Chitosan-based beads, preparations, compositions and uses

Abstract

The present invention relates to a composition comprising beads and an aqueous phase comprising a plurality of beads, wherein the beads comprise a hydrogel matrix comprising or consisting of at least one carboxyalkylchitosan having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkylchitosan is crosslinked by covalent bonds between the carboxyalkylchitosan chains and / or co-crosslinked by covalent bonds with one or more other polymers. The present invention also relates to methods for their preparation and their application.

Description

Technical Field

[0001] The present invention relates to beads that can be administered to humans or animals, particularly injectable, transplantable or ophthalmic drops, comprising or consisting of at least one crosslinked carboxyalkyl chitosan. The present invention relates to compositions that can be administered to humans or animals, particularly injectable, transplantable or ophthalmic drops, comprising beads or consisting of a hydrogel matrix comprising at least one crosslinked carboxyalkyl chitosan.

[0002] The present invention also relates to methods for preparing these beads, compositions, particularly compositions containing them in the form of suspensions or dispersions, and their uses, particularly their use by administration to humans or animals, particularly their use by injection, transplantation, infusion or other routes adapted to targeted applications by means of an injection needle or an endoscopic system.

[0003] More specifically, the present invention relates to beads that can be administered to humans or animals, compositions containing them, methods for their manufacture, and their different uses, particularly in the fields of therapy, rheumatology, orthopedics, ophthalmology, aesthetic medicine, plastic surgery, internal medicine, dermatology, gynecology or beauty, comprising or consisting of a hydrogel matrix containing at least one carboxyalkyl chitosan crosslinked by covalent bonds.

Background Art

[0004] Products that can be administered to humans or animals made of bioabsorbable polymer particles are already on the market, particularly for the indication of volumization of skin tissue for aesthetic purposes and drug delivery by injection. However, these products need improvement, particularly in terms of resistance, biocompatibility, and volume effect.

[0005] It is currently recognized that the body's reaction to particle systems administered to tissues depends on several parameters, particularly composition, shape, size, biodegradability, and surface.

[0006] Therefore, there are commercially available products based on bioabsorbable beads that, while not in the form of hydrogels, pose a risk of inducing reactions to foreign bodies or granulomas. Consequently, their use is limited to deep tissue layers, and for example, they cannot be administered to the dermis. Such microspheres can be injected via an intraocular route (Sustained treatment of retinal vascular disease with autoaggregating sunitinib microparticles, Nature Comm 11, 694, 2020).

[0007] Furthermore, the beads according to the present invention should be suitable for use in humans or animals, particularly in terms of cohesiveness, safety, immunocompatibility, bioabsorption, biomechanical properties, ease of administration, and lifespan or activity duration. However, state-of-the-art compositions do not adequately provide such properties and therefore would not be in line with the present invention.

[0008] Certain filler products are obtained by extruding hydrogels formed from a matrix of cross-linked hyaluronic acid (HA). However, because the shape obtained by extrusion is non-spherical and irregular, it generally carries a higher risk of foreign body and granuloma reaction than spherical hydrogels, as specifically described by Lemperle (Biocompatibility of injectable microspheres, Biomed J Sci Tech Res 2, 1, 2018). Hydrogel beads obtained by extrusion are also known to require greater force to inject through finer needles than spherical hydrogel beads. To prioritize appropriate short- and long-term tolerance and easy injection with fine needles, the use of spherical hydrogel beads is recommended.

[0009] Literature, particularly patent applications, describes spherical hydrogel beads of bioabsorbable polymers formed by crosslinking via non-covalent bonds. For example, international application WO2011089173 (Biopharmex) describes an injectable composition for tissue filling of soft tissue. More specifically, this application describes a suspension of microspheres with an average volume diameter of 5–50 μm, wherein the microspheres, comprising at least one bioabsorbable polysaccharide in an amount of 50–90% by weight relative to the total weight of the microspheres, induce a second filling by tissue induction and further comprising at least one second bioabsorbable polymer. These hydrogel microspheres have low stability due to the absence of covalent bonds; therefore, their integrity in their initial shape and size is shortened after injection or implantation. Their suspension storage stability must also be short-lived.

[0010] Other documents describe the preparation of bioabsorbable polymer hydrogel beads obtained by crosslinking via covalent bonds, for example, the publication by Luo et al., which relates to carboxymethyl chitosan in the form of dehydrated beads (Development of Carboxymethyl Chitosan Hydrogel Beads in Alcohol-Aqueous Two-Component Solvent for Nutrient Delivery Applications, Food Hydrocolloids 31, 332, 2013). However, Luo et al. used the crosslinking agent glutaraldehyde, which is not suitable for injection into humans or animals. More specifically, Luo et al. do not describe any of the following properties: mechanical strength, elasticity, volume, immunocompatibility, injectability with a needle, or the ability to remove residues (salts and crosslinking agents) by a washing process without damaging the beads. Above all, however, we have found that the method of Luo et al. is not suitable for the preparation of hydrogel beads starting with strongly substituted and / or acetylated chitosan derivatives and the crosslinking agent BDDE (preferably more than glutaraldehyde). Furthermore, the chitosan derivatives used by Luo et al. undergo some degree of acetylation and substitution, and therefore cannot provide acceptable immunoreactivity. Consequently, it is impossible for those skilled in the art to learn sufficiently from this document to develop beads according to the present invention. [Overview of the project] [Problems that the invention aims to solve]

[0011] Therefore, no injectable beads exist that fully satisfy the objectives set by the inventors. The field of preparing spherical, fully acceptable hydrogel beads based on bioabsorbable polymers, particularly those that can be easily administered by injection, is complex, and research on this problem has not yet been developed. Accordingly, the inventors attempted to develop this technique, but it had not been sufficiently investigated. [Means for solving the problem]

[0012] One objective of the present invention is to solve the technical problem of providing injectable beads that have appropriate resistance to targeted adaptations after administration to humans or animals, particularly by injection or implantation, and more specifically in the fields of therapeutic, surgical, and cosmetic applications.

[0013] One objective of the present invention is to solve the technical problem of providing such injectable beads for volume-enhancing effects (e.g., volume-enhancing / remodeling effects of facial / body contours in cosmetic medicine) or for drug delivery.

[0014] In particular, one objective of the present invention is to solve the technical problem of providing bioabsorbable beads that are acceptable in terms of biomechanical properties, lifespan or in-situ activity, adapted for use in contact with human or animal tissue, seeking appropriate health safety, including acceptable short-term and long-term immune and / or foreign body reactions, and providing beneficial effects, particularly in the fields of regenerative medicine or anti-aging medicine, for example, in the fields of therapy, rheumatology, orthopedics, gynecology, ophthalmology, cosmetic medicine, plastic surgery, internal medicine surgery, dermatology or cosmetic medicine.

[0015] One objective of the present invention is to solve the technical problem of providing such injectable beads that exhibit sufficient stability during storage and are suitable for the targeted indication.

[0016] In particular, one objective of the present invention is to solve the technical problem of providing injectable beads that offer satisfactory resistance, physicochemical properties, and volume-enhancing effects, and that can be used for a sufficient period of time after subcutaneous implantation.

[0017] One objective of the present invention is to solve the technical problem of providing modular properties to beads according to the target indication.

[0018] In particular, one objective of the present invention is to solve the technical problem of providing beads that can withstand a purification process by washing them in an aqueous environment to remove undesirable residues from the process and balance the pH and osmotic pressure of the beads with those of a physiological medium.

[0019] Another object of the present invention is to solve the technical problem of providing bioabsorbable injectable beads that have a sustained effect over time, particularly with respect to volume-enhancing effects and / or delivery of at least one compound (therapeutic active ingredient, nutrient, etc.) targeted for an indication.

[0020] One object of the present invention is also to solve the technical problem of providing (injectable) beads that are essentially spherical and have a smooth, non-rough surface.

[0021] In particular, one objective of the present invention is to solve the technical problem of providing a satisfactory injection device for such beads when intended for delivery by injection, without altering the integrity of the beads at the outlet of the injection system.

[0022] Another objective of the present invention is to solve the technical problem of providing beads that are suspendable in an aqueous phase and remain intact during their storage in such a suspension.

[0023] One object of the present invention is also to solve the technical problem of providing beads that can be suspended in an aqueous phase and that remain integral during their sterilization in such a suspension, particularly by a wet heat sterilization process.

[0024] One object of the present invention is to solve the above technical problem by providing chitosan derivative beads.

Embodiments for Carrying Out the Invention

[0025] To provide beads that can be injected into humans or animals, they must be biocompatible with the tissues into which they are injected or implanted. Preferably, to provide biocompatible beads, they preferably contain a non-toxic and non-immunoreactive polymer, preferably a biopolymer matrix. The polymers of the bead matrix according to the present invention are advantageously bioabsorbable and neither toxic nor immunoreactive. Carboxyalkyl chitosan derivatives described in PCT / EP2018 / 080763 and PCT / EP2018 / 080767 applications (KIOMEDPHARMA) are preferred because their matrix is ​​sufficiently tolerant of being non-toxic or immunoreactive. In fact, it is not possible to form hydrogels that are acceptable for use in humans or animals, particularly in terms of immunocompatibility (or immunoreactivity), using chitosan derivatives. Immunocompatibility means a substance that is foreign to the human body or other organism that does not stimulate cells or the immune system and elicits a specific immune response to an antigen. PCT / EP2020 / 064159 application relates to the use of hydrogel matrices prepared by crosslinking such chitosan derivatives. To prepare beads that satisfy the objectives of the present invention, it has been considered for some time to use a matrix of such cross-linked chitosan derivatives in accordance with this international application PCT / EP2020 / 064159. However, preparing beads from these cross-linked chitosan derivative matrices requires, for example, extruding or grinding a hydrogel. However, the inventors have found that such beads are unsatisfactory because their shape is non-spherical and irregular (i.e., angular parts, surface roughness, and poor control of size distribution) (Lemperle 2018, Biocompatibility of injectable microspheres, Biomed J Sci Tech Res 2, 1, 2018), increasing the risk of foreign body reactions when injected or implanted in human or animal tissue. Similarly, they are not easy to inject due to their irregular shape.

[0026] Based on this observation, the inventors devised a research program. More specifically, they considered preparing spherical hydrogel beads based on carboxyalkyl chitosan according to the various methods described in the literature for carboxyalkylated chitosan derivatives, according to PCT / EP2018 / 080763 and PCT / EP2018 / 080767.

[0027] Thus, for example, the methods of forming ionic bridges or multi-electron complexes described by Anitha et al. (Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N,O-carboxymethyl chitosan nanoparticles, Carbohydrate Polymers 78, 672, 2009), Anitha et al. (Curcumin-loaded N,O-carboxymethyl chitosan nanoparticles for anticancer drug delivery, J Biomater Sci 23, 1381, 2012), Lin and Lin (Preparation of N,O-carboxymethyl chitosan nanoparticles as insulin carriers, 2009), Kalliola et al. (pH-sensitive properties of carboxymethyl chitosan nanoparticles crosslinked with calcium ions, Colloids Surfaces B, 153, 229, 2017) and Feng et al. (Chitosan / O-carboxymethyl chitosan nanoparticles for efficient and safe oral anticancer drug delivery, Int J Pharma 457, 158, 2013) did not provide satisfactory results. In fact, in most cases, hydrogel beads were not formed, i.e., the polymer precipitated in spherical form but was not stabilized and was not viscoelastic and hydrated like hydrogel beads, or if they were formed, they did not remain integrated and stable when incorporated into the continuous phase.

[0028] Surprisingly, as a result of numerous developments by the inventors, it has been discovered that the present invention makes it possible to provide beads that satisfy the objects of the present invention.

[0029] More specifically, the present invention relates to a composition comprising an aqueous phase containing a plurality of beads, wherein the beads comprise a hydrogel matrix comprising or consisting of at least one carboxyalkyl chitosan having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkyl chitosan is crosslinked by covalent bonds between carboxyalkyl chitosan chains and / or co-crosslinked by covalent bonds with one or more other polymers.

[0030] The present invention also relates to one or more injectable beads for human or animal use, wherein the beads comprise or comprise a hydrogel matrix comprising at least one carboxyalkyl chitosan having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkyl chitosan has a degree of acetylation between 30% and 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 and / or co-crosslinked with one or more other polymers.

[0031] Carboxyalkyl chitosan Advantageous chitosan derivatives and their families, such as carboxyalkyl chitosan, described in the patent applications filed by Kiomed Pharma under the numbers PCT / EP2018 / 080763 and PCT / EP2018 / 080767, are known and incorporated herein by reference. Furthermore, an international application by Kiomed Pharma, PCT / EP2020 / 064159 (whose contents are incorporated herein by reference), describes, for example, carboxyalkyl chitosan as hyaluronan, either alone or crosslinked with another polymer.

[0032] The matrix according to the present invention may be characterized by a starting carboxyalkyl chitosan that is crosslinked and / or co-crosslinked with one or more polymers to form the matrix according to the present invention.

[0033] According to the first aspect, a carboxyalkyl chitosan having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with a carboxyalkyl group is used (of fungal origin), wherein the carboxyalkyl chitosan has a degree of substitution with carboxyalkyl groups of more than 20%, expressed as the number of moles of substituents relative to the number of moles of total units.

[0034] This is also called chitosan or a substituted chitosan derivative.

[0035] Carboxyalkyl chitosans are prepared by chitosan substitution. Typically, carboxyalkyl chitosans are prepared in accordance with the patent applications of Kiomed Pharma filed under the numbers PCT / EP2018 / 080763 and its family (particularly FR 1761314 and EP18799772.1), PCT / EP2018 / 080767 and its family (particularly FR 1761323 and EP18799773.9), and PCT / EP2020 / 064159 and its family (particularly FR1905504), which are incorporated herein by reference in particular to illustrate the preparation of carboxyalkyl chitosans.

[0036] For example, chitosan is referenced by CAS number 9012-76-4.

[0037] The chitosan used in this invention is advantageously of fungal origin, and preferably derived from ascomicete-type fungi, particularly the mycelium of Aspergillus niger and / or Basidiomycete fungi, and especially from Lentinula edodes (shiitake mushroom) and / or Agaricus bisporus (cultivated mushroom). Preferably, the chitosan is derived from Agaricus bisporus. The chitosan is preferably very pure, that is, it contains little impurities from its fungal origin or manufacturing process and is of microbiological quality suitable for use as an implant or pharmaceutical composition. The method for preparing the chitosan is the method described in International Publication WO 03 / 068824 (EP1483299; US7556946).

[0038] Generally, chitin is placed in an aqueous suspension 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 in an acidic medium, precipitated in an alkaline medium, washed, and dried.

[0039] Preferably, the chitosan is of a grade that is sufficiently pure for pharmaceutical use.

[0040] Chitosan is advantageously purified and then preferably dried. After purification, the method of the present invention may include the step of drying the carboxyalkyl chitosan and then grinding it to obtain a powder. For example, chitosan can be dried by evaporation of water, for example, spray drying (atomization), fluidized bed, or by a vacuum or atmospheric pressure heating drying process, or by freeze-drying.

[0041] Next, this chitosan can be substituted to produce carboxyalkyl chitosan, for example, as described in the present invention.

[0042] Next, beads according to the present invention are prepared using such carboxyalkyl chitosan.

[0043] For simplicity, DA and DS are represented according to the carboxyalkyl chitosan before bead formation.

[0044] The degree of acetylation (DA) of chitosan is determined by potentiometric titration, as described, for example, in International Publications WO2017009335 and WO2017009346. The DA of chitosan and carboxyalkylated chitosan can be measured by known methods for chitosan, such as liquid-phase proton NMR, solid-phase carbon-13 NMR, infrared spectroscopy, and UV-Vis spectroscopy.

[0045] According to one alternative method, carboxyalkyl chitosan has a degree of acetylation of less than 30%, for example, 5% to 30%, expressed as the number of moles of N-acetyl-glucosamine units per total mole.

[0046] Advantageously, carboxyalkyl chitosans have a degree of acetylation between 30 and 80%, expressed as the number of moles of N-acetyl-glucosamine units relative to the total mole 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).

[0047] Advantageously, carboxyalkyl chitosans have a degree of acetylation between 30 and 75%, expressed as the number of N-acetyl groups relative to the total number of glucosamine units.

[0048] According to one alternative method, the degree of acetylation is in the range of 35-50%.

[0049] According to one alternative method, the degree of acetylation is in the range of 40-60%.

[0050] According to one alternative method, the degree of acetylation is in the range of 50-75%.

[0051] The degree of acetylation of chitosan carboxyalkyl can be determined by solid-phase carbon-13 NMR or liquid-phase proton NMR. Carboxyalkyl chitosan is advantageous when its degree of acetylation is controlled. "Chitosan with controlled degree of acetylation" refers to a product in which the degree of acetylation, i.e., the ratio of N-acetyl-glucosamine units, can be adjusted in a manner controlled particularly by the acetylation reaction.

[0052] Preferably, the carboxyalkyl chitosan is reacetylated.

[0053] According to one alternative method, the method for preparing carboxyalkyl chitosan according to the present invention comprises preparing chitosan of fungal origin, reacetylating the chitosan, and carboxyalkylating the reacetylated chitosan. Thus, the present invention relates to reacetylated carboxyalkyl chitosan. More specifically, the present invention relates to anionic carboxyalkyl chitosan.

[0054] Therefore, according to one embodiment, chitosan can be dissolved in an aqueous medium, preferably slightly acidified (e.g., pH 6). Acetyl anhydride can be added to the chitosan solution in one or more steps. Next, basic agents such as sodium and urea are added. Then, an alkylating agent such as sodium monochloroacetate (i.e., sodium salt of chloroacetic acid) or chloroacetic acid is added. Next, the substituted chitosan is purified, recovered, and dried.

[0055] According to one alternative method, the carboxyalkyl chitosan preparation method according to the present invention comprises 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, more specifically, obtaining a high degree of acetylation, for example, greater than 40%. Accordingly, the present invention relates to reacetylated chitosan, and subsequently carboxyalkylated or reacetylated carboxyalkyl chitosan.

[0056] According to one alternative method, a method for preparing carboxyalkyl chitosan according to the present invention comprises preparing chitin of fungal origin, carboxyalkylating the chitin, and possibly reacetylating the carboxyalkylated chitin to obtain carboxyalkyl chitosan according to the present invention.

[0057] According to one alternative method, a method for preparing carboxyalkylated chitosan according to the present invention comprises preparing chitin of fungal origin, deacetylating the chitin, carboxyalkylating the chitin, and possibly reacetylating the carboxyalkylated chitin to obtain carboxyalkyl chitosan according to the present invention.

[0058] According to one alternative method, carboxyalkyl chitosan has an average molecular weight of less than 500,000.

[0059] According to one specific alternative method, the substituted chitosan preferably has an average molecular weight of 50,000 to 400,000.

[0060] The average molecular weight is the molecular weight of carboxyalkyl chitosan before bead formation and crosslinking. Here, preferably, the average molecular mass is the average molecular mass (Mv) of the viscosity calculated from the intrinsic viscosity. This expression is common to experienced practitioners. The intrinsic viscosity (η) is measured by capillary viscometery using an Ubbelohde type capillary viscometer according to the method of monography 2.2.9 of the European Pharmacopoeia. Using an automatic viscometer I-Visc (Lauda), the flow time of the solution through a suitable capillary tube (Lauda, ​​e.g., a 0.53 mm diameter capillary tube Ubbelohde 510 01) is measured. Then, to calculate the average viscometric mass of carboxyalkyl chitosan, the Mark-Houwink formula (η = K * Mv) is used. α ) is applied here -Mv is the average molecular mass of the viscosity of carboxyalkyl chitosan. -η is the intrinsic viscosity of carboxyalkyl chitosan. - The values ​​of the constants K and α are 0.0686 and 0.7638, respectively, and were determined in advance for (unsubstituted) chitosan by stereoexclusion chromatography using a MALLS detector.

[0061] The intrinsic viscosity of carboxylazole chitosan can usually be expressed to assess its average molecular weight.

[0062] Chitosan can be hydrolyzed to reduce its molecular weight.

[0063] Typically, in non-crosslinked carboxyalkyl chitosans, the glucosamine units are D-glucosamine units (D-glucosamine units, N-acetyl-D-glucosamine units, and units in which at least one of the D-glucosamine units and N-acetyl-D-glucosamine units is substituted).

[0064] According to one alternative method, the substituted chitosan has substitutions only in the D-glucosamine units.

[0065] According to another alternative method, the substituted chitosan simultaneously substitutes D-glucosamine and N-acetyl-D-glucosamine units, and according to the first alternative method, the carboxyalkyl group is covalently bonded to the amine group of chitosan alone, or according to another alternative method, it is simultaneously bonded to the amine group and hydroxyl group of chitosan.

[0066] Substitutions are generally partial, and not all units are necessarily replaced.

[0067] According to one embodiment, the degree of substitution of D-glucosamine units, expressed as the number of moles of D-glucosamine units relative to the number of moles of total units of substituted chitosan (D-glucosamine and N-acetyl-D-glucosamine units, whether substituted or not), is The range is 30% to 250%.

[0068] 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.

[0069] According to one embodiment, the degree of substitution by carboxyalkyl groups exceeding 50% is expressed as the number of moles of substituents relative to the number of moles of total units.

[0070] According to one embodiment, the degree of substitution of D-glucosamine units, expressed as the number of moles of D-glucosamine units relative to the number of moles of total units of substituted chitosan (D-glucosamine and N-acetyl-D-glucosamine units, whether substituted or not), is in the range of 50% to 200%, and more preferably higher than 70%.

[0071] According to one embodiment, the degree of substitution by carboxyalkyl groups of less than 80% is expressed as the number of moles of substituents relative to the number of moles of total units.

[0072] Typically, substitutions are carried out by covalent bonds.

[0073] According to one alternative method, carboxyalkyl chitosan is N,O-carboxyalkyl chitosan. The proportion of units substituted with carboxyalkyl groups at the O position (O3 and / or O6 of glucosamine and / or N-acetyl-glucosamine units) and / or the N position (glucosamine unit) varies. Therefore, the degree of substitution can be greater than 100%. Advantageously, the degree of substitution (DS) and degree of acetylation (DA) of carboxyalkyl chitosan are measured by solid-phase carbon-13 magnetic resonance spectroscopy (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 64–512 scans. The area of ​​the carbon signal is determined after deconvolution. The carbons considered are: "acetyl CH3" (methyl carbon of the acetyl group of the N-acetyl-D-glucosamine unit, whether substituted or not), "Cx" (carbon at position x of the glucosamine and N-acetyl-glucosamine units, x in the range of 1 to 6), and "C=O" (carbonyl carbon of the carboxyalkyl substituent and carbonyl C=O carbon of the acetyl group of the N-acetyl-glucosamine unit, whether substituted or not). To determine the DS of a given carboxyalkyl chitosan, the NMR spectrum of carbon 13 of the precursor chitosan of this carboxyalkyl chitosan must also be recorded. From the spectrum of the precursor chitosan, the "CSU ratio," i.e., the ratio of the signal area of ​​the "CH3 acetyl" group (methyl carbon of the acetyl group of the N-acetyl-glucosamine unit) to the signal area of ​​"C=O" (carbonyl carbon of the acetyl group of the N-acetyl-D-glucosamine unit), is calculated. The DA of 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 being considered.

number

number

[0074] Preferably, the degree of substitution (DS) range is expressed in relation to the results of solid-phase carbon-13 NMR analysis in this description, as it is easily carried out.

[0075] DA and DS can also be determined using other known methods for carboxyalkyl chitosan, for example by aqueous medium proton NMR, by magnetic resonance spectroscopy, or by adding a solution of deuterated hydrochloric acid, for example, according to the method described by Liu et al. (Carb Polym 137, 600, 2016).

[0076] If another NMR method is more advantageous for estimating DA and / or DS in a reliable manner, such a method should be used. The above methods should be adapted by those skilled in the art with respect to the sample preparation and signals to be integrated, in particular depending on the resolution, robustness, and proton positions of the signals used in calculating the degree of substitution.

[0077] The degree of carboxyalkylation (DS) of chitosan is expressed as the number of moles of carboxyalkyl relative to the total number of moles of units, and can be in the range of 20-250%, preferably 50-200%, for example, 70-170%.

[0078] According to one alternative method, the degree of carboxyalkylation (DS) of chitosan is expressed as the number of moles of carboxyalkyl relative to the total number of moles of units, and can favorably range from 40 to 130%, for example, from 70 to 130%.

[0079] The degree of substitution of chitosan is usually correlated with the amount of reagents related to chitosan at the start of the reaction. As carboxyalkylating agents, acidic chlorides (or their salts, e.g., sodium monochloroacetate) can be mentioned, for example, those having one or more carboxymethyl, carboxyethyl, carboxypropyl, or carboxybutyl groups.

[0080] According to one alternative method, the present invention relates to carboxyalkyl chitosan in which the alkyl portion of the carboxyalkyl is linear or branched C1-C5.

[0081] According to one alternative method, the present invention relates to carboxymethyl chitosan.

[0082] According to this alternative method, substituted chitosan is N-carboxyalkylated chitosan.

[0083] According to this alternative method, the substituted chitosan is O-carboxyalkylated chitosan.

[0084] According to this alternative method, substituted chitosans are N-carboxyalkylated and O-carboxyalkylated chitosans.

[0085] According to a second aspect, the present invention relates to chitosan derivatives having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with carboxyalkyl groups, wherein the carboxyalkyl chitosan having a zeta potential is measured at pH 7.5 and is -10 mV or less, preferably -15 mV or less. More specifically, such chitosan derivatives enable the chitosan derivative or a composition having the same to limit the immune response of a subject to which it is typically administered by intravenous infusion, injection, or transplantation.

[0086] Advantageously, the zeta potential measured at pH 7.5 is below -18mV.

[0087] Advantageously, carboxyalkyl chitosan has a zeta potential of -22mV or less, preferably -24mV or less, as measured at pH 7.5.

[0088] According to one particular alternative method, the substituted chitosan has a degree of substitution (DS) of 20-80%, preferably 40-60%, and a degree of acetylation (DA) of 30-80%, preferably 30-75%.

[0089] According to one particular alternative method, the substituted chitosan has a degree of substitution (DS) of 50-200%, preferably 70-200%, and a degree of acetylation (DA) of 30-80%, preferably 30-75%.

[0090] According to one particular alternative method, the substituted chitosan has a degree of substitution (DS) of 90-200%, preferably 90-150%, and a degree of acetylation (DA) of 30-80%.

[0091] According to one specific alternative method, the substituted chitosan has a degree of substitution (DS) of 90-200%, preferably 90-150%, and a degree of acetylation (DA) of 30-60%.

[0092] According to one particular alternative method, the substituted chitosan has a degree of substitution (DS) of 90-200%, preferably 90-150%, and a degree of acetylation (DA) of 40-75%.

[0093] By substituting chitosan, it is possible to prepare a solution of one carboxyalkyl chitosan that is soluble in aqueous solutions with a wide range of pH variations, whereas unsubstituted chitosan is soluble only at pH levels lower than approximately 6. Carboxyalkyl chitosan certainly has the ability to solubilize at different pH levels, more specifically at the physiological pH or other pH levels when physiological fluids are modified by pathological conditions, such as inflammatory conditions, due to the presence of the carboxyalkyl group which alters its solubility profile.

[0094] "Water solubility" means that carboxyalkyl chitosan does not exhibit visible cloudiness when placed in an aqueous solution. More specifically, the solubility, i.e., lack of turbidity, of a 1% (m³ / m³) solution or buffer of carboxyalkyl chitosan in water, such as a 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, relative to a reference tank containing only the aqueous solvent used in the sample being measured, but without substitution. Another method consists of a visual inspection according to the monograph 2.9.20 of the European Pharmacopoeia. 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.

[0095] According to one alternative method, carboxyalkyl chitosan is sterile.

[0096] "Crosslinked by covalent bonds between carboxyalkyl chitosan chains" means that a main chitosan chain (also called a chitosan backbone) is covalently bonded to one or more main chitosan chains. Therefore, a three-dimensional network of chitosan molecules is advantageously obtained. While the present invention is not limited to a specific covalent crosslinking method, a preferred method uses a chemical molecule used as a crosslinking agent, also called a crosslinking agent. The concept of "crosslinked by covalent bonds between carboxyalkyl chitosan chains" particularly relates to covalent bonds involving a crosslinking agent that binds the carboxyalkyl chitosan molecules. However, these are covalent bonds between carboxyalkyl chitosan chains by atomic chains (usually part of the crosslinking agent located between their reaction functions).

[0097] According to the present invention, carboxyalkyl chitosan is crosslinked.

[0098] beads Advantageously, according to the present invention, the beads contain or consist of a hydration matrix in the form of a hydrogel.

[0099] The term "hydrogel" is intended to have the conventional meaning of the word, and more specifically, to refer to a three-dimensional (3D) network of at least one hydrophilic polymer that, through cross-linking of individual polymer chains, swells in water and can hold a considerable amount of water while maintaining its structure. Hydrogel beads are hydrated beads containing at least 70% by mass of water relative to their total wet mass. Typically, hydrogel beads can retain this water content under ambient conditions or after administration to tissue.

[0100] According to one alternative method, the matrix comprises at least one polymer, for example, at least one hyaluronan, which is possibly crosslinked by covalent bonds and / or by covalent bonds with carboxyalkyl chitosan.

[0101] "By itself" or "by itself" means that the molecule can contain covalent bonds of intra-chain and / or inter-chain molecular bridges.

[0102] Advantageously, the hydrogel matrix of the present invention comprises or is composed of one or more bioabsorbable polymers, each containing at least one carboxyalkyl chitosan.

[0103] According to one alternative method, the matrix according to the present invention that forms the hydrogel consists of carboxyalkyl chitosan.

[0104] According to one alternative method, the matrix according to the present invention that forms a hydrogel comprises or consists of carboxyalkyl chitosan, which is crosslinked by itself and / or co-crosslinked with another polymer, and possibly one or more other biopolymers that are crosslinked or co-crosslinked with carboxyalkyl chitosan.

[0105] Therefore, the present invention includes several alternative methods, including the following:

[0106] An alternative method comprising or consisting of a hydrogel matrix that is crosslinked by itself and contains no other polymers in the hydrogel matrix, or carboxyalkyl chitosan.

[0107] An alternative method in which the hydrogel matrix comprises or is composed of self-crosslinked carboxyalkyl chitosan and one or more polymers, whether or not they are self-crosslinked within the hydrogel matrix.

[0108] An alternative method in which the hydrogel matrix contains or is composed of one carboxyalkyl chitosan co-crosslinked with one or more other polymers, and the carboxyalkyl chitosan and / or other polymers can be crosslinked by themselves.

[0109] Therefore, according to one alternative method, the carboxyalkyl chitosan is co-crosslinked with another polymer.

[0110] According to one alternative method, the bioabsorbable polymers according to the present invention are natural biopolymers, for example, naturally present in human or animal tissues, and may be polysaccharides, proteins, and any other polymers present at the tissue and cellular levels. According to one embodiment, biopolymers other than carboxyalkyl chitosan are selected from polysaccharides or proteins, and are preferably selected along any of the following biopolymers or combinations thereof: elastin, hyaluronan, chondroitin, fibrin, collagen, dextran, cellulose, carboxyalkylcellulose, lubricin, mucin, silk, albumin, and any derivatives thereof.

[0111] Biopolymers can be natural because they possess modified natural structures.

[0112] According to one alternative method, the other biopolymer is selected from polysaccharides crosslinked by covalent or non-covalent bonds, whether oxidized or not, such as glycosaminoglycans, more specifically, hyaluronans, such as hyaluronic acid, sodium hyaluronate or any salt thereof, or derivatives thereof.

[0113] According to one alternative method, bioabsorbable polymers are synthetic polymers.

[0114] According to one alternative method, the matrix comprising the hydrogel beads according to the present invention comprises or consists of carboxyalkyl chitosan and hyaluronan, wherein the carboxyalkyl chitosan is crosslinked by itself and / or co-crosslinked with hyaluronan. These two polymers are advantageously crosslinked by themselves and / or co-crosslinked together.

[0115] One preferred object of the present invention is to combine these two polymers to adapt the intended properties of the beads, for example, to control the water content of the beads, their biomechanical properties and / or bioabsorption dynamics.

[0116] According to one alternative method, the hyaluronan used to prepare the beads has an average molecular weight of less than 5,000,000, as determined by capillary viscometery or stereoexclusion chromatography. The molecular weight of hyaluronan can be expressed by its intrinsic viscosity determined by capillary viscometery, which correlates well with the molecular weight by Mark Houwink's relationship. Therefore, the hyaluronan used to prepare the beads should be "high" (approximately 3-4.25 m) according to one alternative method, depending on the intended properties of the beads. 3 ( / kg), according to another alternative method, "medium" (1.5~3m 3 ( / kg) or by another alternative method, "low" (0.1~1.5m 3 It may exhibit an intrinsic viscosity ( / kg).

[0117] According to one alternative method, hyaluronan can be obtained by fermentation using, for example, Streptococcus equii. According to another alternative method, it is produced by initial extraction from cock's combs.

[0118] According to one alternative method, the matrix contains at least one hyaluronane cross-linked by covalent bonds.

[0119] Therefore, cross-linked hyaluronanes contain covalent bonds between different hyaluronane chains.

[0120] Different types of hyaluronanes, such as hyaluronanes with different molecular weights or different hyaluronane salts, can be crosslinked together.

[0121] Accordingly, the present invention relates to a hydrogel in the form of beads comprising or consisting of at least one hyaluronan cocrosslinked by covalent bonding with carboxyalkyl chitosan.

[0122] Preferably, the beads according to the present invention have a water content of more than 80%, preferably 85% or more, for example, more than 90%, relative to the total mass of the beads.

[0123] Advantageously, the beads according to the present invention are non-toxic in humans or animals; that is, they are free of toxic components or do not decompose into toxic components, and are checked, for example, whether they are medical devices, pharmaceuticals, or veterinary products, depending on the standards applied to the product and the indications they are intended for, and are checked, for example, using in vitro cytotoxicity models and / or animal models, for the absence of chronic toxicity after oral or systemic administration. Advantageously, the beads are biocompatible and do not have local resistance issues or unacceptable immunoreactivity for the targeted indications in or over the tissues to which they are administered.

[0124] According to one favorable alternative, the beads according to the present invention do not contain any animal-derived compounds.

[0125] According to one favorable alternative, the beads according to the present invention do not contain alginates, gelatin, carrageenan, or natural chitosan, which are not chitosan derivatives.

[0126] According to one favorable alternative, the beads according to the present invention comprise at least one carboxyalkyl chitosan and possibly hyaluronan. The designation of hyaluronan includes hyaluronic acid and its salts.

[0127] Advantageously, the beads according to the present invention are bioabsorbable, preferably have a long residence time, or are adaptable to the targeted indication. "Bioabsorbable" means a polymer that is gradually broken down / absorbed by natural biological / physiological phenomena, usually over several days, preferably several weeks or months (maximum time depending on the target indication), into low molecular weight compounds that can be removed by subcutaneous or intradermal injection into the body of warm-blooded animals or humans.

[0128] Advantageously, the beads according to the present invention can be sterilized, preferably by wet heat sterilization. Advantageously, the beads according to the present invention retain their characteristics and properties in essence, particularly after sterilization by wet heat or filtration. It is preferable that the beads be sterilized once in a suspension state in the final stage.

[0129] Advantageously, the beads according to the present invention are injectable via a needle conforming to the instructions; that is, the force applied to inject them must be acceptable, the injection action must not cause a jerk, and no change should occur in the discharged beads. The injection force ensures a simple, accurate, and well-controlled medical procedure. Injections must be performed regularly and smoothly. Therefore, according to the present invention, "easy" injection of the beads or composition is preferred. "Easy" injection means that the force applied to the syringe to flow the composition according to the present invention through a needle defined for the target application at an appropriate piston discharge rate (e.g., 10 mm / min) is less than 50 Newtons, preferably less than 40 Newtons. For example, in dermatological applications, beads injectable through needles in the diameter range of 25G to 34G are preferred. For example, in ophthalmic applications, beads that can be easily injected through needles in the range of 27G to 34G are preferred. For example, in rheumatic applications, beads that can be easily injected through needles in the range of 18G to 25G are preferred.

[0130] Advantageously, the beads according to the present invention have the desired properties after injection, implantation, or instillation via the administration device used.

[0131] According to one alternative method, the hydrogel beads according to the present invention have an average diameter of 20 μm to 450 μm in their hydrated state. The average diameter of the beads is measured by particle size analysis methods, for example, by laser diffraction (average volume diameter), or by microscopy techniques, for example, by analysis of images recorded by optical, scanning electron sweep, nuclear, or confocal scanning microscopes. These techniques can also be used to measure the diameter of the beads in a dehydrated state.

[0132] According to one alternative method, the beads according to the present invention have an average volume diameter of 25 μm to 250 μm in a hydrated form, particularly to minimize the response to foreign matter and prevent the beads from migrating within the tissue.

[0133] According to one alternative method, the hydrogel beads according to the present invention have an average volume diameter between 1 μm and 20 μm in their hydrated form.

[0134] According to one alternative method, the hydrogel beads according to the present invention have an average volume diameter of less than 1000 nm, preferably less than 500 nm. These are nanobeads, hydrogels, or nanoparticles of nanogels.

[0135] Typically, the distribution of bead sizes varies depending on the application being targeted.

[0136] The beads are spherical, minimizing the foreign body reaction after injection or implantation and ensuring suitability for the target indication.

[0137] Advantageously, the beads according to the present invention exhibit virtually no roughness and preferably a smooth surface morphology in their hydrated form.

[0138] An advantage is that smooth or non-rough surfaces minimize the reaction to foreign matter.

[0139] Advantageously, the beads according to the present invention have a negative or neutral surface charge, particularly as measured by the zeta potential. Advantageously, the negative or neutral surface charge minimizes reaction with foreign matter.

[0140] For many bead applications, homogeneous hydrogel beads that are not fragmented and have no solid components inside or on the surface of the beads are preferred.

[0141] An advantage is that the beads are transparent.

[0142] Hydrogel beads remain homogeneous and integrated when incorporated into the final phase (intermediate), such as the aqueous phase, and their final packaging, such as syringes, and their entire storage life are also preferable for many applications. These are called "integrated" and "homogeneous" beads.

[0143] Mechanical properties Advantageously, the beads according to the present invention are flexible in their hydrated state, meaning their shape can be altered without breaking when subjected to some deformable force (e.g., compression or shear), and they can return to their original shape when released from the deformable force.

[0144] Advantageously, the beads according to the present invention exhibit appropriate resistance to the mechanical stress imposed by the implantation site.

[0145] Advantageously, the beads according to the present invention have the appropriate ability to increase, fill, and / or modify the tissue into which they are injected or implanted.

[0146] Physiological properties Advantageously, the beads according to the present invention are suitable for use in humans or animals, particularly in terms of safety, immunocompatibility, bioabsorbability, biomechanical properties, and lifespan or activity duration. However, state-of-the-art compositions do not adequately provide such properties and therefore would not be in line with the present invention.

[0147] Preparation method The present invention also relates to a method for preparing beads according to the present invention.

[0148] The present invention relates more specifically to a method for preparing a plurality of beads as defined in the present invention, wherein the method is Prepare an aqueous solution of carboxyalkyl chitosan, preferably at an alkaline pH, in the presence or absence of at least one other polymer and in the presence of at least one crosslinking agent; This solution forms droplets in the form of multiple beads; Carboxyalkyl chitosan and, if present, at least other polymers crosslinked with a crosslinking agent; and The present invention includes obtaining multiple beads.

[0149] In particular, the method according to the present invention includes forming droplets based on a bioabsorbable polymer and then stabilizing them by covalent crosslinking of the polymer.

[0150] Typically, the method according to the present invention involves dissolving carboxyalkyl chitosan and possibly other polymers, forming droplets of this solution, then covalently crosslinking them in the form of hydrogel beads, and possibly sorting them to select the desired bead size.

[0151] According to a preferred alternative method, the beads are incorporated into an aqueous phase, possibly containing one or more other polymers.

[0152] According to one embodiment, one or more activators are added to an aqueous solution containing carboxyalkyl chitosan and optionally one or more other polymers before droplet formation. Thus, a solution or suspension containing at least one activator can be prepared, regardless of whether each activator is soluble or insoluble in this aqueous solution, and then beads according to the present invention can be formed to encapsulate one or more activators in the beads.

[0153] dissolve According to a preferred embodiment, carboxyalkyl chitosan and possibly one or more other polymers are dissolved in an aqueous solution containing a crosslinking agent, preferably having an alkaline pH. Advantageously, the aqueous solution also contains one or more crosslinking agents.

[0154] The alkaline agent is typically, for example, soda with a mass concentration of 0.1-5%.

[0155] According to one embodiment, in this dissolution step, carboxyalkyl chitosan can be mixed with other water-soluble polymers, such as biopolymers, polysaccharides, or hyaluronane.

[0156] droplet formation According to one embodiment, the droplet is placed in the presence of an aqueous phase, which may contain an organic solvent, such as an alcohol, such as ethanol.

[0157] According to one embodiment, this method involves solidifying droplets in the form of multiple beads before they are crosslinked, in the presence of at least one coagulant.

[0158] Advantageously, this method involves forming carboxyalkyl chitosan droplets and, if present, possibly one or more other polymers, and then coagulating the droplets, for example, by ionic gelation (non-covalent bonding).

[0159] Conventional methods can be used to form droplets of solutions or polymers. Polymer droplets can be formed by emulsification in hydrophilic lipids, oil-in-water, or water-in-oil media.

[0160] Droplets can be formed by passing the solution through any tube with a diameter suitable for the desired bead diameter at an appropriate flow rate.

[0161] This can also be done by controlling the flow rate and / or pressure, for example, by using a binary nozzle and a spray (also called a nebulizer) of a specific diameter. For example, a binary nozzle with a diameter of 2.8 mm (Buchi) is used to obtain beads with a diameter mostly of less than 400 μm (D0.9). To change the diameter of the droplets, the droplet formation system, for example, the inner diameter of the nozzle, is adapted. According to one alternative method, the droplet formation system or its parameters are adapted according to the viscosity of the polymer solution. Other methods also exist, such as laminar jet electromagnetic (e.g., using the VAR-D continuous device sold by Nisco), or continuous extrusion by cutting the solution jet using electrostatic, coaxial airflow, dynamic airflow, ultrasonic, or rotary tools (e.g., Jettutter, Genialab).

[0162] In one embodiment, the solidification of the droplets takes place in a solution called a "solidification bath" containing a coagulant. In one alternative method, the agent is an alkaline earth metal chloride, typically calcium chloride, which induces gelation through ionic interactions with carboxyalkyl chitosan.

[0163] According to one embodiment, the coagulation bath is an aqueous solution in the presence of an alcohol-type solvent, such as ethanol.

[0164] Preferably, the coagulation bath is made with a water / solvent volume ratio of 90 / 10 to 10 / 90, typically 30 / 70, 20 / 80, or 80 / 20.

[0165] Preferably, the concentration of calcium chloride in the coagulation bath is 10 to 200 mg / mL.

[0166] Preferably, solidification is achieved at room temperature, i.e., without heating, preferably at a temperature of 20-25°C.

[0167] According to one embodiment, the process includes a step of classifying solidified beads and controlling their distribution.

[0168] Bridge According to one alternative method, the beads obtained after crosslinking are subjected to a purification process by washing and equilibrating the pH and osmotic pressure in a physiologically acceptable medium.

[0169] Advantageously, this method involves crosslinking the polymer, which has been formed in the form of droplets, possibly by solidification, via a crosslinking agent present in the polymer solution, preferably early in the first step.

[0170] According to one alternative method, the crosslinking is formed by a crosslinking agent that forms the covalent bond.

[0171] Therefore, some chitosan chains are used as crosslinking agents, for example, to crosslink biopolymers, especially polysaccharides, such as 1,4-butanediol diglycidyl ether, 1-bromo-3,4-epoxybutane, 1-bromo-4,5-epoxypentane, 1-chloro-2,3-epithiopropane, 1-bromo-2,3-epithiopropane, 1-bromo-3,4-epitiobane, 1-bromo-4,5-epithiopentane, 2,3-dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanthol, It can be crosslinked by reaction with one or more crosslinking agents selected from 2,4-dibromobutanethiol, 2,5-dibromopentan-thiol epichlorohydrin, 2,3-dibromopropanol, 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 again divinyl sulfone.

[0172] Jenipine is a naturally derived crosslinking agent used to crosslink polysaccharides, particularly carboxymethyl chitosan (Yang et al. Ophthalmic drug-loaded N,O-carboxymethyl chitosan hydrogels, Acta Pharmacol Sin 31, 1625, 2010). Jenipine colors the hydrogels from dark blue to black, which may be advantageous in some indications.

[0173] Preferably, the crosslinking agent is a polyepoxide-type agent, for example, a bifunctional agent. Preferably, 1,4-butanediol diglycidyl ether (BDDE) or ethylene glycol diglycidyl ether (EGDE) are used as crosslinking agents because they have already been used in the preparation of biomaterials applied to hyaluronic acid hydrogels for human, particularly intradermal, intra-articular, or intraocular administration. According to one alternative method, the crosslinking agent is divinyl sulfone.

[0174] Typically, crosslinking occurs at an appropriate pH, depending on the crosslinking agent and the type of bond being considered.

[0175] According to one alternative method, crosslinking is carried out in the alkaline aqueous phase, for example, in the presence of a sodium hydroxide (NaOH) solution. Advantageously, initially, the concentration of carboxyl kyl chitosan in the aqueous phase is in the range of 1-30% by mass of carboxyl kyl chitosan with respect to the volume of the alkaline aqueous phase, preferably 5-20% (m / v).

[0176] Advantageously, the amount of crosslinking agent is 0.001 moles to 0.1 moles of BDDE per gram of polymer.

[0177] Preferably, when using BDDE, the amount of crosslinking agent is 0.005 to 0.02 moles per gram of polymer.

[0178] Typically, crosslinking occurs at temperatures and durations appropriate for each case, depending on the crosslinking agent, polymer, and desired biomechanical properties.

[0179] Generally, after crosslinking, the culture medium is neutralized by adding an acid, such as acetic acid or hydrochloric acid.

[0180] purification Advantageously, this method involves purifying the crosslinked beads. Purification significantly removes excess coagulant and unreacted crosslinking agent, while also balancing the hydrogel of the beads with a selected medium, such as a physiologically acceptable medium including a buffer, in terms of pH and osmotic pressure.

[0181] According to one embodiment, purification involves one or more washes in a solution containing a buffer, preferably a saline phosphate buffer (often abbreviated as PBS, representing phosphate-buffered saline) or a medium containing beads, depending on the target product.

[0182] Preferably, the purification process involves one or more washes with pharmaceutical-grade water, followed by a physiological solution, i.e., a solution having a pH and osmotic pressure suitable for a single injection or implantation into tissue.

[0183] Advantageously, purification is carried out by a series of filtration / washing steps on a filtration membrane, or by dialysis using a selected solution and an appropriate cutoff dialysis membrane.

[0184] According to one embodiment, the hydrogel beads are first neutralized by adding hydrochloric acid; then collected on a filtration membrane under vacuum. This operation can be repeated several times. Next, the beads can be suspended in a buffer (composition, osmotic pressure, and pH selected according to the instructions) and collected by filtration according to the conventional technique. This process is renewed until a suspension of hydrogel beads is obtained having a residue content in the beads that is lower than the limits specified in advance for the target product, in relation to the osmotic pressure and pH of the desired buffer, as well as the amount delivered and its indication. Thus, it is checked that the coagulant content and the residual crosslinking agent content in the beads are lower than the specified limits. The coagulant content is measured by the whole ash method of the European Pharmacopoeia in the case of calcium chloride. The crosslinking agent content is determined by LC-MS in the case of BDDE, based on the method described by Fidalgo et al. (Detection of a new reaction by-product in BDE crosslinked autoclaved hyaluronic acid hydrogels by LC-MS analysis, Medical Devices: Evidence Res 11, 367, 2018).

[0185] Sorting / Classification According to one alternative method, multiple beads are classified so that the beads are selected according to their dimensions.

[0186] If necessary, smaller beads are removed by allowing larger beads to settle. The hydrogel beads are then sorted by size according to, for example, a conventional sorting method ("sieving") and placed on a suitable porous sieve that has been agitated by ultrasonic waves, vacuum filtration with a reducing porous filter, or other sorting methods.

[0187] keep According to one embodiment, the resulting hydrogel beads are stored by incorporating them into an aqueous solution, such as a buffer such as phosphate buffer or any other aqueous solution, until they are finally formulated, sterilized, and packaged.

[0188] According to one embodiment, the obtained hydrogel beads are dehydrated, for example, by freeze-drying or drying in a vacuum oven, and stored in a dry state.

[0189] According to one particular embodiment, the bead preparation method according to the present invention includes: -A mixture of carboxyalkyl chitosan mix, another polymer, preferably hyaluronan, and at least one crosslinking agent is solubilized in an alkaline phase; - Form droplets of carboxyalkyl chitosan and other polymers, preferably hyaluronane, and then possibly solidify them in a coagulation bath containing a coagulant, for example, via ionic gelation; - Perform a cross-linking reaction on the droplets; - Obtain a co-crosslinked matrix of carboxyalkyl chitosan and other polymers, preferably hyaluronan beads.

[0190] Therefore, for example, the method according to the present invention includes: - Prepare a solution of carboxyalkyl chitosan (and possibly another polymer, preferably hyaluronan) and 1,4-butanediol diglycidyl ether (BDDE) in a basic solution containing NaOH; and then -The solution is dispersed in droplets at room temperature in a coagulation bath containing a coagulant, usually calcium chloride in a water / ethanol mixture, causing coagulation in the form of spherical beads; and then, -BDDE is used to crosslink carboxyalkyl chitosan (and possibly other polymers) to provide hydrogel beads of crosslinked carboxyalkyl chitosan; - To remove excess coagulant and BDDE and balance the hydrogel of the beads to the desired pH and osmotic pressure, the cross-linked carboxyalkyl chitosan hydrogel beads are neutralized and washed; -Optionally, select hydrogel beads according to their size.

[0191] In this way, beads in the form of a hydrogel containing the matrix according to the present invention are obtained.

[0192] According to one alternative method, the matrix according to the present invention is sterile.

[0193] Providing hydrogels in the form of beads from the matrix of the present invention by this method is beneficial.

[0194] composition Advantageously, the compositions of the present invention may also contain polymers other than crosslinked carboxyalkyl chitosan, particularly in the phase related to beads. Thus, if the composition includes an aqueous phase, according to one alternative method, the aqueous phase and / or beads may contain one or more polymers.

[0195] According to one favorable alternative, the polymer (of the associated phase and / or beads) is a biopolymer, for example, a polysaccharide crosslinked by covalent or non-covalent bonds, whether oxidized or not, such as glycosaminoglycans, and more specifically, one of hyaluronans, such as sodium hyaluronate or its derivatives. According to one alternative, the phase associated with the hydrogel beads also contains carboxyalkyl chitosan, whether crosslinked or not.

[0196] One of the advantages of combining or crosslinking carboxyalkyl chitosan with several other polymers is that it can add to their properties or create synergistic effects.

[0197] According to one alternative method, multiple beads are associated with an aqueous phase, a lipophilic phase, a hydrophilic lipid phase, or another solid phase, and may contain one or more polymers, such as carboxyalkyl chitosan, hyaluronan, or any combination thereof.

[0198] According to one favorable alternative, the aqueous phase composition and / or beads are in the form of a hydrogel having a pH and osmotic pressure balanced with the physiological medium.

[0199] According to one alternative method, the step in which the beads are bound is specific to injection into a human or animal body.

[0200] According to one favorable alternative, the aqueous phase and / or beads are forms specific to injection into the body of a human or animal.

[0201] The present invention also relates to a composition that can be administered to humans or animals, the composition comprising a plurality of beads as defined in accordance with the present invention.

[0202] According to one preferred alternative, the hydrogel beads according to the present invention are formulated in the form of a dispersion or suspension in an injectable aqueous medium (possibly slightly viscous) for human or animal use. This is generally referred to as the composition according to the present invention.

[0203] According to one alternative method, the composition is formulated as an injectable suspension, ready for ophthalmic administration or implantable in humans or animals.

[0204] According to one advantageous alternative method, particularly for products intended for skin injection, sorted or unsorted beads are incorporated into a solution of one or more hydrophilic biopolymers, preferably hyaluronan.

[0205] According to one alternative method, the bead formulation is prepared in a device suitable for delivery, such as a syringe, and then properly sterilized, for example by autoclaving, by applying a cycle aimed at ensuring sterility. Thus, according to one alternative method, the present invention relates to a sterile syringe containing a formulation of hydrogel beads based on cross-linked carboxyalkyl chitosan, ready for delivery by injection.

[0206] The mass ratio [carboxyalkyl chitosan / hyaluronan] is, for example, 5 / 9 to 95 / 5, for example, 10 / 90 to 90 / 10, for example, 20 / 80 to 80 / 20, and also, for example, 30 / 70 to 70 / 30. According to an alternative method, the mass ratio [carboxyalkyl chitosan / hyaluronan] is 1 / 1 (i.e., 50% chitosan and 50% hyaluronan).

[0207] The aqueous medium may be water or an aqueous solution with a pH, and the osmotic pressure is adjusted using a buffer solution to which, for example, salts and / or possibly polyols, such as sorbitol, mannitol, glycerol and / or trehalose are added.

[0208] According to one embodiment, the matrix composition has an osmotic pressure of 100 to 700 mosm / kg, preferably 120 to 500 mosm / kg.

[0209] According to one embodiment, the matrix composition has an osmotic pressure of 100 to 500 mosm / kg, preferably 120 to 270 mosm / kg.

[0210] According to one embodiment, the matrix composition has an osmotic pressure of 250-400 mosm / kg, preferably 280-350 mosm / kg.

[0211] According to one alternative method, the matrix composition has an osmotic pressure appropriate for joint movement.

[0212] According to one alternative method, the matrix composition has an osmotic pressure that is compatible with the eye or the intraocular surface.

[0213] According to one alternative method, the matrix composition has an osmotic pressure compatible with the skin and subcutaneous fat.

[0214] According to one alternative method, the osmotic pressure of the matrix composition is preferably in the range of 100 to 400, more specifically in the range of 120 to 350 mosm / kg.

[0215] According to one alternative method, the composition according to the present invention is sterile.

[0216] Advantageously, the compositions according to the present invention can be contained in an injection, implantation, or infusion device, such as a syringe or vial. Advantageously, an injection device, such as a syringe, can then be steam sterilized. The device, such as a syringe, can then be packaged, preferably aseptically or in an aseptic manner. This could also be a pouch, capsule, or vial that allows for the infusion of the compositions according to the present invention, which is aseptically filled after sterilization of the formulation or sterilized immediately after filling.

[0217] According to one alternative method, the composition according to the present invention, more specifically the composition comprising hydrogel beads according to the present invention, is sterilized by filtration and / or steam sterilization before filling an injection, implantation, or infusion device, such as a syringe or vial.

[0218] Those skilled in the art know the sterilization techniques for hydrogels to obtain the desired sterile hydrogel. Those skilled in the art can utilize several types of apparatus for sterilization by heat or steam, and several types of cycles to remove microbial charge.

[0219] More specifically, the present invention relates to an injectable composition comprising hydrogel beads according to the present invention.

[0220] The present invention also relates to a pharmaceutical composition comprising at least hydrogel beads according to the present invention.

[0221] According to one alternative method, the compositions according to the present invention are used as injectable, implantable or ready-to-inject pharmaceutical compositions, or as medical devices that are ready for injection, implantable or ready-to-inject.

[0222] The present invention also covers compositions according to the present invention in a dried form, particularly in a freeze-dried form. Freeze-dried products can be (re)dispersed more specifically before use.

[0223] More specifically, the present invention relates to compositions according to the present invention for use in therapeutic purposes, and for example, to methods including injection of the compositions subcutaneously, intracutaneously, intraocularly, or intraarticularly, intramucosally, intraperitoneally, or intramuscularly, for the purpose of repairing, regenerating, or filling at least one body tissue / fluid that is in need of repair or filling.

[0224] It is advantageous to use chitosan of sufficient purity for the intended application.

[0225] It is advantageous to use hyaluronan of sufficient purity for the intended application.

[0226] The biomechanical properties required by the compositions according to the present invention may vary in nature and amplification according to instructions, for example, the tissue in which the hydrogel must be incorporated, the mechanism of action, or the effect and duration of the effect intended to benefit the patient.

[0227] Advantageously, the properties of the compositions according to the present invention, and more specifically the properties of the hydrogel beads according to the present invention, are conformed to the instructions. To adapt these properties, the final concentration of the polymer (carboxyalkyl chitosan and / or other polymers such as hyaluronan) and / or the crosslinking rate are adjusted, for example, particularly through the crosslinker / polymer mass ratio and / or the properties and / or amount of the ions and / or the initial molecular mass of the polymer and / or the method for preparing the polymer droplets.

[0228] In particular, the present invention relates to hydrogel beads according to the present invention that are elastic, especially when a durable volume increase is required at the skin, subcutaneous (subcutaneous) or periosteal level. More specifically, the present invention relates to beads suspended in a continuous phase. This phase can also provide variable viscoelasticity or other advantageous properties, such as a lubricating effect, to the target product. More specifically, in the case of a product used in a joint, this allows for shock absorption and both the cartilage and the beads to benefit from the lubricating effect. Compositions of the present invention can have a variable level of elasticity, which can be adjusted according to the instructions and characterized by measurement of the elastic modulus by rheometry.

[0229] The present invention also relates to a composition comprising a phase containing a plurality of hydrogel beads according to the present invention. At this stage, the beads can be said to be incorporated.

[0230] According to one alternative method, the phase containing the beads is the aqueous phase.

[0231] According to one alternative method, the phase containing the beads is a lipophilic phase.

[0232] According to one alternative method, the phase containing the beads is a hydrophilic lipid phase, more specifically, an emulsion such as direct, inverse, single, or multiple.

[0233] According to one alternative method, the phase containing the beads is a solid phase.

[0234] According to one specific alternative method, the phase containing the beads is a continuous aqueous phase.

[0235] The present invention relates to an injectable composition characterized by comprising hydrogel beads according to the present invention and a phase in which the beads are incorporated.

[0236] The present invention relates to a pharmaceutical composition comprising hydrogel beads according to the present invention.

[0237] According to one alternative method, the composition is formulated as a pharmaceutical composition or a composition for implementation in a therapeutic treatment.

[0238] According to one alternative method, the composition may be used as a pharmaceutical composition for use in therapeutic methods, for example, for repairing or filling at least one body tissue requiring repair or filling, including intracutaneous, intracutaneous, mucous membrane, ocular, intraocular, or intra-articular infusion, local delivery, or injection of the composition.

[0239] According to one alternative method, the compositions according to the present invention are used in a method for treating, repairing or filling at least one bodily fluid or tissue that requires repair or filling, wherein the bodily tissue is selected from any of the following: vocal cords, muscles, ligaments, tendons, mucous membranes, sexual organs, bones, joints, eyes, skin, or any combination thereof, more specifically, tissues belonging to the surface of skin, cartilage, synovial membrane, skin wounds, or eyes.

[0240] The present invention relates to a composition for the treatment of arthropathy, or for use, for example, by injection into a biological fluid, such as synovial fluid, or by mixing with a biological fluid, such as blood, and implanting it into cartilage, for the repair of cartilage deficiency. Biological fluids are bodily-derived fluids that have been or have not been treated to alter their composition.

[0241] 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.

[0242] The present invention relates more specifically to the present invention for use in treatment, surgery or cosmetic treatment, and more specifically to treatment in dermatology for the prevention of postoperative tissue adhesion, and in rheumatology, ophthalmology, gynecology, cosmetic medicine, plastic surgery, internal medicine surgery, orthopedic surgery, and gynecological surgery.

[0243] The present invention also relates to compositions according to the present invention for use in the eye, for example, for the treatment of dry eye syndrome, corneal lesions, or intraocular delivery in any other ocular tissue.

[0244] The present invention also relates to compositions that are applied to the surface of the eye, particularly to lubricate or regenerate the ocular surface, or to prevent or treat corneal lesions or dry eye syndrome.

[0245] In fact, the present invention also relates to a composition of eye drops comprising carboxyalkyl chitosan as defined in accordance with the present invention.

[0246] According to one alternative method, the subject is affected by inflammatory pathologies of the joints (e.g., osteoarthritis, arthritis, etc.).

[0247] More specifically, the present invention relates to compositions for the treatment of arthropathy, arthritis, or for the repair of cartilage deficiency by injection into the synovial space or transplantation at the level of cartilage deficiency.

[0248] 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.

[0249] Accordingly, according to one preferred alternative method, the present invention relates to a medical device comprising a chamber containing a composition according to the present invention in a dry form, particularly under freeze-drying conditions, and possibly one or more other chambers containing one or more active substances, additives or excipient products.

[0250] The compositions according to the present invention may also comprise one or more activators for desired indications, and / or one or more additives or excipients that enable modification of the properties of the compositions according to the present invention.

[0251] According to one alternative method, the beads according to the present invention contain or, in other words, encapsulate one or more activators for a desired indication, and / or one or more additives or excipients that allow for modification of the properties of the composition according to the present invention. For example, they may be pharmaceutically active ingredients or nutrients for a delivery zone, more specifically for injection, intravenous infusion, or implantation zone.

[0252] The present invention also relates to compositions according to the present invention formulated as compositions for implementation in pharmaceutical compositions or therapeutic methods.

[0253] The present invention also relates to compositions according to the present invention for the preparation of pharmaceutical compositions or medical devices.

[0254] The present invention also relates to compositions of the present invention for use in the treatment of arthropathy, or for use in repairing cartilage deficiency by injection into synovial pockets, for example, or after mixing with biological fluids, such as blood, and by transplantation into cartilage / bone.

[0255] The present invention also relates to compositions according to the present invention for use in methods of therapeutic or cosmetic care by filling skin tissue. More specifically, this means, for example, injecting the compositions according to the present invention subcutaneously, intracutaneously, intramucosally, or intramuscularly.

[0256] The present invention also relates to compositions according to the present invention for use in methods of treating the skin by multiple injections via an intradermal route or other tissue. Such compositions can typically be used in dermatology as treatments for cosmetic purposes.

[0257] The present invention also relates to compositions of mucus replenishment agents for use in therapeutic methods. This means, for example, injecting the compositions of the present invention at the intra-articular level to limit friction, particularly on the cartilage surface of the joint.

[0258] The present invention also relates to compositions according to the present invention for use as cell vectors of one or more cell types and / or vectors of one or more activators. These must be effective agents from a pharmaceutically or biological standpoint. The compositions of the present invention may, in fact, be compatible with the presence of cells, preferably living cells. Among living cells of interest, for example, the following can be mentioned: 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 used as therapeutic vectors for targeted local delivery and / or controlled release of at least one therapeutic agent.

[0259] According to one alternative, the beads according to the present invention contain one or more activators, e.g., water-soluble molecules (whole or partially) and / or non-water-soluble molecules. According to one alternative, the beads according to the present invention contain one or more anti-inflammatory molecules, e.g., corticosteroids, particularly glucocorticoids. An example of a water-soluble module is ascorbic acid. Another example of a water-soluble activator is the anti-inflammatory agent dexamethasone. Another example of a non-water-soluble activator is the anti-inflammatory agent triamcinolone. According to one alternative, the activators encapsulated in the beads are high molecular weight (e.g., higher than 10 kDa) water-soluble biomolecules and, for example, proteins.

[0260] The mechanism and release of the activator depend on its dissolution profile, either by diffusion in the case of water solubility, or by the decomposition of the beads, such as hydrolysis or mechanical decomposition.

[0261] Advantageously, the compositions and beads according to the present invention provide a system having the localized and controlled release of one or more activators. Advantageously, the bead polymer compositions according to the present invention can be adapted to a targeted release profile (which is dependent on indication and molecule), and thus the actual versatility can be adapted to the encapsulation and release of different activators.

[0262] According to one alternative method, blood, or plasma, or platelet lysate, or platelet-rich plasma, or any other biological fluid is added together with the composition of the present invention, for example, to enhance the performance of the product.

[0263] According to one alternative method, the composition according to the present invention is formulated in solid form, for example, under a film, tube, or porous foam, which can release beads upon delivery.

[0264] According to one alternative method, the composition is formulated in the form of a spray.

[0265] The present invention also relates to compositions according to the present invention for use in methods of cosmetic treatment or care of one or more tissues or organs affected by excessive temperature, such as in the case of burns.

[0266] The present invention also relates to compositions according to the present invention for use in therapeutic methods of cartilage repair (for example, by transplanting them into cartilage defects to support their regeneration).

[0267] The present invention relates to a physiologically acceptable composition intended to be delivered topically by injection, infusion, or implantation to one or more biological tissues subjected to oxidative stress: - 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 enhance the repair of cartilage defects; - Intraosseous grafting to enhance bone repair (bone induction / bone conduction); - Subcutaneous, subcutaneous and / or periosseous injections to fill or regenerate skin or hair follicles, to increase volume in cases of lipodystrophy, or to deliver active ingredients; - Intravenous infusion into the eye to alleviate symptoms on the surface of the eye or prevent changes. For example, treatment of dry eyes or corneal lesions, or delivery of active ingredients; - Intraocular injection, for example, as an adjunct to ophthalmic surgery to optimize the effectiveness of glaucoma surgery or vitreous replacement, for the regeneration of anterior or posterior segment tissue, and / or intraocular delivery of active ingredients; - Administration to internal tissues and organs to prevent postoperative adhesion; - Administration to wounds, tears, lacerations, and cavities of tissues and organs such as skin, bone, cartilage, cornea, tendons, and meniscus to enhance repair or regeneration; - Injections into the vulvar mucus level for the treatment of vulvar pain; - For cell culture, as a support or scaffold for cells, especially in bioreactors, for example, tissue engineering or remodeling, or for the production of desired substances by cells.

[0268] The present invention also relates to compositions according to the present invention that form intra-articular mucus supplements.

[0269] Generally, the osmotic pressure and pH ranges of the compositions are adapted and typically close to the osmotic pressure and pH values ​​of the tissue in contact with the composition according to the present invention.

[0270] Advantageously, the compositions according to the present invention are sterile. A significant advantage is that the compositions according to the present invention are sterilized by moist heat.

[0271] According to one alternative method, the composition of the present invention is transparent or translucent.

[0272] More specifically, the present invention relates to a preferably sterile item or package comprising one or more drip infusion or injection devices pre-filled with the composition according to the present invention, more specifically in the form of a hydrogel. These devices typically allow the product to be injected in liquid or pre-filled syringe form.

[0273] Advantageously, one composition of the present invention can be stored for several months, preferably in an item or package suitable for its labeling.

[0274] The compositions and beads according to the invention can advantageously be sterilized. Thus, the invention relates to a sterilized crosslinked carboxyalkyl chitosan. Thus, the crosslinked carboxyalkyl chitosan is sterile, especially in applications where this is required.

[0275] According to one alternative, the beads or compositions of the invention are sterilized by steam, based on methods known to those skilled in the art and / or recommended by the European Pharmacopoeia.

[0276] According to another alternative, the composition can be sterilized by filtration using a suitable filter, for example a filter having a porosity of 0.2 μm or less.

[0277] The invention also covers a method of therapeutic treatment comprising injecting a composition according to the invention.

[0278] The invention also covers the use of a composition according to the invention for the preparation of a pharmaceutical composition, more specifically for a therapeutic treatment as more specifically defined by the invention, for example.

[0279] The invention also covers a method of cosmetic care, i.e. non-therapeutic care, comprising injecting a composition according to the invention. For example, this can be the treatment of lipodystrophy or fillers, or one or more areas of visible damaged tissue for cosmetic purposes, for example after an accident or surgical procedure.

[0280] Tissue is a set of similar cells of the same origin grouped into a functional set that participates in the same function. Among the tissues are skin tissue (e.g. epithelial tissue), connective tissue, muscle tissue, adipose tissue, and nervous tissue.

[0281] "The composition according to the invention" or equivalent terms means, in the context of the invention, the composition defined in the invention, including any one of its alternatives, by a particular or particular embodiment, independently or by any combination thereof that includes desirable properties.

[0282] Other objects, features, and advantages of the present invention will become readily apparent to those skilled in the art by reading the description, which refers to the examples given for illustrative purposes only and which should not in any way limit the scope of the present invention.

[0283] The examples are entirely part of the present invention, and any feature that appears new with respect to any prior art state from the description taken as a whole, including the examples, is entirely part of the present invention in terms of its function and generality.

[0284] Therefore, each example has a general range.

[0285] On the other hand, unless otherwise specified, in the examples all percentages are given in mass, temperature is expressed in °C unless otherwise specified, and pressure is atmospheric pressure unless otherwise specified. [Examples]

[0286] The force of ejection using a thin needle and the integrity of the beads after ejection. Measurements are performed using a load cell (Instron 500N, Mecmesin) and a compression bench (Inston 5566, Mecmesin) equipped with a sample holder fitted to the syringe. A 1 mL BD glass syringe containing the formulation to be tested is set to room temperature. A 27 G needle (1 / 2" length, TSK) is attached to the syringe, and the syringe is placed in the sample holder. The compression cell is lowered at a speed of 10 mm / min for 1 minute, collecting a homogeneous gel / phase mixture in a watch glass. The ejection force is determined as the average value of the plateau in the force curve displacement at a constant speed. Bead integrity is checked after ejection using an optical microscope (shape, surface).

[0287] Optical microscope (size, sphericity, and visual inspection) Images of the beads are recorded using an Olympus SC-50 camera and an Olympus CKX-41 microscope equipped with Olympus Stream image capture software, and processed using CellSens Dimension Desktop software. To do this, one to two drops of the bead suspension are placed on a borosilicate glass slide and covered with an object cover. The slide is then immediately placed on a microscope plate.

[0288] To determine the dimensions and sphericity of the beads, a 4x magnification is selected, and a minimum of 20 beads are displayed in each image. Ten images are saved and analyzed with CellSens software to determine the dimensions (width, length, and diameter) of the beads.

[0289] The sphericity of a bead is equal to the square exponent of its width over its entire length. The final reported value is the average of the measurements for each sample after analyzing 10 images (a minimum of 200 beads in total). A perfectly spherical bead has a sphericity of 1.

[0290] To perform a visual inspection aimed at confirming the absence of undesirable insoluble species on the surface of the heart or beads, place 1-2 drops of the bead suspension onto two slides and observe the beads at 4x, 10x, and 40x magnification.

[0291] water content Place approximately 500 mg of hydrated beads on a piece of paper and remove any excess unbound water. Place the beads on a Moisture Analyzer MA37 (Sartorius) moisture scale plate according to the method of European Pharmacopoeia 2.2.32 and allow them to dry. Moisture content is calculated by the difference between the wet mass and the dry mass.

[0292] ash Ash content reflects the ability to remove excess mineral substances during the washing process, primarily calcium chloride during the coagulation process. Measurement is performed according to European Pharmacopoeia Method 2.4.16 (Total Ash), by first measuring the moisture content on pre-dehydrated beads in a vacuum incubator, followed by calcination at 600°C. Ash content is based on the mass of the dry beads.

[0293] Contents of BDDE and by-products of residual BDDE Unreacted BDDE crosslinking agents may be found in their natural form or as by-products. The concentration of BDDE or its residual by-products is determined by LC-MS according to a method conforming to that of Fidalgo et al. (Detection of novel reaction by-product BDDE crosslinked autoclaved hyaluronic acid hydrogel by LC-MS analysis, Medical Devices: Evidence Res 11, 367, 2018). For CC hydrogel bead-based products for cutaneous injection, an upper limit of 2 ppm of BDDE and its by-products (total content) is imposed (De Boulle et al., Review of the metabolism of 1,4-butanediol diglycidyl ether crosslinked hyaluronic acid dermal fillers, Dermatol Surg 39, 1758, 2013).

[0294] Determination of bead size (diameter) by laser diffraction method Laser diffraction measurements are performed using a Mastersizer 2000 (Malvern) equipped with a "Hydro Unit" (Hydro 2000SM). The amount of the bead suspension to be tested is introduced into the Hydro Unit containing pharmaceutical-grade water using a pipette, and a darkening titration is performed beforehand to determine the optimal darkening level, until a darkening level of 3%–10% is obtained. The Hydro Unit is stirred at a rate that ensures uniform dispersion of the sample and the absence of bubbles in the system. The optical parameters (refractive index) used are 1.52 for the sample (bead suspension) and 1.33 for the water. Each measurement is performed three times. The bead size distribution (diameter) is calculated and supplied by the instrument (average of the three measurements). The instrument calculates the bead size distribution, and the software is set to obtain the following data: average volume diameter, diameter at 10% (D0,1), 50% (D0,5), and 90% (D0,9) of the volume distribution.

[0295] Biomechanical properties (elastic modulus) determined by rheometry The biomechanical profile of the continuous-phase bead suspension is characterized using a DHR-2 Hydrod Rheometer (TA Instrument) with a 20 mm planar shape spaced 700 μm apart, at a temperature of 37°C, a frequency of 0.7 Hz, and a deformation amplitude of 0.1 at 1000%. The instrument provides a value of the elastic modulus (G'). Since each measurement is performed three times, the average value of G' is calculated from the three measurements.

[0296] Surface roughness of beads as measured by a confocal laser scanning microscope. The support is specially designed to be able to lock the bead suspension during analysis by a confocal laser scanning microscope. This is a disk based on a hydrated polyacrylate type biomaterial (described in International Publication WO2006063994), which contains wells where the beads are placed. The beads are analyzed in a suspension of phosphate buffered saline. One or two drops of the bead suspension are dripped, and measured at a magnification of 20 using a VK-X (Keyence) confocal laser scanning microscope. To evaluate the relative roughness of the beads, the image of the beads is compared with the image of the surface of a "mirror-polished" polyacrylate slide photographed as a "very smooth" control and the image of freeze-dried beads as a "very rough" control.

[0297] Qualitative evaluation of the compressive strength of the beads Three beads are arranged flat in a triangle, covered with a glass slide, and a weight with a mass increasing from 10 g to 50 g is placed thereon. If the beads resist a mass of 10 g, the mechanical compressive strength is considered "good"; and if they resist 50 g, it is considered "very good". If it is less than 10 g, the strength is considered unacceptable.

[0298] Method for preparing carboxyalkyl chitosan (according to the international application filed under PCT / EP2020 / 064159) For the carboxymethyl chitosan (CC) of the examples, reference CC1, CC2 and CC3, the carboxymethylation reaction of chitosan is carried out according to the following method. A lump of 30 g of chitosan derived from Agaricus bisporus is dispersed at 50% (m / v) in 600 mL of isopropanol, 41 mL of water, and 163 mL of sodium hydroxide. 135 g of monochloroacetic acid (MCA) is dissolved in isopropanol, and the solution is added to the chitosan suspension. After the reaction, the polymer is recovered by precipitation in ethanol, and then purified by solubilization cycles in water and precipitation in ethanol. CC is recovered after drying.

[0299] For CC1 and CC2 reference CC, one additional step is taken according to the following method: Disperse a 21g mass of CC from step 1 in 570mL of water and adjust the pH of the solution to approximately 8. Add 10mL of acetic anhydride and stir the solution at room temperature. Adjust the pH of the solution and then add 10mL of acid anhydride. The pH is adjusted to approximately 7.0-7.5. The CC is collected after drying.

[0300] In Examples 1-5, the polymers used are those listed in Tables 1 and 2. [Table 1]

[0301] [Table 2]

[0302] Example 1 - Preparation of beads formed by ion gelation and subsequent covalent crosslinking with BDDE In this example, we are attempting to form CC beads using a process in which polymer droplets are first coagulated by calcium chloride ion gelation, and then stabilized by covalent crosslinking by adding the crosslinking agent 1,4-butanediol diglycidyl ether (BDDE, Alfa Aesar, CAS [2425-79-8]). The success of this method is determined by evaluating the beads' ability to withstand an aqueous media washing process, and then, if they withstand washing, their ability to withstand slight manual compression.

[0303] The three CC reference solutions (Table 1) are prepared in water at a concentration of 40 mg / mL. The pH of the solution is adjusted to 3, 6, or 13 by adding 1N hydrochloride and / or 1N sodium hydroxide to the desired pH, diluted to 30 mg / mL with water, and filled into a 1 mL syringe. In parallel, a 30 mg / mL calcium chloride solution is prepared in a 30 / 70 (v / v) ethanol / water mixture.

[0304] In each CC solution, droplets are formed by slowly extruding CC from a 27G needle (2 mL). The droplets fall into a calcium chloride solution (10 mL) and solidify into jelly-like beads. After suspending the beads in the calcium chloride solution for 1 hour, 80 μL of BDDE (BDDE / CC ratio 1.33 μL / mg) is added. After stirring for 1 hour, the beads are collected on a filtration membrane. The beads are washed by resuspending them in water and filtering to remove excess unreacted calcium chloride and BDDE. This process is repeated three more times, after which the beads are suspended in water for 1 hour.

[0305] The formation of jelly-like beads was observed in the calcium chloride solution and during the crosslinking and washing processes. However, after 15 minutes in the final aqueous solution (after four washing processes), all beads were solubilized regardless of the pH of the initial solution. This indicates that covalent crosslinking of the CC chain of BDDE did not occur.

[0306] Example 2 - Hydrogel beads based on carboxymethyl chitosan (CC) This example provides cross-linked CC hydrogel beads according to the present invention, which are spherical (sphericity greater than 0.70), smooth, resistant to compression, have a diameter of approximately 20 μm to 400 μm, and have a mass water content exceeding 85%. The beads are washed and purified in an aqueous medium and evaluated for their ability to balance pH and osmotic pressure. It is also required that the beads be classified according to their diameter.

[0307] The beads are manufactured according to the following general method using the special conditions and parameters shown in Table 3. The beads are then dispersed in a continuous phase, the mixture is packaged, and optionally sterilized according to the specifications of the target product, for example, as described in Example 3. Alternatively, the beads may be dried, for example, by freeze-drying or other suitable drying method, and prepared in a dry form.

[0308] In this example, a solution of CC1 (Table 1) (30 mg / mL) is prepared in a 1% NaOH solution, and BDDE is added at a rate of 3 μL (0.0162 mM) per mg of CC. This solution is dispersed into droplets using a mini-Buchi spray via a 2.8 mm diameter binary nozzle at an air pressure of 0.3 bar. The droplets are dropped at room temperature into a solidification bath based on calcium chloride (50 mg / mL) in a water / ethanol 30 / 70 (v / v) mixture and solidify as spherical beads. Next, a crosslinking step of CC with BDDE is performed while controlling the temperature and time.

[0309] Next, the suspension is neutralized by adding a 1N hydrochloric acid solution, the beads are collected on a filtration membrane under vacuum, they are suspended again in hydrochloric acid and then collected on a filtration membrane. They are then suspended in physiological saline phosphate buffer and collected by filtration, and this process is repeated until a suspension of gel beads is obtained with the correct osmotic pressure and buffer pH.

[0310] By allowing the larger beads to settle, the smaller beads are removed. Finally, the gel beads are sorted by size by placing them on a 400 μm porous sieve, then filtering them under vacuum using a reduced porous filter. Thus, three fractions of hydrogel beads with reference SB2-A, B, and C are obtained, which are stored in suspension in physiological saline phosphate buffer.

[0311] [Table 3]

[0312] Stable beads are actually obtained after the washing, sorting, and suspension processes. The three parts of the beads are characterized according to the methods detailed in the description: size distribution by laser diffraction (Mastersizer 2000, Malvern); moisture content using a humidity scale (Moisture Analyzer MA37, Sartorius); compressive strength by qualitative methods (weight of 50g mass); sphericity and visual inspection with an optical microscope; and surface roughness by scanning confocal microscopy. The results are shown in Table 4.

[0313] [Table 4]

[0314] This bead preparation method allows for the production of CC hydrogel beads according to the properties required for the present invention. Furthermore, these hydrogel beads withstand the washing process in aqueous media, removing excess substances, particularly calcium chloride and BDDE, and balancing the media with respect to pH and osmotic pressure, as can be seen from the formulation of these beads in Example 3.

[0315] Example 3 - Preparation of sterile formulations based on CC hydrogel beads Fractions of CC hydrogel beads reference SB2-A or SB2-B from Example 2 were collected and then mixed with a 2% (m / m) sodium hyaluronate solution (reference HA2 in Table 2) in physiological saline phosphate buffer at a mass ratio of 70 / 30 to separate the formulations. The final target pH was 7.2 ± 0.2 and the final osmotic pressure was 315 ± 35 mOsm / kg.

[0316] The formulations are slowly agitated at room temperature for approximately 12 hours. Next, they are packaged into 1 mL glass syringes (Hypak, BD) and closed with stoppers (Hypak, BD). The syringes are then transferred to an autoclave (Systec, DX65). This yields sterile syringes containing two formulations of CC hydrogel beads (reference FSB1-A and FSB1-B) ready for delivery by injection.

[0317] The final formulation is characterized (pH, osmotic pressure, residual BDDE content, elastic modulus G', ease of dispensing the composition from a 27G fine needle (TSK, 1 / 2")). Next, the beads are collected to confirm that they have not been altered by the method (shape, surface, compressive strength).

[0318] The properties and composition of the beads are shown in Table 5. For reference, the properties of two commercially available products are reported in the skin volume-enhancing claims for cosmetic purposes: Restylane LYFT (Galderma) and Ellanse-M (Sinclair Pharma).

[0319] [Table 5]

[0320] From this example, it can be concluded that the CC hydrogel beads according to the present invention maintain homogeneity, sphere shape, smooth surface, and compressive strength, and can therefore be formulated as a solution of HA, then packaged in a 1 mL syringe and sterilized without modification.

[0321] The two formulations, FSB2-A and FSB2-B, conform in terms of pH balance and osmotic pressure with the aqueous phase used. They also conform in terms of residual BDDE content and BDDE by-products (<2 ppm).

[0322] The formulation is easily injectable via a 27G needle and requires a force of 9-10N to release into the air, making it perfectly satisfactory, especially for subcutaneous injection. As confirmed by visual inspection using an optical microscope, the beads maintain their integrity even after release. Therefore, the formulation would be suitable for skin volumization applications for cosmetic purposes, particularly by subcutaneous injection.

[0323] The modulus of elasticity of these two bead formulations, SB2-A and B, is significantly higher than that of the reference commercial product, and this is due to the beads themselves, not the phase in which the beads are suspended. In fact, non-crosslinked HA solutions without beads are known to have a significantly lower G' value. This high elasticity of the beads is advantageous for signs of tissue volumeization, as they can deform during tissue migration without being compressed or losing their volume-generating ability.

[0324] Example 4 - Variable composition CC and HA hydrogel beads In this example, the properties of CC hydrogel beads are to be adjusted while maintaining manual compressive strength, a smooth surface, and spherical shape. To achieve this, hyaluronan is added to the CC solution (reference CC2 in Table 1) and BDDE in the form of sodium hyaluronate (reference HA1 in Table 2). Two different CC / HA mass ratios (50 / 50 and 75 / 25) are used for the same total concentration of polymer at 40 mg / mL. The goal is to form beads with a diameter of approximately 10 μm to 400 μm. The same general method as in Example 2 is applied, adjusting the parameters as described in Table 6.

[0325] [Table 6]

[0326] Resistant beads are obtained at the end of the purification, compounding, and sterilization processes. The properties of the beads and the final sterile formulation are shown in Table 7.

[0327] [Table 7]

[0328] CC and HA beads were obtained, with the moisture content (93% and 97%) increasing only in Example 1 (90%) compared to the CC beads. They were more deformable, elastic, and sticky than the beads of Example 1.

[0329] It can be concluded that the properties of CC hydrogel beads can be adjusted by changing the composition of the polymer solution, for example, by adding HA in various ratios. The same general method can be applied, but several parameters should be considered.

[0330] Example 5 - Hydrogel beads based on CC with variable molecular structure In this example, by adjusting the parameters, it is required to form hydrogel beads from Agaricus bisporus-derived CC (see CC3 in Table 1) with a DA of less than 30% following the same general method as in Example 2 (Table 8).

[0331] Depending on the facility, droplets are formed by passing the polymer solution through a 30G needle instead of the spraying process of Examples 2-3. Consequently, the diameter of the beads is larger than that of the beads in Examples 1-3 (approximately 1 mm). Nevertheless, this method allows for the determination of the bead suitability in terms of sphericity, smooth surface, and adequate resistance to manual compression after the washing process.

[0332] [Table 8]

[0333] Beads that resisted washing and suspension in buffer solution were successfully obtained. Their characteristics are shown in Table 9.

[0334] [Table 9]

[0335] It can be concluded that spherical, smooth, and manually compressible beads from carboxymethyl chitosan with a DA of less than 30% can be formed according to general methods.

[0336] Example 6 - CC Hydrogel Beads for Volume Enhancement of Rat Skin Tissue In this example, local tolerance and volumetric capacity of CC hydrogel beads are evaluated after subcutaneous injection into rats for 3 months. Two formulations based on CC hydrogel beads from Example 3, ready for injection, are used: FSB2-A (average diameter at 90 μm volume) and FSB2-B (average diameter at 200 μm volume).

[0337] For comparison, we simultaneously study the following two commercially available injectable products intended for cosmetic skin volumization: Ellanse-M (Sinclair Pharma), based on solid and unhydrated microspheres of polycaprolactone in carboxymethylcellulose gel; and Restylane® LYFT (Galderma), a hydrogel of crosslinked and extruded hyaluronic acid in the form of non-spherical particles.

[0338] In this example, the general term "particles" is used to simultaneously refer to CC hydrogel beads, polycaprolactone solid microspheres, and extruded cross-linked hyaluronic acid particles.

[0339] Protocol. Each Wistar female rat receives a subcutaneous injection of 200 μL of each product using a 27G needle (a total of four injection sites per animal, two on the left flank and two on the right flank). Regularly over a 3-month period, any signs of reaction or skin irritation (erythema, edema) attributable to the tested product are scored and evaluated. Each time, for the skin tissue at each transplant site, the dimensions of the relief created at the level are measured and the volume is estimated (height x width x length, mm). 3 The average volume of the six transplant sites is calculated for each product and each time. Table 10 shows the average volume over time that represents the short, medium, and long term of the study.

[0340] Evaluation of local tolerance. For the four products tested, the scores for erythema and edema were zero throughout the first week. They remained zero throughout the 3-month observation period. Therefore, no clinical signs of skin irritation were observed for any of the four products tested, and their tolerance is considered excellent.

[0341] Evaluation of volume-enhancing effect. Injecting the two CC hydrogel bead-based products immediately resulted in skin volumization, with a slight increase in volume during the first few days. The volume-enhancing stabilized from day 7 and remained at a nearly constant level throughout the approximately 3-month follow-up period (day 85). Therefore, these products function as expected for skin volume enhancement. No significant volume difference was observed between the two products.

[0342] The volume-enhancing effect of the commercially available Ellanse-M product is characterized by significant relaxation during the first two weeks, after which it stabilizes at the same level as the two base products using CC hydrogel beads until the end of the study.

[0343] The average relief size produced by the commercially available Restylane® LYFT product was the same as that of other products during the first two weeks. Then, due to a gradual decrease from day 14 to the end of the study (day 85), it was significantly lower than at all times.

[0344] [Table 10]

[0345] In this example, two products based on CC hydrogel beads with average diameters of 90 μm and 200 μm are concluded to be readily injectable subcutaneously, well-tolerated, and do not cause clinically visible irritation or inflammatory reactions during the 3-month study period, as expected as an indication of skin volumization for cosmetic purposes. Their subcutaneous injections allow for the creation of a certain and expanded relief of skin tissue in a localized manner, as desired.

[0346] With equivalent injection volumes, the resulting relief exhibits stable volume relief over time, reaching a level similar to that produced by Ellanse-M products. This is higher than that produced by Restylane® LYFT products and gradually decreases over time. Therefore, CC hydrogel beads confirm the safety and efficacy of CC hydrogel beads as a volume-enhancing product for tissue, more specifically, skin.

[0347] Example 7 - Evaluation of local reactions after injection of CC hydrogel beads into rats: Histopathological analysis at 1 month and 3 months. In this example, local reactions to the two formulations FSB2-A and B, including inflammation and foreign bodies, are characterized by histopathological analysis of skin tissue at the injection site level of the study in Example 6, at 1 month and 3 months post-injection. The injection site tissues for the two reference commercial products are handled similarly.

[0348] By observing tissue sections, mounted on glass slides and stained with hematoxylin, under a microscope, various aspects of the local response are evaluated according to the semi-quantitative evaluation system described in ISO 10993 standard - Part 6 (2016). A response score is assigned to each site for each parameter, and then the average score for all sites is calculated. Table 11 shows the results obtained in two trials, at 1 month and 3 months.

[0349] [Table 11]

[0350] Within one month, local responses occurred appropriately for products based on hydrogel particles (CC beads and Restylane® LYFT), with responses scoring "none to low" for all parameters. After three months, cellular responses did not increase or decrease, and tissue penetration within the CC hydrogel beads was observed (however, Restylane particles were not observed). In the case of the Ellanse-M product, a higher cellular response was observed after one month, with macrophage scores being "moderate to prominent," and lymphocyte and giant cell scores being higher than with the other products. The presence of necrosis was observed at one site. The response remained significant after three months, as it consistently scored higher than with the other products as a result of a recurrent inflammatory response characterized by persistent lymphocytes, macrophages ("moderate to prominent" scores), and giant cells.

[0351] From this example, it can be concluded that the two products based on CC hydrogel beads according to the present invention elicit appropriate local responses one month and three months after subcutaneous injection. Encapsulation of the beads by cells or fibrous tissue is not observed, or is hardly observed; rather, the presence of collagen fibers in the material, signs of good biointegration, and minimization of the risk of long-term granuloma are observed, as required by the present invention.

[0352] Example 8 - Incorporation of ascorbic acid into CC hydrogel beads In this example, pre-formed CC hydrogel beads incorporate ascorbic acid, a model molecule (water-soluble in this case), demonstrating their ability to function as tanks for substances such as active ingredients and nutrients. Reference beads SB2-C (average volume diameter 373 μm) from Example 2 are used, having been collected in their hydrated form beforehand. The beads are suspended in a 50 mg / ml solution of ascorbic acid in saline phosphate buffer, then gently stirred at room temperature for 12 hours. The beads are then separated from the solution by filtration under vacuum across a membrane and left in a suspension in saline phosphate buffer for 7 days. A portion of the beads are washed and rinsed in buffer, lyophilized and dehydrated, and then characterized by FTIR.

[0353] The same method was applied to SB2-C beads suspended in the same buffer solution without ascorbic acid, and their FTIR spectra were recorded. The FTIR spectra of the beads suspended in ascorbic acid solution were 862 cm⁻¹ compared to the spectra of the beads suspended in buffer solution only. -1 and 530cm -1 It has two additional bands, confirming the presence of ascorbic acid in the CC beads.

[0354] From this example, it can be concluded that CC hydrogel beads can function as a reservoir for water-soluble molecules of ascorbic acid by incorporating it into pre-formed beads via a simple diffusion process of ascorbic acid. The fact that ascorbic acid is still present in the phosphate buffer and washing solution after 7 days supports the idea that incorporation is actually occurring not only on the surface of the beads but also in the center.

[0355] Example 9 - Encapsulation of triamcinolone hexacetonide into hydrogel beads Considering the topical and gradual delivery of triamcinolone, a corticosteroid-type anti-inflammatory agent, it is required to encapsulate it in hydrogel beads. The beads must be implantable or injectable into tissues or organs.

[0356] The encapsulation method allows for the encapsulation of triamcinolone without alteration. The beads contain at least 80% water, are spherical, and resistant to compression. Because it is not water-soluble, the triamcinolone is dispersed in the hydrogel of the beads. The triamcinolone powder, present in particles with a diameter of 1 μm to 10 μm, is visible to the naked eye. This is acceptable as a smooth surface, and the transparency of the beads is not required for local delivery applications.

[0357] Triamcinolone is suspended in the initial polymer solution (see CC1 and HA1 in Tables 1 and 2) during the first bead preparation step. Next, beads are formed at the start of the solution containing the polymer, triamcinolone, and crosslinking agent (BDDE) according to the general method of Examples 2 and 4. The beads are collected without division according to size. The beads are characterized according to the method described in previous examples (Table 12).

[0358] The triamcinolone content in the beads was evaluated using the following method: A certain amount of beads was introduced into a centrifuge tube and suspended in ethanol / aqueous solution (30:20, v / v) to ensure dissolution and diffusion of triamcinolone outside the beads. After 24 hours, the suspension was centrifuged at 2,500 rpm for 10 minutes. The absorbance of the supernatant was then measured at 242 nm using a spectrophotometer (Multiskan Sky High, Thermo Scientific). Calibration curves were prepared under the same conditions using solutions of 0–0.5 mg / mL of triamcinolone. The triamcinolone content is expressed as the mass of triamcinolone per unit mass of wet beads (Table 12).

[0359] [Table 12]

[0360] Thus, we successfully encapsulated a substantial amount of triamcinolone, ranging from 6 to 15 mg, depending on the CC and HA composition of the beads. FTIR confirmed that the structure of the triamcinolone remained unchanged. The following two observations have been reported: The beads become opaque because the triamcinolone is incorporated in the form of particles dispersed within the hydrogel of the beads. Note also that the beads are not smooth and are slightly rough due to the presence of these particles. These two observations are consistent with the solubility profile of triamcinolone. Furthermore, the beads conform to the present invention in terms of water content, sphericity, and resistance to compression.

[0361] Example 10 - Encapsulation of triamcinolone from sterile hydrogel beads Sterile formulations are prepared using the three hydrogels and triamcinolone beads of Example 9. To do this, the beads are suspended in hyaluronan solution (see HA2 in Table 2) according to the same general method as in Example 3. The formulations are packaged in 3 mL syringes (Hypak, BD) and sterilized by autoclaving.

[0362] Next, by applying mechanical stress to the beads through agitation, it can be confirmed that triamcinolone is released from the beads. This evaluation is performed on beads separated and rinsed from a sterile suspension. For each study up to 27 days, a centrifuge tube is reserved and filled with 1 g of wet beads, 8 mL of PBS, and three stainless steel beads with a diameter of 4.5 mm. The tube is subjected to orbital agitation (190 rpm) at room temperature. This can induce mechanical degradation through movements that mimic the movement of a joint, such as a knee.

[0363] To evaluate the triamcinolone concentration released into the supernatant each time, the beads are allowed to settle by centrifugation at 600 rpm for 5 minutes. 0.4 mL of the supernatant is collected, and 0.6 mL of ethanol is added. The absorbance of the solution is then measured at a wavelength of 242 nm using a spectrophotometer (Multiskan Sky High, Thermo Scientific). Calibration curves are created in parallel under the same operating conditions using a solution containing only triamcinolone. The triamcinolone concentration in the supernatant is then calculated each time (for each tube). Finally, the total amount of triamcinolone released is calculated in mg per g of wet beads (Table 13).

[0364] [Table 13]

[0365] Triamcinolone is actually released gradually from the beads into the supernatant at a certain point in time, and then gradually throughout the entire study. Under the conditions of this test, the release of SB8-1 and SB8-2 beads begins on day 2. For SB8-3 beads, it begins on day 7. The majority of the initial triamcinolone content is released on the final day of the test for all three types of beads. This indicates that there is no blocking of release in the aqueous medium when the beads are subjected to mechanical stress.

[0366] It is concluded that triamcinolone can be gradually released from CC or CC / HA hydrogel beads set under mechanical stress. Furthermore, the time to trigger release and the release rate can be adjusted by changing the polymer composition of the beads. The polymer composition of the beads can be adjusted according to the release profile and target indications of the activator used.

Claims

1. A composition administerable to humans or animals, wherein the composition comprises an aqueous phase containing a plurality of beads, the beads comprising or consisting of a hydrogel matrix containing at least one N,O-carboxyalkyl chitosan having glucosamine units, N-acetyl-glucosamine units, and glucosamine units substituted with a carboxyalkyl group, the N,O-carboxyalkyl chitosan having a degree of acetylation of more than 30% and up to 80%, expressed as the number of moles of N-acetyl groups relative to the number of moles of total glucosamine units, the N,O-carboxyalkyl chitosan having a degree of substitution with carboxyalkyl groups of more than 50%, expressed as the number of moles of substituents relative to the number of moles of total units, the N,O-carboxyalkyl chitosan being crosslinked by covalent bonds between the N,O-carboxyalkyl chitosan chains and / or co-crosslinked by covalent bonds with one or more other polymers, and the composition being sterilized, and the beads being integral with the aqueous phase.

2. The composition according to claim 1, characterized in that the N,O-carboxyalkyl chitosan has a degree of substitution with a carboxyalkyl group of more than 70% and less than 200%, expressed as the number of moles of substituents relative to the number of moles of total units.

3. The composition according to claim 1 or 2, wherein the matrix comprises at least one polymer.

4. The composition according to claim 3, wherein the polymer is hyaluronan.

5. The composition according to claim 3, wherein the polymer is crosslinked by covalent bonds or by covalent bonds with N,O-carboxyalkyl chitosan.

6. The composition according to claim 1, wherein the crosslinking is formed by a crosslinking agent that forms the covalent bond.

7. The composition according to claim 1, wherein the aqueous phase comprises at least one polymer.

8. The composition according to claim 1, wherein N,O-carboxyalkyl chitosan is co-crosslinked with other polymers in the beads.

9. The composition according to claim 1, wherein the aqueous phase and / or the beads exist in the form of a hydrogel having a pH and osmotic pressure balanced with the physiological medium.

10. The composition according to claim 1, wherein the aqueous phase and / or the beads are injectable into a human or animal.

11. The composition according to claim 1, wherein the composition is formulated as an injectable suspension.

12. The composition according to claim 1, wherein the composition is formulated as an intravenous infusion composition.

13. The composition according to claim 1, wherein the composition is formulated as an implantable composition.

14. The composition according to claim 1, wherein the composition is formulated as a pharmaceutical composition or a composition for use in a therapeutic method.

15. The composition according to claim 14, wherein the beads contain at least one activator.

16. The crosslinking agent is 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-epitiobane, 1-bromo-4,5-epithiopentane, 2,3-dibromopropanol, 2,4-dibromobutanol, 2,5-dibromopentanol, 2,3-dibromopropanthol, The composition according to claim 1, selected from 2,4-dibromobutanethiol, 2,5-dibromopentanethiol, epichlorohydrin, 2,3-dibromopropanol, 1-chloro-2,3-epithiopropane, dimethylaminopropylcarbodiimide, gallic acid, epigallocatechin gallate, curcumin, tannic acid, genipin, diisocyanate, hexamethylene diisocyanate, toluene diisocyanate, or divinyl sulfone.

17. Use of the composition for preparing a pharmaceutical composition for a therapeutic method comprising administering the composition according to any one of claims 1 to 16 by intravenous infusion, local administration, or injection.

18. The use according to claim 17, wherein the pharmaceutical composition is administered via a subcutaneous, intracutaneous, mucous membrane, eye, intraocular, or intra-articular route.

19. Use of the composition according to any one of claims 1 to 16 for preparing a pharmaceutical composition for repairing or filling at least one body tissue that is in need of repair or filling.

20. Use of the composition according to any one of claims 1 to 16 for preparing a pharmaceutical composition for the treatment of inflammatory joint diseases, the treatment of arthritis, or the repair of cartilage deficiency.

21. Use of the composition according to any one of claims 1 to 16 for preparing a pharmaceutical composition for treating osteoarthritis.

22. Use of the composition according to any one of claims 1 to 16 for preparing a pharmaceutical composition for treating corneal lesions or dry eye syndrome.

23. A medical device characterized by comprising or consisting of the composition described in any one of claims 1 to 16.

24. A method for preparing a composition comprising an aqueous phase and a plurality of beads as defined in any one of claims 1 to 16, the method comprising the following steps A step of preparing an aqueous solution of N,O-carboxyalkyl chitosan in the presence of at least one crosslinking agent; The process of forming droplets of this solution in the form of multiple beads; A step of crosslinking N,O-carboxyalkyl chitosan with a crosslinking agent; The process of obtaining multiple beads; A step of adding the plurality of beads to the aqueous phase; and A step of obtaining a composition comprising an aqueous phase and a plurality of beads as defined in any one of claims 1 to 16; A method that includes this.

25. The method according to claim 24, wherein the step of preparing an aqueous solution of N,O-carboxyalkyl chitosan and at least one crosslinking agent is carried out in the presence of at least one other polymer.

26. The method according to claim 24 or 25, characterized in that the droplet is placed in the presence of an aqueous phase.

27. The method according to claim 24 or 25, characterized by comprising solidifying droplets in the form of a plurality of beads before crosslinking the droplets in the presence of at least one coagulant.

28. The method according to claim 24 or 25, characterized in that the multiple beads obtained after crosslinking are subjected to a purification step by balancing the pH and osmotic pressure in a physiologically acceptable culture medium and washing them.

29. The method according to claim 24 or 25, characterized by classifying the plurality of beads and selecting beads according to their dimensions.

30. The method according to claim 24 or 25, wherein the plurality of beads are combined with an aqueous phase, a lipophilic phase, a hydrophilic lipid phase, or a solid phase.

31. The method according to claim 24 or 25, wherein the plurality of beads are combined with an aqueous phase, a lipophilic phase, a hydrophilic lipid phase, or a solid phase containing one or more N,O-carboxyalkyl chitosan, hyaluronan, or a combination thereof.

32. The method according to claim 30, wherein the phase is capable of being injected into the body of a human or animal.

33. The method according to claim 24 or 25, wherein the composition comprising an aqueous phase and a plurality of beads is sterilized.

34. The method according to claim 33, wherein the composition comprising an aqueous phase and a plurality of beads is steam-sterilized.

35. The method according to claim 33, wherein the composition comprising an aqueous phase and a plurality of beads is filtered and sterilized.

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