Chitosan and its uses

Abstract

The present invention relates to a matrix-forming crosslinked carboxyalkylchitosan, a composition comprising the same, a method for producing the same, and a method for preparing the same, particularly for therapeutic applications in rheumatology, ophthalmology, cosmetic medicine, plastic surgery, internal surgery, dermatology, gynecology, or cosmetics. The invention particularly relates to a matrix comprising 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% to 80%, and the carboxyalkylchitosan being crosslinked by covalent bonds between the carboxyalkylchitosan chains.

Description

Technical Field

[0001] The present invention relates to crosslinked carboxyalkyl chitosan forming a matrix, a composition containing the same, a method for producing the same, and particularly to the fields of therapy, rheumatology, ophthalmology, aesthetic medicine, plastic surgery, laparotomy, dermatology, gynecology or cosmetics.

Background Art

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

[0003] According to the present inventors, it would be advantageous to be able to adjust the biomechanical behavior of carboxyalkyl chitosan compositions or to increase the duration or effect of treatment due to the presence of carboxyalkyl chitosan. However, it is not obvious to those skilled in the art to provide such compositions with improved biomechanical properties, especially when it is desired to prepare hydrogels. One of the technical problems of biopolymer-based compositions, and particularly of 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 and thus forms particles, fragments. This is also known as fragmented gel or hydrogel.

[0004] Such non-aggregating hydrogels are recognized as undesirable for many medical applications because they carry a risk of long-term inflammatory nodule formation or granulomatous reactions when the product is implanted in human or animal tissue (Bergerey-Galley, Aesth Surf J 24, 33, 2004). Therefore, from the standpoint of health safety for the subject or patient, it is important to avoid the formation of separate fragments and to obtain compositions in the form of aggregated hydrogels.

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

[0006] Therefore, in many applications, a cohesive hydrogel that remains in a single mass when an aqueous medium is added to it is preferred. This is also called a “homogeneous” hydrogel. Furthermore, in most applications, a hydrogel called a “smooth” hydrogel is preferred due to its visual appearance, which is lumpy or minimal.

[0007] In addition to cohesiveness, the compositions according to the present invention, particularly the hydrogels, should be suitable for use in humans or animals, especially in terms of safety, immunocompatibility, bioabsorbability, biomechanical properties, and duration of existence or activity. However, not all compositions in the art exhibit such properties to a sufficient degree and therefore would not conform to the present invention.

[0008] Various methods are known for encapsulating carboxylalkyl chitosan in hydrogel form. In particular, Rufato et al. (Intechopen 81811, 2018), Upadhyaya et al. (J Controlled Release 2014), and Fonseca-Santos et al. (Mater Sci Engineering C 77, 1349, 2017) have identified several chitosan-based hydrogels containing carboxylalkyl chitosan for medical or pharmaceutical applications. However, none of these hydrogels meet the expectations of the inventors, particularly in terms of cohesiveness, safety, immunocompatibility, biomechanical properties, bioabsorption, and / or duration of existence or activity, and therefore do not meet the inventors' expectations. With the exception of the compositions of Kiomed Pharma in the aforementioned patent applications PCT / EP2018 / 080763 and PCT / EP2018 / 080767, none of the carboxyalkyl chitosans used to prepare known hydrogels according to the latest technology exhibit good immunocompatibility, according to the inventors. Not all chitosans can be used to form hydrogels suitable for use in humans or animals.

[0009] Chitosan-based hydrogels known to date are prepared by combining chitosan or one of its derivatives with other polymers, such as alginates, isopropylacrylamide, polyurethanes, polyacrylonitriles, gelatin, polyethylene glycol (PEG), and polyvinyl alcohol (PVA). However, these polymers are either non-biodegradable or immunoreactive and therefore do not satisfy 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 not accepted by 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 group of carboxymethyl chitosan and the aldehyde of hyaluronan. However, in our experience, the carboxymethyl chitosan used does not have the molecular structure necessary to satisfy the objectives of the present invention. In particular, the described hydrogels are reabsorbed very rapidly according to the described in vitro and in vivo tests. Therefore, such hydrogels need to be improved, especially with respect to their duration of existence, for use in a wide range of indications.

[0011] Furthermore, previous products often lacked versatility to meet the needs of various indications, particularly diverse therapeutic applications. Therefore, there is a need to provide products that are sufficiently versatile in terms of properties, especially biomechanical properties, and can be easily adapted to various applications.

[0012] For example, in regenerative medicine or surgery, the goal is generally to repair altered tissue or fluid, and / or prevent further tissue changes, fill tissue, or separate tissue to avoid adhesions. Tissue changes can be caused by natural aging, external attacks (trauma, UV radiation, surgery, etc.), inflammation, autoimmune conditions, and other pathological conditions. However, most tissue changes are associated with oxidating stress, sometimes called oxidative stress, characterized by high concentrations of free radical species that can damage tissue or cells. By reducing the amount of free radical species, tissue aging can be prevented / slowed, and its harmful consequences can be mitigated. There are several ways to reduce the amount of free radical species in tissue, for example, by administering antioxidants such as vitamins C, B, E, and / or ubiquinone. Alternatively, compositions capable of removing free radicals can be used, thereby reducing their content and proliferation within the tissue.

[0013] Chitosan and some of its derivatives exhibit the ability to scavenge oxidative free radical species, as described in the review by Ngo et al. (Adv Food Nutrition Res 73, 15, 2014) and in many formulations for biomedical use. For example, carboxymethyl chitosan, which differs in structure and molecular weight, has been studied for its ability to scavenge various types of free radicals using in vitro assays, particularly as described by Ujang et al. (The Development, Characterization and Application of Water Soluble Chitosan; in Biotechnology of Biopolymers, InTech, 2011. ISBN: 978-953-307-179-4).

[0014] However, it is difficult to provide compositions for applying the beneficial effects of chitosan, particularly its ability to scavenge free radicals, in the form of a treatment that allows for the reduction of the effects of oxidative stress on tissues and better regulation of the biomechanical behavior of the product, or both to further enhance the duration or therapeutic effect due to the presence of this exogenous polymer.

[0015] Therefore, the latest technology does not obviously enable those skilled in the art to provide a satisfactory composition in order to overcome the problems set forth in the present invention. [Overview of the Initiative]

[0016] Objective of the present invention One of the objectives of the present invention is to solve the technical problem of providing a chitosan derivative or a composition containing the same that is suitable for use in humans or animals, particularly in the fields of therapeutic, surgical, and cosmetic applications.

[0017] One of the objectives of the present invention is to solve a technical problem by providing chitosan derivatives or compositions containing the same, for applying the beneficial effects of chitosan, particularly its ability to scavenge free radicals, in the form of a therapy that reduces the effects of oxidative stress on tissues, better modulates biomechanical behavior, and enhances either the duration of life or the therapeutic effect through the presence of this exogenously derived polymer.

[0018] In particular, one of the objectives of the present invention is to solve the technical problem of providing a composition that is acceptable in terms of biomechanical properties, in situ duration or activity, and good health safety (especially the absence of immune and / or foreign body reactions in the short and long term), and that is suitable for use in contact with human or animal tissue, particularly in the form of a bioabsorbable hydrogel, and that provides beneficial effects in the context of regenerative medicine or anti-aging medicine, such as therapeutic, rheumatology, orthopedics, gynecological ophthalmology, cosmetic medicine, plastic surgery, laparotomy, dermatology, or cosmetic medicine.

[0019] One of the objectives of the present invention is to solve the technical problem of providing a composition having good biomechanical properties, particularly biomechanical properties that can be adjusted according to its indications.

[0020] One of the objectives of the present invention is to solve the technical problem of providing chitosan derivative-based products that enable the preparation of a series of products having various biomechanical properties suited to each intended indication.

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

[0022] One of the objectives of the present invention is to solve the technical problems described in the present invention by providing chitosan derivatives or compositions containing them in grades acceptable to humans or animals in the intended indications. [Modes for carrying out the invention]

[0023] Detailed description of the invention To solve the technical problems described in the present invention, the inventors attempted to develop a chitosan that possesses both good antioxidant properties and good mechanical properties (referred to as biomechanical properties) for intended applications in humans or animals.

[0024] The inventors have insights from their own experience regarding the advantages of substituted chitosans, particularly 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 problems set forth in the present invention.

[0025] The inventors have noticed that carboxyalkyl chitosan hydrogels formed by ionic (i.e., non-covalent) crosslinking do not retain their biomechanical properties long enough after implantation for some intended uses; in particular, in this technique, a wide adjustment of the duration of survival or the active time is not possible. Furthermore, carboxyalkyl chitosan hydrogels formed by enzymatically catalyzed crosslinking have a risk of enzyme immunoreactivity due to their proteinaceous nature, which makes the final purification of the resulting crosslinked product difficult.

[0026] Patent application CN107325306 (lmeik Technology Development) describes the preparation of gels based on carboxymethyl chitosan 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 criteria of the present invention, especially because the resulting hydrogel is formed by particles of a crosslinked chitosan derivative dispersed in a solution of carboxymethyl chitosan and is not cohesive as 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 thus may have an adverse effect on immunocompatibility after contact with the human or animal body, which is exactly what the present invention seeks to avoid. The present invention is further advantageous in that it allows for a greater diversity of indications, especially when a cohesive (i.e., remaining in one piece without fragmentation upon contact with water, for example) and / or "smooth" appearance hydrogel is desired. According to CN107325306, the DA of the carboxymethyl chitosan used is low (the degree of deacetylation is 60-99%, preferably 80-95%, and in fact the degree of acetylation (DA) is much lower than 40%). Carboxymethyl chitosan hydrogels with a low degree of acetylation are also described by Czechowska-Biskup et al. (D01: 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 carboxyalkyl chitosan matrix or composition according to the present invention, particularly the hydrogel containing the same, can solve at least one, preferably all, of the technical problems described in the present invention.

[0028] Therefore, the present invention relates to a matrix comprising at least one carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkyl chitosan is represented as the molar number of N-acetyl groups relative to the molar number of total glucosamine units and has a degree of acetylation in the range of greater than 40% and up to 80%, and the carboxyalkyl chitosan is crosslinked by covalent bonds between carboxyalkyl chitosan chains.

[0029] In fact, crosslinked carboxyalkyl chitosan with a DA of less than 40% cannot obtain a hydrogel with desired cohesiveness in that it fragments into separate pieces during humidification, which has been found to be 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 the methods conventionally used to characterize hydrogels for intradermal use, such as the methods described by Micheels et al (J Clin Aesth Dermatol 10, 29, 2017 and J Drugs Dermatol 15, 1092, 2016):

[0031] Place a 1g mass of the hydrogel to be tested in the center of a 5cm diameter petri dish. Add 1mL of distilled water around the dish. Gently shake the petri dish until the water covers the hydrogel, then return it to a horizontal position. Observe the hydrogel immediately after contact with the matrix and water, preferably after 15-25 seconds of contact, and preferably after at least 30 seconds of contact, i.e., whether it forms a single fragment and is aggregated, or whether it spontaneously separates into distinct parts, or whether it forms particles visible to the naked eye and is non-aggregated.

[0032] Furthermore, it was advantageously found that the matrix according to the present invention can remove free radical species. The retention of this chitosan property was not obvious to those skilled in the art. While it is known that the molecular structure (DS) and molecular weight of carboxyalkyl chitosan affect its ability to remove free radicals, conflicting results have been published. Therefore, it was not clear that crosslinked carboxyalkyl chitosan exhibits free radical scavenging ability.

[0033] Furthermore, the hydrogel according to the present invention exhibits such antioxidant activity while possessing appropriate cohesiveness, biomechanical profile, longevity, and safety.

[0034] Furthermore, it was not clear that cross-linked carboxyalkyl chitosan formulated as a hydrogel is cohesive, preferably smooth, i.e., free from distinct, visible or perceptible fragments to the touch, and possesses appropriate 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. For the cross-linked matrix to be immunocompatible, i.e., non-immunoreactive and not substantially activate an immune response, it should be prepared from at least one or more non-immunoreactive polymers. Certain standardized tests are used to confirm that the polymers are non-immunoreactive, e.g., human whole blood tests (in vitro) and subcutaneous injection into mouse airbags.

[0035] The hydrogel formed by the matrix according to the present invention is not perfectly smooth and, for example, under the condition that it is cohesive according to the aforementioned water test, it may be observed to have visible or perceptible lumps when touched.

[0036] The matrix according to the present invention can be characterized by a starting carboxyalkyl chitosan that is crosslinked to form the matrix according to the present invention.

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

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

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

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

[0041] The chitosan used in the present invention is advantageously of fungal origin, preferably from ascomycete fungi, particularly Aspergillus piger, and / or basidiomycete fungi, particularly Lentinula edodes (shiitake) and / or Agaricus bisporus (white mushroom). Preferably, the chitosan is derived from Agaricus bisporus (white mushroom). The chitosan is preferably of high purity, containing little to no impurities from its fungal origin or manufacturing process, and is of microbiological grade suitable for use as an implant or pharmaceutical composition. One method for preparing chitosan is described in International Patent Publication No. 03 / 068824 (EP1483299; US7,556,946).

[0042] Generally, chitin is suspended in an aqueous medium in the presence of sodium hydroxide, and the medium is then heated at a high temperature for a time that varies depending on the desired molecular weight. Next, the chitosan is purified by solubilizing it in an acidic medium, precipitated in an alkaline medium, washed, and dried.

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

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

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

[0046] Next, such carboxyalkyl chitosan is crosslinked to prepare the matrix according to the present invention.

[0047] The DA and DS of crosslinked carboxyalkyl chitosan remain substantially unchanged during crosslinking and can therefore be expressed as functions of the DA and DS of uncrosslinked carboxyalkyl chitosan. However, if the crosslinking agent provides N-acetyl or carboxyalkyl groups, these groups, being exogenous to the starting uncrosslinked carboxyalkyl chitosan, are not considered in the DA and DS of the crosslinked carboxyalkyl chitosan. As described below, the values ​​of DA and DS are known to those skilled in the art. Therefore, DA and DS refer to both before and after crosslinking.

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

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

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

[0051] According to one alternative method, the degree of acetylation is in the range of 40-50%. According to another alternative method, 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 carboxyalkyl chitosan can be determined by solid-phase carbon-13 NMR, solid-phase carbon-13 NMR, or liquid-phase proton NMR. Carboxyalkyl chitosan advantageously has a controlled degree of acetylation. The term "controlled degree of acetylation chitosan" refers to a product in which the degree of acetylation, i.e., the proportion of N-acetyl-glucosamine units, can be adjusted in a manner controlled in particular by the acetylation reaction.

[0054] Preferably, the carboxyalkyl chitosan is reacetylated.

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

[0056] Therefore, 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. Then, the substituted chitosan is purified, recovered, and dried.

[0057] According to one alternative method, a process for preparing carboxyalkyl chitosan 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 for precise control of the degree of acetylation of the final carboxyalkyl chitosan, and in particular, a high degree of acetylation of, for example, more than 40% can be obtained. Thus, the present invention relates to carboxyalkylated chitosan after reacetylation, or reacetylated carboxyalkyl chitosan.

[0058] According to one alternative method, a process for preparing carboxyalkyl chitosan according to the present invention includes the steps of preparing chitin of fungal origin, carboxyalkylating the chitin, and optionally reacetylating the carboxyalkylated chitin to obtain carboxyalkyl chitosan according to the present invention.

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

[0060] According to one alternative method, the average molecular weight of carboxyalkyl chitosan is less than 400,000.

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

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

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

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

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

[0066] Here, preferably, the average molecular weight is the viscosity-average molecular weight (Mv) calculated from the intrinsic viscosity. This expression is customary to those skilled in the art. The intrinsic viscosity (n) is measured by a capillary viscometer using an Ubbelohde-type capillary viscometer according to the method in the European Pharmacopoeia study 2.2.9. The flow time of the solution is measured using an automated I-Visc viscometer (Lauda) via a fitted capillary tube (Lauda, ​​e.g., an Ubbelohde 510 01 capillary tube with a diameter of 0.53 mm). Next, the Mark-Houwink equation (η=K*Mva) is applied to calculate the average viscometric mass of carboxyalkyl chitosan. Here, -Mv is the viscosity-average molecular weight of carboxyalkyl chitosan. -η is the intrinsic viscosity of carboxyalkyl chitosan, - The constants K and α have values ​​of 0.0686 and 0.7638, respectively, as previously determined for (unsubstituted) chitosan by stereoexclusion chromatography with a MALLS detector.

[0067] Therefore, the intrinsic viscosity of carboxyalkyl chitosan can usually be expressed as follows:

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

[0069] Typically, in non-crosslinked carboxyalkyl chitosan, the glucosamine unit is a D-glucosamine unit (a D-glucosamine unit, an N-acetyl-D-glucosamine unit, and at least one of the substituted D-glucosamine units and N-acetyl-D-glucosamine units).

[0070] According to one alternative method, the substituted chitosan has only the D-glucosamine unit substituted.

[0071] According to another alternative method, the substituted chitosan has simultaneous substitution of D-glucosamine and N-acetyl-D-glucosamine units, where a carboxyalkyl group is covalently bonded, either according to an alternative method with only the amine group of chitosan, or according to another alternative method with both the amine and hydroxyl groups of chitosan.

[0072] Substitutions are 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%, as the number of moles of D-glucosamine units relative to the number of moles of the total units of substituted chitosan (substituted or unsubstituted D-glucosamine and N-acetyl-D-glucosamine units).

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

[0075] According to one embodiment, the degree of substitution by a carboxyalkyl group is greater than 50% and is expressed as the number of moles of substituents relative to the number of moles of total units.

[0076] According to one embodiment, the degree of substitution of D-glucosamine units, expressed as the number of moles of the 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 more preferably higher than 70%.

[0077] According to one embodiment, the degree of substitution by a carboxyalkyl group is less than 80%, and is expressed as the number of moles of substituents relative to the number of moles of total units.

[0078] Typically, substitutions are achieved through covalent bonding.

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

[0080] Advantageously, the degree of substitution (DS) and degree of acetylation (DA) of carboxyalkyl chitosan are measured by solid-phase 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 1–8 seconds and scan counts of 64–512. The signal area for carbons is determined after inverse superposition integration. The carbons considered are "CH3 acetyl" (methyl carbon of the acetyl group in N-acetylglucosamine units, whether substituted or not), "Cx" (carbon at position x of glucosamine and N-acetylglucosamine units, where x ranges from 1 to 6), and "C=O" (carbonyl carbon of carboxyalkyl substituents and C=O carbonyl carbon of the acetyl group in N-acetylglucosamine units, whether substituted or not). To determine the signal-to-signal (DS) of a given carboxyalkyl chitosan, the carbon-13 NMR spectrum of its precursor chitosan must also be recorded. From the precursor chitosan spectrum, the "CSU ratio," that is, the ratio of the signal area of ​​the "CH3 acetyl" group (methyl carbon of the acetyl group in the N-acetylglucosamine unit) to the signal area of ​​"C=O" (carbonyl carbon of the acetyl group in the N-acetyl-D-glucosamine unit), is calculated. The DA of the carboxyalkyl chitosan is calculated according to Equation 1, and the DS is calculated according to Equation 2, where I represents the signal area of ​​the carbon of interest.

[0081] Formula 1:

[0082]

number

[0083] Formula 2:

[0084]

number

[0085] DA and DS can be determined using other known methods for carboxyalkyl chitosan, for example by proton NMR in an aqueous medium using magnetic resonance spectroscopy, or, for example, by following the method described by Liu et al. (Carb Polym 137, 600, 2016), for example by hydrolyzing the carboxyalkyl chitosan 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 a method is suitable for use. The above methods should be adapted by those skilled in the art with respect to sample preparation and the integrated signals, particularly with respect to the resolution, intensity, and proton positions of the signals used to calculate the degree of substitution.

[0087] The degree of carboxyalkylation of chitosan is advantageously 20-250%, preferably 50-200%, and may also be in the range of, for example, 70-170%, and is expressed as the number of moles of carboxyalkyl relative to the number of moles of total units.

[0088] According to one alternative method, the degree of carboxyalkylation of chitosan may be advantageously in the range of 40–130%, for example, 70–130%, and is expressed as the number of moles of carboxyalkyl relative to the number of moles of total units.

[0089] The degree of substitution of chitosan is usually correlated with the mass ratio of the reactants to the chitosan at the start of the reaction. Examples of carboxyalkylating agents include acid chlorides (or their salts, such as sodium monochloroacetate) that have one or more carboxymethyl, carboxyethyl, carboxypropyl, or carboxybutyl groups.

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

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

[0092] According to this alternative method, the substituted chitosan is N-carboxyalkyl chitosan.

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

[0094] According to this alternative method, the substituted chitosan is either N-carboxyalkyl chitosan or O-carboxyalkyl chitosan.

[0095] According to a second aspect, the present invention relates to chitosan derivatives having glucosamine units, N-acetylglucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkyl chitosan is measured at pH 7.5 and is -10 mV or less, preferably -15 mV or less. In particular, such chitosan derivatives enable the chitosan derivative or a composition containing the same to limit the immune response of a subject to which it is typically administered by drop, injection, or transplantation.

[0096] Advantageously, the zeta potential measured at pH 7.5 is -18mV or less.

[0097] Advantageously, carboxyalkyl chitosan has a zeta potential of -22mV or less, preferably -24mV or less, as measured at pH 7.5.

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

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

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

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

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

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

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

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

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

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

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

[0109] According to a specific alternative method, the substituted chitosan has a degree of substitution in the range of 90-200%, preferably 90-150%, and a degree of acetylation of 40-60%, preferably 50-60%.

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

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

[0112] By substituting chitosan, it was possible to prepare a solution of carboxyalkyl chitosan that is soluble in aqueous solutions with a wide range of pH variations, whereas unsubstituted chitosan is soluble only at pH levels below 5.5-6.5. Therefore, carboxyalkyl chitosan exhibits the ability to be solubilized at different pH levels, particularly at physiological pH or at pH levels altered by pathology, such as inflammatory conditions, due to the presence of carboxyalkyl groups that alter its solubility profile.

[0113] "Water solubility" means that carboxyalkyl chitosan does not show visible turbidity 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, such as phosphate buffer, can be confirmed by an optical density of less than 0.5, preferably less than 0.2, measured by UV-Vis spectroscopy at a wavelength of 500 nm, with reference to a reference cell containing only the aqueous solvent used for the sample but no substituted chitosan. Another method is visual inspection according to European Pharmacopoeia Research Book 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] The covalent bonding of the chitosan backbone (also called the chitosan skeleton) to one or more main chitosan chains is particularly understood by the fact that they are "crosslinked by covalent bonds between carboxyalkyl chitosan chains." Advantageously, this thus provides a three-dimensional network of chitosan molecules. The present invention is not limited to specific covalent crosslinking methods, and also includes methods using 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 method, crosslinking is formed by a crosslinking agent that forms the aforementioned covalent bond.

[0118] Therefore, some chitosan chains are crosslinking agents used to crosslink polysaccharides, for example, 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-epitiobtan, 1-bromo-4,5-epithiopentane, 2,3-dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanitol It can be crosslinked by reaction with one or more crosslinking agents selected from ru-1,2,4-dibromobutanethiol and 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 crosslinking agent used to crosslink polysaccharides, particularly carboxymethyl chitosan (Yang et al. Acta Pharmacol Sin 31, 1625, 2020). Genipin colors hydrogels from dark blue to black, which may be advantageous in some indications.

[0120] Preferably, the crosslinking agent is of the polyepoxy type, for example, a bifunctional substance. Preferably, 1,4-butanediol diglycidyl ether (BDDE) or ethylene glycol diglycidyl ether (EGDE) is used as a crosslinking agent because it has already been used in the preparation of biomaterials applied to humans, particularly hyaluronane hydrogels for intradermal, intra-articular, or intraocular administration. According to one alternative, the crosslinking agent is divinyl sulfone.

[0121] Advantageously, the compositions of the present invention may also contain biopolymers other than crosslinked carboxyalkyl chitosan. According to an advantageous alternative, the biopolymers are, for example, glycosaminoglycans, particularly hyaluronans such as hyaluronic acid or sodium hyaluronate, whether oxidized or not, and whether covalently crosslinked or not.

[0122] The advantage of combining or crosslinking crosslinked carboxyalkyl chitosan with several other polymers is that it adds to or creates synergistic effects on their biological and physicochemical properties.

[0123] According to one alternative method, the matrix according to the present invention comprises crosslinked carboxyalkyl chitosan and hyaluronan, chondroitin sulfate and / or carboxymethylcellulose. To date, there are no hydrogels of crosslinked carboxyalkyl chitosan (as defined in the present invention) combined with hyaluronan. For example, one of the objectives of the present invention is to combine these two polymers so that the recognized moisturizing properties of hyaluronan can be combined with the protective properties against oxidative stress of chitosan.

[0124] According to one alternative method, the matrix contains at least one hyaluronane.

[0125] Advantageously, the matrix according to the present invention comprises crosslinked carboxymethyl chitosan alone, or crosslinked carboxymethyl chitosan combined with hyaluronane, whether or not it is crosslinked. This allows for the adaptation of desired properties.

[0126] The above matrix contains at least one carboxymethyl chitosan and hyaluronan.

[0127] According to one alternative method, hyaluronan has an average molecular weight of less than 5 million, preferably more than 1 million, and preferably more than 2 million, as determined by a capillary viscometer. The molecular weight of hyaluronan is sometimes expressed as density because it correlates via a linear relationship. Hyaluronan has a maximum molecular weight of 4.25 m 3 It may have a density of / kg, for example, a low density (for example, about 1-2m 3 ( / kg) or high density (for example, about 2-4 m 3 It can be specified as / kg.

[0128] According to one alternative method, hyaluronan can be obtained by fermentation using, for example, streptococci. According to another alternative method, it can be produced by extraction from rooster peaks.

[0129] According to one alternative method, the matrix contains at least one hyaluronane cross-linked by covalent bonds.

[0130] Therefore, cross-linked hyaluronanes contain covalent bonds between different hyaluronane chains.

[0131] Different types of hyaluronanes can be crosslinked with hyaluronanes having different molecular weights or different hyaluronane salts.

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

[0133] According to one alternative method, the process for preparing the matrix according to the present invention is: The process involves contacting carboxyalkyl chitosan with at least one crosslinking agent, preferably carried out in an alkaline phase; A step of crosslinking carboxyalkyl chitosan with a crosslinking agent; and Steps to obtain a matrix containing crosslinked carboxyalkyl chitosan. Includes.

[0134] According to one alternative method, carboxyalkyl chitosan is crosslinked in the presence of an alkaline aqueous phase, such as a sodium hydroxide (NaOH) solution.

[0135] Advantageously, the initial concentration of carboxyalkyl chitosan in the aqueous phase is in the range of 1 to 30% by weight, preferably 5 to 20% by weight (w / v), relative to the volume of the alkaline aqueous phase.

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

[0137] Preferably, the mass ratio between the crosslinking agent and the polymer is 0.5% to 20%, and in particular when BDDE is used, it is expressed as the weight of the crosslinking agent relative to the weight of the polymer.

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

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

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

[0141] On the other hand, carboxyalkyl chitosan is an exogenous molecule and is more resistant to degradation than hyaluronan after transplantation / injection / eye drop administration into the body.

[0142] Therefore, the present invention relates to a matrix comprising a three-dimensional network based on these two polymers having different molecular weights. Thus, advantageously, a range of biomechanical properties, in situ product duration, and therapeutic duration are provided while retaining the free radical scavenging power of carboxyalkyl chitosan.

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

[0144] According to one alternative method, the process for preparing a matrix containing another biomolecule, and preferably carboxyalkyl chitosan co-crosslinked with hyaluronane, preferably as described in the present invention, is: A mixture of carboxyalkyl and other biopolymers, and preferably hyaluronane, is brought into contact with at least one crosslinking agent; this contact is preferably carried out in an alkaline phase. A step of crosslinking carboxyalkyl chitosan and other biopolymers, and preferably hyaluronan, with a crosslinking agent; Steps to obtain a matrix of co-crosslinked carboxyalkyl chitosan and other biopolymers, and preferably hyaluronan. Includes.

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

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

[0147] Therefore, the present invention relates to a hydrogel, and advantageously, to forming an aggregated hydrogel.

[0148] Therefore, the present invention relates to a crosslinked carboxyalkyl chitosan hydrogel in which the carboxyalkyl chitosan has a high degree of acetylation (DA) (greater than 40%) and preferably 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 in accordance with the present invention.

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

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

[0152] According to one alternative method, the concentration (m / m) of the polymer (e.g., carboxyalkyl chitosan, with or without other biomacromolecules such as hyaluronan) relative to the total mass of the composition, particularly 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%, for example, 10-90%, and further, for example, 30-70%. The mass ratio (m / m) [hyaluronan / carboxyalkyl chitosan] is, for example, 5-95%, for example, 10-90%, and further, for example, 30-70%. According to one alternative method, 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 or an aqueous solution, and its pH and osmotic pressure are adjusted, for example, using an acid / base buffer system to which salts and / or optionally polyols (sorbitol, mannitol, glycerol) are added.

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

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

[0157] Advantageously, the osmotic pressure of the matrix composition is 250-400 mosm / kg, preferably 270-330 mosm / kg.

[0158] According to one alternative method, the matrix composition has an osmotic pressure suitable for the joint.

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

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

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

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

[0163] Advantageously, the compositions according to the present invention are contained in injection, implantation, or eye drop devices such as syringes or vials.

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

[0165] According to one alternative method, the compositions according to the present invention, in particular the hydrogels according to the present invention, are sterilized by filtration and / or steam sterilization before filling injection, implantation, or eye drop devices such as syringes or vials.

[0166] Those skilled in the art know techniques for sterilizing hydrogels to obtain desired sterile hydrogels. These techniques involve several types of equipment for heat or steam sterilization and several types of number of cycles for removing microbial loads.

[0167] More specifically, the present invention relates to an injectable composition comprising a matrix, preferably in the form of a hydrogel, according to the present invention.

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

[0169] According to one alternative method, the compositions according to the present invention may be used as injectable, implantable, or ophthalmic pharmaceutical compositions, or as injectable, implantable, or ophthalmic medical devices.

[0170] The present invention further covers compositions according to the present invention in a dried form, particularly in a freeze-dried form. Freeze-dried products can be (re)dispersed and preferably solubilized before use.

[0171] More specifically, the present invention relates to compositions according to the present invention for use in therapeutic treatment, including, for example, injection via subcutaneous, intradermal, intraocular, or intraarticular, intramucosal, or intramuscular routes, the compositions being for, for example, the repair, regeneration, or filling of at least one body tissue / fluid that requires repair or filling.

[0172] It is advantageous to use chitosan that has sufficient purity for the intended application.

[0173] It is advantageous to use hyaluronan of sufficient purity for the intended application.

[0174] The biomechanical properties required for the composition according to the present invention may vary in range depending on the indication, for example, the tissue in which the hydrogel is incorporated, the mechanism of action, or the effect intended to ensure the patient's benefit, and the duration of the effect.

[0175] Advantageously, the properties of the compositions according to the present invention, and in particular the properties of the hydrogels according to the present invention, are suitable for the indications. To adapt these properties, the final concentration of the polymer (carboxyalkyl chitosan and / or other biopolymers such as hyaluronan), and / or the proportion of crosslinking via the mass ratio of the crosslinking agent / polymer, and / or the properties and / or the amount of ions, and / or the initial molecular weight of the polymer are changed, for example.

[0176] Specifically, the present invention relates to highly elastic hydrogels where a sustained increase in volume must be ensured, particularly at the skin, subcutaneous, or periosteal level (for projection or remodeling), or to viscoelastic gels that enable both shock absorption and lubrication effects, particularly at the joint level. The present invention also relates to lubricating hydrogels where friction needs to be reduced, particularly between two biological surfaces, for example, the two cartilage surfaces of a joint, or between the surface of the eye and the eyelid. The compositions of the present invention may have a variable elasticity level adjusted according to the indication and can be characterized by measuring the elastic modulus by a flow meter.

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

[0178] The present invention relates to an injectable composition characterized by comprising at least one matrix defined according to the present invention.

[0179] The present invention relates to a pharmaceutical composition characterized by comprising at least one matrix defined in accordance with the present invention.

[0180] According to one alternative method, the compositions according to the present invention may be used, for example, as injectable, implantable, or ophthalmic, or topically administered pharmaceutical compositions or injectable, or implantable, or ophthalmic, or topically administered medical devices for use in therapeutic methods, for example, comprising topically instilling, administering, or injecting the compositions via subcutaneous, intradermal, mucosal, ocular, intraocular, or intra-articular, intraosseous routes for the repair or filling of at least one body tissue requiring repair or filling.

[0181] According to one alternative method, the compositions according to the present invention are used in methods for treating, repairing or filling at least one bodily fluid or tissue that requires repair or filling, for example, the bodily tissue being selected from the vocal cords, muscles, ligaments, tendons, mucous membranes, genitals, bones, joints, eyes, skin, or any combination thereof, in particular from tissues belonging to the surface of the skin, cartilage, synovial membrane, skin wounds, or eyes.

[0182] The present invention relates to compositions for use in methods for treating osteoarthritis or repairing cartilage defects, for example, by injection into a bodily fluid such as synovial fluid, or after mixing with a bodily fluid such as blood, and after implantation into cartilage. Bodily fluids refer to bodily fluids, whether or not they have received treatment to alter their composition.

[0183] The present invention relates to a medical device, such as a medical implant, characterized by comprising or consisting of a composition defined in accordance with the present invention.

[0184] The present invention specifically relates to compositions according to the present invention for use in therapeutic, surgical, or cosmetic treatments, particularly in rheumatology, ophthalmology, gynecology, cosmetic medicine, plastic surgery, abdominal surgery, orthopedic surgery, and dermatology, for the prevention of postoperative tissue adhesion.

[0185] The present invention also relates to compositions according to the present 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 present invention by instilling them into the surface of the eye, particularly for the purpose of lubricating or regenerating the surface of the eye, in order to prevent or combat corneal damage or dry eye syndrome.

[0187] Therefore, the present invention also relates to an eye drop composition comprising carboxyalkyl chitosan as defined in accordance with the present invention.

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

[0189] More specifically, the present invention relates to compositions according to the present invention for the treatment of arthropathy, arthritis, or repair of cartilage defects, for example, by injection into the synovial space or implantation into the cartilage defect.

[0190] More specifically, the present invention relates to a medical device, such as a medical implant, characterized by comprising or consisting of a composition according to the present invention.

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

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

[0193] The present invention also relates to compositions according to the present invention for use in therapeutic treatment methods.

[0194] The present invention also relates to compositions for use in methods for treating arthritis or repairing cartilage defects, for example, by injection into the synovial sac, or after mixing with bodily fluids such as blood, and after implantation into cartilage / bone.

[0195] The present invention also relates to compositions according to the present invention for use in a method of cosmetic treatment or care by dermal filling ("dermal filling") or lip filling. This particularly includes, for example, injecting the compositions according to the present invention subcutaneously, intradermally, intramucosally or intramuscularly.

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

[0197] The present invention also relates to compositions according to the present invention for use in therapeutics in which the composition is a mucus replenishment agent. Here, for example, the compositions of the present invention are injected intra-articularly to limit friction on the cartilage surface of the joint.

[0198] The present invention also relates to compositions according to the present invention for use as cell vectors, one or more cell types and / or one or more activators. These may be activators from a pharmaceutical or biological standpoint. The compositions of the present invention may actually be compatible with the presence of cells, preferably living cells. Examples of living cells to be targeted include chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligamentous fibroblasts (ligaments), dermal fibroblasts (skin), tendinocytes (tendons), myofibroblasts (muscle), mesenchymal stem cells, erythrocytes (blood), and keratinocytes (skin). The compositions of the present invention can also be targeted as therapeutic vectors for the targeting and / or controlled-release delivery of at least one therapeutic agent.

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

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

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

[0202] The present invention also relates to compositions according to the present invention for use in methods for the treatment or cosmetic care of one or more tissues or organs that develop due to excessive temperature, such as in the case of burns.

[0203] The present invention also relates to compositions according to the present invention for use in methods for treating cartilage repair (for example, by transplanting them into cartilage defects for the purpose of promoting their regeneration).

[0204] The present invention also relates to compositions according to the present invention for use in a method for the prophylactic treatment of postoperative tissue adhesions: The product is applied at the end of surgery to tissues such as gynecological, abdominal, visceral, orthopedic, etc.

[0205] The present invention relates to a physiological composition administered topically by injection or implantation for contact with one or more biological tissues exposed to oxidative stress, for example: - Intra-articular injections for the treatment of osteoarthritis (through synovial fluid replenishment, cartilage lubrication, shock absorption at the joint level, and synovial regeneration); intra-articular grafts to promote the repair of cartilage defects; - Intraosseous grafting (bone induction / bone conduction) to promote bone repair; - Subcutaneous and / or intradermal injections to fill or regenerate skin or hair follicles to increase volume in cases of lipodystrophy; - Eye drops to relieve symptoms on the ocular surface or prevent degeneration, such as for the treatment of dry eye and corneal lesions, and administration of active ingredients; - Intraocular injection, for example, to optimize the effectiveness of glaucoma surgery or vitreous replacement, as an adjuvant in cataract surgery, for the regeneration of anterior or posterior segment tissue, and for intraocular administration of active ingredients; - Administration (film) to internal tissues and organs to prevent postoperative adhesions; - Administration to wounds, cracks, lacerations, and cavities of tissues and organs such as skin, bone, cartilage, cornea, tendons, and meniscus, for the purpose of promoting repair or regeneration; - Injection into the vulvar mucosa for the treatment of vulvar pain.

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

[0207] The compositions according to the present invention can mimic healthy synovial fluid or improve healthy or defective synovial fluid, for example, by improving its lubrication ability to reduce joint friction and / or by pursuing its shock absorption properties (identifiable by its modulus of elasticity G'), while also being easily injectable, for example, by filling a syringe or injecting it into the body of a human or animal. As an indicator, the modulus of elasticity G' of healthy synovial fluid is between 40 and 100 Pa, and its loss modulus of elasticity G'' is between 1 and 10 Pa.

[0208] Advantageously, in the case of intra-articular injection, the compositions according to the present invention can be readily injected at room temperature through a fine needle, for example, a 21-gauge diameter needle. "Easy" injection means, preferably, that the force applied to such a syringe is less than 50 Newtons (at a rate of 10 mm / min), and preferably less than 20 Newtons, to flow the composition according to the present invention through the 21-gauge needle.

[0209] Advantageously, in the case of intradermal injection, the compositions according to the present invention can be readily injected at room temperature through a fine needle, for example, a needle with a diameter of 25 gauge or less. "Easy" injection means, preferably, 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), and preferably less than 20 Newtons, to flow the composition according to the present invention through a 27 gauge needle.

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

[0211] Generally, the osmotic pressure and pH range of the composition are adapted and generally close to the osmotic pressure and pH of the tissue in contact with the composition according to the present invention.

[0212] Advantageously, the compositions according to the present invention are sterile. More advantageously, the compositions according to the present invention are sterilized by temperature rise, preferably under autoclaving conditions.

[0213] According to one embodiment, and according to tests of the embodiment of the present invention, the matrix has a low coefficient of friction (COF), for example less than 20, and a lubrication capacity, for example less than 10.

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

[0215] "Translucent" means that the composition can distinguish the object when placed between the observer's eye and the object. "Transparent" means that the alphanumeric characters can distinguish when the composition is placed between the observer's eye and the observed characters. Generally, this evaluation is performed with a composition thickness of about 1 cm. The method of visual inspection described in European Pharmacopoeia Research Book 2.9.20 can also be employed. The optical density of the composition can also be measured, for example, by UV-Vis spectroscopy at 500 nm, and it should be confirmed that the optical density is less than 0.5, preferably less than 0.2, relative to the reference solvent.

[0216] According to one alternative method, the composition of the present invention is not milky white, or is slightly milky white.

[0217] "Opal" means that the solution causes diffraction of visible light to the naked eye, for example, by visual inspection using methods such as those described in Section 2.9.20 of the European Pharmacopoeia study, and by comparison with reference solutions of different opacity levels in the European Pharmacopoeia. According to one alternative method, the composition of the present invention is colorless, i.e., a person observing it with the naked eye will not perceive any particular color in the composition. According to another alternative method, the opacity is below the maximum acceptable value for the intended use.

[0218] The present invention relates more particularly to sterile articles or packaging comprising one or more eye drop or injection devices pre-filled with the compositions according to the present invention, especially in the form of hydrogels. These are typically devices for administering the product in the form of drops or pre-filled syringes.

[0219] The compositions of the present invention can, advantageously, be stored for several months, preferably in articles or packaging suitable for their indications.

[0220] Advantageously, the compositions of the present invention can be sterilized. Therefore, the present invention relates to sterilized crosslinked carboxyalkyl chitosan. Thus, crosslinked carboxyalkyl chitosan is sterile, especially in applications where it is required.

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

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

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

[0224] The present invention also covers methods for therapeutic treatment, which include injecting compositions according to the present invention.

[0225] The present invention also covers the use of compositions according to the present invention for the preparation of pharmaceutical compositions, particularly for therapeutic treatment, as more specifically defined by the present invention.

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

[0227] Tissue is a group of similar cells of the same origin, assembled into functional units; that is, they all contribute to the same function. Tissues mentioned may include skin tissue (e.g., epithelial tissue), connective tissue, muscle tissue, and nerve tissue.

[0228] "Composition according to the present invention" or equivalent terms mean a composition as defined in the present invention, including any alternative method, a particular or specific embodiment, alone or in any combination thereof, and including those with preferred properties.

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

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

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

[0232] On the other hand, in the examples, unless otherwise specified, all percentages are expressed in mass, temperatures are expressed in Celsius unless otherwise specified, and pressures are atmospheric pressure unless otherwise specified. [Examples]

[0233] Method for measuring zeta potential The formulation to be analyzed is diluted in 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 0.1N sodium hydroxide or 0.1N hydrochloric acid. The zeta potential of each nuclear molecule is measured using a "Nano-Z" instrument (Zeta-Sizer range, Malvern Instruments).

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

[0235] Biomechanical profiles obtained using flow meters The biomechanical profile of the sample is characterized by a temperature of 37°C, a frequency of 3.98 rad / s, and a deformation amplitude in the range of 0.1–10%, using a DHR-2 Hydrid Rheometer (TA Instrument) with a 20 mm planar shape positioned 700 μm apart from the Peltier element. Each measurement is performed three times, and the average values ​​of the elastic modulus (G'), viscosity (G''), and tanδ (G'' / G') from 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, with the parameters selected according to the product and application.

[0237] - Methods for intra-articular replacement drugs Two discs based on polyacrylate biomaterial used in the manufacture of a 16.15 mm diameter hydrophobic intraocular lens (described in patent EP1830898) were pre-hydrated by immersion in 60°C water for approximately 2 hours, and then fixed in the upper and lower geometry configurations of a DHR-2 rheometer (TA Instruments). Approximately 100 μL of the sample to be tested was placed on the lower disc, and then the upper configuration was lowered to an imposed normal force of 5 Newtons until both discs were in contact. The coefficient of friction was measured at 25°C for 150 seconds under a constant normal force (5N), an oscillation frequency of 1.256 rad / s, and a deformation angle of approximately 0.05 radians, following a protocol adapted from the protocol described by Waller et al. (in: J 47 Rheumatol 39, 7, 1473, 2012). The option "Adhesion to the zero starting point of the oscillation 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, the measurement is repeated five times. The value of the coefficient of friction is reported by extrapolating the initial intercept of each COF-time curve (COF0).

[0238] - Methods for artificial tears Two discs based on polyacrylate biomaterial used in the manufacture of a 16.15 mm diameter hydrophobic intraocular lens (described in patent EP1830898) were pre-hydrated by immersion in 60°C water for approximately 2 hours, and then fixed in the upper and lower geometry configurations of a DHR-2 rheometer (TA Instruments). Approximately 100 μL of the sample to be tested was placed on the lower disc, and then the upper configuration was lowered to an imposed normal force of 5 Newtons until both discs were in contact. The coefficient of friction was measured at 25°C for 150 seconds under a constant normal force (5N), an oscillation frequency of 1.256 rad / s, and a deformation angle of approximately 0.05 radians, following a protocol adapted from the protocol described by Waller et al. (in: J 47 Rheumatol 39, 7, 1473, 2012). The option "Adhesion to the zero starting point of the oscillation 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, the measurement is repeated five times. The value of the coefficient of friction is reported by extrapolating the initial intercept of each COF-time curve (COF0).

[0239] Pressing force by the needle The measurement is performed using a MultiTest 2.5-i compression tester (Mecmesin) equipped with a 100N compression cell. A suitable needle is fitted to the syringe containing the sample. The syringe is placed in the tester, and the plunger of the syringe is pushed at a constant speed (e.g., 10 or 80 mm / min) to measure the force required for extrusion. The maximum force that the device can handle is approximately 70 Newtons.

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

[0241] Each series of tests is conducted 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 MilliQ water) to a concentration of 7 mM ABTS. Protect the mixture from light and stir at room temperature for 24 hours. This is the time required to generate a defined amount of ABTS·1 radical cations. A diluted standard solution of ABTS·1 is finally obtained by taking 600 μL of the latter mixture and diluting this amount with MilliQ water to a concentration of 415 μM.

[0243] 2) The calibration curve for free radical scavenging capacity is established by comparison with the reference antioxidant molecule (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Torolox. Solutions of Torolox at concentrations of 30, 60, 90, 120, 150, 180, and 210 μM are obtained by diluting a stock solution of Torolox (15 mg in 5 mL of 100% methanol) with Milli-Q water. Absorbance measurements are performed 1 hour after mixing 50 μL of ABTS·1 working solution with 50 μL of each Torolox solution at a wavelength of 734 nm. The relationship between absorbance and Torolox concentration in the linearity zone is read. 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 Milli-Q water (defined according to the product to be tested such that the absorbance of the mixture with ABTS·1 solution is above the detection limit). Mix 50 μL of reference dilution solution with 50 μL of the product to be tested solution. After incubation at room temperature for 1 hour, measure the absorbance at a wavelength of 734 nm. If the absorbance value is within the detection range of the instrument, it is retained, and the Tororox equivalent is calculated via a calibration curve labeled as "Tororox Equivalent Antioxidant Capacity" TEAC.

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

[0246] Example 1 Carboxymethyl chitosan is produced via carboxymethylation and acetylation reactions according to the following method, using the reaction parameters given as examples in Table 1a. Furthermore, the molecular structure of carboxymethyl chitosan can be modified using other reaction parameters. 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 at 35°C for 23 hours. The polymer is recovered by precipitation with ethanol, and then purified by a cycle of solubilization in water and precipitation with ethanol. Carboxymethyl chitosan (see CC4, Table 1b) is collected after drying in a ventilated oven.

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

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

[0249] [Table 1]

[0250] [Table 2]

[0251] Example 2 - Matrix of carboxymethyl chitosan Synthetic studies were conducted to provide a carboxymethyl chitosan matrix by covalent crosslinking using the crosslinking agent 1,4-butanediol diglycidyl ether (CAS 245-79-8, BDDE). Several carboxymethyl chitosans derived from Agaricus bisporus (white mushroom), prepared by Kiomed Pharma according to the method of Example 1, were used. Their properties are shown in Table 1. BDDE (96%, specific gravity 1.049) is supplied by Alfa Aesar (ThermoFischer, Kandel, Germany).

[0252] Example 2a After adjusting the reaction parameters, a crosslinking matrix is ​​prepared from carboxymethyl chitosan CC3 (Table 2a, see M1-A). The degree of acetylation of CC3 is 55%, and the degree of carboxymethylation is 87%, as measured by carbon-13 NMR (Equation 2). After dialysis, the hydrogel formed by the matrix is ​​transferred to a 3 mL glass syringe and steam-sterilized in a SYSTEC-DX-65 autoclave (condition "A2") for a short cycle. The final polymer concentration of the resulting sterile hydrogel (M1-A) is determined by mass balance. The aggregation properties of the hydrogel are analyzed by water testing, and its viscoelasticity level (on a scale of 1-4) is determined by a flow meter. A higher score indicates higher viscoelasticity of the matrix forming the hydrogel. After adjusting the reaction parameters, water testing concludes that it is possible to obtain a BDDE crosslinked carboxyalkyl chitosan matrix that forms an aggregation hydrogel. The elasticity score of the hydrogel is 1. The hydrogel can be injected through an intradermal needle (27G 13 mm).

[0253] Next, these same reaction parameters were applied to two carboxymethyl chitosans with different molecular structures and less than 40% acetylation (CC4 from fungi (Kiomed Pharma) and CC7 from crustaceans (Kraeber)).

[0254] [Table 3]

[0255] Matrixes obtained under the same conditions as matrix M1-A, M1-B, and M1-C (Table 2a) did not form a cohesive hydrogel according to water tests. In contrast, matrix M1-A was able to form a cohesive hydrogel and therefore achieves this objective of the present invention.

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

[0257] [Table 4]

[0258] It appears that the biomechanical properties, particularly viscoelasticity, of crosslinked carboxyalkyl chitosan-based hydrogels can be altered by changing reaction parameters (especially the initial concentration of carboxyalkyl chitosan or the crosslinking agent / carboxyalkyl chitosan ratio, in this case the BDDE / carboxymethyl chitosan ratio) and the molecular weight of the carboxyalkyl chitosan.

[0259] Example 3 - Matrix of co-crosslinked carboxymethyl chitosan and hyaluronan The matrix is ​​explored by crosslinking a mixture of carboxymethyl chitosan with over 40% DA derived from fungi (Table 1b) and a mixture of hyaluronan and BDDE ("co-crosslinking"). Hyaluronan (HA) with average viscosity molecular weights of 2.2 million or 2.3 million (HA1 type) and 4.3 million (HA2 type) is used (Table 3a).

[0260] [Table 5]

[0261] The drug (BDDE), medium, temperature, and duration of the crosslinking reaction are the same as in Example 2 for matrix M1-A, as well as the neutralization and purification conditions. The hydrogel formed by the matrix is ​​sterilized by autoclaving according to cycle A1 or A2 as described in Example 2. Several hydrogels are described as examples, as other combinations and / or parameters may also result in agglomerating hydrogels. All of these hydrogels can be readily injected through an intradermal needle of size 27 gauge, length 13 mm.

[0262] Example 3a It is required to demonstrate that carboxyalkyl chitosan (CC) can be co-crosslinked with hyaluronan (HA) to form an aggregated hydrogel. For this purpose, a matrix is ​​prepared from a mixture of CC and HA with a CC / HA mass ratio of 75:25 (Table 3a). The CC reference is the same as in the previous example. Furthermore, it is required to adjust the elasticity level from 1 to 3 (on a scale of 0 to 4) by adjusting the parameters of the crosslinking reaction.

[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 was observed to have higher elasticity than the hydrogel M1-A of CC alone in Example 2. Furthermore, it can be concluded that the viscoelastic properties of the co-crosslinked carboxyalkyl chitosan and HA hydrogels can be altered by changing the molecular weight of HA and, in this case, the proportion of the crosslinking agent, which is BDDE.

[0265] Example 3b There is a need to obtain agglomerating hydrogels from carboxyalkyl chitosan and HA co-crosslinked in various ratios.

[0266]

Table 7

[0267] Co - crosslinked carboxyalkyl chitosan and HA cohesive hydrogels can be obtained in various ratios, and their elastic levels appear to depend on the carboxyalkyl chitosan / HA ratio.

[0268] Example 4 - Matrix of Crosslinked Carboxymethyl Chitosan Combined with Hyaluronic Acid In this example, it is required to evaluate the possibility of forming a cohesive hydrogel from the matrix of crosslinked carboxyalkyl chitosan combined with HA. The carboxyalkyl chitosan is first crosslinked with BDDE according to the method of Example 1, and then a solution of HA (HA1 type) is added thereto. The resulting hydrogel is autoclaved through cycle A2 (Table 4).

[0269]

Table 8

[0270] It is easy to incorporate HA into the hydrogel based on the crosslinked carboxyalkyl chitosan matrix. The resulting hydrogel is cohesive according to the water test, and the viscoelastic score is 3, but it can be easily injected from a 27 - gauge intradermal needle.

[0271] Example 5 - Biomechanical Properties of Hydrogels In this example, the biomechanical properties of some representative CC hydrogels from Example 2 to 4 are characterized by a flowmeter (Table 5). The hydrogels are cohesive, injectable through a 27G needle, and the elastic levels are 1 - 3. These are compared with three commercially available crosslinked hyaluronic acid - based products for intradermal injection for cosmetic purposes (Table 5, Refs. B1 - B3). According to the water test, B1 is a viscous solution (tan delta > 1), and B2 and B3 are cohesive gels (tan delta < 1).

[0272] [Table 9]

[0273] The cross-linked carboxyalkyl chitosan-based hydrogel according to the present invention has been confirmed to possess biomechanical properties, particularly an elastic modulus (G'), comparable to commercially available cross-linked HA-based products intended for intradermal injection in cosmetic medicine.

[0274] Example 6 - Ability to remove ABTS°1 free radicals (in vitro) It is required to confirm that the cross-linked carboxyalkyl chitosan (CC) matrix can scavenge oxidative free radicals using a standard in vitro test known as "ABTS," where the free radical ABTS°1 is formed and calibrated with the antioxidant "Tororox." Each test product is diluted to obtain total concentrations of polymer Cp(CC, HA, or CC and HA) at 8 mg / mL, 4 mg / mL, and 1 mg / milk. The results are checked to ensure they are within the detection zone of the test, and the ability to scavenge free radicals ABTS°1 is expressed in Torox equivalents. The antioxidant capacity of a 20 μg / mL ascorbic acid solution (positive control) is also measured. The antioxidant capacity of each product tested is normalized according to the following formula: Normalized antioxidant capacity = TEAC(product) / TEAC(ascorbic acid 20 μg / mL).

[0275] For comparison, non-crosslinked carboxyalkyl chitosan polymer (CC2) in solution and a non-crosslinked HA solution-based commercial product (reference B6) are tested. Four commercial products intended for intradermal injection for cosmetic purposes are also characterized: references B1-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 can significantly remove free radicals ABTS°1, and therefore function as antioxidants even in non-crosslinked CC solutions (S1) or crosslinked CC hydrogels (M1-E and M2-A). At the same polymer concentrations, commercially available 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) exhibited the highest antioxidant capacity among all products tested, compared to solution S1 of non-crosslinked CC. Both hydrogels had similar antioxidant capacity to ascorbic acid at 20 μg / milk.

[0280] Among commercially available HA-based products, only B4 can significantly remove the radical ABTS°1, but its capacity is half that of M1-E and M2-A. In fact, B4 is a cross-linked hyaluronan associated with a complex of several small molecules, including antioxidants, which is responsible for the observed effect. However, because these substances are water-soluble small molecules, they may rapidly diffuse from the B4 hydrogel after intradermal injection, potentially leading to a loss of the hydrogel's antioxidant capacity.

[0281] Example 7 - Ability of hydrogel to reduce oxidative stress in in vitro skin cell culture The ability of two hydrogels based on cross-linked CC (see References M1-E, Example 2) and co-cross-linked CC / HA (see References M2-A, Example 3) to protect human skin cells from damage caused by ROS (reactive oxygen species) free radicals, which are radical species encountered in skin tissue under oxidative stress, is evaluated in standard in vitro tests. This is compared to solutions of non-cross-linked carboxyalkyl chitosan solutions and commercially available products based on cross-linked hyaluronic acid intended for intradermal injection for cosmetic purposes (see References B3, Example 5).

[0282] Human dermal fibroblasts (NHDFs) are cultured at approximately 40% of their in vitro proliferation capacity in a monolayer of DMEM (Dulbeccoo's modified Eagle 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 fractionated into wells. The product to be tested 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 product). After contact with the product for 72 hours, a 2'-7'-dichlorodihydrofluorescein diacetate probe that 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 product to be tested, the cells are returned to HBSS, and 12.5 J / cm³ is added to all wells. 2 ROS is generated by irradiating with UVA for 20 minutes.

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

[0284] [Table 11]

[0285] Under the in vitro culture conditions of this test, it is concluded that the CC-based composition, whether cross-linked (M1-E) or non-cross-linked (S2), has an excellent ability to reduce the ROS content, that is, to reduce oxidative stress that may change cells and skin tissues. This ability is at the same level as ascorbic acid (504 / mL, vitamin C) and is much higher than that of commercially available cross-linked HA products. Since CC is 75%, the co-cross-linked CC / HA composition M2-A also has an excellent ability to reduce oxidative stress. <00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​It is concluded that a cohesive, fluid, and drip-able cross-linked CC hydrogel with lubrication capabilities comparable to commercially available products for the treatment of the ocular surface can be obtained.

[0292] Example 9 - Local effect (short term) after intradermal transplantation in rabbits Three CC matrix-based hydrogels are evaluated by intradermal administration to rabbits: M1-A (cross-linked CC, see Example 1), M2-A, and M2-B (co-cross-linked CC / HA, see Example 2). These formulations were bottled in 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 good. Two commercially available cross-linked hyaluronic acid-based products intended for intradermal injection for cosmetic purposes are also evaluated (B1 and B2, see Example 5).

[0293] A 200 μL dose of the formulation was administered to rabbits by intradermal injection via a 27-gauge needle, following a protocol that met the ISO10993-10 criteria for evaluating primary irritation induced by intradermal implants. A total of 12 injections per product were given to 6 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 0-4). Attention was also paid to whether papules were visible on day 7 (score on a scale of 0-4). The presence of the product was assessed using macroscopic or microscopic analysis (dermatohistochemistry) of the injection site of animals euthanized 7 days post-injection.

[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 0–4. This corresponds to a mild level of erythema comparable to that observed with two commercially available products. Furthermore, the presence of the product in the dermis was demonstrated when the animals were euthanized and histological analysis was performed on day 7.

[0297] Example 10 - Hydrogel for joint mucus replenishment In this embodiment, the viscoelastic properties and lubrication capabilities of two hydrogels based on crosslinked CC (M1-E) and co-crosslinked CC / HA (M2-B) were evaluated and compared with two commercially available crosslinked HA-based products intended for the treatment of osteoarthritis with intra-articular replacement therapy (see compositions B9 and B10, Table 10). The lubrication properties of the hydrogels are determined by their ability to reduce the coefficient of friction between two polyacrylate polymer discs attached to a flow meter, according to the intra-articular replacement therapy method.

[0298] [Table 15]

[0299] Both the crosslinked CC and the co-crosslinked CC / HA hydrogels were observed to have the same elastic modulus G' as B9, while B10 had a higher elastic modulus. Both the CC and CC / HA hydrogels were observed to exhibit significant lubrication capabilities characterized by a low coefficient of friction between the two surfaces, comparable to the coefficient of friction of the crosslinked HA intra-articular replacement B10 and superior to the coefficient of friction of the crosslinked HA intra-articular replacement B11.

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

[0301] [Table 16]

[0302] [Table 17]

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

[0304] In CC8, the delta-tangent value (tan delta measured by a flow meter) is used. In fact, 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 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 cyanoacrylate (CC) based hydrogels for restoring facial volume or for filling large skin depressions via subcutaneous injection or into the deep dermis. For these two indications, a level 4 viscoelastic hydrogel is required. This is characterized by an elastic modulus G' exceeding approximately 150 Pa, cohesiveness according to water tests, and ease of injection 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 reference.

[0307] Hydrogel M2-J is 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 maintaining cohesiveness and being easily injectable, consistent with expectations for the intended indication (Table 12).

[0308] [Table 19]

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

[0310] Following a protocol similar to that of Example 9, the same amounts of Hydrogel M2-K and commercial product B12 (see Table 12, viscoelasticity level 4) were intradermally injected into rabbits via a 27-gauge needle. Local reactions were evaluated at regular intervals for 26 days post-injection, and the volume of papules formed by the injected product and visible on the skin surface was estimated by assigning a score on the following scale. The volume of the papules indicates the presence of the product, as well as its ability to locally increase the volume of skin tissue.

[0311] Injections of both products did not induce significant local reactions during the follow-up period. Immediately after injection, papules formed with an average volume score of 3±0 for both products (out of 20 injection sites evaluated). Over the following few days, the papules slightly subsided but remained present. At 26 days post-injection, the papules were still present, with average score volumes of 2.0±0.0 for M2-L and 2.4±0.5 for B12 (out of 20 evaluated sites), consistent with relative elasticity levels. The difference in volume scores provided by hydrogels M2-K and B12 was not significant at this point.

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

[0313] Example 14 - Storage of co-crosslinked CC / HA hydrogel The potential for preserving co-crosslinked CC / HA hydrogels is assessed by placing them under accelerated degradation conditions in a 40°C oven and monitoring the progression of their biomechanical properties. Hydrogels are considered acceptable from a biomechanical standpoint if they are cohesive according to water tests, readily injectable, exhibit gel-like behavior (tan delta value less than 1), and have viscoelastic levels at the initial level at t0, consistent with their intended indications.

[0314] To obtain viscoelasticity level 2, reference hydrogel M2-L was prepared according to the reaction conditions of Example 12 by co-crosslinking CC9 (see Table 11a) and HA2 at a CC / HA ratio of 70:30. This product was packaged in a 1 mL glass syringe (Hypak, BD Medical) and sterilized in the same manner as in Example 9. The syringe was 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 degradation conditions at 40°C, product M2-L remained a hydrogel (tan delta < 1), maintaining its cohesiveness, ease of injection, and viscoelasticity level 2. Therefore, by extrapolation, it is estimated that this co-crosslinked CC / HA hydrogel should maintain acceptable properties for its intended indications for at least 12 months at room temperature.

Claims

1. A matrix for forming an aggregated hydrogel, comprising at least one carboxyalkyl chitosan having glucosamine units, N-acetylglucosamine units, and carboxyalkyl-substituted glucosamine units, wherein the carboxyalkyl chitosan has a degree of substitution by carboxyalkyl groups ranging from 70% to 250%, expressed as the number of moles of substituents relative to the number of moles of total units, and has a degree of acetylation ranging from greater than 40% to a maximum of 80%, expressed as the number of moles of N-acetyl groups relative to the number of moles of total glucosamine units, and the carboxyalkyl chitosan is crosslinked by covalent bonds between carboxyalkyl chitosan chains.

2. The matrix according to claim 1, characterized in that the carboxyalkyl chitosan has a degree of acetylation of at least 50%.

3. The matrix according to claim 1 or 2, characterized in that the carboxyalkyl chitosan is N,O-carboxyalkyl chitosan.

4. The matrix according to claim 1 or 2, characterized in that the chitosan is derived from the mycelium of an ascomycete.

5. The matrix according to claim 1 or 2, characterized in that the chitosan is derived from the mycelium of the fungus Aspergillus piger.

6. The matrix according to claim 1 or 2, characterized in that the chitosan is derived from Lentinula edodes (shiitake mushroom) and / or Agaricus bisporus (white mushroom).

7. The matrix according to claim 1 or 2, characterized in that the carboxyalkyl chitosan is reacetylated.

8. The matrix according to claim 1 or 2, characterized in that the matrix is ​​sterile.

9. The matrix according to any one of claims 1 to 8, characterized in that the matrix contains at least one hyaluronan.

10. The matrix according to any one of claims 1 to 9, characterized in that the matrix contains at least one hyaluronan obtained by fermentation.

11. The matrix according to any one of claims 1 to 10, characterized in that the matrix comprises at least one hyaluronan crosslinked by covalent bonds.

12. The matrix according to any one of claims 1 to 11, characterized in that the matrix comprises at least one hyaluronan co-crosslinked by covalent bonding with carboxyalkyl chitosan.

13. The matrix according to any one of claims 1 to 12, characterized in that the crosslinks are formed by a crosslinking agent that forms the covalent bonds.

14. The matrix according to claim 13, characterized in that the crosslinking agent is selected from crosslinking agents used for crosslinking polysaccharides.

15. The matrix is ​​characterized by having antioxidant capacity by capturing free radicals, the antioxidant capacity of each product tested herein being normalized by the formula: Normalized antioxidant capacity = TEAC (product) / TEAC (ascorbic acid 20 μg / mL), where TEAC is the Tororox equivalent antioxidant capacity, according to any one of claims 1 to 14.

16. The matrix according to claim 15, characterized in that the matrix has a normalized antioxidant capacity greater than 0.

30.

17. A composition comprising at least one matrix defined according to any one of claims 1 to 16.

18. An injectable composition comprising at least one matrix as defined in any one of claims 1 to 16.

19. A pharmaceutical composition comprising at least one matrix defined according to any one of claims 1 to 16.

20. The composition according to claim 18 or 19, characterized in that the composition is used as an injectable, implantable, or instillable pharmaceutical composition, or as an injectable, implantable, or instillable medical device.

21. The composition according to claim 17, characterized in that it is the composition for use in a therapeutic method comprising topical instillation or administration, or injection, via a subcutaneous, intradermal, mucous membrane, eye, intraocular, or intra-articular route.

22. The composition according to claim 19, characterized for use in a method for treating, repairing or filling at least one body tissue that requires repair or filling, wherein the body tissue is selected from tissues belonging to the vocal cords, muscles, ligaments, tendons, mucous membranes, genitals, bones, joints, eyes, skin, cartilage, synovial membrane, skin wounds, or the surface of the eye or any combination thereof.

23. The composition according to claim 18 or 19, for use in a method for treating osteoarthritis or a method for repairing cartilage defects.

24. A medical device comprising or consisting of the composition described in any one of claims 17 to 23.

25. A process for preparing the matrix according to any one of claims 1 to 16, wherein the process is: A step of contacting the carboxyalkyl chitosan with at least one crosslinking agent in an alkaline phase; A step of crosslinking the carboxyalkyl chitosan with the crosslinking agent; Steps to obtain a matrix containing the crosslinked carboxyalkyl chitosan A process that includes this.

26. A process for preparing a matrix comprising a carboxyalkyl chitosan according to any one of claims 1 to 16, which is co-crosslinked with another biopolymer, the process being: A step of contacting a mixture of carboxyalkyl chitosan and other biopolymers with at least one crosslinking agent in an alkaline phase; A step of crosslinking the carboxyalkyl chitosan and other biopolymers with the crosslinking agent; Steps to obtain a matrix of co-crosslinked carboxyalkyl chitosan and other biomolecules. A process that includes this.

27. The process according to claim 26, characterized in that the other biopolymer is hyaluronan.

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