyalkylchitosan of fungal origin is used having carboxyalkyl-substituted glucosamine units, N-acetylglucosamine units, and glucosamine units, said carboxyalkylchitosan preferably having a degree of substitution with carboxyalkyl groups, expressed as the number of moles of substituents relative to the number of moles of total units, of more than 20%.

[0038] It is also called a chitosan derivative or a substituted chitosan.

[0039] Carboxyalkyl chitosan is prepared by substitution of chitosan.Usually, carboxyalkyl chitosan is prepared according to the patent applications of Kiomed Pharma filed under PCT / EP2018 / 080763 and its family (especially FR1761314 and EP18799772.1) and PCT / EP2018 / 080767 and its family (especially FR1761323 and EP18799773.9).These are incorporated herein by reference to illustrate the preparation of carboxyalkyl chitosan in particular.

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

[0041] The chitosan used in the present invention is advantageously of fungal origin, preferably derived from ascomycete fungi, particularly Aspergillus niger, and / or basidiomycete fungi, particularly Lentinula edodes (shiitake mushroom) and / or Agaricus bisporus (white mushroom). Preferably, the chitosan is derived from Agaricus bisporus (white mushroom). The chitosan is preferably highly pure, substantially free of impurities from its fungal origin or manufacturing process, and of microbiological grade, making it suitable for use as an implant or pharmaceutical composition. One method for preparing chitosan is described in International Patent Publication No. 03 / 068824 (EP 1483299; U.S. Pat. No. 7,556,946).

[0042] Generally, chitin is suspended in an aqueous medium in the presence of sodium hydroxide, and the medium is then heated at high temperature for a variable period of time depending on the desired molecular weight. Chitosan is then purified by solubilization in an acidic medium, precipitated in an alkaline medium, washed, and dried.

[0043] Preferably the chitosan is of a grade sufficiently pure for pharmaceutical use.

[0044] The chitosan is advantageously purified and then preferably dried. After purification, the process of the present invention may include a step of drying the carboxyalkylchitosan and then optionally grinding it into a powder. The carboxyalkylchitosan can be dried, for example, by evaporating the water, for example, by spray drying (atomization), a fluidized bed process, or by thermal drying under vacuum or atmospheric pressure, or by freeze-drying.

[0045] The carboxyalkyl chitosan can be solubilized in aqueous solutions, for example, pharmaceutical grade water that is acceptable for injection or implantation into the body, particularly the human body.

[0046] Such carboxyalkylchitosan is then crosslinked to prepare the matrix according to the present invention.

[0047] The DA and DS of crosslinked carboxyalkyl chitosans do not change substantially upon crosslinking, and can therefore be expressed as a function of the DA and DS of uncrosslinked carboxyalkyl chitosans. However, if the crosslinker provides N-acetyl or carboxyalkyl groups, these groups, which are exogenous to the starting non-crosslinked carboxyalkyl chitosan, are not taken into account in the DA and DS of the crosslinked carboxyalkyl chitosan. As explained below, the values ​​of DA and DS are known to those skilled in the art. Therefore, DA and DS refer both before and after crosslinking.

[0048] The degree of acetylation (DA) of chitosan is determined, for example, by potentiometric titration as described in patent applications WO 2017009335 and WO 2017009346. Alternatively, DA can be measured by other methods known for chitosan, such as liquid phase proton NMR, solid phase carbon-13 NMR, infrared spectroscopy, etc.

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

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

[0051] According to one alternative, the degree of acetylation is in the range of 40-50%. According to another alternative, the degree of acetylation is in the range of 50-60%.

[0052] In one embodiment, the degree of acetylation is in the range of 60-75%.

[0053] The degree of acetylation of carboxyalkylchitosan can be determined by solid-state carbon-13 NMR, solid-state carbon-13 NMR, or liquid-phase proton NMR. Carboxyalkylchitosan advantageously has a controlled degree of acetylation. The term "chitosan with a controlled degree of acetylation" refers to a product in which the degree of acetylation, i.e., the proportion of N-acetyl-glucosamine units, can be adjusted in a controlled manner, particularly by an acetylation reaction.

[0054] Preferably, the carboxyalkyl chitosan is reacetylated.

[0055] According to one alternative, the process for preparing carboxyalkylchitosan according to the present invention comprises the steps of preparing chitosan of fungal origin, reacetylating the chitosan, and carboxyalkylating the reacetylated chitosan. Thus, the present invention relates to reacetylated carboxyalkylchitosan. In particular, the present invention relates to anionic carboxyalkylchitosan.

[0056] Thus, according to one embodiment, chitosan can 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. Next, a basic agent, such as soda and / or urea, is added. Next, an alkylating agent, such as sodium monochloroacetate (i.e., the sodium salt of chloroacetic acid) or chloroacetic acid, is added. The substituted chitosan is then purified, recovered, and dried.

[0057] According to one alternative, the process for preparing carboxyalkylchitosan according to the present invention comprises the steps of preparing chitosan, carboxyalkylating the chitosan, and then reacetylating the carboxyalkylated chitosan. Advantageously, such a method allows precise control of the degree of acetylation of the final carboxyalkylchitosan, and in particular, high levels of acetylation, for example, greater than 40%, can be obtained. Thus, the present invention relates to chitosan that has been reacetylated and then carboxyalkylated, or to reacetylated carboxyalkylchitosan.

[0058] According to one alternative, the process for preparing carboxyalkylchitosan according to the invention comprises the steps of preparing chitin of fungal origin, carboxyalkylating the chitin, and optionally reacetylating the carboxyalkylated chitin to obtain carboxyalkylchitosan according to the invention.

[0059] According to one alternative, the process for preparing carboxyalkylated chitosan according to the invention comprises the steps of preparing chitin of fungal origin, deacetylating the chitin, carboxyalkylating the chitin, and optionally reacetylating the carboxyalkylated chitin to obtain carboxyalkylchitosan according to the invention.

[0060] According to one alternative, the carboxyalkylchitosan has an average molecular weight of less than 400,000.

[0061] According to one alternative, the average molecular weight is between 20,000 and 60,000.

[0062] According to another alternative, the average molecular weight is between 60,000 and 120,000.

[0063] According to another alternative, the average molecular weight is between 100,000 and 400,000.

[0064] According to another alternative, the average molecular weight is between 120,000 and 400,000.

[0065] According to another alternative, the average molecular weight is between 180,000 and 400,000.

[0066] Preferably, the average molecular weight here is the viscosity average molecular weight (Mv) calculated from the intrinsic viscosity. This expression is conventional for those skilled in the art. The intrinsic viscosity (n) is measured by capillary viscosimetry using an Ubbelohde-type capillary viscometer according to the method of the European Pharmacopoeia, monograph 2.2.9. The flow time of the solution is measured using an automatic I-Visc viscometer (Lauda) through a suitable capillary tube (Lauda, ​​for example, an Ubbelohde 510 01 capillary tube with a diameter of 0.53 mm). The Mark-Houwink equation (η=K*Mva) is then applied to calculate the average viscometric mass of the carboxyalkylchitosan, where: - Mv is the viscosity average molecular weight of the carboxyalkyl chitosan; -η is the intrinsic viscosity of the carboxyalkyl chitosan, - The constants K and α have values ​​of 0.0686 and 0.7638, respectively, as previously determined for (unsubstituted) chitosan by steric exclusion chromatography equipped with a MALLS detector.

[0067] Therefore, the intrinsic viscosity of a carboxyalkylchitosan can generally be expressed as:

[0068] Chitosan can be hydrolyzed to reduce its molecular weight.

[0069] Typically, in non-crosslinked carboxyalkyl chitosans, the glucosamine units are D-glucosamine units (at least one of D-glucosamine units, N-acetyl-D-glucosamine units, and substituted D-glucosamine units and N-acetyl-D-glucosamine units).

[0070] According to one alternative, the substituted chitosan is substituted only at the D-glucosamine units.

[0071] According to another alternative, the substituted chitosan has simultaneous substitution of D-glucosamine and N-acetyl-D-glucosamine units, to which carboxyalkyl groups are covalently attached, according to an alternative with only amine groups of chitosan, or according to another alternative with simultaneous amine and hydroxyl groups of chitosan.

[0072] The substitution is generally partial and not all units are necessarily substituted.

[0073] According to one embodiment, the degree of substitution of D-glucosamine units is in the range of 30% to 250% in terms of the number of moles of D-glucosamine units relative to the number of moles of total substituted chitosan units (substituted or unsubstituted D-glucosamine and N-acetyl-D-glucosamine units).

[0074] According to one embodiment, the carboxyalkylchitosan has a degree of substitution with carboxyalkyl groups of more than 20%, for example more than 50%, for example less than 200%, expressed as moles of substituents relative to moles of total units.

[0075] According to one embodiment, the degree of substitution with carboxyalkyl groups is greater than 50%, expressed as moles of substituents relative to moles of total units.

[0076] According to one embodiment, the degree of substitution of D-glucosamine units, expressed as the number of moles of total units of substituted chitosan (substituted or unsubstituted D-glucosamine and N-acetyl-D-glucosamine units), is in the range of 50% to 200%, and even more preferably higher than 70%.

[0077] According to one embodiment, the degree of substitution with carboxyalkyl groups is less than 80%, expressed as moles of substituents relative to moles of total units.

[0078] Typically, the substitution is achieved by a covalent bond.

[0079] According to one alternative, the carboxyalkylchitosan is an N,O-carboxyalkylchitosan. The percentage of units substituted with carboxyalkyl groups at the O-position (either O3 or O6 of the glucosamine and / or N-acetylglucosamine units) and / or at the N-position (of the glucosamine units) varies. Thus, the degree of substitution can exceed 100%.

[0080] Advantageously, the degree of substitution (DS) and degree of acetylation (DA) of carboxyalkylchitosans 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, spectra are recorded at room temperature, with relaxation times of 1 to 8 seconds and scans of 64 to 512. The areas of the carbon signals are determined after deconvolution. The carbons considered are "CH3acetyl" (the methyl carbon of the acetyl group of the N-acetylglucosamine unit, whether substituted or not), "Cx" (the carbon at the x-position of the glucosamine and N-acetylglucosamine units, where x ranges from 1 to 6), and "C=O" (the carbonyl carbon of the carboxyalkyl substituent and the C=O carbonyl carbon of the acetyl group of the N-acetylglucosamine unit, whether substituted or not). To determine the DS of a given carboxyalkylchitosan, the carbon-13 NMR spectrum of its precursor chitosan must 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 (the methyl carbon of the acetyl group in the N-acetylglucosamine unit) to the signal area of ​​the "C=O" (the carbonyl carbon of the acetyl group in the N-acetyl-D-glucosamine unit). The DA of the carboxyalkylchitosan is calculated according to Equation 1, and the DS is calculated according to Equation 2, where I represents the signal area of ​​the carbon of interest.

[0081] Formula 1:

[0082]

number

[0083] Formula 2:

[0084]

number

[0085] DA and DS can be determined using other methods known for carboxyalkylchitosans, 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 by hydrolyzing the carboxyalkylchitosan beforehand and adding a concentrated solution of deuterated hydrochloric acid before analysis.

[0086] If another NMR method is more advantageous for reliably estimating DA and / or DS, such method is suitable for use. The above methods should be adapted by those skilled in the art with regard to sample preparation and signals to be integrated, particularly with regard to the resolution, intensity, and proton position of the signals used to calculate the degree of substitution.

[0087] The degree of carboxyalkylation of chitosan may advantageously range from 20 to 250%, preferably from 50 to 200%, for example from 70 to 170%, expressed as moles of carboxyalkyl relative to moles of total units.

[0088] According to one alternative, the degree of carboxyalkylation of chitosan may advantageously range from 40 to 130%, for example from 70 to 130%, expressed as moles of carboxyalkyl relative to moles of total units.

[0089] The degree of substitution of chitosan is usually a function of the mass ratio of reactants to chitosan at the start of the reaction. Examples of carboxyalkylating agents include acid chlorides (or salts thereof, e.g., sodium monochloroacetate), such as those bearing one or more carboxymethyl, carboxyethyl, carboxypropyl, carboxybutyl groups, etc.

[0090] According to one alternative, the present invention relates to C1-C5 carboxyalkylchitosans, in which the alkyl moiety of the carboxyalkyl is linear or branched.

[0091] According to one embodiment, the present invention relates to carboxymethyl chitosan.

[0092] According to this alternative, the substituted chitosan is an N-carboxyalkylchitosan.

[0093] According to this embodiment, the substituted chitosan is an O-carboxyalkylchitosan.

[0094] According to this alternative, the substituted chitosan is an N-carboxyalkylchitosan or an O-carboxyalkylchitosan.

[0095] According to a second aspect, the present invention relates to a chitosan derivative having a glucosamine unit, an N-acetylglucosamine unit, and a glucosamine unit substituted with a carboxyalkyl group, wherein the carboxyalkylchitosan has a pH of −10 mV or less, preferably −15 mV or less, measured at pH 7.5. In particular, such a chitosan derivative enables the chitosan derivative or a composition containing the same to limit the immune response in a subject to which the chitosan derivative or a composition containing the same is administered, typically by instillation, injection, or implantation.

[0096] Advantageously, the zeta potential measured at pH 7.5 is less than or equal to −18 mV.

[0097] Advantageously, the carboxyalkylchitosan has a zeta potential measured at pH 7.5 of less than or equal to -22 mV, preferably less than or equal to -24 mV.

[0098] According to one particular alternative, the substituted chitosan preferably has an average molecular weight of 150,000 to 220,000 and a degree of substitution ranging from 50 to 200%, the molecular weight preferably being expressed before substitution.

[0099] According to another particular alternative, the substituted chitosan has an average molecular weight of 120,000 to 150,000 and a degree of substitution ranging from 70 to 200%, the molecular weight preferably being expressed before substitution.

[0100] According to one particular alternative, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000 and a degree of substitution ranging from 70 to 200%, the molecular weight preferably being expressed before substitution.

[0101] According to another particular alternative, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution ranging from 50 to 200%, the molecular weight preferably being expressed before substitution.

[0102] According to another particular alternative, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution ranging from 50 to 200%, the molecular weight preferably being expressed before substitution.

[0103] According to another particular alternative, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution ranging from 70 to 200%, the molecular weight preferably being expressed before substitution.

[0104] According to one particular alternative, the substituted chitosan preferably has an average molecular weight of 120,000 to 150,000 and a degree of substitution ranging from 20% to 50%, the molecular weight preferably being expressed before substitution.

[0105] According to another particular alternative, the substituted chitosan has an average molecular weight of 220,000 to 300,000 and a degree of substitution ranging from 20% to 50%, the molecular weight preferably being expressed before substitution.

[0106] According to another particular alternative, the substituted chitosan has an average molecular weight of 300,000 to 500,000 and a degree of substitution ranging from 20% to 50%, the molecular weight preferably being expressed before substitution.

[0107] According to a particular alternative, the substituted chitosan has a degree of substitution ranging from 20 to 80%, preferably from 40 to 60%, and a degree of acetylation ranging from 40 to 80%, preferably from 50 to 75%.

[0108] According to another particular alternative, the substituted chitosan has a degree of substitution ranging from 90 to 200%, preferably from 90 to 150%, and a degree of acetylation of from 40 to 80%, the molecular weight preferably being expressed before substitution.

[0109] According to a particular alternative, the substituted chitosan has a degree of substitution ranging from 90 to 200%, preferably from 90 to 150%, and a degree of acetylation of from 40 to 60%, preferably from 50 to 60%.

[0110] According to one particular alternative, the substituted chitosan has a degree of substitution ranging from 90 to 200%, preferably from 90 to 150%, and a degree of acetylation of from 50 to 75%.

[0111] According to one particular alternative, the substituted chitosan preferably has an average molecular weight of 220,000 to 300,000, a degree of substitution ranging from 90 to 200%, preferably from 90 to 150%, and a degree of acetylation of 50 to 75%, the molecular weight preferably being expressed before substitution.

[0112] By substituting chitosan, it was possible to prepare solutions of carboxyalkylchitosan that were soluble in aqueous solutions over a wide range of pH values, whereas unsubstituted chitosan was soluble only at pH values ​​below 5.5-6.5. Thus, carboxyalkylchitosan exhibits the ability to be solubilized at different pH values, particularly physiological pH or the pH of body fluids altered by pathologies, such as inflammatory conditions, due to the presence of the carboxyalkyl group, which alters its solubility profile.

[0113] "Water-soluble" means that the carboxyalkyl chitosan does not exhibit turbidity visible to the naked eye when placed in an aqueous solution. More specifically, the solubility, i.e., the absence of turbidity, of a 1% (m / m) solution of carboxyalkyl chitosan in water or a buffer solution, e.g., phosphate buffer, can be confirmed by an optical density of less than 0.5, preferably less than 0.2, measured by UV-visible spectroscopy at a wavelength of 500 nm with reference to a reference cell containing only the aqueous solvent used in the measured sample but no substituted chitosan. Another method is visual inspection according to European Pharmacopoeia Monograph 2.9.20. If the chitosan is not sufficiently substituted, the composition will not dissolve at room temperature within a satisfactory pH range, e.g., pH 5.5 to pH 8.5.

[0114] In one embodiment, the carboxyalkyl chitosan is sterile.

[0115] By "covalently crosslinked by the carboxyalkylchitosan chains," it is specifically understood that the chitosan backbone (also called the chitosan backbone) is covalently bonded to one or more main chitosan chains. Advantageously, a three-dimensional network of chitosan molecules is obtained in this manner. The present invention is not limited to a specific covalent crosslinking method, and includes the use of chemical molecules as crosslinking agents, also known as crosslinking agents.

[0116] According to the present invention, the carboxyalkyl chitosan is crosslinked.

[0117] According to one alternative, the crosslinks are formed by a crosslinking agent which forms the covalent bonds described above.

[0118] Thus, some chitosan chains may be crosslinked by crosslinking agents used to crosslink polysaccharides, such as 1,4 butanediol diglycidyl ether, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxypentane, 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. The crosslinking can be effected by reaction with one or more crosslinking agents such as selected from 2,5-dibromopentanethiol epichlorohydrin, 2,3-dibromopropanol-1,1-chloro-2,3-epithiopropane, dimethylaminopropylcarbodiimide, gallic acid, epigallocatechin gallate, curcumin, tannic acid, genipin, or diisocyanate compounds such as hexamethylene diisocyanate or toluene diisocyanate, or divinyl sulfone.

[0119] Genipin is a naturally occurring crosslinker used to crosslink polysaccharides, particularly carboxymethyl chitosan (Yang et al. Acta Pharmacol Sin 31, 1625, 2020). Genipin imparts a deep blue to black color to the hydrogel, which may be advantageous for some indications.

[0120] Preferably, the crosslinker is of the polyepoxy type, such as a bifunctional substance. Preferably, 1,4-butanediol diglycidyl ether (BDDE) or ethylene glycol diglycidyl ether (EGDE) are used as crosslinkers, since they are already used in the preparation of biomaterials for human use, in particular hyaluronan hydrogels for intradermal, intraarticular, or intraocular administration. According to one alternative, the crosslinker is divinyl sulfone.

[0121] Advantageously, the compositions of the invention may also contain biopolymers other than cross-linked carboxyalkylchitosans. According to an advantageous alternative, the biopolymer is, for example, a glycosaminoglycan, in particular a hyaluronan such as hyaluronic acid or sodium hyaluronate, whether oxidized or not, whether covalently cross-linked or not.

[0122] The advantage of combining or crosslinking crosslinked carboxyalkyl chitosan with some other polymers is to add or create synergistic effects on their biological and physicochemical properties.

[0123] According to one alternative, the matrix according to the invention comprises cross-linked carboxyalkylchitosan and hyaluronan, chondroitin sulfate and / or carboxymethylcellulose. To date, there are no hydrogels of cross-linked carboxyalkylchitosan (as defined in the present invention) combined with hyaluronan. One of the aims of the present invention is to combine these two polymers, for example, to be able to combine the recognized moisturizing properties of hyaluronan with the protective properties of chitosan against oxidative stress.

[0124] According to one alternative, the matrix comprises at least one hyaluronan.

[0125] Advantageously, the matrix according to the invention comprises cross-linked carboxymethylchitosan alone or in combination with hyaluronan, whether cross-linked or not, so that the desired properties can be tailored.

[0126] The matrix comprises at least one of carboxymethylchitosan and hyaluronan.

[0127] According to one alternative, the hyaluronan has an average molecular weight of less than 5 million, preferably greater than 1 million, and preferably greater than 2 million, as determined by capillary viscosimetry. The molecular weight of hyaluronan is sometimes expressed as density, as it is correlated via a linear relationship. Hyaluronan has a maximum molecular weight of 4.25 m 3 / kg, for example, low density (e.g., about 1-2 m 3 / kg) or high density (e.g., about 2-4 m 3 / kg).

[0128] According to one alternative, hyaluronan is obtained by fermentation, for example with streptococci, according to another alternative, it is produced by extraction from rooster peak.

[0129] According to one alternative, the matrix comprises at least one covalently cross-linked hyaluronan.

[0130] Thus, cross-linked hyaluronan contains covalent bonds between different hyaluronan chains.

[0131] Different types of hyaluronan can be cross-linked to each other, such as hyaluronan having different molecular weights or different hyaluronan salts.

[0132] The present invention also relates to a process for preparing crosslinked carboxyalkyl chitosan.

[0133] According to one alternative, the process for preparing the matrix according to the invention comprises: contacting the carboxyalkylchitosan with at least one crosslinking agent, the contacting preferably being carried out in an alkaline phase; cross-linking the carboxyalkyl chitosan with a cross-linking agent; and Obtaining a matrix containing crosslinked carboxyalkyl chitosan Includes:

[0134] According to one alternative, the carboxyalkylchitosan is crosslinked in the presence of an alkaline aqueous phase, for example a sodium hydroxide (NaOH) solution.

[0135] Advantageously, the concentration of carboxyalkylchitosan initially present in the aqueous phase ranges from 1 to 30% by weight, preferably from 5 to 20% by weight (w / v) of carboxyalkylchitosan relative to the volume of the alkaline aqueous phase.

[0136] Advantageously, the mass ratio between crosslinker and polymer is between 0.1% and 30%, expressed as the weight of crosslinker relative to the weight of polymer.

[0137] Preferably, the mass ratio between crosslinker and polymer is between 0.5% and 20%, expressed as the weight of crosslinker relative to the weight of polymer, especially when BDDE is used.

[0138] Typically the reaction is carried out with heating, for example at a temperature of 25 to 60° C., for example 50° C., for a period of time of, for example, 30 minutes to 48 hours, for example 1 hour to 5 hours. Generally, crosslinking is stopped, for example by adding an acid, and by neutralization and dilution, for example by adding acetic acid or hydrochloric acid.

[0139] Advantageously, the reaction residues are removed by dialysis using a phosphate buffer.

[0140] In this way, a hydrogel comprising a matrix according to the invention is obtained.

[0141] On the other hand, carboxyalkylchitosan is an exogenous molecule and is more resistant to degradation than hyaluronan after implantation / injection / ocular instillation.

[0142] The present invention therefore relates to matrices comprising three-dimensional networks based on these two polymers with different molecular weights, thus advantageously providing a range of biomechanical properties, in situ product duration, and therapeutic duration, while retaining the free radical scavenging power of carboxyalkylchitosan.

[0143] The present invention relates to a matrix comprising at least one hyaluronan co-crosslinked by covalent bonds with a carboxyalkylchitosan.

[0144] According to one alternative, the process for preparing a matrix comprising a carboxyalkylchitosan, preferably according to the invention, co-crosslinked with another biopolymer, and preferably hyaluronan, comprises the steps of: contacting the mixture of carboxyalkyl and other biopolymers, and preferably hyaluronan, with at least one crosslinker, the contacting preferably being carried out in an alkaline phase; cross-linking the carboxyalkyl chitosan and other biopolymers, and preferably hyaluronan, with a cross-linking agent; Obtaining a matrix of co-crosslinked carboxyalkyl chitosan and other biopolymers, and preferably hyaluronan. Includes:

[0145] According to one alternative, the matrix according to the invention is sterile.

[0146] It is advantageous to provide a hydrogel from a matrix according to the invention.

[0147] The present invention therefore relates to hydrogels, which advantageously form cohesive hydrogels.

[0148] Thus, the present invention relates to crosslinked carboxyalkylchitosan hydrogels in which the carboxyalkylchitosan 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 usually less than 200%).

[0149] The present invention relates to a composition comprising at least one matrix as defined according to the invention.

[0150] According to one preferred alternative, the matrix according to the invention is formulated in an aqueous medium to form a composition in the form of a hydrogel.

[0151] Advantageously, the concentration (m / m) of polymer (e.g. carboxyalkylchitosan with or without another biopolymer such as hyaluronan) relative to the total mass of the composition, in particular the hydrogel, is less than 10% by mass, for example 5% or less.

[0152] According to one alternative, the concentration (m / m) of the polymer (e.g., carboxyalkylchitosan, with or without another biopolymer such as hyaluronan) relative to the total mass of the composition, in particular the hydrogel, is less than 4% by mass, for example 3% or less.

[0153] The mass ratio (m / m) [carboxyalkyl chitosan / hyaluronan] is, for example, 5-95%, such as 10-90%, further such as 30-70%. The mass ratio (m / m) [hyaluronan / carboxyalkyl chitosan] is, for example, 5-95%, such as 10-90%, further such as 30-70%. According to one alternative, the mass ratio (m / m) [carboxyalkyl chitosan / hyaluronan] is 1:1 (i.e., 50% chitosan and 50% hyaluronan).

[0154] The aqueous medium may be water, an aqueous solution, the pH and osmolality of which are adjusted using, for example, an acid / base buffer system with the addition of salts and / or optionally polyols (sorbitol, mannitol, glycerol).

[0155] According to one alternative, the matrix according to the invention is formulated in a hydrophilic lipid vehicle that allows the formation of one or more direct or reverse emulsions.

[0156] According to one embodiment, the matrix composition has an osmolality of 100 to 700 mosm / kg, preferably 120 to 500 mosm / kg.

[0157] Advantageously, the osmolality of the matrix composition is between 250 and 400 mosm / kg, preferably between 270 and 330 mosm / kg.

[0158] According to one alternative, the composition of the matrix has an osmolality suitable for the joint.

[0159] According to one alternative, the composition of the matrix has an osmolality that is compatible with the eye or intraocular surface.

[0160] According to one alternative, the composition of the matrix has an osmotic pressure that is compatible with the dermis or mucous membranes.

[0161] According to one alternative, the osmolality of the matrix composition is preferably between 100 and 400 mosm / kg, more particularly between 120 and 380 mosm / kg.

[0162] According to one alternative, the composition according to the invention is sterile.

[0163] Advantageously, the composition according to the invention is contained in an injection, implant or eye drop device such as a syringe or a vial.

[0164] Advantageously, the injection device, such as a syringe, can then be steam sterilized. This device, such as a syringe, can then be packaged, preferably in an aseptic or sterile manner. It can also be a bag, flapula, or vial for instilling the composition according to the invention, which is filled aseptically after sterilizing the formulation or is sterilized directly after filling.

[0165] According to one alternative, the composition according to the invention, in particular the hydrogel according to the invention, is sterilized by filtration and / or steam sterilization before filling the injection, implant or eye drop device, such as a syringe or a vial.

[0166] Those skilled in the art are aware of techniques for sterilizing hydrogels to obtain the desired sterile hydrogels, which are equipped with several types of equipment for heat or steam sterilization and can use several types of times to eliminate the microbial load.

[0167] The invention more particularly relates to an injectable composition comprising a matrix according to the invention, preferably in the form of a hydrogel.

[0168] The present invention also relates to a pharmaceutical composition comprising at least one matrix according to the invention, preferably in the form of a hydrogel.

[0169] According to one alternative, the composition according to the invention is used as an injectable, implantable or ophthalmic pharmaceutical composition or as an injectable, implantable or ophthalmic medical device.

[0170] The present invention further covers compositions according to the invention in dry form, in particular in lyophilized form. Lyophilized products can in particular be (re)dispersed and preferably solubilized before use.

[0171] The present invention more particularly relates to a composition according to the invention for use in therapeutic treatment, including, for example, injection by subcutaneous, intradermal, intraocular, or intra-articular, intramucosal, or intramuscular route, said composition being for example for repair, regeneration or filling of at least one body tissue / fluid in need of repair or filling.

[0172] It is advantageous to use chitosan that is sufficiently pure for the intended use.

[0173] It is advantageous to use hyaluronan that is sufficiently pure for the intended use.

[0174] The biomechanical properties required for the compositions according to the invention may vary in nature depending on the indication, for example, on the tissue into which the hydrogel is to be incorporated, the mechanism of action, or the effect and duration of effect aimed to ensure the benefit of the patient.

[0175] Advantageously, the properties of the compositions according to the invention, in particular the properties of the hydrogels according to the invention, are adapted to the indication: to adapt these properties, the final concentration of the polymer (carboxyalkylchitosan and / or other biopolymers such as hyaluronan) and / or the degree of cross-linking, in particular via the cross-linker / polymer mass ratio, and / or the nature and / or amount of ions and / or the initial molecular weight of the polymer are, for example, varied.

[0176] Specifically, the present invention relates to highly elastic hydrogels, particularly when sustained volume growth must be ensured at the cutaneous, subcutaneous, or periosteal levels (due to projection or remodeling), or to viscoelastic gels that provide both shock absorption and lubrication, particularly at the joint level. The present invention also relates to lubricating hydrogels, particularly when friction must be reduced between two biological surfaces, such as two cartilage surfaces in a joint, or between the surface of the eye and the eyelid. The compositions of the present invention can have variable elasticity levels tailored to the indication and can be characterized by measuring the elastic modulus with a flow meter.

[0177] Preferably, the matrix has an antioxidant capacity by scavenging free radicals, in particular a normalized antioxidant capacity of greater than 0.30, preferably greater than 0.50, even more preferably greater than 0.80, for example greater than 0.90.

[0178] The present invention relates to an injectable composition, characterized in that it comprises at least one matrix as defined according to the invention.

[0179] The present invention relates to a pharmaceutical composition, characterized in that it comprises at least one matrix as defined according to the invention.

[0180] According to one alternative, the compositions according to the invention are used as an injectable, implantable or ophthalmic or topically administrable pharmaceutical composition or an injectable or implantable or ophthalmic or topically administrable medical device, for example for use in a method of therapeutic treatment, comprising topically instilling or administering or injecting said composition by subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular, intraosseous route, for example for the repair or filling of at least one body tissue in need of repair or filling.

[0181] According to one alternative, the composition according to the invention is used in a method for the treatment, repair or filling of at least one body fluid or tissue in need of repair or filling, for example the body tissue being selected from the vocal cords, muscles, ligaments, tendons, mucous membranes, genitals, bones, joints, eyes, skin or any combination thereof, in particular tissue belonging to the skin, cartilage, synovium, skin wounds or the surface of the eye.

[0182] The present invention relates to a composition according to the invention for use in a method for treating osteoarthritis or for repairing cartilage defects, for example by injection into a body fluid such as synovial fluid or after mixing with a body fluid such as blood and after implantation into cartilage. By body fluid is meant a fluid from the body, whether or not it has undergone treatment to alter its composition.

[0183] The present invention relates to a medical device, such as a medical implant, characterized in that it comprises or consists of a composition as defined according to the invention.

[0184] The invention particularly relates to compositions according to the invention for use in therapeutic, surgical or cosmetic treatment, including in particular treatment in rheumatology, ophthalmology, gynecology, aesthetic medicine, plastic surgery, laparotomy, orthopedics, gynecology, for the prevention of post-operative tissue adhesions, and in dermatology.

[0185] The present invention also relates to a composition according to the invention for use in the therapeutic treatment of dry eye syndrome, corneal damage or inflammation of the eye or joints.

[0186] The present invention further relates to the administration of compositions according to the invention by instillation onto the surface of the eye to prevent or combat corneal damage or dry eye syndrome, particularly for the purpose of lubricating or regenerating the ocular surface.

[0187] Therefore, the present invention also relates to an ophthalmic composition comprising a carboxyalkylchitosan as defined according to the present invention.

[0188] According to one alternative, the subject suffers from an inflammatory condition (eg, osteoarthritis, arthritis, dry eye syndrome).

[0189] The invention more particularly relates to compositions according to the invention for the treatment of arthrosis, arthritis or for repairing cartilage defects, for example by injection into the synovial cavity or implantation into the cartilage defect.

[0190] The invention more particularly relates to medical devices, such as medical implants, characterized in that they comprise or consist of a composition according to the invention.

[0191] According to one preferred alternative, the present invention therefore relates to a medical device comprising a chamber containing the composition according to the invention in dry form, in particular in lyophilized form, and optionally one or more other chambers containing one or more active products, additives or excipients.

[0192] The compositions according to the invention may also contain one or more active agents for a desired indication, and / or one or more additives or excipients for adjusting the properties of the compositions according to the invention.

[0193] The present invention also relates to a composition according to the invention for use in a therapeutic method of treatment.

[0194] The present invention also relates to a composition according to the invention for use in a method for treating arthropathy or for repairing cartilage defects, for example by injection into the synovial sac or after mixing with a body fluid such as blood and after implantation into the cartilage / bone.

[0195] The present invention also relates to a composition according to the invention for use in a dermal filling ("dermal filling") or lip filling aesthetic treatment or care method, which in particular comprises, for example, subcutaneous, intradermal, intramucosal or intramuscular injection of a composition according to the invention.

[0196] The present invention also relates to a composition according to the present invention for use in a method for surface treatment of skin or other tissues by multiple intradermal injections using conventional mesotherapy methods well known to those skilled in the art.Such compositions can usually be used in dermatology as a treatment for cosmetic purposes.The purpose of such a method is, for example, to plump up the skin and eliminate the appearance of wrinkles (treatment of wrinkles and / or fine lines).Such treatment can be intended for subjects who want to give their skin a rejuvenated appearance.

[0197] The present invention also relates to a composition according to the invention for use in therapy, wherein the composition is a viscosupplement, for example, where the composition of the invention is injected intra-articularly to limit friction on the cartilage surfaces of the joints.

[0198] The present invention also relates to compositions according to the present invention for use as a cell vector for one or more cell types and / or one or more active agents. These may be active agents from a pharmaceutical or biological perspective. The compositions of the present invention may in fact be compatible with the presence of cells, preferably live cells. Examples of live cells of interest include chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligaments), dermal fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscles), mesenchymal stem cells, erythrocytes (blood), and keratinocytes (skin). The compositions of the present invention may also be targeted as therapeutic vectors for targeted and / or controlled-release delivery of at least one therapeutic agent.

[0199] According to one preferred alternative, blood, or plasma, or platelet lysate, or platelet-rich plasma, or any body fluid is added with the composition of the present invention, for example to enhance the performance of the product.

[0200] According to one alternative, the composition according to the invention is formulated in a solid form (e.g., a film or porous foam) which swells / moistens when implanted (e.g., tear plug, dressing).

[0201] According to one alternative, the composition is formulated in the form of a sprayable composition (spray).

[0202] The present invention also relates to a composition according to the invention for use in a method for the therapeutic or cosmetic care of one or more tissues or organs affected by excessive temperature, such as in the case of burns.

[0203] The present invention also relates to a composition according to the invention for use in a method for treating cartilage repair (for example by implantation into a cartilage defect for the purpose of promoting its regeneration).

[0204] The present invention also relates to a composition according to the invention for use in a method for the preventive treatment of post-surgical tissue adhesions: the product is applied to tissues at the end of surgery, for example gynecological, abdominal, visceral, orthopedic, etc.

[0205] The present invention relates to physiological compositions that are administered locally by injection or implantation to contact one or more biological tissues exposed to oxidative stress, such as: -Intra-articular injections for the treatment of osteoarthritis (through synovial fluid replenishment, cartilage lubrication, shock absorption at the joint level, synovial membrane regeneration); intra-articular implants to promote the repair of cartilage defects; -Intraosseous implants to promote bone repair (osteoinduction / osteoconduction); - Subcutaneous and / or intradermal injections to fill or regenerate skin or hair follicles, to increase volume in cases of lipoatrophy; - instillations into the eyes to relieve symptoms or prevent degeneration of the ocular surface, for example, for the treatment of dry eye and corneal lesions, and administration of active ingredients; - intraocular injections, for example to optimize the effectiveness of glaucoma surgery or vitreous replenishment, as an adjuvant in cataract surgery, for the regeneration of anterior or posterior segment tissues, and for intraocular administration of active ingredients; -Administration to internal tissues and organs to prevent postoperative adhesions (films); -Administered to wounds, cracks, tears, cavities in tissues and organs such as skin, bone, cartilage, cornea, tendons, meniscus, etc., to promote repair or regeneration; -Injection into the vulvar mucosa for the treatment of vulvodynia.

[0206] The present invention also relates to compositions according to the invention that form artificial synovial fluid.

[0207] The composition according to the invention makes it possible to mimic healthy synovial fluid or improve healthy or defective synovial fluid, for example by improving its lubricating capacity to reduce friction in the joints and / or by seeking its shock-absorbing properties (identifiable by its elastic modulus G'), while at the same time being easily injectable, for example for filling a syringe or for injection 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.

[0208] Advantageously, for intra-articular injection, the compositions according to the invention are easily injectable at room temperature through a thin needle, for example a needle having a diameter of 21 gauge. By "easy" injection, it is preferably meant that the force applied to such a syringe is less than 50 Newtons (at a rate of 10 mm / min) to cause the compositions according to the invention to flow through a 21 gauge needle, and preferably less than 20 Newtons.

[0209] Advantageously, for intradermal injection, the compositions according to the invention are easily injectable at room temperature through thin needles, for example needles with a diameter of 25 gauge or less. "Easy" injection preferably means that the force applied to such a syringe and released into the air is less than 30 Newtons (at a rate of 10 mm / min) to cause a composition according to the invention to flow through a 27 gauge needle, and preferably less than 20 Newtons.

[0210] The present invention also relates to a composition as an artificial tear comprising the carboxyalkylchitosan according to the present invention.

[0211] Generally, the range of osmolality and pH value of the composition will be adapted and will generally be close to the osmolality and pH value of the tissue in contact with the composition according to the invention.

[0212] Advantageously, the composition according to the invention is sterile. Very advantageously, the composition according to the invention is sterilized by elevated temperature, preferably under autoclave.

[0213] According to one embodiment, the matrix has a low coefficient of friction (COF), such as a lubricating ability of less than 20, such as less than 10, according to testing in the examples of the present invention.

[0214] According to one alternative, the composition of the invention is transparent or translucent.

[0215] "Translucent" means that the composition allows the observer to distinguish the object when placed between the observer's eye and the object. "Transparent" is understood to mean that the observer can distinguish alphanumeric characters when placed between the observer's eye and the observed text. Generally, this evaluation is performed at a composition thickness of approximately 1 cm. The method of European Pharmacopoeia Monograph 2.9.20 for visual inspection can also be adopted. The optical density of the composition can also be measured, for example, by UV-visible spectroscopy at 500 nm, to confirm that the optical density is less than 0.5, preferably less than 0.2, relative to the reference solvent.

[0216] According to one alternative, the compositions of the invention are not milky white or are slightly milky white.

[0217] By "opalescence" is meant that the solution causes diffraction of light visible to the naked eye, as determined by visual inspection, for example, by methods such as European Pharmacopoeia Monograph 2.9.20, and comparison with European Pharmacopoeia reference solutions of different opalescence levels. In one alternative, the compositions of the present invention are colorless, i.e., a person observing with the naked eye does not perceive any particular color in the composition. In another alternative, the opalescence is below the maximum value permitted for the intended use.

[0218] The present invention relates in particular to a preferably sterile article or package comprising one or more eye drop or injection devices pre-filled with a composition according to the invention, in particular in the form of a hydrogel. These are usually devices for administering a product to the eye in the form of drops or pre-filled syringes.

[0219] The compositions of the present invention can advantageously be stored, preferably for several months, preferably in an article or packaging suitable for its indication.

[0220] Advantageously, the compositions of the present invention can be sterilized. Thus, the present invention relates to sterilized cross-linked carboxyalkyl chitosan. Thus, the cross-linked carboxyalkyl chitosan is sterile, especially for applications requiring it.

[0221] According to one alternative, the compositions of the invention are steam sterilized according to methods known to those skilled in the art and / or recommended by the European Pharmacopoeia.

[0222] According to another alternative, the composition may be sterilized by filtration using a filter intended for this purpose, for example a filter with a porosity of 0.2 μm or less.

[0223] Advantageously, according to a preferred embodiment, the cross-linked carboxyalkyl chitosan loses less than 40% of its intrinsic viscosity upon steam sterilization.

[0224] The present invention also covers a method for therapeutic treatment comprising injecting a composition according to the present invention.

[0225] The present invention also covers the use of a composition according to the invention, eg as more specifically defined by the present invention, for the preparation of a pharmaceutical composition, in particular for therapeutic treatment.

[0226] The present invention also covers a method for cosmetic, i.e. non-therapeutic, treatment, comprising injecting a composition according to the invention, said treatment being, for example, the filling of wrinkles or the filling of one or more damaged visible tissue zones for cosmetic purposes, for example as a result of an accident or surgical procedure.

[0227] A tissue is a group of similar cells of the same origin, grouped into a functional unit, i.e., they all contribute to the same function. Some of the tissues mentioned may consist of skin tissue (e.g., epithelial tissue), connective tissue, muscle tissue, and nervous tissue.

[0228] By "composition according to the invention" or equivalent term is meant a composition as defined in the present invention, including in any of the alternative ways, in particular or specific embodiments, alone or in any combination thereof, including by preferred properties.

[0229] Further objects, features and advantages of the present invention will become apparent to those skilled in the art upon reading the illustrative description which refers to examples which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0230] The Examples are an integral part of the present invention, and any feature that appears from the specification as a whole, including the Examples, to be novel with respect to any prior art is an integral part of the present invention in its function and generality.

[0231] Therefore, each example has a general range.

[0232] However, in the examples, all percentages are by weight unless otherwise stated, temperatures are in degrees Celsius unless otherwise stated, and pressures are atmospheric unless otherwise stated. [Example]

[0233] How to measure zeta potential The formulation to be analyzed is diluted with phosphate buffer to a final polymer concentration of 0.05% and gently stirred until homogenized. The solution is then separated into different fractions, and the pH of each fraction is adjusted to the desired value between pH 4 and 8 by adding either 0.1 N sodium hydroxide or 0.1 N hydrochloric acid. The zeta potential of each fraction is measured using a "Nano-Z" device (Zeta-Sizer range, Malvern Instruments).

[0234] Method for determining the solubility range of chitosan polymers The solubility range is established by preparing a solution of the polymer to be tested at a concentration of 1% and a pH of 9, which is then divided into several fractions whose pH is adjusted to various values ​​ranging from 9 to 1. For each fraction, the solubility of the polymer, i.e., the absence of turbidity, is verified according to the visual inspection method of European Pharmacopoeia monograph 2.9.20. The pH ranges in which the polymer is soluble or insoluble are indicated.

[0235] Flow meter biomechanical profile The biomechanical profile of the specimens is characterized using a DHR-2 Hydrid Rheometer (TA Instrument) with a 20 mm planar geometry spaced 700 μm from the Peltier element at a temperature of 37 °C, a frequency of 3.98 rad / s, and a deformation amplitude ranging from 0.1 to 10%. Each measurement is performed in triplicate, and the average values ​​of the elastic modulus (G'), viscosity (G''), and tan δ (G'' / G') of the three measurements are calculated.

[0236] Lubrication ability Lubrication ability is characterized by the coefficient of friction (COF) between two surfaces. The coefficient of friction is measured by the following methods, the parameters of which are selected depending on the product and the intended indication:

[0237] -Method for viscosupplementation Two discs based on polyacrylate biomaterials used in the manufacture of 16.15 mm diameter hydrophobic intraocular lenses (described in patent EP1830898) were pre-hydrated by immersion in water at 60°C for approximately 2 hours and then fixed in the upper and lower geometry of a DHR-2 rheometer (TA Instruments). A volume of approximately 100 μL of the sample to be tested was placed on the lower disc, and then the upper geometry was lowered to an imposed normal force of 5 Newtons until both discs came into contact. The friction coefficient was measured at 25°C for 150 seconds at a constant normal force (5 N), a vibration frequency of 1.256 rad / s, and a deformation angle of approximately 0.05 radians, according to a protocol adapted from that described by Waller et al. (in: J 47 Rheumatol 39, 7, 1473, 2012). The option "Adhesion to zero starting point of vibration motion" was activated. Torque values ​​are recorded at each measurement point, and the coefficient of friction (COF) is calculated according to the following formula: COF = torque / (1 / 3 × disc diameter × normal force). For each formulation, measurements are repeated five times. Coefficient of friction values ​​are reported by extrapolating the intercept at the beginning of each COF vs. time curve (COF0).

[0238] -Methods for artificial tears Two discs based on polyacrylate biomaterials used in the manufacture of 16.15 mm diameter hydrophobic intraocular lenses (described in patent EP1830898) were pre-hydrated by immersion in water at 60°C for approximately 2 hours and then fixed in the upper and lower geometry of a DHR-2 rheometer (TA Instruments). A volume of approximately 100 μL of the sample to be tested was placed on the lower disc, and then the upper geometry was lowered to an imposed normal force of 5 Newtons until both discs came into contact. The friction coefficient was measured at 25°C for 150 seconds at a constant normal force (5 N), a vibration frequency of 1.256 rad / s, and a deformation angle of approximately 0.05 radians, according to a protocol adapted from that described by Waller et al. (in: J 47 Rheumatol 39, 7, 1473, 2012). The option "Adhesion to zero starting point of vibration motion" was activated. Torque values ​​are recorded at each measurement point, and the coefficient of friction (COF) is calculated according to the following formula: COF = torque / (1 / 3 x disc diameter x normal force). For each formulation, measurements are repeated five times. Coefficient of friction values ​​are reported by extrapolation of the intercept at the beginning of each COF vs. time curve (COF0).

[0239] Needle ejection force Measurements are performed using a MultiTest 2.5-i compression tester (Mecmesin) equipped with a 100 N compression cell. An appropriate needle is fitted to the syringe containing the sample. The syringe is placed in the tester, and the syringe plunger is pushed at a constant rate (e.g., 10 or 80 mm / min) and the force required for extrusion is measured. The maximum force allowed by the instrument is approximately 70 Newtons.

[0240] In vitro antioxidant capacity (ABTS test) To measure the antioxidant activity of carboxyalkyl chitosan preparations and compare it with commercially available products, an in vitro "ABTS" test was applied. This test consists of determining the ability of a substance to capture the cation radical of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·1). The absorption maximum of this chromophore in its cation radical form is at 734 nm. The protocol was adapted from the method described by Valyova et al. (Int J Applied Res Nat Prod, 5, 19, 2012) and was performed using Nunclon 96-type polystyrene microplates (Thermo Fisher Scientific) and an Infinite M200 microplate reader (Tecan Life Sciences) for absorbance measurements.

[0241] Each test series is carried out in four steps.

[0242] 1) Dilute 1 g of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with a homogeneous solution of K2S208 (2.45 mM in MilliQ water) to a concentration of 7 mM ABTS. This mixture is protected from light and stirred at room temperature for 24 hours, which is the time required to generate a defined amount of ABTS·1 radical cation. A working solution of ABTS·1 is finally obtained by taking 600 μL of the latter mixture and diluting this volume with MilliQ water to a concentration of 415 μM.

[0243] 2) A calibration curve for free radical scavenging capacity was established by comparison with Trolox, a reference antioxidant molecule (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Trolox solutions at concentrations of 30, 60, 90, 120, 150, 180, and 210 μM were obtained by diluting 15 mg of Trolox stock solution in 5 mL of 100% methanol with MilliQ water. Absorbance measurements were performed at 734 nm one hour after mixing 50 μL of the working solution of ABTS·1 with 50 μL of each Trolox solution. The relationship between absorbance and Trolox concentration was measured within the linearity zone. The minimum absorbance value in the linearity zone corresponds to the detection limit.

[0244] 3) The product to be tested is characterized as such at its initial concentration or diluted with MilliQ water (defined according to the product to be tested so that the absorbance of its mixture with ABTS·1 solution is higher than the detection limit). 50 μL of the reference dilution solution is mixed with 50 μL of the test product solution. After 1 hour of incubation at room temperature, the absorbance is measured at a wavelength of 734 nm. If the absorbance value is within the detection range of the instrument, it is retained and the Trolox equivalent is calculated via a calibration curve labeled TEAC, for "Trolox Equivalent Antioxidant Capacity."

[0245] 4) The positive control is ascorbic acid (vitamin C) in a solution with a concentration of 0.02 mg / mL (20 μg / mL), which is used to represent antioxidant capacity in a normalized manner from one series to another. First, measure the TEAC of 0.005 to 0.05 mg / mL ascorbic acid solutions. Confirm that the absorbance of the 0.02 mg / mL ascorbic acid solution is within the linear zone. Finally, the normalized antioxidant capacity of the tested product is expressed as the ratio of TEAC (product) / TEAC (0.02 mg / mL ascorbic acid).

[0246] Example 1 Carboxymethyl chitosan can be produced via carboxymethylation and acetylation reactions according to the following method, using the reaction parameters in Table 1a as an example. Additionally, other reaction parameters can be used to tailor the molecular structure of the carboxymethyl chitosan. Step 1: Carboxymethylation of chitosan. 30 g of chitosan derived from Agaricus bisporus (white mushroom) is dispersed in 600 mL of isopropanol, 41 mL of water, and 163 mL of 50% sodium hydroxide (m / v). 135 g of the alkylating agent monochloroacetic acid (MCA) is dissolved in 135 mL of isopropanol and added to the chitosan suspension. The reaction is continued for 23 h at 35 °C. The polymer is recovered by precipitation with ethanol and subsequently purified by cycles of solubilization in water and precipitation with ethanol. Carboxymethyl chitosan (reference CC4, Table 1b) is collected after drying in a ventilated oven.

[0247] Step 2: Acetylation of carboxymethyl chitosan. 21 g of CC4 is dispensed into 570 mL of water and the pH of the solution is adjusted to pH > 7. 10 mL of acetic anhydride is added and the solution is stirred at 25 °C for 30 minutes. The pH of the solution is adjusted to pH > 7, and then 10 mL of acid anhydride is added. After homogenization (stirring at room temperature for approximately 30 minutes), the pH is adjusted to approximately pH 7.5. The polymer is recovered by precipitation in ethanol and then purified by a solubilization cycle in water and precipitation. The carboxymethyl chitosan (reference CC3, Table 1b) is collected after drying in a ventilated oven.

[0248] The carboxymethyl chitosans used to prepare the matrices of Examples 2 to 11 are listed in Table 1b. CC1 to CC6 are carboxymethyl chitosans derived from fungal chitosans and prepared according to the method described above. CC7 is a commercially available shellfish-derived carboxymethyl chitosan provided by Kraeber Company (product code 5313009900, Ellerbek, Germany).

[0249] [Table 1]

[0250] [Table 2]

[0251] Example 2 - Carboxymethylchitosan matrix Synthetic studies were conducted to provide a matrix of carboxymethylchitosan by covalent crosslinking using the crosslinker 1,4-butanediol diglycidyl ether (CAS 245-79-8, BDDE). Several carboxymethylchitosans derived from Agaricus bisporus (white mushroom) were used, manufactured by Kiomed Pharma according to the method described in Example 1. Their properties are listed in Table 1. BDDE (96%, specific gravity 1.049) was provided by Alfa Aesar (ThermoFischer, Kandel, Germany).

[0252] Example 2a After adjusting the reaction parameters, a crosslinked matrix was prepared from carboxymethyl chitosan CC3 (Table 2a, see M1-A). The degree of acetylation of CC3 was 55% and the degree of carboxymethylation was 87%, as determined by carbon-13 NMR (Equation 2). After dialysis, the hydrogel formed by the matrix was transferred to a 3 mL glass syringe and steam sterilized in a short cycle in a SYSTEC-DX-65 autoclave (condition "A2"). 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 water test, and its viscoelasticity level (on a scale of 1 to 4) was determined by flow meter. The higher the score, the more viscoelastic the matrix that formed the hydrogel. After adjusting the reaction parameters, it was concluded that a matrix of BDDE-crosslinked carboxyalkyl chitosan that formed a cohesive hydrogel was obtained according to the water test. The hydrogel's elasticity score was 1. The hydrogel could be injected through an intradermal needle (27G, 13 mm).

[0253] These same reaction parameters were then applied to two carboxymethylchitosans with different molecular structures and a degree of acetylation below 40%: fungal-derived CC4 (Kiomed Pharma) and crustacean-derived CC7 (Kraeber).

[0254] [Table 3]

[0255] The matrices obtained under the same conditions as matrices M1-A, M1-B, and M1-C (Table 2a) did not form cohesive hydrogels according to the water test. In contrast, matrix M1-A was able to form cohesive hydrogels, thus achieving this objective of the present invention.

[0256] Example 2b Attempts have been made to modulate the biomechanical properties of crosslinked carboxyalkylchitosan-based hydrogels, especially their viscoelasticity (measured on a scale of 0 to 4). For this purpose, matrices are prepared from CC1, CC5, and CC6 with a DA greater than 40% by varying the molecular weight of the carboxyalkylchitosan (expressed as intrinsic viscosity) and the crosslinking reaction parameters (Table 1b). The crosslinker (BDDE), medium, temperature, and reaction time are the same as for matrix M1-A, as are the neutralization and purification conditions.

[0257] [Table 4]

[0258] It appears that the biomechanical properties, especially the viscoelasticity, of crosslinked carboxyalkylchitosan-based hydrogels can be altered by varying the reaction parameters (especially the initial concentration of carboxyalkylchitosan or the crosslinker / carboxyalkylchitosan ratio, here the BDDE / carboxymethylchitosan ratio) as well as the molecular weight of the carboxyalkylchitosan.

[0259] Example 3 - Co-crosslinked carboxymethyl chitosan and hyaluronan matrix Matrices are explored by crosslinking mixtures of fungal-derived carboxymethyl chitosan with over 40% DA (Table 1b) and mixtures of hyaluronan and BDDE ("co-crosslinking") with hyaluronan (HA) of average viscosity molecular weights of 2.2 million or 2.3 million (HA type 1) and 4.3 million (HA type 2) (Table 3a).

[0260] [Table 5]

[0261] The drug (BDDE), medium, temperature, and duration of the crosslinking reaction were the same as those for matrix M1-A in Example 2, as were the neutralization and purification conditions. The hydrogels formed by the matrix were sterilized by autoclaving as described in Example 2, following cycles A1 or A2. Because other combinations and / or parameters may also result in cohesive hydrogels, several hydrogels are listed as examples. All of these hydrogels can be easily injected through a 27-gauge, 13-mm-long intradermal needle.

[0262] Example 3a We aim to demonstrate that carboxyalkylchitosan (CC) can be co-crosslinked with hyaluronan (HA) to form a cohesive hydrogel. To this end, matrices are prepared from mixtures of CC and HA at a CC / HA mass ratio of 75:25 (Table 3a). The CC references are based on the previous example. Furthermore, we aim to adjust the elasticity level from 1 to 3 (on a scale of 0 to 4) by adjusting the crosslinking reaction parameters.

[0263] [Table 6]

[0264] At the same BDDE / polymer ratio (18%) and the same final polymer concentration of 23 mg / mL, the hydrogel M2-B of CC co-crosslinked with 25% HA is observed to be more elastic than the hydrogel M1-A of CC alone in Example 2. It is also concluded that it is possible to alter the viscoelastic properties of the hydrogels of co-crosslinked carboxyalkylchitosan and HA by varying the molecular weight of HA and the proportion of the crosslinker, here BDDE.

[0265] Example 3b It is desired to obtain cohesive hydrogels from carboxyalkyl chitosan and HA co-crosslinked in various ratios.

[0266] [Table 7]

[0267] Cohesive hydrogels of co-crosslinked carboxyalkylchitosan and HA can be obtained in various ratios, and their elasticity level appears to depend on the carboxyalkylchitosan / HA ratio.

[0268] Example 4 - Matrix of cross-linked carboxymethyl chitosan combined with hyaluronan This example seeks to evaluate the possibility of forming a cohesive hydrogel from a matrix of crosslinked carboxyalkylchitosan combined with HA. The carboxyalkylchitosan is first crosslinked with BDDE according to the method of Example 1, and then a solution of HA (HA type 1) is added to it. The resulting hydrogel is autoclaved via cycle A2 (Table 4).

[0269] [Table 8]

[0270] It is easy to incorporate HA into hydrogels based on a cross-linked carboxyalkyl chitosan matrix. The resulting hydrogels are cohesive by water testing, have a viscoelasticity score of 3, yet are easily injectable through a 27-gauge intradermal needle.

[0271] Example 5 - Biomechanical properties of hydrogels In this example, the biomechanical properties of several representative CC hydrogels from Examples 2 to 4 are characterized by rheometry (Table 5). The hydrogels are cohesive, injectable through a 27G needle, and have elasticity levels of 1 to 3. They are compared with three commercially available cross-linked hyaluronan-based products intended for intradermal injection for cosmetic purposes (Table 5, see B1 to B3). By water testing, B1 is a viscous solution (tan delta > 1), while B2 and B3 are cohesive gels (tan delta < 1).

[0272] [Table 9]

[0273] The cross-linked carboxyalkyl chitosan-based hydrogels according to the present invention have been confirmed to have biomechanical properties, in particular elastic modulus (G'), comparable to commercially available cross-linked HA-based products intended for intradermal injection in aesthetic medicine.

[0274] Example 6 - Ability of ABTS°1 to Scavenge Free Radicals (in vitro) The ability of a cross-linked carboxyalkylchitosan (CC) matrix to scavenge oxidative free radicals was confirmed using a standard in vitro test known as "ABTS," in which the free radical ABTS°1 is formed and calibrated using the antioxidant "Trolox." Each test product was diluted to obtain total concentrations of polymer Cp (CC, HA, or CC and HA) of 8 mg / mL, 4 mg / mL, and 1 mg / mL. Results were checked to ensure they were within the detection zone of the test, and their ability to scavenge the free radical ABTS°1, expressed in Trolox equivalents, was also measured. The antioxidant capacity of a 20 μg / mL ascorbic acid solution (positive control) was also measured. The antioxidant capacity of each tested product was normalized according to the following formula: Normalized antioxidant capacity = TEAC (product) / TEAC (ascorbic acid 20 μg / mL).

[0275] For comparison, a non-crosslinked carboxyalkyl chitosan polymer in solution (CC2) and a commercial product based on a non-crosslinked HA solution (reference B6) are tested.Four commercial products intended for intradermal injection for cosmetic purposes are also characterized: references B1 to B3 (based solely on crosslinked HA, see Table 5 in Example 5), and B4, a hydrogel based on crosslinked HA combined with a complex of several small molecules, including antioxidant molecules.

[0276] Table 6 shows the results obtained for all products at the same total polymer concentration (Cp) of 4 mg / mL.

[0277] [Table 10]

[0278] All CC-based compositions function as antioxidants, even the non-crosslinked CC solution (S1) or crosslinked CC hydrogels (M1-E and M2-A), because they are able to significantly scavenge the free radical ABTS°1. At the same concentrations of polymer, commercial HA-only products (B6, B1, B2, and B3) do not exhibit this ability.

[0279] Surprisingly, hydrogels M1-E (CC) and M2-A (CC / HA 75:25) show the highest antioxidant capacity of all the products tested, compared to solution S1 of non-crosslinked CC. Both hydrogels have an antioxidant capacity similar to that of ascorbic acid at 20 μg / milk.

[0280] Among commercially available HA-based products, only B4 is able to significantly scavenge the radical ABTS°1, although its capacity is half that of M1-E and M2-A. Indeed, B4 is a cross-linked hyaluronan complex containing several small molecules, including antioxidants, responsible for the observed effects. However, because these substances are water-soluble small molecules, they may rapidly diffuse out of the B4 hydrogel after intradermal injection, potentially resulting in a loss of the hydrogel's antioxidant capacity.

[0281] Example 7 - Ability of hydrogels to reduce oxidative stress in skin cell cultures in vitro The ability of two hydrogels based on cross-linked CC (Reference M1-E, see Example 2) and co-cross-linked CC / HA (Reference M2-A, see Example 3) to protect human skin cells from damage caused by "ROS" (reactive oxygen) free radicals, radical species encountered in skin tissue under oxidative stress, is evaluated in a standard in vitro test and compared with a solution of non-cross-linked carboxyalkyl chitosan and a solution of a commercial product based on cross-linked hyaluronic acid intended for intradermal injection for cosmetic purposes (Reference B3, see Example 5).

[0282] Human dermal fibroblasts (NHDFs) are cultured at approximately 40% of their in vitro proliferation capacity in monolayers in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 atmosphere. The cultures are transferred to DMEM without fetal bovine serum and aliquoted into wells. The test product is diluted in DMEM to a total polymer concentration of 0.6 mg / mL and 0.2 mg / mL and added to the wells (three wells per test product). After 72 hours of contact with the test product, a 2'-7'-dichlorodihydrofluorescein diacetate probe, which fluoresces under the influence of free radicals, is added for 30 minutes. Next, the cultures in each well are rinsed with HBSS to remove the test product, and the cells are returned to HBSS. All wells are then exposed to 12.5 J / cm. 2 UVA irradiation for 20 minutes to generate ROS.

[0283] Untreated, non-irradiated cultures are used as references. Untreated and irradiated cultures are used as negative controls, and ascorbic acid-treated (504 / mL) and irradiated cultures are used as positive controls. At the end of UVA irradiation, the fluorescence intensity (excitation wavelength 485 nm, emission 520 nm), which is proportional to the ROS content, is measured, and the relative ROS content to the non-irradiated reference is then calculated (Table 7). The reduction in ROS content compared to the untreated and irradiated control is then calculated, characterizing the product's ability to reduce oxidative stress.

[0284] [Table 11]

[0285] We conclude that under the in vitro culture conditions of this study, CC-based compositions, whether crosslinked (M1-E) or non-crosslinked (S2), have excellent ability to reduce ROS content, i.e., oxidative stress that can alter cells and skin tissue. This ability is at the same level as ascorbic acid (50 μg / mL, vitamin C) and is significantly higher than commercially available crosslinked HA products. Because CC is 75%, the co-crosslinked CC / HA composition M2-A also has excellent ability to reduce oxidative stress.

[0286] Example 8 - Carboxyalkylchitosan matrix-based liquid hydrogel for ocular administration In this example, it is sought to obtain a crosslinked CC hydrogel that, thanks to its viscosity, can be easily injected in the form of well-defined droplets, while having excellent lubricating ability suitable for the indication of artificial tears for the treatment of the ocular surface.

[0287] For this purpose, cohesive crosslinked CC hydrogels are prepared by targeting dynamic viscosities (at a shear rate of 10 s) in the range of 1–60 mPa s (M8-B, Table 8a). Their instillability is verified, and their lubrication ability between two polyacrylate surfaces is measured according to the artificial tear method, expressed as a friction coefficient.

[0288] The properties of this hydrogel are compared with those of two commercially available non-crosslinked HA-based products intended for the treatment of the ocular surface (see B7 and B8, Table 8b). Their lubrication ability is measured using the same series of tests as M8-B.

[0289] [Table 12]

[0290] [Table 13]

[0291] It is concluded that a cohesive, flowable, and injectable crosslinked CC hydrogel is obtained with lubricating capabilities comparable to commercial products for the treatment of the ocular surface.

[0292] Example 9 - Local effects after intradermal implantation in rabbits (short term) Three CC matrix-based hydrogels were evaluated by intradermal administration in rabbits: M1-A (crosslinked CC, see Example 1), M2-A, and M2-B (co-crosslinked CC / HA, see Example 2). These formulations were dispensed into 1 mL glass syringes (Hypak, BD Medical) and sterilized. Their endotoxin content, measured according to Method D of the European Pharmacopoeia (EP2.6.14), was satisfactory. Two commercially available crosslinked hyaluronic acid-based products intended for intradermal injection for cosmetic purposes were also evaluated (B1 and B2, see Example 5).

[0293] A 200 μL volume of the formulation was administered intradermally to rabbits via a 27-gauge needle, following a protocol that met the IS010993-10 standard for the evaluation of primary irritation induced by intradermal implants. A total of 12 injections per product were administered to six rabbits. Local effects were observed daily at all injection sites, particularly at the erythema level.

[0294] Table 9 reports the mean level of erythema at 7 days post-injection (score on a scale of 0 to 4). It also notes whether a papule is visible at 7 days (score on a scale of 0 to 4). Macroscopic or microscopic analysis (skin histology) of the injection site of animals euthanized 7 days post-injection is used to assess the presence of product.

[0295] [Table 14]

[0296] Intradermal injection of the hydrogel was associated with the appearance of a mild local effect characterized by erythema, with a maximum score of 1 on average over 7 days on a scale of 0 to 4. This corresponds to a mild level of erythema comparable to that observed with two commercially available products. Furthermore, when animals were euthanized and histological analysis was performed on day 7, the presence of the product in the dermis was demonstrated.

[0297] Example 10 - Hydrogel for Joint Mucosupplementation In this example, the viscoelastic properties and lubricating ability of two hydrogels based on cross-linked CC (M1-E) and co-cross-linked CC / HA (M2-B) were evaluated and compared with two commercially available products based on cross-linked HA (B9 and B10, see compositions in Table 10) intended for the treatment of osteoarthritis by viscosupplementation of joints. The lubricating properties of the hydrogels were determined by their ability to reduce the coefficient of friction between two polyacrylate polymer discs attached to a flow meter, following the method for viscosupplementation.

[0298] [Table 15]

[0299] It is observed that both the cross-linked CC and co-cross-linked CC / HA hydrogels have elastic moduli G' in the same range as B9, while B10 has a higher elastic modulus. It is observed that both the CC and CC / HA hydrogels exhibit significant lubricating ability, characterized by a low coefficient of friction between the two surfaces, comparable to that of cross-linked HA viscosupplement B10 and superior to that of cross-linked HA viscosupplement B11.

[0300] In Examples 11 to 14, the polymers CC and HA used are those described in Tables 11a and 11b.

[0301] [Table 16]

[0302] [Table 17]

[0303] Example 11 - Test of co-crosslinking CC and HA with an acetylation degree of less than 40% It was sought to verify whether cohesive hydrogels could be obtained by crosslinking CC with a DA of less than 40% (CC8, Table 11a) and HA of type HA1 (Table 11b) using the same conditions as in Table 3 of Example 3. The conditions and the properties of the resulting formulation are shown in Table 11c (Reference M2-I) and compared with those of the reference hydrogel M2-A of Example 3 (according to the invention).

[0304] In CC8, the delta tangent value (tan delta measured by a rheometer) was measured. Indeed, the M2-I formulation contains a tan delta value of 1.6, which is higher than 1 and indicates the behavior of a viscous solution rather than a gel. Conversely, the tan delta value of the hydrogel M2-A is 0.4, which is less than 1 and indicates the behavior of a gel, according to the present invention.

[0305] [Table 18]

[0306] Example 12 - Hydrogel for volume restoration or filling of large skin depressions This example illustrates the use of cross-linked CC-based hydrogels for facial volume restoration or for filling large skin depressions via subcutaneous injection or deep into the dermis. These two indications require a level 4 viscoelastic hydrogel, which has an elastic modulus G' of greater than approximately 150 Pa, is cohesive according to water testing, and can be easily injected with a 27-gauge, 13-mm long needle. For these indications, two commercially available products, B11 and B12 (Table 12), which are cross-linked hyaluronan-based cohesive hydrogels with elasticity level 4, are used as references.

[0307] Hydrogel M2-J was obtained by crosslinking CC5 and HA type HA1 (CC / HA ratio 25:75) with 13% BDDE overnight at room temperature. It has an elastic modulus of 295 Pa, corresponding to the desired elasticity level 4, while remaining cohesive and easily injectable, consistent with expectations for the intended indication (Table 12).

[0308] [Table 19]

[0309] Example 13 - Volume retention after intradermal injection of co-crosslinked CC / HA hydrogel for 1 month The hydrogel is prepared by co-crosslinking CC9 (see Table 11a) and HA2 at a CC / HA mass ratio of 40:60 according to the reaction conditions of Example 12. The resulting hydrogel (reference M2-K) is packaged in a 1 mL glass syringe (Hypak, BD Medical) and sterilized in the same manner as in Example 9. The final polymer concentration is 23 mg / mL, is cohesive, injectable through a 27 G needle, and has a viscoelasticity level of 3.

[0310] Following a similar protocol as in Example 9, equal amounts of hydrogel M2-K and commercial product B12 (see Table 12, viscoelasticity level 4) are intradermally injected into rabbits via a 27-gauge needle. At regular intervals and for 26 days after injection, the local reaction is evaluated, and the volume of the papule formed by the injected product and visible on the surface of the skin is estimated by assigning a score on the following scale: The volume of the papule indicates the presence of the product and its ability to locally increase the volume of skin tissue.

[0311] Injections of both products do not induce significant local reactions during the follow-up period. Immediately after injection, papules form with a mean volumetric score of 3 ± 0 for both products (out of 20 injection sites evaluated). Over the following days, the papules resolve slightly but remain present. 26 days after injection, the papules are still present, with a mean volumetric score of 2.0 ± 0.0 for M2-L and 2.4 ± 0.5 for B12 (out of 20 sites evaluated), consistent with their relative elasticity levels. The difference in volumetric scores provided by the hydrogels M2-K and B12 is not significant at this time point.

[0312] It is therefore established that the hydrogel M2-K indeed remains present in the dermis and maintains a significant volume effect around the injection site, as expected for its indication of filling skin depressions, for at least 26 days after intradermal injection in rabbits.

[0313] Example 14 - Storage of co-crosslinked CC / HA hydrogels The possibility of preserving the co-crosslinked CC / HA hydrogel is evaluated by subjecting it to accelerated degradation conditions in an oven at 40°C and monitoring the course of its biomechanical properties. Hydrogels are considered acceptable from a biomechanical point of view as long as they are cohesive according to the water test, easily injectable, have gel-like behavior (tan delta value less than 1), and have viscoelasticity levels at initial levels at t0, consistent with the intended indication.

[0314] To obtain a viscoelasticity level of 2, a reference hydrogel M2-L is prepared according to the reaction conditions of Example 12 by co-crosslinking CC9 (see Table 11a) and HA2 in a CC / HA ratio of 70:30. The product is packaged in 1 mL glass syringes (Hypak, BD Medical) and sterilized in the same manner as in Example 9. The syringes are placed in a 40°C oven for 6 months. The properties measured after a 3-month storage period are shown in Table 13.

[0315] [Table 20]

[0316] After 3 months under accelerated aging conditions at 40 °C, product M2-L remains a hydrogel (because tan delta < 1) and maintains its cohesiveness, ease of injection, and viscoelasticity level 2. Therefore, by extrapolation, we estimate that this co-crosslinked CC / HA hydrogel should maintain acceptable properties for its intended indication for at least 12 months at room temperature.

Claims

1. 1. A matrix comprising at least one carboxyalkylchitosan having glucosamine units, N-acetylglucosamine units, and carboxyalkyl group-substituted glucosamine units, the carboxyalkylchitosan having a degree of acetylation, expressed as moles of N-acetyl groups relative to moles of total glucosamine units, ranging from greater than 40% to 80%, and the carboxyalkylchitosan being crosslinked by covalent bonds between the carboxyalkylchitosan chains.

2. 2. The matrix according to claim 1, characterized in that the carboxyalkylchitosan has a degree of substitution with carboxyalkyl groups, expressed as the number of moles of substituents relative to the number of moles of total units, of more than 20%, for example more than 50%, for example less than 200%.

3. 3. A matrix according to claim 1 or 2, characterized in that the chitosan is derived from an ascomycete fungus, in particular Aspergillus niger, and / or a basidiomycete fungus, in particular Lentinula edodes (shiitake mushroom) and / or Agaricus bisporus (white mushroom).

4. A matrix according to any one of claims 1 to 3, characterized in that the carboxyalkylchitosan is reacetylated.

5. The matrix according to any one of claims 1 to 4, characterized in that the matrix is ​​sterile.

6. The matrix according to any one of claims 1 to 5, characterized in that the matrix forms a cohesive hydrogel.

7. The matrix according to any one of claims 1 to 6, characterized in that the matrix comprises at least one hyaluronan.

8. The matrix according to any one of claims 1 to 7, characterized in that the matrix comprises at least one hyaluronan obtained by fermentation.

9. The matrix according to any one of claims 1 to 8, characterized in that the matrix comprises at least one covalently cross-linked hyaluronan.

10. The matrix according to any one of claims 1 to 9, characterized in that it comprises at least one hyaluronan co-crosslinked by covalent bonds with a carboxyalkylchitosan.

11. A matrix according to any one of claims 1 to 10, characterized in that the cross-links are formed by a cross-linking agent which forms the covalent bonds.

12. Crosslinking agents used to crosslink polysaccharides, such as 1,4 butanediol diglycidyl ether, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxypentane, 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-dibromobutanol, 12. A matrix according to claim 11, characterized in that the crosslinking agent is chosen from bromobutanethiol, 2,5-dibromopentanethiol epichlorohydrin, 2,3-dibromopropano 1,1-chloro-2,3-epithiopropane, dimethylaminopropylcarbodiimide, gallic acid, epigallocatechin gallate, curcumin, tannic acid, genipin, or diisocyanate compounds such as hexamethylene diisocyanate or toluene diisocyanate, or divinyl sulfone.

13. A matrix according to any one of claims 1 to 12, characterized in that it forms a hydrogel.

14. A matrix according to any one of claims 1 to 13, characterized in that it forms a cohesive hydrogel.

15. 15. A matrix according to any one of claims 1 to 14, characterized in that the matrix has an antioxidant capacity by scavenging free radicals, in particular a normalized antioxidant capacity of more than 0.30, preferably more than 0.50, even more preferably more than 0.80, for example more than 0.

90.

16. A composition, characterized in that it comprises at least one matrix as defined according to any one of claims 1 to 15.

17. Injectable composition, characterized in that it comprises at least one matrix as defined according to any one of claims 1 to 15.

18. Pharmaceutical composition, characterized in that it comprises at least one matrix as defined according to any one of claims 1 to 15.

19. 19. The composition of claim 17 or 18, characterized in that the composition is used in a therapeutic method of treatment, for example for repair or filling of at least one body tissue in need of repair or filling, as an injectable, implantable, instillable, or topically administrable pharmaceutical composition or an injectable, implantable, instillable, or topically administrable medical device, for example by topical instillation or administration or injection of the composition via subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular routes.

20. 20. The composition according to claim 19, for use in a method for the treatment, repair or filling of at least one body tissue in need of repair or filling, characterized in that the body tissue is selected from tissue belonging to the vocal cords, muscles, ligaments, tendons, mucous membranes, genitals, bones, joints, eyes, skin or any combination thereof, in particular skin, cartilage, synovium, skin wounds or the surface of the eye.

21. 19. A method according to claim 17 or 18 for use in a method for treating osteoarthritis or for repairing cartilage defects, for example by injection into a body fluid such as synovial fluid or after mixing with a body fluid such as blood and after implantation into cartilage.

22. A medical device, such as a medical implant, characterized in that it comprises or consists of a composition according to any one of claims 16 to 21.

23. A process for preparing a matrix according to any one of claims 1 to 15, the process comprising: contacting said carboxyalkyl chitosan with at least one crosslinking agent, said contacting preferably being carried out in an alkaline phase; cross-linking the carboxyalkyl chitosan with the cross-linking agent; obtaining a matrix comprising said crosslinked carboxyalkyl chitosan. The process includes:

24. 16. A process for preparing a matrix comprising a carboxyalkylchitosan, preferably according to any one of claims 1 to 15, co-crosslinked with another biopolymer, and preferably hyaluronan, said process comprising: contacting the mixture of carboxyalkyl and other biopolymers, and preferably hyaluronan, with at least one cross-linking agent, the contacting preferably being carried out in an alkaline phase; cross-linking the carboxyalkyl chitosan and other biopolymers, and preferably hyaluronan, with the cross-linking agent; Obtaining a matrix of co-crosslinked carboxyalkyl chitosan and other biopolymers, and preferably hyaluronan. The process includes:

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