Hydrogels for soft tissue filling

Abstract

The present invention relates to a process for the preparation of hydrogels comprising cross-linked polysaccharides, in particular to a process for the preparation of injectable hydrogels comprising cross-linked hyaluronic acid. The invention also relates to a hydrogel, preferably an injectable hydrogel, obtainable by said process, to a composition comprising said hydrogel and to the use of said hydrogel.

Description

Technical Field

[0001] The present invention relates to a method for preparing a hydrogel comprising cross-linked polysaccharides, in particular a method for preparing an injectable hydrogel comprising cross-linked hyaluronic acid. The present invention also relates to a hydrogel (preferably an injectable hydrogel) obtainable by the method, a composition comprising the hydrogel and the use of the hydrogel. Background Art

[0002] Polysaccharide hydrogels (especially hyaluronic acid hydrogels) are widely used for soft tissue filling, especially for example, skin with volume defects (e.g., wrinkles, scars), or for increasing its volume. Hydrogels suitable for soft tissue filling are usually cross-linked hydrogels, that is, polysaccharides are cross-linked by one or more cross-linking agents. Such cross-linking can obtain hydrogels with the desired mechanical properties for soft tissue filling.

[0003] Although hyaluronic acid hydrogels are biodegradable and biocompatible, they may cause adverse reactions (with a low incidence) that occur between 6 and 24 months after the application of the filler. In particular, migration of the filler to areas away from the area in which it is applied in or on the body may occur (Chae SY, Lee KC, Jang YH, Lee SJ, Kim DW, Lee WJ. A Case of the Migration of Hyaluronic Acid Filler from Noseto Forehead Occurring as Two Sequential Soft Lumps. Ann Dermatol. 2016; 28 (5): 645-647 and Mosleh, Rasha & Mukari, Abed & Krausz, Judit & Hartstein, Morris & Hamed Azzam, Shirin. (2019). Orbit mass secondary to migration of dermal hyaluronic acidfiller. JAAD Case Reports. 5. 488-490). Various reasons have been proposed to explain this phenomenon, such as poor injection technique, injection volume that is too large, physical activity after injection (muscle activity, exercise), displacement caused by pressure (e.g. massage), vascular injection, lymphatic invasion or gravity. In order to reduce the risk of migration of polysaccharide gels, certain methods have been described; in particular, it has been proposed to apply a repelling electric field to at least a portion of the edge around the treatment site (WO2020 / 172371).

[0004] However, there is still a need to provide a hydrogel based on cross-linked polysaccharides, in particular a hydrogel based on cross-linked hyaluronic acid, which retains the hyaluronic acid chains, has mechanical properties suitable for soft tissue augmentation, and does not migrate in the tissue after administration. Summary of the invention

[0005] The present invention relates to a method for preparing a hydrogel, preferably an injectable hydrogel, comprising the following steps:

[0006] a) providing at least one polysaccharide;

[0007] b) providing at least one cross-linking agent, the cross-linking agent comprising at least two functional groups Z, the functional groups Z being the same or different and selected from the group consisting of isocyanate groups, amino groups, epoxide groups, carboxyl groups, N-succinimidyloxycarbonyl groups, N-sulfosuccinimidyloxycarbonyl groups, halocarbonyl groups, isothiocyanate groups, vinyl groups, formyl groups, hydroxyl groups, thiol groups, hydrazino groups, acylhydrazino groups, aminooxy groups, carbodiimide groups and anhydride residues;

[0008] c) preparing a cross-linking reaction medium comprising a polysaccharide, a cross-linking agent and a solvent, wherein the total amount of the cross-linking agent ranges from 0.001 mol to less than 0.02 mol per 1 mol of polysaccharide repeating unit, and the duration of the reaction medium preparation step does not exceed 5 hours;

[0009] d) placing the reaction medium obtained at the end of step c) at a pressure P and a temperature T for 2 to 17 weeks, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the eutectic temperature of the reaction medium measured at the pressure P and less than the freezing point of the reaction medium measured at the pressure P.

[0010] The invention also relates to a hydrogel obtainable by the method described herein, as well as to a cosmetic or pharmaceutical composition comprising such a hydrogel and its use in filling and / or replacing tissue, for preventing and / or treating changes in the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, in particular for filling wrinkles, fine lines and scars; for example, to reduce nasolabial folds and frown lines, to increase the volume of the cheekbones, chin or lips, to restore facial volume (in particular the cheeks, temples, facial contours and periocular area); or to reduce the appearance of wrinkles and fine lines; or to stimulate, regenerate, moisturize, tighten or restore the radiance of the skin, in particular by mesotherapy.

[0011] Other aspects of the invention are described below and in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows the hydrogel according to the present invention compared with conventional hydrogels on metal ( Figure 1A ),Glass( Figure 1B),plastic( Figure 1C ) and collagen ( Figure 1D ) surface stickiness.

[0013] definition

[0014] The term "gel" denotes a polymer network in which a fluid extends throughout its volume. This means that a gel consists of two media dispersed in one another, one solid and the other liquid. The so-called solid medium consists of long polymer molecules linked together by weak bonds (for example hydrogen bonds) or covalent bonds (crosslinks). The liquid medium consists of a solvent. A gel generally corresponds to a product having a phase angle δ at 1 Hz less than or equal to 45° when deformed by 0.1% or at a pressure of 1 Pa, advantageously a phase angle δ of 2° to 45° or 20° to 45°.

[0015] The term "hydrogel" means a gel as defined above in which the solvent constituting the liquid medium is predominantly water (e.g. at least 90% by weight, in particular at least 95% by weight, in particular at least 99% by weight of the liquid medium). Preferably, the liquid medium comprises, in particular consists of, a buffer solution advantageously bringing the pH of the liquid medium to between 6.8 and 7.8, in particular a saline phosphate buffer.

[0016] The term "injectable gel" means a gel that can flow and can be manually injected through a syringe equipped with a needle with a diameter of 0.1 mm to 0.5 mm (e.g., a 30G, 27G, 26G or 25G hypodermic needle). Preferably, the injectable gel is a gel having an average extrusion force of less than or equal to 25 N, preferably 5 N to 25 N, and more preferably 8 N to 15 N when measured at room temperature using a dynamometer at a fixed speed of about 12.5 mm / min in a syringe with an outer diameter greater than or equal to 6.3 mm and a needle with a diameter less than or equal to 0.4 mm (27G) and a length of 1 / 2 inch.

[0017] The extensibility of a product represents its ability to stretch between the two surfaces to which it is attached. Extensibility can be determined using a texture analyzer, a sensory analysis performed by a panel of experts, or rheological and mechanical measurements (particularly measurements including phase angle (δ) or stretching tests). In particular, this feature can be measured as described by P. Micheels et al. (Micheels et al., Comparison of two Swiss-designed hyaluronic acid gels: Six-month clinical follow-up, Journal of Drugs in Dermatology, 2017, 16: 154-161, "Resistance to stretching"), or by performing a viscosity test and measuring the length of the gel line when pulled.

[0018] The stickiness of a product represents its ability to adhere to a surface. It can be determined qualitatively using a sensory analysis by a panel of experts or by moving a pellet over a surface. It can also be determined quantitatively by measuring the adhesion to a surface, by a traction machine or by mechanical analysis.

[0019] The term "polysaccharide" denotes a polymer composed of monosaccharides (preferably the D enantiomers) linked together by glycosidic bonds.

[0020] The term "monosaccharide" refers to unmodified or modified monosaccharides.

[0021] "Unmodified monosaccharide" means a monosaccharide of the formula H-(CHOH) x -CO-(CHOH) y -H compound, wherein x and y are independently integers from 0 to 5, provided that 2≤x+y≤5, the monosaccharide may be in a linear form represented by the above formula, or may be in a cyclized form in which a hemiacetal or hemiketal group is formed by the reaction of a CO functional group (aldehyde or ketone) with an OH group. Preferably, the monosaccharide is in a cyclized form. There are two types of monosaccharides: aldoses with an aldehyde functional group (when x or y is 0) and ketoses with a ketone functional group (when both x and y are not 0). Monosaccharides are classified by the number of carbons. For example, a monosaccharide with 6 carbons (x+y=5) is of the formula C6H 12 O6 hexose, and can be allose, altrose, glucose, mannose, gulose, idose, galactose or talose. A monosaccharide with 5 carbons (x+y=4) is of the formula C5H 10 The monosaccharide is a pentose of O5 and may be ribose, arabinose, xylose or lyxose. Preferably, the monosaccharide is a hexose, i.e. x+y=5. The monosaccharide also contains an x+y asymmetric carbon, thus containing 2 (x+y-1)Each pair of enantiomers is represented by a different name, and the same pair of enantiomers is called D enantiomers and L enantiomers respectively.

[0022] "Modified monosaccharides" means unmodified monosaccharides as defined above, for example:

[0023] - one or more OH functions are replaced by another functional group, for example:

[0024] (i) an OR group in which R represents (C1-C6)alkyl (e.g. methyl or ethyl), hydroxy-(C1-C6)alkyl (e.g. hydroxyethyl (-CH2CH2OH) or hydroxypropyl (-CH2-CH(OH)-CH3)), carboxy-(C1-C6)alkyl (e.g. carboxymethyl (-CH2COOH)) or CO-(C1-C6)alkyl (e.g. acetyl); and / or

[0025] (ii) NR'R" groups, wherein R' and R" independently represent H, (C1-C6)alkyl or CO-(C1-C6)alkyl (eg acetyl); and / or

[0026] (iii) OSO3H group; and / or

[0027] - the terminal CH2OH functional group is replaced by a COOH or CHO group; the -CH(OH)-CH(OH)- bond is oxidized to produce two terminal -CHO (aldehyde) groups in place of the bond; and / or

[0028] -The terminal CH2OH functional group combines with the OH functional group to form an -O-CH2- chain.

[0029] The expression "repeating unit" of a polysaccharide refers to a structural unit composed of one or more (usually 1 or 2) monosaccharides, the repetition of which produces the complete polysaccharide chain.

[0030] Some or all of the monosaccharides may be in a modified form. When modified, the monosaccharides may be in different modified forms.

[0031] The term "physiologically acceptable" means a material that is generally safe, nontoxic, and neither biologically nor otherwise undesirable and is acceptable for cosmetic (i.e., nontherapeutic) or therapeutic human or veterinary use, including use by injection into the human or animal body or for topical application to the skin.

[0032] Salts which can be used in the context of the present invention are preferably physiologically acceptable salts. The term "physiologically acceptable salts" refers in particular to:

[0033] (1) a pharmaceutically acceptable acid addition salt formed with a pharmaceutically acceptable inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), or a pharmaceutically acceptable acid addition salt formed with a pharmaceutically acceptable organic acid (e.g., formic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethylsulfonic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethylsulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, hexanedioic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, ditoluoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid, etc.), and

[0034] (2) Pharmaceutically acceptable base addition salts formed when the acidic protons present in the parent compound are replaced by metal ions (e.g., alkali metal ions (e.g., Na, K), alkaline earth metal ions (e.g., Ca, Mg), zinc ions, silver ions or aluminum ions), or coordinated with a pharmaceutically acceptable organic base (e.g., diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, etc.) or coordinated with a pharmaceutically acceptable inorganic base (e.g., aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc.).

[0035] The "degree of modification" (MOD) of a polysaccharide (e.g., hyaluronic acid) is the molar amount of cross-linking agent bound to the polysaccharide through one or more ends of the polysaccharide, expressed per 100 moles of repeating units of the polysaccharide. It can be determined by methods known to those skilled in the art, such as nuclear magnetic resonance spectroscopy (NMR).

[0036] The "molar crosslinking ratio" (CR) (expressed in %) represents the molar ratio of the amount of crosslinking agent relative to the amount of repeating units of polysaccharide introduced into the crosslinking reaction medium (expressed per 100 moles of repeating units of polysaccharide in the crosslinking medium).

[0037] The expression "therapeutically active ingredient" means a substance that treats or alleviates symptoms and / or prevents disease, a substance that has therapeutic or preventive properties for human or animal diseases, and any substance that can be used or administered to humans or animals with the intention of establishing a medical diagnosis or restoring, correcting or changing their physiological function by exerting a pharmacological, immunological or metabolic action.

[0038] The expression "cosmetic active ingredient" denotes any non-therapeutic substance intended in particular to come into contact with the various superficial parts of the human body (e.g. the epidermis, hair, nails, lips, breasts and teeth) with the intention of exclusively or primarily cleansing, protecting, perfumed, preserving in good condition or altering their appearance or odor.

[0039] The term "about" means that the relevant value may be 10%, in particular 5%, especially 1% less or greater than the indicated value.

[0040] The expression "spacer group" denotes a moiety comprising at least one atom intended to bond together two chemical groups within the same molecule. Preferably, the spacer group comprises at least one carbon atom.

[0041] The term "halogen" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[0042] An "epoxide" group is an ethylene oxide residue bonded to the rest of the molecule through one of its carbon atoms.

[0043] An "N-succinimidyloxycarbonyl" group is a group of the following formula GR1:

[0044]

[0045] An "N-sulfosuccinimidyloxycarbonyl" group is a group of the following formula GR2:

[0046]

[0047] "Halocarbonyl" is a radical of the formula -CO-Hal, wherein Hal represents a halogen, such as Cl or Br.

[0048] A "carbodiimide" group is a group comprising a -N=C=N- unit, more particularly a group of the formula -N=C=NR a A group in which R a represents an aliphatic hydrocarbon group having 1 to 20 carbon atoms, preferably a (C1-C6)alkyl group, one or more carbon atoms of which are optionally substituted by a heteroatom selected from O, S and N, in particular N.

[0049] An "anhydride residue" is a group comprising -C(O)-OC(O)- units, more particularly a monovalent cyclic group comprising -C(O)-OC(O)- units, for example a monocyclic monovalent saturated hydrocarbon group comprising 5 to 10 carbon atoms, in particular 5 or 6 carbon atoms, wherein three consecutive carbon atoms are substituted by -C(O)-OC(O)-, and optionally one or more (in particular one) additional carbon atoms (preferably not consecutive to the three carbon atoms substituted by -C(O)-OC(O)-) are each substituted by a heteroatom (for example N, O or S, in particular N). The anhydride residue may in particular correspond to the following formula GR3:

[0050]

[0051] The anhydride residues may also be selected from maleic anhydride residues or succinic anhydride residues.

[0052] The expressions "aliphatic hydrocarbon chain" or "aliphatic hydrocarbon group" denote linear, branched and / or cyclic, saturated or unsaturated and nonaromatic hydrocarbon groups, advantageously containing 1 to 50 carbon atoms, in particular 1 to 20 carbon atoms, for example 1 to 12 carbon atoms or 1 to 6 carbon atoms. They will in particular be alkyl groups.

[0053] The expression "branched aliphatic hydrocarbon chain" particularly denotes a main aliphatic hydrocarbon chain comprising at least one secondary aliphatic hydrocarbon chain.

[0054] The expression "star-shaped aliphatic hydrocarbon chain" means a branched aliphatic hydrocarbon chain comprising a plurality of secondary aliphatic hydrocarbon chains all starting from a single branching point.

[0055] The expression "C1-Cx alkyl" or "(C1-Cx)alkyl" or alternatively "alkyl having 1 to x carbon atoms" denotes a saturated, linear or branched, monovalent hydrocarbon radical having 1 to x carbon atoms, wherein x is an integer, for example methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropyl, cyclohexyl, etc.

[0056] The expression "(C1-Cx)alkylene" denotes a saturated, linear or branched, divalent hydrocarbon radical having 1 to x carbon atoms, where x is an integer, for example methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, pentane-1,5-diyl, hexane-1,6-diyl, hexane-1,5-diyl, heptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, decane-1,10-diyl, etc. It is especially a methane-1,1-diyl or propane-1,3-diyl radical.

[0057] The expression "hydroxy-(C1-Cx)alkyl" denotes a (C1-Cx)alkyl group as defined above substituted by a hydroxy (OH) group, for example hydroxyethyl (-CH2CH2OH) or hydroxypropyl (eg -CH2-CH(OH)-CH3).

[0058] The expression "carboxy-(C1-Cx)alkyl" denotes a (C1-Cx)alkyl group as defined above substituted by a carboxy (COOH) group, such as carboxymethyl (-CH2COOH).

[0059] The expression "aryl" denotes a monovalent aromatic hydrocarbon radical comprising one or more rings, preferably having 6 to 10 carbon atoms, such as phenyl or naphthyl.

[0060] The expression "arylene" denotes a divalent aromatic hydrocarbon radical comprising one or more rings, preferably having 6 to 10 carbon atoms, such as phenylene.

[0061] The expression "aryl-(C1-Cx)alkyl" denotes an aryl group as defined above linked to the rest of the molecule via a (C1-Cx)alkyl chain as defined above, wherein x is an integer, for example benzyl or phenethyl.

[0062] The expression "polyvalent group" means a group that can form multiple covalent bonds with other groups of the same compound or two different compounds. The bonds with other groups can be formed by the same atom of the polyvalent group or by different atoms of the polyvalent group, preferably by different atoms of the polyvalent group. In particular, the polyvalent group is a divalent group and can therefore form two covalent bonds with two other groups of the same compound or two different compounds. The number of covalent bonds that can be formed represents the valence of the polyvalent group. DETAILED DESCRIPTION

[0063] The present inventors have developed a method that enables the preparation of hydrogels that meet the needs expressed. The viscosity of the hydrogels obtained allows the hydrogels to adhere to the tissue and not migrate in the tissue after their administration / injection.

[0064] Currently available fillers create volume to fill soft tissue, but cannot counteract tissue sagging. However, chronological aging of the skin is associated with structural changes in the skin (appearance of wrinkles), but also with sagging of fat areas. Interestingly, in certain embodiments, due to its ductility, the hydrogel according to the present invention can be used to retain tissue and prevent age-related tissue sagging, especially when applied and arranged in a grid form. Therefore, the hydrogel according to the present invention is most particularly useful in filling and / or replacing soft tissue.

[0065] Furthermore, the hydrogel obtained is highly biocompatible. In fact, its preparation method uses a very small amount of cross-linking agent (0.001 mol to less than 0.02 mol of cross-linking agent per 1 mol of polysaccharide repeating unit). Therefore, the hydrogel obtained is weakly cross-linked.

[0066] Furthermore, the hydrogel obtained has high mucoadhesive properties, which allow it to adhere to the mucosa, thus giving the hydrogel a good persistence at the site of application. This can be seen by qualitative ex vivo or in vitro tests to track the marker components.

[0067] The method for preparing a hydrogel, preferably an injectable hydrogel, as developed by the present inventors comprises the following steps:

[0068] a) providing at least one polysaccharide;

[0069] b) providing at least one cross-linking agent, the cross-linking agent comprising at least two functional groups Z, the functional groups Z being the same or different and selected from the group consisting of isocyanate groups, amino groups, epoxide groups, carboxyl groups, N-succinimidyloxycarbonyl groups, N-sulfosuccinimidyloxycarbonyl groups, halocarbonyl groups, isothiocyanate groups, vinyl groups, formyl groups, hydroxyl groups, thiol groups, hydrazino groups, acylhydrazino groups, aminooxy groups, carbodiimide groups and anhydride residues;

[0070] c) preparing a cross-linking reaction medium comprising a polysaccharide, a cross-linking agent and a solvent, wherein the total amount of the cross-linking agent ranges from 0.001 mol to less than 0.02 mol per 1 mol of polysaccharide repeating unit, and the duration of the reaction medium preparation step does not exceed 5 hours;

[0071] d) placing the reaction medium obtained at the end of step c) at a pressure P and a temperature T for 2 to 17 weeks, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the eutectic temperature of the reaction medium measured at the pressure P and less than the freezing point of the reaction medium measured at the pressure P.

[0072] In the above method, the polysaccharide and / or the cross-linking agent may be in the form of a salt.

[0073] It is readily understood that, in the process of the present invention, cross-linking of the polysaccharide occurs primarily in step d), ie when the reaction medium is in a frozen state.

[0074] The properties of the hydrogels obtained by the method of the present invention are particularly surprising because, when it comes to the optimal cross-linking temperature, the prior art recommends no less than 10°C and is silent on the use of temperatures below 0°C (Jaeuk Baek, Yingfang Fan, Seol-Ha Jeong, Ho-Yong Lee, Hyun-do Jung, Hyoun-Ee Kim, Sukwha Kim, Tae-Sik Jang. Facile strategy involving low-temperature chemical cross-linking to enhance the physical and biological properties of hyaluronic acid hydrogel, Carbohydrate Polymers 202 (2018) 545-553).

[0075] Furthermore, when cryogelation is involved, the amount of crosslinker used is still very high (Anna Strom, Anette Larsson, Oguz Okay, Preparation and physical properties of hyaluronic acid-based cryogels, J. Appl. Polym. Sci. 2015, 42194; WO201029344). Otherwise, the prepared product cannot withstand the heat sterilization conditions usually applied to this type of product (autoclave sterilization). These unstable sterilized products end up with a major viscous component and / or a large portion of polymers with a low weight-average molecular weight, which is undesirable for soft tissue augmentation applications. However, for obvious safety reasons, it is always desirable to propose products that contain as little chemical modification as possible.

[0076] The hydrogels of the invention differ from hydrogels prepared with the same ingredients but which cannot be frozen by their macroporous structure, i.e. by having interconnected macropores, the size of which varies from less than one micrometer to hundreds of micrometers, depending on the synthesis conditions, i.e. by the identity and amount of the reactants, as well as by the temperature and the freezing rate. These macropores are visible in morphological analysis by scanning electron microscopy in the frozen state or by confocal laser scanning microscopy after staining the gel, for example with rhodamine B.

[0077] A hydrogel in a frozen state that reaches room temperature after the cross-linking step may be referred to by the term "cryogel".

[0078] Furthermore, although cryogels are known to produce only small amounts of hyaluronic acid fragments, it is still surprising that the method of the present invention retains the structure of polysaccharides with high weight average molecular weights, in particular the structure of hyaluronic acid with high weight average molecular weights. The method according to the present invention enables the preparation of cross-linked polysaccharide gels without degradation into fragments with low weight average molecular weights.

[0079] The polysaccharide with high weight average molecular weight represents a polysaccharide with a weight average molecular weight corresponding to the weight average molecular weight of a naturally synthesized polysaccharide (biologically). For example, the hyaluronic acid polysaccharide with high weight average molecular weight represents a polysaccharide with a weight average molecular weight greater than or equal to 1MDa, preferably in the range of 1MDa to 5MDa, such as 1MDa to 3MDa or 1.5MDa to 3MDa; the chondroitin sulfate or chondroitin polysaccharide with high weight average molecular weight represents a polysaccharide greater than or equal to 30kDa, preferably in the range of 30kDa to 150kDa; high weight average molecular weight chitosan polysaccharide represents a polysaccharide with an average molecular weight greater than or equal to 100kDa, preferably in the range of 100kDa to 250kDa. The polysaccharide provided in the step a) of the method is generally a polysaccharide with a high weight average molecular weight.

[0080] The low weight average molecular weight polysaccharide refers to a polysaccharide having an average molecular weight significantly lower than the average molecular weight of biologically synthesized polysaccharides, more specifically, the low weight average molecular weight polysaccharide refers to a polysaccharide having a weight average molecular weight less than or equal to one third of the weight of the high molecular weight polysaccharide provided in step a) of preparing the hydrogel.

[0081] For example, low weight average molecular weight hyaluronic acid polysaccharide refers to a polysaccharide with a weight average molecular weight range of 0.04MDa to 0.3MDa, preferably 0.08MDa to 0.20MDa, and more preferably 0.08MDa to 0.15MDa; low weight average molecular weight chondroitin sulfate or chondroitin polysaccharide refers to a polysaccharide with a weight average molecular weight less than or equal to 50kDa; low weight average molecular weight chitosan polysaccharide refers to a polysaccharide with a weight average molecular weight less than or equal to 30kDa.

[0082] The absence of low molecular weight fragments is advantageous. In fact, although hyaluronic acid is known to be biocompatible, it has been reported in the literature that hyaluronic acid with a low weight average molecular weight may cause long-term adverse reactions after being injected into a patient (Cyphert JM, Trempus CS, Garantziotis S. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology. International Journal of Cell Biology 2015; 2015: 563818).

[0083] Ingredients used in the preparation of hydrogels

[0084] Polysaccharides

[0085] A polysaccharide may be any polymer composed of monosaccharides linked together by glycosidic bonds.

[0086] Preferably, the polysaccharide is selected from pectin and pectin substances, chitosan, chitin, cellulose and its derivatives, agarose, mucopolysaccharides (such as hyaluronic acid, heparin precursors, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate) and mixtures thereof.

[0087] Pectic substances, including pectin, are polysaccharides composed of a backbone of D-galacturonic acid in the acid form (which can be esterified using methanol) and L-rhamnose which can be branched with other monosaccharides.

[0088] Chitosan and chitin are each a polysaccharide composed of D-glucosamine repeating units linked to each other by β-(1,4) bonds, a portion of which is N-acetylated. More specifically, the degree of acetylation of chitosan is less than 50%, while the degree of acetylation of chitin is greater than 50%.

[0089] Cellulose is a polysaccharide composed of linear chains of D-glucose molecules.

[0090] Cellulose derivatives include methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and carboxymethylcellulose (CMC).

[0091] Agarose is a polysaccharide containing a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose as repeating units.

[0092] Mucopolysaccharides are linear polysaccharides composed of repeating units of disaccharides, which contain a hexosamine (glucosamine (GlcN) or galactosamine (GalN)) and another monosaccharide (glucuronic acid (GlcA), iduronic acid (IdoA) or galactose (Gal)). The hexosamine and other monosaccharides may be optionally sulfated and / or acetylated. The mucopolysaccharide may in particular be hyaluronic acid, a heparin precursor, dermatan sulfate, keratan sulfate, chondroitin or chondroitin sulfate.

[0093] Hyaluronic acid is a mucopolysaccharide whose repeating unit is a disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine that are linked together by alternating glycoside β-(1,4) and β-(1,3) keys. When hyaluronic acid is in the form of a salt, it is also referred to as hyaluronate or hyaluronic acid (hyaluronan). In the context of the present invention, the weight-average molecular weight of hyaluronic acid can be 0.05MDa to 10MDa, preferably 0.5MDa to 5MDa, even more preferably greater than 0.05MDa, for example, 0.07MDa to 10MDa or 0.07MDa to 5MDa, or 0.5MDa to 5MDa or 1MDa to 5MDa or 2MDa to 4MDa. Hyaluronic acid can be in salt form, particularly physiologically acceptable salt form, such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof. More particularly, hyaluronic acid is in acid form or sodium salt form (NaHA).

[0094] Heparin precursor is a mucopolysaccharide whose repeating unit is a disaccharide composed of glucuronic acid (GlcA) linked to N-acetylglucosamine (GlcNAc) through an α-(1,4) bond. Each disaccharide repeating unit is linked to another disaccharide repeating unit through a β-(1,4) bond.

[0095] Chondroitin sulfate is a mucopolysaccharide whose repeating units are disaccharides composed of glucuronic acid linked to sulfated N-acetylgalactosamine via a β-(1,3) bond, i.e., it contains at least one sulfate ester substituent. Each disaccharide repeating unit is linked to another disaccharide repeating unit via a β-(1,4) bond.

[0096] Dermatan sulfate is a mucopolysaccharide whose repeating unit is a sulfated disaccharide (i.e., comprising at least one sulfate ester substituent) of L-iduronic acid and N-acetylgalactosamine connected by α-(1,3) bonds. Advantageously, the disaccharide is sulfated at the C-4 position of N-acetylgalactosamine, the C-6 position of N-acetylgalactosamine, the C2-position of L-iduronic acid, or a combination of these positions. Each disaccharide repeating unit is connected to another disaccharide repeating unit by a β-(1,4) bond.

[0097] Keratan sulfate is a mucopolysaccharide whose repeating units are a sulfated disaccharide (ie, containing at least one sulfate ester substituent) composed of D-galactose and N-acetylglucosamine linked by alternating β-(1,4) and β-(1,3) bonds.

[0098] The polysaccharide may be in the form of a salt, in particular a physiologically acceptable salt, such as sodium, potassium, zinc, calcium, magnesium, silver salts and mixtures thereof, more particularly a sodium or potassium salt.

[0099] Advantageously, the polysaccharide is a mucopolysaccharide or a salt thereof, preferably hyaluronic acid or a salt thereof, more preferably hyaluronic acid or a physiologically acceptable salt thereof, such as sodium, potassium, zinc, silver salts and mixtures thereof, even more preferably hyaluronic acid or its sodium salt.

[0100] The weight average molecular weight of the polysaccharide is generally from 0.03 MDa to 10 MDa.

[0101] Preferably, if the polysaccharide is hyaluronic acid, its weight average molecular weight (MW) ranges from 0.05 MDa to 10 MDa, preferably in the range of 0.5 MDa to 5 MDa, even more preferably greater than 0.05 MDa, for example in the range of 0.07 MDa to 10 MDa or 0.07 MDa to 5 MDa, or 0.5 MDa to 5 MDa or 1 MDa to 5 MDa or 2 MDa to 4 MDa.

[0102] Crosslinking agent

[0103] Cross-linking agents are compounds comprising at least two functional groups capable of covalently bonding with functional groups present on the polysaccharide (e.g. OH, CHO, NH2 or COOH groups carried by the polysaccharide), thus being able to induce bonding (cross-linking) between polysaccharide chains and / or bonding within the same polysaccharide chain.

[0104] The crosslinking agents useful in the context of the present invention contain at least two, preferably 2 to 8, in particular 2 functional groups (denoted as "groups Z"), said groups Z preferably being independently selected from isocyanate groups (-N = C = O), amino groups (-NH2), epoxide groups, carboxyl groups (-COOH), N-succinimidyloxycarbonyl groups, N-sulfosuccinimidyloxycarbonyl groups, halocarbonyl groups, isothiocyanate groups (-N = C = S), vinyl groups (-CH = CH2), formyl groups (-CH = O), hydroxyl groups (-OH), mercapto groups (-SH), hydrazines (-NH-NH2), acylhydrazines (-CO-NH-NH2), aminooxy groups (-O-NH2), carbodiimide groups and anhydride residues. Preferably, the functional groups are identical.

[0105] Isocyanate groups can react with OH or NH2 groups of polysaccharides to form carbamate or urea functional groups. Amino groups can react with COOH groups of polysaccharides to form amide functional groups. Epoxide groups can react with OH or COOH groups of polysaccharides to form ether or ester functional groups. Carboxyl groups can react with OH or NH2 groups of polysaccharides to form ester or amide functional groups. N-succinimidyloxycarbonyl and N-sulfosuccinimidyloxycarbonyl can react with OH or NH2 groups of polysaccharides to form ester or amide functional groups. Halocarbonyl groups can react with OH or NH2 groups of polysaccharides to form ester or amide functional groups. Isothiocyanate groups can react with OH or NH2 groups of polysaccharides to form thiocarbamate or thiourea functional groups. Vinyl groups can react with OH groups of polysaccharides to form ether functional groups. The formyl group can react with the OH or NH2 group of the polysaccharide to form a hemiacetal or hemiamine aldehyde functional group. The hydroxyl group can react with the COOH group of the polysaccharide to form an ester functional group. The sulfhydryl group can react with the COOH group of the polysaccharide to form a thioester functional group. The hydrazine group (-NH-NH2) can react with the CHO group of the polysaccharide to form a hydrazone functional group. The acylhydrazine group can react with the CHO group of the polysaccharide to form a carbonylated hydrazone =NNHC(O)-functional group. The aminooxy group can react with the CHO group of the polysaccharide to form an oxime =NO-functional group. The carbodiimide group can react with the COOH group of the polysaccharide to produce a CO-NR a -CO-NH functional groups, and the anhydride residues can react with OH or NH2 groups of the polysaccharide to form ester or amide functional groups.

[0106] Preferably, the functional groups Z are identical and represent an epoxide group or a vinyl group, more preferably an epoxide group.

[0107] According to another advantageous embodiment, the functional groups Z are identical and are selected from amino, vinyl, formyl and carbodiimide groups, preferably amino groups.

[0108] In particular, the crosslinking agent is selected from hexamethylene diisocyanate, diphenylmethylene 4,4'-diisocyanate, 4-arm PEG20K-isocyanate, spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), poly(ethylene glycol) diamine, ethylenediamine, 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3- glycidyloxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with diglycidyl ether (CAS No. 130167-23-6), poly(ethylene glycol) diacid, disuccinimidyl suberate, di(sulfosuccinimidyl) suberate, sebacic acid chloride, 1,4-butanedithioisocyanate, divinyl sulfone (DVS), glutaraldehyde, polyethylene glycol, 1,5-pentanedithiol, adipic acid dihydrazide, bisaminooxy-poly(ethylene glycol), diethylenetriaminepentaacetic dianhydride, and mixtures thereof.

[0109] When the functional group Z is an epoxide group, the crosslinking agent is preferably selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated with diglycidyl ether at each end (CAS No.: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups, and mixtures thereof.

[0110] More preferably, the crosslinking agent is selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

[0111] When the functional group Z is an amino group, the cross-linking agent is preferably a polyamine selected from spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), salts thereof or mixtures thereof, more preferably, the cross-linking agent is a polyamine selected from spermine, spermidine and mixtures thereof.

[0112] The cross-linking agent may be selected from hydroxyapatite beads modified to carry epoxy groups (such as the compound of formula I described below) and mixtures thereof.

[0113] Preferably, the crosslinking agent is a compound of formula I:

[0114] Y-(Z) n

[0115] wherein the functional groups Z are the same or different and are as defined above,

[0116] n is an integer greater than or equal to 2, in particular 2 to 8, preferably 2,

[0117] Y is a polyvalent hydrocarbon radical, in particular aliphatic, having a valence of n and having 1 to 150 carbon atoms:

[0118] - wherein one or more (e.g., 1 to 150, or 1 to 50, or 1 to 15, or 1 or 2) CH2 units are optionally substituted by one or more divalent units selected from arylene, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )- and -[SiR 2 R 3 O] m -SiR 2 R 3 -,in:

[0119] R 1 represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl group;

[0120] m is an integer between 1 and 20, and

[0121] R 2 and R 3 are the same or different and represent a hydrogen atom, a halogen atom, -OR 11 Group (where R 11 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms), an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms which is optionally substituted by one or more groups selected from halogen atoms, aryl groups or hydroxyl groups,

[0122] - the polyvalent group is unsubstituted or substituted by one or more monovalent groups selected from halogen atoms, hydroxyl groups and aryl-(C1-C6)alkyl groups, preferably unsubstituted.

[0123] In particular, n is an integer from 2 to 8, preferably, n represents 2, 3 or 4, even more preferably, n is equal to 2.

[0124] Advantageously, R 1 represents a hydrogen atom or a (C1-C6) alkyl group.

[0125] In particular, R2 and R 3 are identical or different and represent an aliphatic hydrocarbon group having 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0126] Preferably, in the definition of Y, the polyvalent hydrocarbon radical may be an aliphatic or aromatic polyvalent hydrocarbon radical, preferably aliphatic and in particular saturated, having a valence of n and having 1 to 150 carbon atoms, preferably 1 to 50 carbon atoms, more preferably 1 to 20 carbon atoms, even more preferably 2 to 20 carbon atoms.

[0127] In particular, in the definition of Y, the polyvalent hydrocarbon group is a saturated, especially linear, aliphatic polyvalent hydrocarbon group.

[0128] Preferably, Y is a polyvalent hydrocarbon group as described above, wherein one or more CH2 units are optionally substituted by one or more divalent units selected from -O-, -SO2-, -[SiR 2 R 3 O] m -SiR 2 R 3 - and -NH-, where R 2 , R 3 and m are as described above.

[0129] In particular, Y is a polyvalent hydrocarbon radical as described above, preferably aliphatic and saturated, in particular linear, branched or star-shaped, and optionally wherein:

[0130] - at least two CH2 units are substituted by -O-, in particular between 1 and 50 CH2 units, more in particular between 1 and 15 CH2 units, or

[0131] - at least one, preferably one or two CH2 units are replaced by -NH- units, or

[0132] - at least one, preferably one, CH2 unit is replaced by a -SO2- unit, or

[0133] - at least two, preferably two CH2 units are replaced by -O-, and at least one, preferably one CH2 unit is replaced by -[SiR 2 R 3 O] m -SiR 2 R 3 -unit substitution, where R 2 , R 3 and m are as described above.

[0134] More particularly, when one or more CH2 units are replaced by -O-, the one or more units replaced make Y contain one or more -CH2-CH2-O-units. In particular, Y contains 1 to 50 -CH2-CH2-O-units, advantageously 2 to 25 -CH2-CH2-O-units, more advantageously 2 to 15 -CH2-CH2-O-units. Y may contain only -CH2-CH2-O-units.

[0135] More preferably, Y is an alkyl group comprising 1 to 150 carbon atoms, in particular 1 to 50 carbon atoms, in particular 1 to 20 carbon atoms, for example 1 to 12 carbon atoms, in particular 1 to 6 carbon atoms, preferably linear, wherein optionally one or more CH2 units are substituted by one or more divalent units selected from -O- and -NH-, more in particular between 1 and 50, in particular between 1 and 15, for example 1 or 2 divalent units selected from -O- and -NH-.

[0136] According to a first embodiment, R 2 and R 3 Same or different, and means -OR 11 Group, where R 11 As mentioned above. In particular, R 11 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0137] According to a second embodiment, R 2 and R 3 may be the same or different and represent an aliphatic hydrocarbon group having 1 to 6 carbon atoms, optionally substituted (preferably unsubstituted) by one or more groups selected from halogen atoms, aryl groups or hydroxyl groups, more preferably an unsubstituted (C1-C6) alkyl group, such as methyl or ethyl.

[0138] Advantageously, the crosslinking agent is a compound of formula Ia below:

[0139] Z 1 -Y 1 -Z 2

[0140] The same or different groups Z 1 and Z 2 is selected from the group consisting of an isocyanate group, an amino group, an epoxide group, a carboxyl group, an N-succinimidyloxycarbonyl group, an N-sulfosuccinimidyloxycarbonyl group, a halocarbonyl group, an isothiocyanate group, a vinyl group, a formyl group, a hydroxyl group, a mercapto group, a hydrazine group, an acylhydrazine group, an aminooxy group, a carbodiimide group and an anhydride residue,

[0141] And Y 1denotes a divalent hydrocarbon chain having 1 to 50 carbon atoms, in particular aliphatic:

[0142] - wherein one or more (e.g., 1 to 15 or 1 or 2) CH2 units are optionally substituted by one or more divalent units selected from arylene, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )- and -[SiR 2 R 3 O]m-SiR 2 R 3 -, where R 1 represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms or an aryl-(C1-C6)alkyl group,

[0143] m is an integer between 2 and 20, and

[0144] R 2 and R 3 are the same or different and represent a hydrogen atom, a halogen atom, -OR 11 Group (where R 11 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms), an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms which is optionally substituted by one or more groups selected from halogen atoms, aryl groups or hydroxyl groups,

[0145] - the chain is unsubstituted or substituted by one or more monovalent groups selected from halogen atoms, hydroxyl groups or aryl(C1-C6)alkyl groups.

[0146] Group Z 1 and Z 2 has the same meaning as the group Z defined above.

[0147] Y 1 has the same definition as Y defined above, wherein the valence n is equal to 2. In particular, Y 1 It may contain only -CH2-CH2-O- units, as defined above.

[0148] Preferably, the crosslinking agent of Formula I or Ia does not contain -[SiR 2 R 3 O] m -SiR 2 R 3 -unit.

[0149] Other ingredients

[0150] The additional ingredients may be lubricants, cosmetic active ingredients (such as antioxidants, coenzymes, amino acids, vitamins, minerals and nucleic acids), therapeutic active ingredients (such as anesthetics, antibiotics, antifungal agents and adrenaline and its derivatives) and mixtures thereof.

[0151] Polysaccharides (particularly uncrosslinked hyaluronic acid or uncrosslinked heparin precursor) can be exemplified as lubricants.

[0152] Examples of anesthetic agents include, by way of non-limiting example, amocaine, amicaine, aprindine, atocaine, articaine, benzocaine, betocaine, bupivacaine, butacaine, butamben, butancaine, chlorobutanol, chloroprocaine, cinchocaine, chlordacaine, cocaine, cleflurane, cyclomethicaine, dexicaine, diaminocaine, dipedoporin, dyclonine, etidocaine, euprocaine, febuvirine, formocaine, guanifecanol, encapcaine, hexacane, hydroxyprocaine, hydroxytetracaine, butamben, leuprocaine, levobupivacaine, and selenocaine. The invention can be used in combination with the following: oxadiazine, ...

[0153] Examples of antioxidants include, by way of non-limiting example, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavanols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and derivatives thereof, and mixtures thereof.

[0154] Examples of amino acids include, in a non-limiting manner, arginine (e.g., L-arginine), isoleucine (e.g., L-isoleucine), leucine (e.g., L-leucine), lysine (e.g., L-lysine or L-lysine monohydrate), glycine, valine (e.g., L-valine), threonine (e.g., L-threonine), proline (e.g., L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, derivatives thereof (e.g., N-acetylated derivatives, such as N-acetyl-L-cysteine), and mixtures thereof.

[0155] Examples of vitamins and their salts include, in a non-limiting manner, vitamins E, A, C, B, in particular vitamins B6, B8, B4, B5, B9, B7, B12, and even better pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0156] Examples of minerals include, in a non-limiting manner, zinc salts (e.g. zinc acetate, particularly zinc acetate dehydrate), magnesium salts, calcium salts (e.g. hydroxyapatite, particularly in bead form), potassium salts, magnesium salts, sodium salts, copper salts (e.g. copper sulfate, particularly copper sulfate pentahydrate) (optionally in aqueous form), and mixtures thereof.

[0157] Examples of nucleic acids include, in a non-limiting manner, adenosine, cytidine, guanosine, thymidine, cytosine, derivatives thereof, and mixtures thereof.

[0158] Coenzymes include coenzyme Q10, CoA, NAD, NADP and mixtures thereof.

[0159] Adrenaline derivatives include adrenaline, norepinephrine, and mixtures thereof.

[0160] The amount of additional compound will obviously depend on the nature of the compound in question, the desired effect and the destination of the composition described herein.

[0161] method

[0162] The steps of the method of the present invention can be described as follows.

[0163] Providing at least one polysaccharide or a salt thereof (step a))

[0164] Step a) of the process according to the invention comprises providing at least one polysaccharide or a salt thereof, in particular a physiologically acceptable salt.

[0165] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably a sodium salt.

[0166] The polysaccharide may be provided in a fully or partially hydrated form, or in a dry form, such as a powder or a fiber form. More particularly, in step a), the polysaccharide is provided in a dry form, such as a powder or a fiber form.

[0167] When the polysaccharide is provided in a hydrated form, it is in the form of an uncrosslinked gel or solution.

[0168] In particular, when the polysaccharide is in hydrated form, it is an aqueous non-crosslinked gel or aqueous solution. More particularly, the polysaccharide is mixed with water, optionally with the addition of a phosphate buffer or a supplemented phosphate buffer, i.e. may contain additional ingredients as defined in the additional step. Thus, it should be understood that the aqueous non-crosslinked gel or aqueous polysaccharide solution does not contain sodium hydroxide.

[0169] Providing at least one cross-linking agent or a salt thereof (step b)

[0170] Step b) of the method according to the invention comprises providing at least one cross-linking agent or a salt thereof, in particular a physiologically acceptable salt.

[0171] The cross-linking agent is as described above.

[0172] Preparation of cross-linking reaction medium (step c)

[0173] Step c) of the method according to the invention comprises preparing a cross-linking reaction medium. The reaction medium comprises a polysaccharide, a cross-linking agent and a solvent.

[0174] The solvent is typically water, or a mixture comprising water and an organic solvent (typically a mixture comprising at least 90% by weight of water, or at least 95% by weight, or at least 99% by weight of water relative to the total weight of the solvent). For example, an organic solvent (e.g., an alcohol (particularly ethanol) or DMSO) may be used to dissolve the crosslinker, for example when it is a poly(dimethylsiloxane) terminated with a diglycidyl ether at each end (CAS No.: 130167-23-6), before addition to the aqueous reaction medium.

[0175] The reaction medium may also contain salts, pH adjusters (e.g., Bronsted bases, more preferably hydroxide salts, such as sodium hydroxide or potassium hydroxide), additional ingredients as described above, and mixtures thereof. 1 or Z 2 ) represents an epoxide group or a vinyl group, the addition of a Bronsted base may be particularly necessary. In these cases, crosslinking occurs at a pH greater than or equal to 10, more advantageously greater than or equal to 12, which requires the addition of a Bronsted base (e.g. sodium hydroxide) to the reaction medium, generally at a concentration between 0.10 M and 0.30 M.

[0176] The reaction medium is usually prepared from one or more polysaccharides in dry form. When the reaction medium is prepared from one or more polysaccharides in hydrated form, the aqueous uncrosslinked gel or polysaccharide aqueous solution used to prepare the reaction medium does not contain sodium hydroxide. Therefore, before step d) begins, the maximum contact time of the polysaccharide with sodium hydroxide (regardless of whether the polysaccharide is provided in dry or hydrated form) is 5 hours, for example 15 minutes to 4 hours or 30 minutes to 2 hours.

[0177] The total amount of crosslinking agent in the reaction medium varies from 0.001 mol to less than 0.02 mol per 1 mol of repeating unit of polysaccharide, for example 0.001 mol to 0.015 mol per 1 mol of repeating unit of polysaccharide, preferably 0.001 mol to 0.01 mol per 1 mol of repeating unit of polysaccharide, even more preferably 0.001 mol to 0.008 mol or 0.001 mol to 0.005 mol per 1 mol of repeating unit of polysaccharide. When the polysaccharide is a mucopolysaccharide (e.g., hyaluronic acid), the repeating unit is a disaccharide unit.

[0178] The mass concentration of the polysaccharide or polysaccharide salt in the reaction medium is advantageously in the range of 50 mg / g solvent to 300 mg / g solvent, preferably 100 mg / g to 200 mg / g.

[0179] Step c) of the process according to the invention generally comprises a step of homogenizing the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with blades or stirring with a spatula.

[0180] Step c) is generally carried out at a temperature of 4°C to 35°C, preferably 15°C to 25°C.

[0181] The duration of the step of preparing the reaction medium does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours. The reaction medium obtained at the end of step c) is advantageously placed directly under the conditions of step d) according to the present invention.

[0182] Crosslinking at temperature T and pressure P (step d)

[0183] Then, the reaction medium obtained at the end of step c) is placed at a pressure P and a temperature T for 2 to 17 weeks, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the eutectic temperature of the reaction medium (i.e., a mixture comprising polysaccharides, cross-linking agents and solvents and optionally salts, pH adjusters and additional ingredients) measured at the pressure P and less than the freezing point of the reaction medium measured at the pressure P.

[0184] The freezing point temperature of the reaction medium represents the temperature at which the mixture of the components of the reaction medium solidifies (i.e. becomes non-fluid) on a macroscopic scale. Below the freezing point, the mixture is in a frozen state characterized by the coexistence of components in solid and liquid form. The frozen state is maintained up to the eutectic temperature of the reaction medium.

[0185] The eutectic temperature of a reaction medium refers to the temperature below which a mixture of components of the reaction medium changes from a frozen state (liquid and solid phases coexist) to a completely solid state (i.e., a state in which all components of the mixture are in solid form).

[0186] The freezing point and eutectic point of a mixture depend on the pressure to which the mixture is subjected, and therefore the freezing point and eutectic point are measured at pressure P.

[0187] The freezing point and the eutectic point can be determined by differential scanning calorimetry. This method allows the determination of phase transitions. To do this, the product to be investigated is gradually cooled until a phase transition is observed.

[0188] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably in the range of -35°C to -10°C. Still more preferably, the temperature T is about -20°C.

[0189] The pressure P is preferably atmospheric pressure. Atmospheric pressure is the pressure exerted by the air constituting the atmosphere on any surface in contact with it. It varies according to the altitude. At an altitude of 0m, the average atmospheric pressure is 101325Pa. Preferably, the pressure P is atmospheric pressure, and the temperature T is greater than or equal to -55°C and less than or equal to -5°C; preferably, T varies from -35°C to -10°C, or is about -20°C.

[0190] Preferably, the reaction medium obtained at the end of step c) is left at said pressure P and said temperature T for 2 to 15 weeks, preferably 2 to 13 weeks, preferably 2 to 11 weeks, preferably 2 to 9 weeks, more preferably 3 to 6 weeks or about 4 to 5 weeks or about 30 days.

[0191] As mentioned above, the cross-linking of the polysaccharides mainly occurs in step d) (although it can start from step c)). Therefore, this step enables the polysaccharide chains to be cross-linked to each other. The functional groups of the cross-linking agent react with the functional groups present on the polysaccharide to bind the polysaccharide chains to each other and cross-link them by forming intermolecular bonds. The cross-linking agent can also react with the functional groups present on the same polysaccharide molecule to form intramolecular bonds. In particular, the functional groups of the cross-linking agent react with the -OH or -COOH or -CHO groups present on the polysaccharide (e.g. hyaluronic acid). Thereby a cross-linked polysaccharide comprising at least one cross-linked chain between two polysaccharide chains is obtained, wherein the cross-linked chain is a residue of the cross-linking agent. In particular, after step d), the cross-linked polysaccharide comprises at least one cross-linked bond between two polysaccharide chains, and the cross-linked bond more particularly comprises a multivalent group Y as described above, preferably a divalent group Y as described above. 1 Certain functional groups Z of the crosslinking agent (e.g. Z 1 and Z 2 ) may not react with the polysaccharide chain. In particular, when the crosslinking agent has two functional groups Z 1 and Z 2 When a functional group Z 1 can react with polysaccharides, and another functional group Z 2 It does not react with any polysaccharide. Then, dangling chains are formed.

[0192] Crosslinking can be carried out in the presence of a variety of crosslinking agents. When crosslinking is carried out in the presence of a variety of crosslinking agents, the crosslinking agent can be added to the reaction medium simultaneously or individually over time. Therefore, step b) can include repeated crosslinking steps. Then, crosslinking is carried out in the presence of a total amount of a crosslinking agent of 0.1 mol to less than 2 mol, preferably 0.1 mol to 1.5 mol or 0.1 mol to 1 mol or 0.1 mol to 0.8 mol or 0.1 mol to 0.5 mol of crosslinking agent (or its salt) per 100 mol polysaccharide repeating units. The crosslinking conditions (particularly the content of the crosslinking agent, the duration and the temperature and the weight average molecular weight (Mw) of the polysaccharide used) are interdependent.

[0193] More specifically, the higher the temperature of the cross-linking reaction, the shorter the reaction time to obtain the same degree of modification of the polysaccharide by the cross-linking agent.

[0194] The lower the content of crosslinking agent, the longer the reaction time must be in order to obtain similar mechanical properties of the obtained gel. In other words, the smaller the molar percentage of crosslinking agent, the fewer reactive functional groups are present in the reaction medium, and the lower the probability of two groups meeting and reacting together, and the longer the reaction time must be in order for the functional groups to react with each other and form crosslinks, thereby obtaining a gel with the desired properties.

[0195] Conversely, too large an amount of cross-linking agent and too long a cross-linking time will result in a gel that becomes brittle and too hard and therefore not interesting for cosmetic applications.

[0196] The higher the weight average molecular weight (Mw) of the polysaccharide, the lower the degree of modification required to obtain a gel with given mechanical properties.

[0197] For the same molar percentage of cross-linking agent, the lower the weight average molecular weight (Mw) of the polysaccharide, the longer the reaction time required to obtain a gel with given mechanical properties.

[0198] When the functional group Z of the crosslinking agent is an amino group, the crosslinking reaction with the polysaccharide is advantageously carried out in the presence of at least one activating agent, if necessary in combination with at least one coupling auxiliary agent.

[0199] In this regard, the activator can be selected from water-soluble carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide hydrochloride (ETC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC), salts thereof and mixtures thereof, preferably represented by EDC.

[0200] As for the coupling aid, when present, it can be selected from N-hydroxysuccinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt) and N-hydroxysulfosuccinimide (sulfoNHS) and mixtures thereof, preferably represented by HOBt.

[0201] Additional Steps

[0202] The method according to the invention may comprise one or more additional steps, such as steps of adding one or more additional ingredients, purification, sterilization, sieving, swelling and / or packaging.

[0203] The method according to the invention may comprise the step of adding at least one additional ingredient. The additional ingredient may be selected from lubricants, cosmetic active ingredients (e.g. antioxidants, coenzymes, amino acids, vitamins, minerals and nucleic acids), therapeutic active ingredients (e.g. anesthetics, antibiotics, antifungals and adrenaline and its derivatives) and mixtures thereof. The additional ingredient may be as described above.

[0204] The method according to the invention may comprise at least one purification step. The purification may be carried out by dialysis.

[0205] The method according to the invention may comprise a step of sterilizing the hydrogel. Sterilization is preferably carried out by heating. Sterilization is generally carried out by raising the temperature of the sterilization medium to a temperature known as the plateau temperature, which is maintained for a specific time known as the plateau time. Sterilization is preferably carried out at a plateau temperature of 121° C. to 135° C., preferably for a plateau time of 1 to 20 minutes, with F0 ≥ 15. The sterilization value F0 corresponds to the time (in minutes) required to inactivate 90% of the number of microorganisms present in the product to be sterilized at 121° C. Alternatively, sterilization may be carried out by gamma radiation, UV radiation or by ethylene oxide.

[0206] The method may comprise a step of sieving the hydrogel, more particularly using a sieve with a porosity between 50 μm and 2000 μm. This sieving step makes it possible to obtain a more uniform hydrogel with an extrusion force that is as constant as possible (i.e. as regular as possible). A person skilled in the art knows how to choose a sieve with a pore size suitable for the mechanical properties of the prepared hydrogel.

[0207] The method may include a step of swelling the hydrogel. In the hydrogel swelling step, the polysaccharide concentration of the hydrogel is adjusted. In particular, a solvent is added, such as water, phosphate buffer or water for injection. More particularly, the pH of the added solvent is near physiological pH (6.8-7.8). The polysaccharide concentration obtained after the swelling step is advantageously from 1 mg / g gel to 50 mg / g hydrogel, more advantageously 5 mg / g hydrogel to 35 mg / g hydrogel, and even more advantageously 10 mg / g hydrogel to 30 mg / g hydrogel.

[0208] The method may include the step of packaging the hydrogel.

[0209] If applicable, the step of adding one or more additional ingredients is preferably performed after the purification step.

[0210] If applicable, the step of adding one or more additional ingredients is preferably performed before the sterilization step.

[0211] In particular, the step of adding one or more additional ingredients may also include adding at least one therapeutic active ingredient or at least one cosmetic active ingredient or a mixture thereof. When adding at least one therapeutic active ingredient and / or at least one cosmetic active ingredient, the step of adding one or more additional ingredients is preferably performed after step d).

[0212] If applicable, a purification step is preferably performed after a step of adding one or more additional ingredients.

[0213] If applicable, a purification step is preferably performed before a sterilization step.

[0214] If applicable, a purification step is preferably performed before the sieving step.

[0215] The sterilization step is preferably performed after steps a) to d) and any additional steps. In particular, the hydrogel is sterilized after it has been packaged in its injection device and the gel is packaged after all steps of the method and before sterilization.

[0216] Hydrogel

[0217] Another object of the present invention is a hydrogel obtainable by the process of the present invention. Such a hydrogel may also be referred to by the term cryogel.

[0218] The hydrogel comprises one or more polysaccharides as defined above. The polysaccharide is cross-linked. The molar degree of cross-linking of the polysaccharide is greater than 0% and less than 2%, preferably less than or equal to 1.5% or less than or equal to 1%, even more preferably less than or equal to 0.8% or 0.5%, in particular between 0.1% and 0.8% or between 0.1% and 0.5% (moles of cross-linking agent per 100 moles of polysaccharide repeating units).

[0219] The one or more cross-linking agents are as defined above.

[0220] The crosslinking agent is preferably a crosslinking agent whose functional group is an epoxide group. Therefore, the crosslinking agent is preferably 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE) and mixtures thereof.

[0221] The hydrogels of the present invention have viscous properties. In some embodiments, the hydrogels of the present invention have extensible properties.

[0222] The hydrogel is preferably an injectable hydrogel.

[0223] It is preferably sterile, in particular heat sterilized at a plateau temperature of 121° C. to 135° C., preferably a plateau time of 1 to 20 minutes, with F0≧15.

[0224] The hydrogel is preferably homogeneous.

[0225] The hydrogel may comprise one or more additional ingredients as described above, in particular one or more additional ingredients selected from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids and mixtures thereof.

[0226] Preferably, the hydrogels according to the invention that are acceptable for the therapeutic and / or cosmetic applications targeted by the invention have a cross stress (or the stress at the intersection of the G' and G" moduli) greater than or equal to 50 Pa, preferably between 50 Pa and 5000 Pa, even more preferably between 100 Pa and 1000 Pa, preferably greater than 150 Pa and an elastic modulus G' greater than or equal to 20 Pa, preferably from 100 Pa to 2000 Pa, still more preferably from 110 Pa to 1000 Pa.

[0227] Preferably, the cohesion of the hydrogel according to the invention, which is acceptable for the therapeutic and / or cosmetic applications for which the invention is intended, is from 1 N to 30 N, preferably greater than 4 N, for example from 6 N to 20 N or from 6 N to 15 N. This cohesion is measured by mechanical compression using a rheometer. For this purpose, the gel is deposited on a Peltier plate with an initial air gap of 2.60 mm; it is then compressed to 70% of the initial air gap at a constant rate of 100 μm / s at 25° C.; finally, the cohesion of the gel is measured at the end of the compression process. The more cohesive the gel is (i.e., it has a high cohesion value), the more it is able to withstand stresses, such as those that may be encountered after application to a subject.

[0228] Preferably, hydrogels according to the invention that are acceptable for therapeutic and / or cosmetic applications contemplated by the invention have a viscosity test result of 1.5 cm to 15 cm, preferably 1.5 cm to 10 cm, even more preferably 2 to 7.

[0229] Preferably, the hydrogels according to the invention that are acceptable for the therapeutic and / or cosmetic applications for which the invention is intended have adhesive properties and will adhere to surfaces such as glass, metal, human tissue, collagen or plastic. Furthermore, they will maintain a cohesive structure as they move over these surfaces.

[0230] Composition

[0231] Another object of the present invention is a composition comprising the hydrogel according to the invention. It is preferably a cosmetic composition or a pharmaceutical composition. It may also contain a physiologically acceptable excipient.

[0232] The hydrogel according to the invention comprises a cross-linked polysaccharide. The composition may also comprise a non-cross-linked polysaccharide.

[0233] Therefore, the composition according to the present invention can include 0.1% to 5% by weight, preferably 1% to 3% by weight of polysaccharide (e.g., hyaluronic acid), relative to the gross weight of the composition, wherein the polysaccharide (e.g., hyaluronic acid) is present in a cross-linked form and an optional uncross-linked form. In particular, the content of uncross-linked polysaccharide (e.g., hyaluronic acid) varies from 0% to 40% by weight, preferably from 1% to 40% by weight, and more preferably from 5% to 30% by weight, relative to the gross weight of the polysaccharide (e.g., hyaluronic acid) present in the composition.

[0234] The polysaccharide of the hydrogel is as defined above.

[0235] The composition according to the invention is preferably a sterile composition, in particular heat sterilized at a plateau temperature between 121° C. and 135° C., preferably with a plateau duration between 1 and 20 minutes, wherein F0≧15.

[0236] It is preferably an injectable composition.The composition according to the invention then preferably comprises a physiologically acceptable medium, preferably a physiologically acceptable aqueous medium.

[0237] The physiologically acceptable aqueous medium may comprise a physiologically acceptable solvent or a mixture of solvents, preferably water.

[0238] The physiologically acceptable medium may also contain isotonic agents, for example, sugars, sodium chloride, and mixtures thereof.

[0239] The physiologically acceptable medium may also comprise at least one isotonic and physiologically acceptable saline solution.

[0240] Preferably, the balanced salt solution is a phosphate buffered saline solution, and in particular a KH2PO4 / K2HPO4 saline solution buffer.

[0241] The composition according to the present invention may further comprise at least one additional compound selected from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, coenzymes, adrenaline derivatives and mixtures thereof. The additional compound may be as described above.

[0242] application

[0243] The hydrogel or composition according to the invention may have therapeutic and / or cosmetic applications.

[0244] Therefore, the present invention also relates to the use of a hydrogel or a composition according to the invention for filling and / or replacing tissue, in particular soft tissue, in particular by injecting the hydrogel or the composition into the tissue.

[0245] In particular, the hydrogel or composition according to the invention is used for oral care, more particularly for treating gum recession, or filling periodontal pockets. More particularly, the hydrogel or composition according to the invention is used for treating defects in the gum structure that may occur in tooth loss, aging, periodontal diseases and disorders, or after the installation of dental implants, crowns or bridges.

[0246] The hydrogel or composition according to the invention may also be used in ophthalmology, more particularly in protecting the ocular structures during ophthalmic surgery, such as surgery on the anterior or posterior segment of the eye, cataract extraction with possible implantation of an intraocular lens, corneal transplantation, glaucoma filtering surgery or implantation of a secondary lens. In this case, the hydrogel or composition according to the invention will more particularly be injected into the eye.

[0247] The hydrogel or composition according to the invention may also be used in orthopedics or rheumatology, for example by injection into the synovial cavity.The hydrogel or composition according to the invention is then used as a viscosupplementation therapy.

[0248] The hydrogel or composition according to the invention may also be used to treat lipodystrophy.

[0249] The hydrogel or composition according to the invention can be used in cosmetic surgery, in particular gynecological plastic surgery and / or penile plastic surgery.

[0250] The hydrogel or composition according to the invention is more particularly applied by injection.

[0251] Advantageously, the hydrogel or composition according to the invention may be used for its mucoadhesive properties, which may be used to treat gingival pain.

[0252] The present invention also relates to a method for treating the above-mentioned conditions, which comprises administering an effective amount of the hydrogel or composition to an individual in need thereof.

[0253] The effective dosage of the hydrogel or composition varies depending on many parameters, such as the chosen route of administration, the body weight, age, sex, the progression of the disease to be treated and the sensitivity of the individual to be treated.

[0254] The present invention preferably aims at the cosmetic (thus non-therapeutic) use of the hydrogel or the composition according to the invention for preventing and / or treating alterations in the viscoelastic or biomechanical properties of the skin, in particular for stimulating, regenerating, moisturizing, tightening or restoring the radiance of the skin (in particular by mesotherapy); for filling volume defects of the skin, in particular filling wrinkles, fine lines or scars (in particular depressed scars); or for reducing the appearance of wrinkles and fine lines.

[0255] For example, the invention relates to the cosmetic use of a hydrogel or a composition according to the invention for reducing nasolabial folds and frown lines; for increasing the volume of the cheekbones, chin or lips; for restoring facial volume, in particular the cheeks, temples, facial contours and periocular area; or for stimulating, regenerating, moisturizing, firming or restoring the radiance of the skin, in particular by mesotherapy.

[0256] In particular, the hydrogel or composition according to the invention is an anti-aging hydrogel or composition.The hydrogel or composition according to the invention is more particularly applied by injection.

[0257] The invention also relates to a cosmetic, preferably anti-aging, treatment method for keratin materials, in particular the skin, comprising at least the step of applying a hydrogel or a composition according to the invention to or through the keratin material, more particularly by injection.

[0258] Administration can be injection, in particular intraepidermal injection and / or intradermal injection and / or subcutaneous injection. Administration according to the invention by intraepidermal injection and / or intradermal injection and / or subcutaneous injection is intended to inject the hydrogel or composition of the invention into the epidermal region, the dermal epidermal region and / or the dermal region. The hydrogel or composition according to the invention can also be administered by periosteal injection.

[0259] The hydrogel or composition according to the invention can be injected using any method known to the skilled person. In particular, the hydrogel or composition according to the invention can be administered by an injection device suitable for intraepidermal injection and / or intradermal injection and / or subcutaneous injection and / or periosteal injection. The injection device can be particularly selected from a syringe, a set of microinjectors, a wire, a laser or hydraulic device, an injection gun, a needle-free injection device or a microneedle roller.

[0260] The injection device can have any injection device that is generally applicable to intradermal injection and / or intradermal injection and / or subcutaneous injection and / or periosteal injection. Preferably, this device can be a hypodermic injection needle or cannula or a set of micro-operation needles.

[0261] The needle or cannula according to the invention may have a diameter varying from 18G to 34G, preferably between 25G and 32G and a length varying from 4mm to 70mm, preferably 4mm to 25mm. The needle and cannula are advantageously disposable.

[0262] Advantageously, the needle or cannula is associated with a syringe or any other device enabling the delivery of said hydrogel or said injectable composition through said needle or cannula.

[0263] According to a variant embodiment, the catheter may be inserted between the needle / cannula and the syringe.In a known manner, the syringe may be activated manually by the practitioner, or by a syringe support (eg a gun).

[0264] Preferably, the injection device is a syringe.

[0265] In a variant embodiment, the injection device may be adapted for the technique of mesotherapy.

[0266] Mesotherapy is a technique of treatment by intraepidermal and / or intradermal and / or subcutaneous injection of a composition or a hydrogel. According to this technique, the composition or the hydrogel is applied by injection in the form of a plurality of small droplets at the epidermis, the dermo-epidermal junction and / or the dermis, in order in particular to achieve a subcutaneous coating. The technique of mesotherapy is described in particular in the book "Traité de mésothérapie [Treatise on mesotherapy]" by Jacques Le Coz (Masson editions, 2004). Mesotherapy performed on the face is also known as meso-resurfacing or meso-peeling.

[0267] Administration may also be topical.

[0268] Preferably, it is a topical application on the skin surface, more particularly the epidermis, even more particularly the facial epidermis.

[0269] Therefore, the present invention also relates to an injection device as described above comprising a hydrogel or a composition according to the present invention.

[0270] The following examples are for illustrative purposes only and are not to be construed as limiting the present invention.

[0271] Example

[0272] 1. Materials

[0273] - 1.5.MDa and 4MDa of uncross-linked sodium hyaluronate (HTL),

[0274] -BDDE (Sigma Aldrich),

[0275] -DVS (Fisher Scientific),

[0276] -0.25M NaOH,

[0277] -1M HCl,

[0278] - Phosphate buffer (Braun),

[0279] -Lidocaine hydrochloride,

[0280] - 3D mixer,

[0281] -DHR-2 Rheometer,

[0282] - Dynamometers and test benches,

[0283] - Paddle type crushing homogenizer,

[0284] -Sterile polyethylene bag.

[0285] 2. Methods

[0286] 2.1 Measurement of viscoelastic properties

[0287] The viscoelastic properties of the obtained hydrogels were measured using a rheometer (DHR-2) with a stainless steel cone with conical geometry (1°-40 mm) and an anodized aluminum Peltier plate (42 mm) (air gap 24 μm).

[0288] 0.5 g of sterile hydrogel was deposited between the Peltier plate and the cone. Then, a stress sweep was performed at 1 Hz and 25° C. The elastic modulus G′, viscous modulus G″ and phase angle δ at 5 Pa stress were reported.

[0289] The stress τ at the intersection of G' and G" is determined at the intersection of the curves of the G' modulus and the G" modulus and is expressed in Pascals.

[0290] 2.2 Measuring extrusion force

[0291] The extrusion force (in Newtons) of the gel packaged in the syringe was measured at room temperature through a 27G, 1 / 2 inch needle at a constant speed of 12.5 mm / min by a test stand equipped with a dynamometer. The extrusion force result is the average of the extrusion forces of at least 2 samples.

[0292] 2.3 Confirm ductility (viscosity test)

[0293] The viscosity test was performed on a DHR-2 rheometer (TA instruments) equipped with a raw steel geometry (40 mm diameter) and a raw steel Peltier plate (40 mm diameter). 1 g of the product was deposited between the two geometries at 25°C, and the upper geometry was quickly approached to the Peltier plate at a speed of 2 mm / s until the air gap was 2 mm. The upper geometry then compressed the gel at an approach speed of 0.1 mm / s until an air gap of 0.1 mm. The upper geometry was then increased to 0.1 mm / s, and the distance at which the gel maintained its ductility characteristics between the two geometries was reported. Therefore, the ductility determined by the length unit is expressed in cm.

[0294] 2.4 Measuring Cohesion

[0295] For the measurement of cohesion (or mechanical strength, expressed in Newtons), the gel was deposited on a Peltier plate with an initial air gap of 2.60 mm. The gel was then compressed at a constant speed of 100 μm / s at 25° C. until 70% of the initial air gap. The cohesion of the gel was measured at the end of the compression process.

[0296] 2.5 Analysis of soluble hyaluronic acid

[0297] Soluble hyaluronic acid or sHA is analyzed using a high performance liquid chromatography (HPLC-SEC-MALS-RI, ASTRA software (Wyatt Technology Corp.)) connected to a multi-angle light scattering detector and a refractive index detector. According to the initial concentration of hyaluronic acid in the SEC mobile phase (filtered solution (pH 7.2) of 150mM sodium nitrate), the sample is diluted about 10 times. The diluted mixture is composed of dense insoluble hyaluronic acid and soluble hyaluronic acid. The soluble hyaluronic acid portion of the sample is released under rotary oscillation for 5 days, thereby avoiding the artificial generation of soluble hyaluronic acid.

[0298] The soluble and insoluble fractions were separated by gentle filtration (syringe equipped with a 0.45 μm filter) and then subjected to SEC analysis.

[0299] Different injection volumes were tested to achieve at least a five to one background-free signal to prevent and avoid overloading the SEC column.

[0300] For a wide hyaluronic acid range from 500 Da to 20 MDa, the HPLC-SEC system uses dual columns to obtain the best peak resolution on the chromatograph.

[0301] In order to have an optimal molecular weight analysis, the refractive index increment value or dn / dc (refractive index change / polysaccharide concentration in the analysis solvent) determined based on an internal sample analyzed under the same conditions as the sample was 0.165 mL / g.

[0302] Chromatograms obtained by SEC were analyzed to quantify the molecular weight, distribution, and ratio of soluble hyaluronic acid in each sample.

[0303] The weight average molecular weight of the sample is the data provided by the HPLC-SEC software. The percentage of the soluble hyaluronic acid fraction (% sHA) is also the direct output of the HPLC-SEC software after inputting the mass concentration (mg / mL) of the total hyaluronic acid of the sample analyzed.

[0304] The percentages may vary from one composition to another depending on the manufacturing technique and the dispersion (w / d) (weight molar mass / number molar mass) of the sample.

[0305] The % sHA for multiple molecular weight limits (<250 kDa, <100 kDa, <50 kDa, <30 kDa and <20 kDa) is also a direct output of the HPLC software. However, this does not take into account the amount of hyaluronic acid actually released in the gel.

[0306] Therefore, the normalized % sHA for the various molecular weight limits was calculated taking into account the amount of hyaluronic acid actually released by the samples analyzed, thereby normalizing the values ​​provided by the software.

[0307] The results can then be compared directly.

[0308] 3. Example

[0309] Example 1: Hyaluronic acid gel crosslinked with BDDE at temperatures below or above the freezing point of the reaction medium

[0310] Prototypes No. 1 to 9 were prepared using the BDDE contents (BDDE molar CR) described in Table 1 in the following manner.

[0311] BDDE and sodium hyaluronate (1.5 MDa, 120 mg / g) were dissolved in 0.25 M aqueous sodium hydroxide solution in a sterile bag. The mixture was then homogenized in a paddle mill at room temperature for 3 15-minute cycles at 210 rpm. The mixture was then maintained at atmospheric pressure and placed at the temperature and duration shown in Table 1. The pH of the mixture was about 13.

[0312] When the process is carried out at temperatures below the freezing point of the reaction medium (BDDE, sodium hyaluronate, aqueous sodium hydroxide), the mixture is thawed before continuing with the preparation.

[0313] Then, 1N HCl solution was added to a sterile bag to obtain a pH of 7.3 ± 0.5. The mixture was diluted to a concentration of 23 mg hyaluronic acid per gram of product using phosphate buffered saline (PBS). The mixture was homogenized using a three-dimensional stirrer for 24 h. The mixture was dialyzed.

[0314] Sodium hyaluronate (4 MDa) was added as a lubricant.

[0315] An aqueous solution of lidocaine hydrochloride was added to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the product obtained.

[0316] The product thus obtained is sieved and then packaged in syringes.

[0317] Finally, the product was sterilized by autoclave (plateau temperature between 121°C and 135°C, with F0≥15).

[0318] After sterilization, the prototypes were analyzed. The elastic modulus G', the phase angle δ and the stress τ at the intersection of G' and G" were determined. The results are shown in Table 1 below.

[0319]

[0320]

[0321] Table 1: Results

[0322] For reference, Table 2 shows the properties of the commercial product Restylane Lyft, which has a modification level of 1% (the lowest known value for cross-linked hyaluronic acid gels for aesthetic applications).

[0323]

[0324] Table 2: Properties of commercial products

[0325] The commercial product Restylane Lyft has a very low cohesion (2.2 N) compared to the gel obtained by the process of the present invention.

[0326] Although crosslinking at a low molar crosslinking rate of 2.2% at 21°C for 3 days failed to produce a gel (prototype 1 with δ greater than 45°), the method of the present invention was able to produce a hydrogel with desired mechanical properties at a molar crosslinking rate of only 0.4% (prototype 6).

[0327] When increasing the cross-linking time, the elastic modulus increases, but the gel has a lower τ. In other words, it may be more brittle (see prototypes 5 vs. 4 and 7 vs. 6).

[0328] Surprisingly, the gels obtained by the process of the invention have rheological properties and cohesion that are at least as interesting, or even more interesting, than gels conventionally obtained at room temperature with a significantly higher degree of crosslinking (Prototype 2) (same except for HA concentration and manufacturing method).

[0329] Example 2: Method for cross-linking hyaluronic acid to DVS according to the present invention

[0330] Prototypes X1 and X2 were prepared using the DVS contents (DVS molar CR) described in Table 3 in the following manner.

[0331] Sodium hyaluronate (1.5 MDa, 120 mg / g) was dissolved in a 0.25 M aqueous solution of DVS and sodium hydroxide in a sterile bag. The mixture was homogenized in a paddle mill at room temperature for 3 15-minute cycles at 210 rpm. The mixture was placed and maintained at the temperature and time shown in Table 3. The pH of the mixture was about 13.

[0332] Thaw the mixture before proceeding with preparation.

[0333] 1 N HCl solution was added to the sterile bag until a pH of 7 ± 0.5 was obtained. The mixture was diluted with phosphate buffered saline (PBS) to a concentration of 23 mg hyaluronic acid per gram of product.

[0334] The mixture was homogenized by a three-dimensional stirrer for 24 h.

[0335] Then, the mixture was dialyzed, and a fixed amount of sodium hyaluronate (4 MDa) was added as a lubricant.

[0336] An aqueous solution of lidocaine hydrochloride was added to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the product obtained.

[0337] The product thus obtained is sieved and then packaged in syringes.

[0338] Finally, the product was sterilized by autoclave (plateau temperature between 121°C and 135°C, with F0≥15).

[0339] Prototype X0 (comparative) was prepared identically to prototype X1. Only the crosslinking step was changed, namely crosslinking at 21°C for 0.75 days.

[0340]

[0341] Table 3: Cross-linking conditions

[0342] After sterilization, the prototypes were analyzed. The elastic modulus G', the phase angle δ and the stress τ at the intersection of G' and G" were determined. The results are shown in Table 4.

[0343] prototype G'(Pa) δ(°) τ(Pa) X0 56±9 47.7±2.5 N / A X1 140±34 34.0±1.6 188±21 X2 433±64 15.8±3.1 147±14

[0344] Table 4: Results

[0345] Prototypes X1 and X2 of hyaluronic acid crosslinked to DVS are gels (phase angle less than 45°C) and have properties suitable for the therapeutic, cosmetic and aesthetic applications targeted by the present invention (stress at the intersection of G' and G" greater than 50 Pa and G' greater than 20 Pa).

[0346] Prototype X0, produced using the same degree of cross-linking as X1 but conventionally (cross-linked at room temperature), did not show the formation of a gel (δ>45°).

[0347] The process of the present invention appears to be very suitable for cross-linking agents having chemical functional groups other than epoxides.

[0348] Example 3: Stability study of the cryogel according to the present invention

[0349] The prototypes shown in Table 5 were placed in an accelerated and forced degradation oven at 40°C to monitor the evolution of rheological properties over a period of 6 months. These conditions simulate a storage period equivalent to 2 years at room temperature. Each prototype was produced according to the synthesis scheme described in Example 1 and the conditions shown in Table 5.

[0350]

[0351] Table 5: Cross-linking conditions

[0352] The change in phase angle over time was measured and expressed as phase angle loss in Table 6: % Phase Angle Loss = (Phase Angle at 40°C for 6 months - Initial Phase Angle) / Initial Phase Angle * 100

[0353]

[0354]

[0355] Table 6: Results

[0356] Prototypes Y1, Y2 and Y3 obtained by the process of the invention with low crosslinking rates show a slight loss of viscoelastic properties after accelerated aging studies simulating storage for 2 years at 25° C. under adverse conditions. This loss is acceptable and is similar to the loss of a conventional gel (Y4) obtained by conventional methods (crosslinking at room temperature) with high molar crosslinking rates.

[0357] Example 4: Analysis of water-soluble hyaluronic acid in cryogels

[0358] The water-soluble hyaluronic acid of the gel of Table 7 (gel prepared according to Example 1) was analyzed by HPLC-SEC equipped with MALS and RI detectors. The gel after sterilization was diluted with gentle stirring at 37°C to extract the soluble hyaluronic acid. The results are shown in Table 8. The weight average molecular weight (MW) and the amount of soluble hyaluronic acid with MW less than 50 kDa are listed.

[0359]

[0360] Table 7: Cross-linking conditions

[0361]

[0362] Table 8: Results

[0363] All these prototypes were formulated with the same raw material of hyaluronic acid with a MW greater than 1 MDa. Therefore, the generation of fragments less than 50 kDa indicates that some degree of degradation of hyaluronic acid occurred during the process.

[0364] It is therefore evident that the 2 prototypes obtained conventionally (K1 and K2) are the only ones with traces of soluble hyaluronic acid with a MW of less than 50 kDa. All the prototypes obtained by the method of the invention, any other parameters being equal, better preserve the integrity of the hyaluronic acid chains and therefore have no traces of sHA less than 50 kDa.

[0365] Example 5: Comparison of the cold temperatures obtained by the method according to the invention or by a freezing method according to the prior art Integrity of soluble hyaluronic acid chains in cryogels

[0366] The size distribution of hyaluronic acid chains in hydrogels prepared according to the method described in Oschlaeger C., Bossler, F., Willenbacher, N., Synthesis, structure and micro-mechanical properties of 3D hyaluronic acid-based cryogel scaffolds (Biomacromolecules 2016) and prepared according to the present invention were compared to evaluate the respective ability of each method to preserve hyaluronic acid chains.

[0367] According to the method of the prior art, the first step of dissolving the hyaluronic acid fibers in sodium hydroxide is carried out for 24 hours at 4° C. Since the cross-linking process has already started within 24 hours before cryogelation, only a cryogelation step of 10 days is required to obtain a gel with interesting properties close to those of the present invention.

[0368] In the method of the invention, the step of dissolving the hyaluronic acid fibers (step a); preparing the reaction medium) is carried out at room temperature for about 1 hour and then frozen for a longer time. Table 9 summarizes the conditions of this comparison.

[0369] In order to compare the impact of every kind of method on the integrity of hyaluronic acid chain, hyaluronic acid fiber carries out two kinds of methods when not using cross-linking agent.Therefore, after each key step (fiber dilution, cryogelation) of method, MW and hyaluronic acid chain distribution can be tracked by HPLC-SEC.Then, the reduction of MW and the increase of the existence of small fragment hyaluronic acid are the synonyms of described method degraded hyaluronic acid.For each HPLC-SEC analysis (above-mentioned system), sample is neutralized to physiological pH, and is diluted to about 0.5mg / mL.

[0370]

[0371] Table 9: Summary of the two methods compared

[0372] Starting from the same raw material with a MW of 859 kDa, using the same aqueous solution for dissolution, neutralization and dilution, and using the same equipment and the same conditions for analyzing the hyaluronic acid chains, we can observe that the method according to the prior art causes a loss of about 100 kDa in MW of the hyaluronic acid compared to the method according to the invention. This also reflects the presence of 0.5% of hyaluronic acid with a MW < 50 kDa, while the method according to the invention does not contain any trace of these hyaluronic acid fragments.

[0373] It is noteworthy that the freezing step of the two methods does not increase the degradation of hyaluronic acid, because the only degradation observed occurs at 4 ° C for 24 h of the fiber dissolution step. Therefore, only this step has an impact on the MW of hyaluronic acid. Compared with the initial raw material, the control sample (dissolved at 21 ° C for 1 h according to the method of the present invention) did not degrade. The reduction of MW will have a negative impact on the final viscoelastic properties of the gel, and the presence of small fragments of hyaluronic acid will have an impact on the local biological inflammatory response after the gel is implanted in vivo.

[0374] Example 6: Evaluation of the extensibility of the gel (viscosity test)

[0375] The extensibility of the gels shown in Table 10 (prepared according to Example 1) was evaluated by the Tack Test by measuring the distance the gel was able to maintain adhesion / cohesion between two surfaces.

[0376]

[0377] Table 10: Cross-linking conditions

[0378] The adhesion test data is shown in Table 11.

[0379]

[0380] Table 11: Results

[0381] FIG. 1 shows the adhesion tests carried out on prototypes Z2 and Z3.

[0382] After compression of the gel, the surfaces were separated.

[0383] Prototypes Z1 and Z3 have the highest ductility. However, according to its rheological properties, Z1 is not a viscoelastic gel (phase angle > 45°) and therefore behaves as a viscous solution.

[0384] In comparison, Z3 is indeed a gel with a larger stretch range than the other prototypes.

[0385] Z2 and Z4 do not have ductile properties. Z2 is a conventionally obtained gel (crosslinked at room temperature), while Z4 is a hydrogel obtained by the process of the present invention, which has a very high molar crosslinking rate (>2%), thus making the gel very elastic, brittle and inextensible at the same time.

[0386] Therefore, the Z3 prototype with a low modification ratio obtained by the process of the present invention gave the best results in terms of obtaining viscoelastic gels and extensible gels with the desired properties (cohesion upon traction).

[0387] Example 7: Evaluation of the viscosity and affinity of gel to surface

[0388] The stickiness of the gel J2 according to the invention (prototype 6 of Example 1) and the conventional gel J1 (prototype 2 of Example 1) was qualitatively observed. The gels are shown in Table 12. After depositing 1 g of gel pellets on the surface, the adhesion or non-adhesion of the tested gels to the model surface was observed. The gels were pushed in 10 cm and the state of the pellets and the behavior of the gels were observed. The four model surfaces were metal surfaces ( Figure 1A ), glass surface( Figure 1B ), plastic surface( Figure 1C ) and collagen surface ( Figure 1D ).

[0389]

[0390] Table 12: Cross-linking conditions

[0391] Prototype J1 obtained by conventional cross-linking technique tended to roll on the model surface when trying to move. In contrast, prototype J2 obtained by cryogelation and with G' relatively close to J1 tended to adhere systematically to the four model surfaces.

[0392] The force required to move J2 across the four model surfaces was also greater due to the sticking of the gel as it moved. It should also be noted that although prototype J2 stuck along its path of movement, it maintained its cohesion (single piece of gel) and did not break apart as it moved across the surface. This can be an important advantage in vivo, as the gel will tend to migrate less by sticking to the surface.

Claims

1. A method for preparing a hydrogel, preferably an injectable hydrogel, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one cross-linking agent, the cross-linking agent comprising at least two functional groups Z, the functional groups Z being the same or different and selected from the group consisting of isocyanate groups, amino groups, epoxide groups, carboxyl groups, N-succinimidyloxycarbonyl groups, N-sulfosuccinimidyloxycarbonyl groups, halocarbonyl groups, isothiocyanate groups, vinyl groups, formyl groups, hydroxyl groups, thiol groups, hydrazino groups, acylhydrazino groups, aminooxy groups, carbodiimide groups and anhydride residues; c) preparing a cross-linking reaction medium comprising a polysaccharide, a cross-linking agent and a solvent, wherein the total amount of the cross-linking agent ranges from 0.001 mol to less than 0.02 mol per 1 mol of polysaccharide repeating unit, and the duration of the reaction medium preparation step does not exceed 5 hours; d) placing the reaction medium obtained at the end of step c) at a pressure P and a temperature T for 2 to 17 weeks, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the eutectic temperature of the reaction medium measured at the pressure P and less than the freezing point of the reaction medium measured at the pressure P.

2. The method according to claim 1, wherein: The polysaccharide is a mucopolysaccharide, and preferably, the polysaccharide is hyaluronic acid or a salt thereof.

3. The method according to claim 1 or 2, wherein: The functional group Z of the cross-linking agent is an epoxide group. Preferably, the cross-linking agent is selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE) and mixtures thereof.

4. A method according to any one of the preceding claims, wherein: The total amount of the cross-linking agent ranges from 0.001 mol to 0.01 mol per 1 mol of the polysaccharide repeating unit, preferably from 0.001 mol to 0.005 mol per 1 mol of the polysaccharide repeating unit.

5. A method according to any one of the preceding claims, wherein: The temperature T is greater than or equal to -55°C and less than or equal to -5°C, preferably about -20°C.

6. A method according to any one of the preceding claims, wherein: The pressure P is equal to atmospheric pressure.

7. A method according to any one of the preceding claims, further comprising a sterilization step.

8. A hydrogel obtainable by a method according to any one of the preceding claims.

9. A cosmetic composition or a pharmaceutical composition comprising the hydrogel according to claim 8.

10. Use of the hydrogel according to claim 8 or the composition according to claim 9 for filling and / or replacing tissue.

11. Cosmetic use of a hydrogel according to claim 8 or a composition according to claim 9 for preventing and / or treating changes in the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, in particular for filling wrinkles, fine lines and scars, to reduce nasolabial folds and frown lines, to increase the volume of the cheekbones, chin or lips, to restore facial volume, in particular the cheeks, temples, facial contours and periocular area, or to reduce the appearance of wrinkles and fine lines, or to stimulate, regenerate, moisturize, tighten or restore the radiance of the skin, in particular by mesotherapy.

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