Hydrogels for soft tissue filling

Abstract

The present invention relates to a method for producing a hydrogel comprising a cross-linked polysaccharide, in particular, a method for producing an injectable hydrogel containing cross-linked hyaluronic acid. The present invention also relates to a hydrogel, preferably injectable, that can be obtained by the method, a composition containing the hydrogel, and the uses of said hydrogel.

Description

[0001] DESCRIPTION

[0002] TITLE: HYDROGELS FOR SOFT TISSUE FILLING

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method for preparing a hydrogel comprising a crosslinked polysaccharide, in particular, to a method for preparing an injectable hydrogel comprising crosslinked hyaluronic acid. The present invention also relates to a hydrogel, preferably injectable, obtainable by the method, a composition comprising the hydrogel, and uses of this hydrogel.

[0005] TECHNOLOGICAL BACKGROUND

[0006] Polysaccharide-based hydrogels, especially hyaluronic acid-based hydrogels, are widely used for filling soft tissues, especially skin, which have, for example, volume defects (e.g., wrinkles, scars) or for increasing their volume. Hydrogels suitable for filling soft tissues are typically crosslinked hydrogels, i.e., the polysaccharide is crosslinked by means of one or more crosslinking agents. This crosslinking results in a hydrogel with the mechanical properties desirable for filling soft tissues.

[0007] Although biodegradable and biocompatible, hyaluronic acid hydrogels can, with a low incidence, cause adverse effects that develop between 6 and 24 months after application of the filler. In particular, the filler may migrate to an area far from its application in or on the body (Chae SY, Lee KC, Jang YH, Lee SJ, Kim DW, Lee WJ. A Case of the Migration of Hyaluronic Acid Filler from Nose to 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 acid filler. JAAD Case Reports. 5. 488-490).Several reasons have been proposed to explain this phenomenon, such as for example poor injection technique, too large an injected volume, post-injection physical activity (muscular activity, exercises), pressure-induced displacement (eg, massage), intravascular injection, lymphatic invasion, gravity. To reduce the risk of migration of polysaccharide-based gels, some methods have already been described, in particular, it has been proposed to apply a repulsive electric field to at least part of the perimeter around the treatment site (W02020 / 172371).

[0008] However, there remains a need for the provision of a cross-linked polysaccharide-based hydrogel, in particular a cross-linked hyaluronic acid-based hydrogel whose hyaluronic acid chains are preserved, having mechanical properties suitable for filling soft tissues and which does not migrate into the tissues after its application.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a method for preparing a hydrogel, preferably injectable, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two functional groups Z, identical or different, chosen from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide groups, and an acid anhydride residue; c) preparing a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total amount of crosslinking agent ranging from 0.001 to less than 0.02 mole per 1 mole of repeating unit of the polysaccharide, the duration of the step of preparing the reaction medium not exceeding 5 hours;d) placing the reaction medium obtained at the end of step c), for a period ranging from 2 weeks to 17 weeks, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P.;

[0011] The present invention also relates to a hydrogel obtainable by a method as described herein, as well as to a cosmetic or pharmaceutical composition comprising such a hydrogel and their uses in filling and / or replacing tissues, for preventing and / or treating the alteration of the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, and in particular for filling wrinkles, fine lines and scars; for example, for reducing nasolabial folds and bitterness folds, for increasing the volume of the cheekbones, chin or lips, for restoring the volumes of the face, in particular the cheeks, temples, the oval of the face, and the area around the eyes; or for reducing the appearance of wrinkles and fine lines; or for stimulating, regenerating, moisturizing, firming or restoring the radiance of the skin, in particular by mesotherapy.

[0012] Other aspects of the invention are as described below and in the claims. FIGURES

[0013] Figure 1 illustrates the stickiness of a hydrogel according to the invention compared to a traditional hydrogel on a metal surface (Fig. 1A), glass (Fig. 1B), plastic (Fig. 1C) and collagen (Fig. 1D).

[0014] DEFINITIONS

[0015] The term "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed of two media, one "solid" and one "liquid," dispersed in each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (e.g., hydrogen bonds) or by covalent bonds (crosslinking). The liquid medium consists of a solvent. A gel generally corresponds to a product that has a phase angle 5 less than or equal to 45° at 1 Hz for a strain of 0.1% or a pressure of 1 Pa, advantageously a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

[0016] The term "hydrogel" designates a gel as defined above in which the solvent constituting the liquid medium is predominantly water (for example at least 90%, in particular at least 95%, in particular at least 99% by weight of the liquid medium). Preferably, the liquid medium comprises, in particular consists of, a buffer solution, advantageously allowing a pH of the liquid medium of between 6.8 and 7.8, in particular a saline phosphate buffer.

[0017] The term “injectable gel” refers to a gel that can flow and be injected manually using a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 30 G, 27 G, 26 G, 25 G hypodermic needle. Preferably, an “injectable gel” is a gel having an average extrusion force of less than or equal to 25 N, preferably ranging from 5 to 25 N, more preferably ranging from 8 to 15 N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm / min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle with an external diameter less than or equal to 0.4 mm (27 G) and a length of 1”, at room temperature.

[0018] The "stringiness" of a product refers to its ability to be stretched between two surfaces to which it has adhered. The stringiness can be determined using a texturometer, sensory analysis carried out by a panel, or rheological and mechanical measurements including phase angle measurement (5) or tensile tests. In particular, this characteristic 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 Drug in Dermatology, 2017, 16:154-161, "Resistance to stretching") or by performing a Tack test and measuring the length of the gel threads under tension. The "stickiness" of a product refers to its ability to adhere to a surface. It can be determined qualitatively using sensory analysis using a panel or by moving a bolus across a surface.It can also be determined quantitatively by measuring the adhesion force to a surface, tensile machine or mechanical analysis.

[0019] The term "polysaccharide" refers to a polymer composed of monosaccharides (preferably D enantiomers) joined together by glycosidic bonds.

[0020] The term “monosaccharide”, also called “ose”, refers to an unmodified or modified monosaccharide.

[0021] An “unmodified monosaccharide” means a compound of the formula H-(CHOH) X-CO- (CHOH)yH with x and y representing, independently of each other, an integer ranging from 0 to 5 provided that 2 < x+y < 5, the monosaccharide being able to be in a linear form represented by the above-mentioned formula or being able to be in a cyclized form by reaction of the CO function (aldehyde or ketone) with one of the OH groups to form a hemiacetal or hemiketal group. Preferably, the monosaccharide is in cyclized form. There are two types of ose: aldoses which carry an aldehyde function (when x or y is 0) and ketoses which carry a ketone function (when neither x nor y is 0). Monosaccharides are classified by number of carbons. For example, 6-carbon monosaccharides (x+y=5) are hexoses with the formula CeH^Oe and can be allose, altrose, glucose, mannose, gulose, idose, galactose or talose.The 5-carbon monosaccharides (x+y=4) are pentoses of formula C5H10O5 and may be ribose, arabinose, xylose, or lyxose. Preferably, the monosaccharide is a hexose, i.e. x+y = 5. A monosaccharide further comprises x+y asymmetric carbons and therefore 2. (x+y-1) pairs of enantiomers. Each pair of enantiomers is designated by a different name and the enantiomers of the same pair are called D and L enantiomers respectively.

[0022] A “modified monosaccharide” means an unmodified monosaccharide as defined above, including, for example:

[0023] - one or more of the OH functional groups have been replaced by another functional group, for example:

[0024] (i) a group OR with R representing a group (Ci-Ci)alkyl such as methyl or ethyl; hydroxy-(Ci-C6)alkyl such as hydroxyethyl (-CH2CH2OH) or hydroxypropyl (-CH2-CH(OH)-CH3); carboxy-(Ci-C6)alkyl such as carboxymethyl (-CH2COOH); or CO-(Ci-C6)alkyl such as acetyl; and / or

[0025] (ii) a group NR'R” with R' and R” representing, independently of each other, H, (Ci-C6)alkyl or CO-(Ci-C6)alkyl such as acetyl; and / or

[0026] (iii) an OSO3H group; and / or - the terminal CH2OH function(s) have been replaced by a COOH or CHO group;

[0027] - a -CH(OH)-CH(OH)- bond is oxidized to give two terminal -CHO (aldehyde) groups in place of this bond; and / or

[0028] - a terminal CH2OH function was condensed with an OH functional group to form an -O-CH2- chain.

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

[0030] Some or all monosaccharides may be in a modified form. Monosaccharides, when modified, may be in different modified forms.

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

[0032] The “salts” useful in the context of the present invention are preferably physiologically acceptable salts. The terms “physiologically acceptable salts” designate in particular:

[0033] (1) pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like;or formed with pharmaceutically acceptable organic acids such as formic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, And;

[0034] (2) pharmaceutically acceptable base addition salts formed when an acid proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion (eg Na, K), an alkaline earth metal ion (eg Ca, Mg), a zinc ion, a silver ion or an aluminum ion; or coordinated with a pharmaceutically acceptable organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like; or with a pharmaceutically acceptable inorganic base such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide and the like.

[0035] The “degree of modification” (MOD) of a polysaccharide, such as hyaluronic acid, corresponds to the molar quantity of crosslinking agent linked to the polysaccharide, by one or more of its ends, 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 (NMR) spectroscopy.

[0036] The “molar crosslinking rate” (TR), expressed in %, designates the molar ratio of the quantity of crosslinking agent to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the crosslinking medium.

[0037] The term "therapeutic active ingredient" means a substance for curing, relieving symptoms of and / or preventing a disease; a substance possessing curative or preventive properties with respect to human or animal diseases, as well as any substance which may be used in humans or animals or which may be administered to them, with a view to establishing a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological, immunological or metabolic action.

[0038] The expression "cosmetic active ingredient" means any non-therapeutic substance intended in particular to be placed in contact with various superficial parts of the human body, such as the epidermis, hair and capillary systems, nails, lips, chest and teeth, with a view, exclusively or mainly, to cleaning them, protecting them, perfuming them, maintaining them in good condition, modifying their appearance or odor.

[0039] The term "approximately" means that the value concerned may be 10% lower or higher, in particular 5%, in particular 1%, than the indicated value.

[0040] The term "spacer group" refers to a fragment comprising at least one atom intended to link together two chemical groups within the same molecule. Preferably, the spacer group contains at least one carbon atom.

[0041] The term "halogen" refers to an atom of fluorine, chlorine, bromine or iodine.

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

[0043] An “N-succinimidyloxycarbonyl” group is a group with the formula Chem. GR1 below: “sulfosuccinimidyloxycarbonyl” is a group with the formula Chem. GR2 below

[0044] A "halocarbonyl" group is a group of formula -CO-Hal with Hal representing a halogen, such as Cl or Br.

[0045] A “carbodiimide” group is a group comprising a -N=C=N- motif, and more particularly a group of formula -N=C=NR a with R a representing an aliphatic hydrocarbon group comprising from 1 to 20 carbon atoms, preferably a (C1-C6)alkyl group, one or more carbon atoms of which are optionally replaced by a heteroatom chosen from O, S and N, in particular N.

[0046] An “acid anhydride residue” is a group comprising a -C(O)-OC(O)- unit, and more particularly a monovalent cyclic group comprising the -C(O)-OC(O)- unit, such as a saturated monovalent hydrocarbon monocyclic group comprising 5 to 10, in particular 5 or 6, carbon atoms of which three successive carbon atoms are replaced by C(O)- OC(O) and optionally of which one or more, in particular one, additional carbon atoms, preferably not consecutive to the three carbon atoms substituted by CO- O-CO, are each replaced by a heteroatom such as N, O or S, in particular N. The acid anhydride residue may correspond in particular to the following Chem. GR3 formula:

[0047] The acid anhydride residue may also be selected from a maleic anhydride residue or a succinic anhydride residue.

[0048] The expression "aliphatic hydrocarbon chain" or "aliphatic hydrocarbon group" designates a linear, branched and / or cyclic hydrocarbon group, saturated or unsaturated but not aromatic, advantageously comprising from 1 to 50, in particular from 1 to 20, for example from 1 to 12 or from 1 to 6 carbon atoms. These will in particular be alkyl groups. The expression "branched aliphatic hydrocarbon chain" specifically designates a main aliphatic hydrocarbon chain comprising at least one secondary aliphatic hydrocarbon chain.

[0049] The term "star-shaped aliphatic hydrocarbon chain" means a branched aliphatic hydrocarbon chain comprising several secondary aliphatic hydrocarbon chains all starting from a single branch point.

[0050] The expression "C1-Cx alkyl" or "(Cl-Cx)alkyl" or "alkyl containing 1 to x carbon atoms" means a saturated, linear or branched monovalent hydrocarbon group containing 1 to x carbon atoms, with x being an integer, such as, for example, a methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropyl, cyclohexyl, etc. group.

[0051] The expression "(Cl-Cx)alkylene" denotes a saturated, linear or branched, divalent hydrocarbon group containing from 1 to x carbon atoms, with x being an integer, such as, for example, a 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. These include a methane-1,1-diyl or propane-1,3-diyl group.

[0052] The expression “hydroxy-(C1-Cx)alkyl” denotes a (Cl-Cx)alkyl group as defined above substituted by a hydroxyl group (OH) such as for example a hydroxyethyl (- CH2CH2OH) or a hydroxypropyl (e.g. -CH2-CH(OH)-CH3).

[0053] The expression “carboxy-(C1-Cx)alkyl” designates a (Cl-Cx)alkyl group as defined above substituted by a carboxyl group (COOH) such as for example a carboxymethyl (-CH2COOH).

[0054] The expression “aryl” denotes a monovalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more cycles, such as for example a phenyl or naphthyl group.

[0055] The term "arylene" means a divalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more rings, such as a phenylene group.

[0056] The expression “aryl-(C1-Cx)alkyl” designates an aryl group as defined above, linked to the rest of the molecule via a (Cl-Cx)alkyl chain as defined above with x an integer, such as for example the benzyl or phenylethyl group.

[0057] The term "polyvalent group" means a group that can form multiple covalent bonds with other groups in the same compound or in two different compounds. The bonds to the other groups can be formed from the same atom of the polyvalent group or from different atoms of the polyvalent group, and preferably from 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 in the same compound or in two different compounds. The number of covalent bonds that can be formed is referred to as the "valence" of the polyvalent group.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The inventors have developed a process for preparing a hydrogel that meets the expressed needs. The resulting hydrogel has a sticky character that allows the hydrogel to adhere to tissues and thus not migrate into the tissues after its application / injection.

[0060] Currently available fillers can create volume to fill soft tissues but do not combat tissue descent. However, chronological aging of the skin is associated with a modification of the structure of the skin with the appearance of wrinkles but also ptosis of fatty areas. Interestingly, in certain embodiments, thanks to its stringy nature, the hydrogel according to the invention can be used to retain tissues and prevent age-related tissue descent, in particular when it is administered and arranged in nets. The hydrogel according to the present invention is therefore particularly useful in filling and / or replacing soft tissues.

[0061] Furthermore, the resulting hydrogel is highly biocompatible. Indeed, its preparation process uses a very small amount of crosslinking agent (from 0.001 to less than 0.02 moles of crosslinking agent per 1 mole of polysaccharide repeating unit). The resulting hydrogel is thus weakly crosslinked.

[0062] In addition, the resulting hydrogel is highly mucoadhesive, which allows adhesion to mucous membranes and thus good persistence of the hydrogel at the administration site(s). This is visible by qualitative ex vivo tests or in vitro tests that trace labeled components.

[0063] The method for preparing a hydrogel, preferably injectable, as developed by the inventors, comprises the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two functional groups Z, identical or different, chosen from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide groups, and an acid anhydride residue; c) preparing a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total amount of crosslinking agent ranging from 0.001 to less than 0.02 mole per 1 mole of repeating unit of the polysaccharide(s), the duration of the step of preparing the reaction medium not exceeding 5 hours;d) placing the reaction medium obtained at the end of step c), for a period ranging from 2 weeks to 17 weeks, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P.;

[0064] In the above process, the polysaccharide(s) and / or the crosslinking agent(s) may be in salt form.

[0065] It will be readily understood that in the process of the present invention, the crosslinking of the polysaccharide mainly takes place during step d), i.e. when the reaction medium is in a frozen state.

[0066] The properties of the hydrogels obtained by the process of the present invention are particularly surprising since when it came to optimal crosslinking temperature, the prior art advised against going below 10°C and remained 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).

[0067] Furthermore, when cryogelation was used, the amounts of crosslinking agent used remained very high (Anna Strom, Anette Larsson, Oguz Okay, Preparation and physical properties of hyaluronic acid-based cryogels, J. A ppi. Polym. Sci. 2015, 42194; WO201029344). Otherwise, the prepared products were not able to withstand the heat sterilization conditions generally applied to this type of product (autoclave sterilization). These sterilization-unstable products ultimately have a predominant viscous component and / or a large fraction of low weight-average molecular weight polymers, which is not desirable for soft tissue filling applications. However, for obvious safety reasons, it is always desirable to offer products with as few chemical modifications as possible.

[0068] The hydrogels of the present invention are distinguished from hydrogels prepared with the same components but without freezing by their macroporous structure, i.e. with interconnected macropores, the size of which varies from less than one micron to several hundred microns, depending on the synthesis conditions, namely depending on the nature and quantity of the reagents as well as on the temperature and freezing speed. These macropores are visible during 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.

[0069] Hydrogels that have been brought back to room temperature after a crosslinking step in a frozen state can be referred to as a “cryogel”.

[0070] Furthermore, although it was known that cryogelation generates only a few hyaluronic acid fragments, it remains surprising that the method of the present invention preserves the structure of high weight-average molecular weight polysaccharides, in particular the structure of high weight-average molecular weight hyaluronic acid. The method according to the invention allows the preparation of a crosslinked polysaccharide gel without degradation into low weight-average molecular weight fragments.

[0071] A high weight average molecular weight polysaccharide means a polysaccharide whose weight average molecular weight corresponds to that of naturally (biologically) synthesized polysaccharides. For example, a high weight average molecular weight hyaluronic acid polysaccharide means a polysaccharide which has a weight average molecular weight greater than or equal to 1 MDa, preferably ranging from 1 MDa to 5 MDa, for example from 1 MDa to 3 MDa or from 1.5 MDa to 3 MDa; a high weight average molecular weight chondroitin sulfate or chondroitin polysaccharide means a polysaccharide which has a weight average molecular weight greater than or equal to 30 kDa, preferably ranging from 30 to 150 kDa; a high weight average molecular weight chitosan polysaccharide means a polysaccharide which has a weight average molecular weight greater than or equal to 100 kDa, preferably ranging from 100 kDa to 250 kDa.The polysaccharide provided in step a) of the process is typically a high weight average molecular weight polysaccharide.

[0072] A low weight average molecular weight polysaccharide means a polysaccharide which has a significantly lower average molecular weight than biologically synthesized polysaccharides, more particularly a low weight average molecular weight polysaccharide means a polysaccharide which has a weight average molecular weight less than or equal to one third of the mass of the high weight molecular weight polysaccharide provided in step a) of the preparation of the hydrogel.For example, a low weight average molecular weight hyaluronic acid polysaccharide designates a polysaccharide which has a weight average molecular weight ranging from 0.04 to 0.3 MDa, preferably from 0.08 to 0.20 MDa, more preferably from 0.08 to 0.15 MDa; a low weight average molecular weight chondroitin sulfate or chondroitin polysaccharide designates a polysaccharide which has a weight average molecular weight less than or equal to 50 kDa; a low weight average molecular weight chitosan polysaccharide designates a polysaccharide which has a weight average molecular weight less than or equal to 30 kDa.

[0073] The absence of low molecular weight fragments is advantageous. Indeed, although hyaluronic acid is known to be biocompatible, it has already been reported in the literature that hyaluronic acid with a low average molecular weight could cause long-term adverse effects after its injection 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).

[0074] COMPONENTS USED IN THE PREPARATION OF HYDROGEL

[0075] Polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds.

[0076] Preferably, the polysaccharide is selected from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof.

[0077] "Pectin" is a polysaccharide composed of a skeleton of D-galacturonic acid in acid form, possibly esterified by methanol, and L-rhamnose capable of forming branches with other oses.

[0078] Chitosan and chitin are each a polysaccharide composed of repeating D-glucosamine units linked together in a B-(1,4) manner, a portion of which is N-acetylated. Chitosan specifically has an acetylation degree of less than 50%, while chitin specifically has an acetylation degree of greater than 50%.

[0079] “Cellulose” is a polysaccharide composed of a linear chain of D-glucose molecules.

[0080] "Cellulose derivatives" include methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and carboxymethylcellulose (CMC).

[0081] "Agarose" is a polysaccharide comprising as a repeating unit a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose.

[0082] Glycosaminoglycans are linear polysaccharides composed of repeating disaccharide units, said disaccharides containing a hexosamine (glucosamine (GIcN) or galactosamine (Gal N)) and another ose (glucuronic acid (GIcA), iduronic acid (IdoA) or galactose (Gal)). The hexosamine and the other ose may optionally be sulfated and / or acetylated. The glycosaminoglycan may be, in particular, hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin or chondroitin sulfate.

[0083] "Hyaluronic acid" is a glycosaminoglycan whose repeating unit is a disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked together by alternating glycosidic bonds |3-(1,4) and |3-(1,3). When hyaluronic acid is in the form of a salt, it is also referred to as "hyaluronate" or "hyaluronan". In the context of the present invention, hyaluronic acid may have a weight-average molecular mass ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, even more preferably greater than 0.05 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa. Hyaluronic acid may be in salt form, particularly in physiologically acceptable salt form such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.More specifically, hyaluronic acid is in its acid form or in the form of sodium salt (NaHA).

[0084] "Heparosan" or "heparosan" is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid (GIcA) linked by an α-(1,4) bond to an N-acetyl glucosamine (GIcNAc). Each disaccharide repeating unit is linked to the next by a β-(1,4) bond.

[0085] Chondroitin sulfate or chondroitin sulfate is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid linked in |3-(1,3) to sulfated N-acetyl galactosamine, i.e. it includes at least one sulfate substituent. Each disaccharide repeating unit is linked to the next by a |3-(1,4) bond.

[0086] "Dermatan sulfate" or "dermatan sulfate" is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e. comprising at least one sulfate substituent, of L-iduronic acid and N-acetyl-galactosamine- linked by a(1-3) bonds. Advantageously, the disaccharide is sulfated at the C-4 position of N-acetyl-galactosamine, at the C-6 position of N-acetyl-galactosamine, at the C-2 position of L-iduronic acid, or at a combination of these positions. Each disaccharide repeating unit is linked to the next by a |3-(1,4) bond.

[0087] "Keratan sulfate" or "keratan sulfate" is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e. comprising at least one sulfate substituent, composed of D-galactose and N-acetylglucosamine linked by alternating P(1-4) and (3(1-3) bonds.

[0088] The polysaccharide may be in salt form, particularly in physiologically acceptable salt form such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt and mixtures thereof, more particularly in sodium or potassium salt form.

[0089] Advantageously, the polysaccharide is a glycosaminoglycan or a salt thereof, preferably hyaluronic acid or a salt thereof, more preferably hyaluronic acid or one of its physiologically acceptable salts such as the sodium salt, the potassium salt, the zinc salt, the silver salt and mixtures thereof, even more preferably hyaluronic acid or its sodium salt.

[0090] The polysaccharide generally has a weight average molecular mass ranging from 0.03 to 10 MDa.

[0091] Preferably, if the polysaccharide is hyaluronic acid, it has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, even more preferably greater than 0.05 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa.

[0092] Crosslinking agent

[0093] The “crosslinking agent” or “crosslinking agent” is a compound comprising at least two functional groups capable of binding covalently with functional groups present on the polysaccharide, such as OH, CHO, NH2 or COOH groups carried by the polysaccharide, and thus inducing bonds between the polysaccharide chains (crosslinking) and / or bonds on the same polysaccharide chain.

[0094] The crosslinking agent useful in the context of the present invention comprises at least two, preferably from 2 to 8, in particular 2, functional groups (designated "Z group") preferably chosen independently from the isocyanate (-N=C=O), amino (-NH2), epoxide, carboxyl (-COOH), N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate (-N=C=S), vinyl (- CH=CH2), formyl (-CH=O), hydroxyl (-OH), sulfhydryl (-SH), hydrazino (-NH-NH2), acylhydrazino (-CO-NH-NH2), aminoxy (-O-NH2), carbodiimide groups, and an acid anhydride residue. Preferably, the functional groups are identical.

[0095] The isocyanate group can react with an OH or NH2 group of the polysaccharide to form a carbamate or urea function. The amino group can react with a COOH group of the polysaccharide to form an amide function. The epoxide group can react with an OH or COOH group of the polysaccharide to form an ether or ester function. The carboxyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The N-succinimidyloxycarbonyl and N-sulfosuccinimidyloxycarbonyl groups can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The halocarbonyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The isothiocyanate group can react with an OH or NH2 group of the polysaccharide to form a thiocarbamate or thiourea function.The vinyl group can react with an OH group of the polysaccharide to form an ether function. The formyl group can react with an OH or NH2 group of the polysaccharide to form a hemiacetal or hemiaminal function. The hydroxyl group can react with a COOH group of the polysaccharide to form an ester function. The sulfhydryl group can react with a COOH group of the polysaccharide to form a thioester function. The hydrazino group (-NH-NH2) can react with a CHO group of the polysaccharide to form a hydrazone function. The acylhydrazino group can react with a CHO group of the polysaccharide to form a carbonylated hydrazone function =NNHC(O)-. The aminoxy group can react with a CHO group of the polysaccharide to form an oxime function =NO-. The carbodiimide group can react with a COOH group of the polysaccharide to give a CO-NR function. a-CO-NH, and an acid anhydride residue can react with an OH or NH2 group of the polysaccharide to form an ester or amide function.

[0096] Preferably, the functional groups Z are identical and represent an epoxide or vinyl group, more preferably epoxide.

[0097] According to another advantageous embodiment, the functional groups Z are identical and chosen from amino, vinyl, formyl, and carbodiimide groups, preferably are amino groups.

[0098] In particular, the crosslinking agent is selected from hexamethylene diisocyanate, 4,4'-diphenylmethylene diisocyanate, PEG20K-4-arm 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-epoxypropoxy)ethane (EGDGE), 1 ,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), poly(ethylene glycol) diacid, disuccinimidyl suberate, bis(sulfosuccinimidyl)suberate, sebacoyl chloride, 1,4-butane diisothiocyanate, divinylsulfone (DVS), glutaraldehyde, polyethylene glycol, 1,5-pentanedithiol, adipic acid dihydrazide,bis-aminooxy-poly(ethylene glycol), diethylenetriaminepentaacetic acid dianhydride, and mixtures thereof.,

[0099] When the functional groups Z are epoxide groups, the crosslinking agent is preferably chosen 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 at each end by a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups and mixtures thereof.

[0100] More preferably, the crosslinking agent is chosen 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.

[0101] When the functional groups Z are amino groups, the crosslinking agent is preferably a polyamine chosen 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), their salts or a mixture thereof, more preferably the crosslinking agent is a polyamine chosen from spermine, spermidine, their salts and their mixtures.

[0102] The crosslinking agent may be selected from hydroxyapatite beads modified to carry epoxy groups, a compound of formula Chem. I as described below, and mixtures thereof.

[0103] Preferably, the crosslinking agent is a compound of formula Chem. I: Y-(Z) nin which the functional groups Z, identical or different, are as defined above, n is an integer greater than or equal to 2, in particular ranging from 2 to 8, preferably equal to 2,

[0104] Y is a polyvalent hydrocarbon group, notably aliphatic, having a valence of n and containing from 1 to 150 carbon atoms:

[0105] - in which one or more (for example 1 to 150, or 1 to 50 or 1 to 15 or 1 or 2) CH2 units are optionally replaced by one or more divalent units chosen from arylenes; -O-; -S-; -S(O)-; -C(=O)-; -SO2-; -N(R 1 )- ; and -[SiR 2 R 3 O] m -SiR 2 R 3 - with :

[0106] R 1 representing a hydrogen atom, an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl; m an integer between 1 and 20 and the R 2 and the Rs3 , identical or different, representing a hydrogen atom; a halogen atom; a -OR group 11 with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl, - said polyvalent group being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, and an aryl-(C1-C6)alkyl, preferably unsubstituted.

[0107] In particular n is an integer ranging from 2 to 8, preferably n represents 2, 3 or 4, even more preferably n is equal to 2.

[0108] Advantageously, R 1 represents a hydrogen atom or a (C1-C6)alkyl group.

[0109] In particular, the R 2 and the Rs 3 , identical or different, represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0110] Preferably, in the definition of Y, the polyvalent hydrocarbon group may be a polyvalent aliphatic or aromatic hydrocarbon group, preferably aliphatic and in particular saturated, having a valence of n and comprising from 1 to 150 carbon atoms, preferably from 1 to 50 carbon atoms, more preferably from 1 to 20 carbon atoms, even more preferably from 2 to 20 carbon atoms.

[0111] In particular, in the definition of Y, the polyvalent hydrocarbon group is a polyvalent aliphatic, saturated, in particular linear, hydrocarbon group.

[0112] Preferably, Y is a polyvalent hydrocarbon group as described above in which one or more CH2 units are optionally replaced by one or more divalent units selected from -O-, -SO2-, -[SiR 2 R 3 O] m -SiR 2 R 3 - and -NH-, with R 2 , R 3 and m as described above.

[0113] In particular, Y is a polyvalent hydrocarbon group as described above, preferably aliphatic and saturated, and in particular linear, branched, or star-shaped, and optionally in which:

[0114] - at least two CH2 units are replaced by -O-, particularly between 1 and 50 CH2 units, more particularly between 1 and 15 CH2 units, or

[0115] - at least one, preferably one or two, CH2 units is replaced by an -NH- unit, or

[0116] - at least one, preferably one, CH2 unit is replaced by an -SO2- unit, or

[0117] - at least two, preferably two, CH2 units are replaced by -O- and at least one, preferably one, CH2 unit is replaced by a -[SiR 2 R 3 O] m -SiR 2 R 3 - with R 2 , R 3 and m as described above.

[0118] More particularly, when one or more CH2 units are replaced by -O-, the replaced unit(s) are such that Y comprises one or more -CH2-CH2-O- units. In particular, Y comprises from 1 to 50 -CH2-CH2-O- units, advantageously from 2 to 25 -CH2-CH2-O- units, more advantageously from 2 to 15 -CH2-CH2-O- units. Y may comprise only -CH2-CH2-O- units.

[0119] More preferably, Y is an alkyl group comprising 1 to 150, in particular 1 to 50, in particular 1 to 20, for example 1 to 12, in particular 1 to 6 carbon atoms, preferably linear, in which optionally one or more CH2 units are replaced by one or more divalent units chosen from -O- and -NH-, more particularly between 1 and 50, in particular between 1 and 15, for example 1 or 2, divalent units chosen from -O- and -NH-.

[0120] According to a first embodiment, the R 2 and the Rs 3 , identical or different, represent a -OR group 11 with R 11 as described above. In particular, R 11 represents an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0121] According to a second embodiment, the R 2 and the Rs 3, identical or different, represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted (preferably unsubstituted) by one or more groups chosen from a halogen atom, an aryl or a hydroxyl, more preferably an unsubstituted (C1-C6)alkyl group such as a methyl or an ethyl.

[0122] Advantageously, the crosslinking agent is a compound of the following Chem formula: Z 1 -Y 1 -Z 2 in which the Z groups 1 and Z 2 , identical or different, are chosen from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide groups, and an acid anhydride residue, and Y 1represents a divalent hydrocarbon chain, in particular aliphatic, comprising from 1 to 50 carbon atoms:

[0123] - in which one or more (e.g.: 1 to 15 or 1 or 2) CH2 units are optionally replaced by one or more divalent units chosen from arylenes, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )- , and -[SiR 2 R 3 O]m-SiR 2 R 3 - with

[0124] R 1 representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl, m an integer between 2 and 20, and the R 2 and the Rs 3 , identical or different, representing a hydrogen atom; a halogen atom; a -OR group 11 with R 11representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl,

[0125] - said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.

[0126] Z groups 1 and Z 2 have the same definition as group Z defined above. Y 1 has the same definition as Y defined above with valence n being equal to 2. In particular Y 1 may comprise only -CH2-CH2-O- units, as defined above.

[0127] Preferably, the crosslinking agent of formula Chem. I or Chem. Ia does not comprise -[SiR 2 R 3 O]m -SiR 2 R 3 -.

[0128] Other components

[0129] The additional components may be lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof.

[0130] Polysaccharides, in particular uncrosslinked hyaluronic acid or uncrosslinked heparosan, can be cited as an example of a lubricating agent.

[0131] Examples of anesthetics include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuvérine, Fomocaine, Guafecainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, Proxymetacaine,Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, a salt of their salts, in particular a hydrochloride, or a mixture thereof.

[0132] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, and a mixture thereof.

[0133] Examples of amino acids include, but are not limited to, 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, their derivatives (e.g., N-acetylated derivatives such as N-acetyl-L-cysteine), and a mixture thereof. Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and more preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0134] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated), magnesium salts, calcium salts (e.g., hydroxyapatite, in particular in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, in particular pentahydrate), optionally in a hydrated form, and mixtures thereof.

[0135] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives, and a mixture thereof.

[0136] As coenzymes, coenzyme Q10, CoA, NAD, NADP, and mixtures thereof can be cited.

[0137] Adrenaline derivatives include adrenaline, noradrenaline, and a mixture of these.

[0138] The amounts of additional compounds depend of course on the nature of the compound in question, the desired effect, and the purpose of the composition as described here.

[0139] PROCESS

[0140] The steps of the method of the present invention may be as described below.

[0141] Providing at least one polysaccharide or a salt thereof (step a))

[0142] Step a) of the method according to the invention comprises providing at least one polysaccharide or a salt thereof, in particular a physiologically acceptable salt thereof.

[0143] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably a sodium salt.

[0144] The polysaccharide may be provided in hydrated form, wholly or partially, or in dry form, such as powder or fiber. More particularly, in step a), the polysaccharide is provided in dry form such as powder or fiber. When the polysaccharide is provided in hydrated form, it is in the form of a non-crosslinked gel or solution.

[0145] In particular, when the polysaccharide is in hydrated form, it is an aqueous non-crosslinked gel or an 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. possibly comprising additional components as defined in the additional steps. It should therefore be understood that the aqueous non-crosslinked gel or the aqueous polysaccharide solution does not comprise sodium hydroxide. Provision of at least one crosslinking agent or a salt thereof (step b))

[0146] Step b) of the method according to the invention comprises providing at least one crosslinking agent or a salt thereof, in particular a physiologically acceptable salt thereof.

[0147] The crosslinking agent is as described above.

[0148] Preparation of the crosslinking reaction medium (step c))

[0149] Step c) of the process according to the invention comprises the preparation of a crosslinking reaction medium. The reaction medium comprises the polysaccharide(s), the crosslinking agent(s) and a solvent.

[0150] 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% or at least 99% by weight of water relative to the total weight of the solvent). For example, an organic solvent such as an alcohol, in particular ethanol, or DMSO, may be used to solubilize the crosslinking agent, for example when it is poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), before its addition to the aqueous reaction medium.

[0151] The reaction medium may further comprise salts, pH adjusters, for example a Bronsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above and mixtures thereof. The addition of a Bronsted base may be particularly necessary when the functional groups Z of the crosslinking agent, such as Z 1 or Z 2 , represent an epoxide group or a vinyl group. In these cases, crosslinking takes place 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 to the reaction medium (for example sodium hydroxide), typically at a concentration between 0.10M and 0.30M.

[0152] The reaction medium is typically prepared from the polysaccharide or polysaccharides in a dry form. When the reaction medium is prepared from the polysaccharide or polysaccharides in a hydrated form, the aqueous uncrosslinked gel or aqueous polysaccharide solution used for the preparation of the reaction medium does not comprise sodium hydroxide. Thus, the maximum contact time of the polysaccharide with sodium hydroxide before engaging in step d), whether the polysaccharide is provided in a dry or hydrated form, is 5 hours, for example from 15 minutes to 4 hours or from 30 minutes to 2 hours.

[0153] The total amount of crosslinking agent in the reaction medium ranges from 0.001 to less than 0.02 moles per 1 mole of repeating unit of the polysaccharide, for example from 0.001 to 0.015 moles per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.01 moles per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.008 moles or from 0.001 to 0.005 moles per 1 mole of repeating unit of the polysaccharide. When the polysaccharide is a glycosaminoglycan such as hyaluronic acid, the repeating unit is a disaccharide unit.

[0154] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 100 to 200 mg / g.

[0155] Step c) of the process according to the invention typically comprises a step of homogenization of the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with blades or stirring with a spatula. Step c) is typically carried out at a temperature ranging from 4 to 35°C, preferably 15°C to 25°C.

[0156] 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 invention.

[0157] Crosslinking at temperature T and pressure P (step d))

[0158] The reaction medium obtained at the end of step c) is then placed, for a period ranging from 2 weeks to 17 weeks, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium (i.e. of the mixture comprising the polysaccharide(s), the crosslinking agent(s), the solvent and any salts, pH adjusters and additional components) as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P.

[0159] The freezing point temperature of the reaction medium refers to the temperature at which the mixture of the components of the reaction medium, on a macroscopic scale, solidifies, i.e. it becomes non-fluid. 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 point temperature of the reaction medium.

[0160] The eutectic point temperature of the reaction medium refers to the temperature below which the mixture of the components of the reaction medium passes from a frozen state (coexistence of liquid and solid phases) to a completely solid state, that is, a state in which all the components of the mixture are in solid form. The freezing point and the eutectic point of a mixture depend on the pressure to which the mixture is subjected, therefore the freezing point and the eutectic point are measured at pressure P.

[0161] The freezing point and eutectic point can be determined by differential scanning calorimetry. This method allows phase transitions to be determined. To do this, the product to be studied is gradually cooled until its phase transitions are observed.

[0162] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C. Even more preferably, the temperature T is approximately -20°C.

[0163] 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 altitude. At an altitude of 0 m, the average atmospheric pressure is 101,325 Pa. 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 approximately -20°C

[0164] Preferably, the reaction medium obtained at the end of step c) is placed for a period ranging from 2 weeks to 15 weeks, preferably from 2 weeks to 13 weeks, preferably from 2 weeks to 11 weeks, preferably from 2 weeks to 9 weeks, more preferably from 3 weeks to 6 weeks or even from approximately 4 to 5 weeks or approximately 30 days at said pressure P and at said temperature T.

[0165] As previously indicated, the crosslinking of the polysaccharide mainly takes place during step d) (it can nevertheless begin as early as step c)). This step therefore makes it possible to crosslink the polysaccharide chains together. The functional groups of the crosslinking agent react with functional groups present on the polysaccharides so as to link the polysaccharide chains together and crosslink them by forming intermolecular bonds. The crosslinking agent can also react with functional groups present on the same polysaccharide molecule so as to form intramolecular bonds. In particular, the functional groups of the crosslinking agent react with the -OH or -COOH groups, or even -CHO, present on polysaccharides such as hyaluronic acid.Crosslinked polysaccharides comprising at least one crosslinking link between two polysaccharide chains, said crosslinking link being the residue of the crosslinking agent are thus obtained. In particular, following step d), the crosslinked polysaccharides comprise at least one crosslinking link between two polysaccharide chains, said crosslinking link more particularly comprising the polyvalent group Y as described above, preferably the divalent group Y. 1 as described above. Certain functional groups Z (such as Z 1 and Z 2 ) of the crosslinking agent may however not react with a polysaccharide chain. In particular, when the crosslinking agent comprises two functional groups Z 1 and Z 2 , one of the Z functional groups 1 can react with a polysaccharide while the other functional group Z 2does not react with any polysaccharide. A dangling bond is then formed.

[0166] Crosslinking can be carried out in the presence of several crosslinking agents. When crosslinking is carried out in the presence of several crosslinking agents, the crosslinking agents can be added simultaneously or separately over time to the reaction medium. Step b) can thus comprise repeated crosslinking steps. Crosslinking is then carried out in the presence of a total amount of crosslinking agents ranging from 0.1 to less than 2 moles, preferably ranging from 0.1 to 1.5 moles or from 0.1 to 1 mole or from 0.1 to 0.8 moles or from 0.1 to 0.5 moles of crosslinking agents (or their salts) per 100 moles of repeating unit of the polysaccharide. The crosslinking conditions, in particular the contents of crosslinking agent, duration and temperatures as well as the weight-average molecular masses (Mw) of the polysaccharide, used are interdependent.

[0167] In particular, the higher the temperature of the crosslinking reaction, the shorter the reaction time can be to obtain the same degree of modification of the polysaccharide by the crosslinking agent.

[0168] The lower the crosslinking agent content, the longer the reaction time must be to obtain similar mechanical properties of the resulting gel. In other words, the lower the molar percentage of crosslinking agent, the fewer reactive functions there are in the reaction medium and the lower the probability that 2 groups meet and react together, thus the longer the reaction time must be to allow the functions to react with each other and form crosslinking bonds, and thus obtain a gel with desirable properties.

[0169] Conversely, too much crosslinking agent and too long a crosslinking time will result in a brittle and too hard gel, therefore not suitable for cosmetic applications. The higher the weight-average molecular mass (Mw) of the polysaccharide, the lower the degree of modification required to obtain gels with given mechanical properties.

[0170] For the same molar percentage of crosslinking agent, the lower the weight-average molecular mass (Mw) of the polysaccharide, the longer the reaction time is required to obtain gels with given mechanical properties.

[0171] When the functional groups Z of the crosslinking agent are amino groups, the crosslinking reaction with the polysaccharide is advantageously carried out in the presence of at least one activator, and where appropriate combined with at least one coupling auxiliary. In this regard, the activator may 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-morphilinoethyl)carbodiimide (CMC), their salts and mixtures thereof, preferably is represented by EDC.

[0172] With respect to the coupling aid, when present, it may be selected from N-hydroxy succinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt) and N-hydroxysylfosuccinimide (sulfo NHS), and mixtures thereof, preferably is represented by HOBt.

[0173] Additional steps

[0174] The method according to the invention may comprise one or more additional steps, such as for example steps of adding one or more additional components, purification, sterilization, swelling screening and / or conditioning.

[0175] The method according to the invention may comprise a step of adding at least one additional component. The additional component may be chosen from lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof. The additional components may be as described above.

[0176] The method according to the invention may comprise at least one purification step. The purification may be carried out by dialysis.

[0177] The method according to the invention may comprise a step of sterilizing the hydrogel. Sterilization is preferably carried out by heat. Sterilization is generally carried out by increasing the temperature of the sterilization medium to a temperature called "plateau temperature", which is maintained for a determined period of time called "plateau time". Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, preferably with a plateau time ranging from 1 minute to 20 minutes with FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized. Alternatively, sterilization may be carried out in particular by gamma ray, UV radiation or by means of ethylene oxide.The method may comprise a step of sieving the hydrogel, more particularly with a sieve with a porosity of between 50 and 2000 μm. This sieving step makes it possible to obtain a more homogeneous hydrogel with the most constant extrusion force possible, i.e., the most regular possible. A person skilled in the art knows how to select a sieve with a suitable pore size depending on the mechanical properties of the hydrogel being prepared.

[0178] The method may comprise a step of swelling the hydrogel. During the step of swelling the hydrogel, the polysaccharide concentration of the hydrogel is adapted. In particular, a solvent is added, for example, water, a phosphate buffer, water for injection. More particularly, the added solvent has a pH around the physiological pH (6.8-7.8). The polysaccharide concentration obtained following the swelling step advantageously varies from 1 mg / g of gel to 50 mg / g of the hydrogel, more advantageously from 5 mg / g to 35 mg / g of the hydrogel, even more advantageously from 10 mg / g to 30 mg / g of the hydrogel gel.

[0179] The method may include a step of conditioning the hydrogel.

[0180] Where appropriate, the step of adding one or more additional components preferably takes place after the purification step.

[0181] Where appropriate, the step of adding one or more additional components preferably takes place before the sterilization step.

[0182] In particular, the step of adding one or more additional components may also comprise the addition of at least one therapeutic active ingredient, or at least one cosmetic active ingredient, or their mixture. When at least one therapeutic active ingredient and / or at least one cosmetic active ingredient is added, the step of adding one or more additional components preferably takes place after step d).

[0183] Where appropriate, the purification step preferably takes place after the step of adding one or more additional components.

[0184] Where appropriate, the purification step preferably takes place before the sterilization step. Where appropriate, the purification step preferably takes place before the screening step.

[0185] The sterilization step is preferably carried out after steps a) to d) and any additional steps. In particular, the hydrogel is sterilized after being packaged in its injection device and the packaging of the gel takes place following all the steps of the process and before sterilization. Tl

[0186] HYDROGEL

[0187] The present invention also relates to a hydrogel capable of being obtained by the method of the present invention. Such a hydrogel may also be designated by the term “cryogel”.

[0188] The hydrogel comprises one or more polysaccharides as defined above. The polysaccharide is crosslinked. The molar crosslinking rate 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%, 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% (number of moles of crosslinking agent(s) per 100 moles of repeating unit of the polysaccharide(s).

[0189] The crosslinking agent(s) are as defined above.

[0190] The crosslinking agent is preferably a crosslinking agent whose functional groups are epoxide groups. Thus, preferably the crosslinking agent is 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.

[0191] The hydrogel of the present invention has a sticky character. In certain embodiments the hydrogel of the present invention has a stringy character.

[0192] The hydrogel is preferably an injectable hydrogel.

[0193] It is preferably sterile, in particular heat sterilized at a plateau temperature of 121°C to 135°C, preferably with a plateau time of 1 minute to 20 minutes with F0 > 15.

[0194] This hydrogel is preferably homogeneous.

[0195] The hydrogel may comprise one or more additional components as described above, in particular one or more additional components selected from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids and mixtures thereof.

[0196] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a cross-over stress (or constraint at the crossing of the moduli G' and G”) greater than or equal to 50 Pa, preferably between 50 and 5000 Pa and more preferably between 100 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, more preferably from 110 Pa to 1000 Pa.

[0197] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a cohesiveness of 1 N to 30 N, preferably greater than 4 N, for example ranging from 6 to 20 N or ranging from 6 to 15 N. This cohesiveness is measured by mechanical compression using a rheometer. For this, the gel is deposited on a Peltier plane with an initial air gap of 2.60 mm; it is then compressed at a constant speed of 100 pm / s up to 70% of the initial air gap, at 25°C; finally, the cohesiveness of the gel is measured at the end of the compression stroke. The more cohesive a gel is, i.e. the higher the cohesiveness value, the more it is able to withstand stresses, such as those it may encounter after its administration to a subject.

[0198] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a Tack test result ranging from 1.5 to 15 cm, preferably from 1.5 to 10 cm, even more preferably from 2 to 7.

[0199] Preferably, a hydrogel according to the present invention acceptable for the therapeutic and / or cosmetic applications targeted by the present invention has a sticky character and will adhere to surfaces such as glass, metal, human tissue, collagen or plastic. In addition, it will maintain a cohesive structure when moving on these surfaces.

[0200] COMPOSITION

[0201] The present invention also relates to a composition comprising a hydrogel according to the present invention. It is preferably a cosmetic or pharmaceutical composition. It may further comprise physiologically acceptable excipients.

[0202] The hydrogel according to the invention comprises a crosslinked polysaccharide. The composition may further comprise a non-crosslinked polysaccharide.

[0203] The composition according to the present invention may thus comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight of polysaccharide (eg, hyaluronic acid), relative to the total weight of said composition, the polysaccharide (eg, hyaluronic acid) being present in crosslinked and optionally non-crosslinked form. In particular, the content of non-crosslinked polysaccharide (eg, hyaluronic acid) varies from 0 to 40% by weight, preferably from 1 to 40% by weight, more preferably from 5 to 30% by weight, relative to the total weight of polysaccharide (eg, hyaluronic acid) present in the composition.

[0204] The polysaccharide of the hydrogel is as defined above.

[0205] The composition according to the present invention is preferably a sterile composition, in particular heat sterilized at a plateau temperature of between 121°C and 135°C, preferably with a plateau time of between 1 minute and 20 minutes with F0 > 15. 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.

[0206] The physiologically acceptable aqueous medium may comprise a physiologically acceptable solvent or mixture of solvents and preferably comprises water.

[0207] The physiologically acceptable medium may also include isotonic agents such as oses, sodium chloride and their mixture.

[0208] The physiologically acceptable medium may further comprise at least one isotonic and physiologically acceptable saline solution.

[0209] Preferably, said balanced salt solution is a phosphate buffered saline solution, and particularly a KH2PO4 / K2HPO4 saline buffer.

[0210] The composition according to the invention may further comprise at least one additional compound chosen from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, coenzymes, adrenaline derivatives, and mixtures thereof. The additional compounds may be as described above.

[0211] APPLICATIONS

[0212] The hydrogel or composition according to the invention may have therapeutic and / or cosmetic applications.

[0213] The present invention therefore also relates to a hydrogel or a composition according to the invention for its use in filling and / or replacing tissues, in particular soft tissues, in particular by injecting the hydrogel or the composition into the tissue.

[0214] In particular, the hydrogel or composition according to the invention is used in oral care and more particularly in the treatment of gingival recession, or to fill periodontal pockets. More particularly, the hydrogel or composition according to the invention is used to treat defects in gingival architecture that may occur with tooth loss, with aging, with periodontal diseases and disorders, or after the placement of tooth implants, crowns or bridges.

[0215] The hydrogel or composition according to the invention can also be used in ophthalmology, more particularly to protect ocular structures during eye surgery such as for example ophthalmic surgery of the anterior or posterior segment, cataract extraction possibly with implantation of an intraocular lens, corneal transplant surgery, glaucoma filtering surgery, or even implantation of a secondary lens. In this case, the hydrogel or composition according to the invention will more particularly be injected into the eye. The hydrogel or composition according to the invention can 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 viscosupplementation.

[0216] The hydrogel or composition according to the invention can also be used in the treatment of lipodystrophy.

[0217] The hydrogel or composition according to the invention can be used in cosmetic surgery, in particular for gynecoplasties and / or penoplasties.

[0218] The hydrogel or composition according to the invention is administered more particularly by injection.

[0219] Advantageously, the hydrogel or composition according to the invention can be used for its mucoadhesive properties useful in the treatment of gum pain.

[0220] The present invention also relates to a method of treating the above-mentioned pathologies which comprises administering, to an individual in need thereof, an effective dose of the hydrogel or composition.

[0221] The effective dose of the hydrogel or composition varies depending on many parameters such as, for example, the chosen route of administration, weight, age, sex, the progress of the pathology to be treated and the sensitivity of the individual to be treated.

[0222] The present invention preferably relates to the cosmetic, and therefore non-therapeutic, use of a hydrogel or a composition according to the invention to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin, and in particular to stimulate, regenerate, moisturize, firm or restore the radiance of the skin, in particular by mesotherapy; to fill volume defects of the skin, and in particular to fill wrinkles, fine lines or scars (in particular hollow scars); or to reduce the appearance of wrinkles and fine lines.

[0223] For example, the subject of the present invention is the cosmetic use of a hydrogel or a composition according to the invention to reduce nasolabial folds and bitterness lines; to increase the volume of the cheekbones, chin or lips; to restore the volumes of the face, in particular the cheeks, temples, oval of the face, and the area around the eyes; or to stimulate, regenerate, hydrate, firm or restore the radiance of the skin, in particular by mesotherapy.

[0224] 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 administered more particularly by injection.

[0225] The present invention also relates to a method for the cosmetic treatment, preferably anti-aging, of keratin materials, in particular of the skin, comprising at least one step of administering a hydrogel or a composition according to the invention on or through said keratin materials, more particularly by injection.

[0226] The administration may be an injection, in particular an intra-epidermal and / or intradermal and / or subcutaneous injection. The administration by intra-epidermal and / or intradermal and / or subcutaneous injection according to the invention aims to inject a hydrogel or a composition of the invention into an epidermal, dermo-epidermal and / or dermal region. The hydrogel or the composition according to the invention may also be administered by a supra-periosteal injection.

[0227] The hydrogel or composition according to the invention may be injected using any of the methods known to those skilled in the art. In particular, a hydrogel or composition according to the invention may be administered by means of an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supra-periosteal injection. The injection device may in particular be chosen from a syringe, a set of microsyringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0228] The injection device may comprise any commonly used injection means suitable for intraepidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. Preferably, such means may be a hypodermic needle or a cannula or a set of microneedles.

[0229] A needle or cannula according to the invention may have a diameter varying from 18 to 34 G, preferably between 25 and 32 G, and a length varying from 4 to 70 mm, and preferably from 4 to 25 mm. The needle or cannula is advantageously for single use.

[0230] Advantageously, the needle or cannula is associated with a syringe or any other device making it possible to deliver said hydrogel or said injectable composition through the needle or cannula.

[0231] According to an alternative embodiment, a catheter can be inserted between the needle / cannula and the syringe. In a known manner, the syringe can be operated manually by the practitioner or by a syringe holder such as guns.

[0232] Preferably, the injection device is a syringe.

[0233] In an alternative embodiment, the injection device can be adapted to the mesotherapy technique.

[0234] Mesotherapy is a treatment technique involving intra-epidermal and / or intradermal and / or subcutaneous injection of a composition or hydrogel. The composition or hydrogel is administered using this technique by injection in the form of multiple small droplets at the level of the epidermis, the dermo-epidermal junction and / or the dermis in order, in particular, to create a subcutaneous coating. The mesotherapy technique is described in particular in the book "Traité de mésothérapie" by Jacques LE COZ, published by Masson, 2004. Mesotherapy performed on the face is also called mesolift, or also under the Anglo-Saxon term "mesoglow".

[0235] Administration can also be topical.

[0236] Preferably, it is a topical application to the surface of the skin, more particularly to the epidermis, even more particularly to the facial epidermis.

[0237] The present invention thus also relates to an injection device as described previously comprising a hydrogel or a composition according to the invention.

[0238] The following examples are given for illustrative purposes, but should in no way be considered as limiting the present invention.

[0239] EXAMPLES

[0240] 1. Materials

[0241] - Non-crosslinked sodium hyaluronate of 1.5MDa and 4MDa (HTL),

[0242] - BDDE (Sigma Aldrich),

[0243] - DVS (Fisher Scientific),

[0244] - NaOH 0.25M,

[0245] - 1 M HCl,

[0246] - Phosphate Buffer (Braun),

[0247] - Lidocaine hydrochloride,

[0248] - Three-dimensional agitator,

[0249] - DHR-2 rheometer,

[0250] - Dynamometer and test bench,

[0251] - Homogenizer Paddle mill,

[0252] - Sterile polyethylene bag.

[0253] 2. Methods

[0254] 2.1 Measurement of viscoelastic properties

[0255] The viscoelastic properties of the obtained hydrogels were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plate geometry and an anodized aluminum peltier plane (42 mm) (air gap 24 μm).

[0256] 0.5 g of sterilized hydrogel is deposited between the Peltier plane and said cone. Then a stress scan is carried out at 1 Hz and 25°C. The elastic modulus G', the viscous modulus G” and the phase angle 5 are reported for a stress of 5 Pa. The stress at the intersection of G' and G”, T, is determined at the intersection of the curves of the moduli G' and G” and is expressed in Pascal.

[0257] 2.2 Measurement of extrusion force

[0258] The extrusion forces (in Newton) of the gels packaged in syringes were conducted using a test bench equipped with a dynamometer at a constant speed of 12.5 mm / min, through a 27G 2” needle and at room temperature. The extrusion force results correspond to the average of the average extrusion forces on at least 2 samples.

[0259] 2.3 Identification of the stringy character (Tack Test)

[0260] The Tack test is conducted on a DHR-2 rheometer (TA Instruments) equipped with a rough steel plane geometry (40 mm diameter) and a rough steel Peltier plane (40 mm diameter). 1 g of product is deposited between the two geometries at 25 °C and the upper geometry is rapidly brought closer to the Peltier plane at a speed of 2 mm / s until a gap of 2 mm. The upper geometry then compresses the gel with a closing speed of 0.1 mm / s until a gap of 0.1 mm. The upper geometry is then recorded at 0.1 mm / s and the distance over which the gel maintains its stringy character between the two geometries is reported. The stringy character thus determined by a unit of length is expressed in cm.

[0261] 2.4 Measurement of cohesiveness

[0262] To measure cohesiveness (or mechanical strength, expressed in Newtons), the gel is placed on a Peltier plate with an initial air gap of 2.60 mm. The gel is then compressed at a constant speed of 100 pm / s to 70% of the initial air gap, at 25°C. The cohesiveness of the gel is measured at the end of the compression stroke.

[0263] 2.5 Analysis of soluble hyaluronic acid

[0264] The analysis of soluble hyaluronic acid or sHA is performed using high-performance liquid chromatography interfaced with a multi-angle light scattering detector and a refractive index detector (HPLC-SEC-MALS-RI, ASTRA software (Wyatt Technology Corp.). Samples are diluted approximately 10 times according to their initial hyaluronic acid concentration in the SEC mobile phase, a filtered 150 mM sodium nitrate solution (pH 7.2). Said diluted mixture consists of compact and insoluble hyaluronic acid and soluble hyaluronic acid. The soluble hyaluronic acid portion of the samples was released for 5 days under orbital shaking to avoid any artificial production of soluble hyaluronic acid. The soluble part is separated from the insoluble part by a gentle filtration method (syringe equipped with a 0.45 µm filter) and then subjected to SEC analysis.

[0265] Varying injection volumes are tested to achieve a background-free signal of at least five to one to prevent and avoid overloading the SEC columns.

[0266] The HPLC-SEC system uses a column duo for wide ranges of hyaluronic acid from 500Da to 20MDa for optimal peak resolution on the chromatograph.

[0267] In order to have an optimal molecular weight analysis, a value of the refractive index increment or dn / dc (refractive index variation / polysaccharide concentration in the analytical solvent) of 0.165 mL / g was determined based on internal samples analyzed under the same conditions as the samples.

[0268] The chromatograms obtained by SEC are analyzed to quantify the molecular weight, distribution and proportion of soluble hyaluronic acid of each sample.

[0269] The mass average molecular weight of the sample is a data provided by the HPLC-SEC software. The percentage of soluble hyaluronic acid fractions (%sHA) is also a direct output of the HPLC-SEC software after entering the mass concentration (mg / ml) of total hyaluronic acid in the analyzed sample.

[0270] The said percentage may differ from one composition to another depending on its manufacturing technique and the dispersion (w / d) (molar mass by weight / molar mass by number) of the sample.

[0271] %sHA for several molecular weight limits (<250kDa, <100kDa, <50kDa, <30kDa and <20kDa) are also a direct output of the HPLC software. However, this does not take into account the amount of hyaluronic acid actually released from the gel.

[0272] Accordingly, a normalized %sHA for the multiple molecular weight limits was calculated to normalize the values ​​provided by the software taking into account the amount of hyaluronic acid actually released from the analyzed sample.

[0273] It is then possible to directly compare the results.

[0274] Prototypes No. 1 to 9 were prepared as follows with the BDDE contents (BDDE molar TR) described in Table 1.

[0275] BDDE and sodium hyaluronate (1.5 MDa, 120 mg / g) were dissolved in a 0.25 M aqueous sodium hydroxide solution in a sterile bag. The mixture was then homogenized in a paddle mill for 3 cycles of 15 min at 210 rpm at room temperature. The mixture was then kept at atmospheric pressure and placed at the temperatures and times shown in Table 1. The pH of the mixture was approximately 13.

[0276] When the process was carried out at temperatures below the freezing point of the reaction medium (BDDE, sodium hyaluronate, aqueous sodium hydroxide solution), the mixture was allowed to thaw before continuing the preparation.

[0277] A 1N HCl solution was then added to the sterile bag until a pH of 7.3 ± 0.5 was obtained. The mixture was diluted to a concentration of 23 mg of hyaluronic acid per gram of product with PBS phosphate buffer. The mixture was homogenized for 24 h using a three-dimensional stirrer. The mixture was dialyzed. Sodium hyaluronate (4 MDa) was added as a lubricant.

[0278] An aqueous solution of lidocaine hydrochloride was added to obtain 0.3% by weight of lidocaine hydrochloride based on the weight of the resulting product.

[0279] The product thus obtained was sieved and then packaged in a syringe.

[0280] Finally, the product was sterilized in an autoclave (plate temperature between 121°C and 135°C with FO > 15).

[0281] After sterilization, the prototypes were analyzed. The elastic modulus G', the phase angle 5 and the stress at the intersection of G' and G”, T were determined. The results are shown in Table 1 below.

[0282] Table 1: Results

[0283] For reference, the properties of a commercial product, Restylane Lyft, which has a degree of modification of 1% (lowest known value of cross-linked hyaluronic acid gels for aesthetic application), are presented in Table 2.

[0284] Table 2: Properties of a commercial product

[0285] The commercial product, Restylane Lyft, has a very low cohesiveness (2.2 N) compared to the gels obtained using the process of the present invention.

[0286] While crosslinking for 3 days at 21°C with a low molar crosslinking rate of 2.2% did not allow the preparation of a gel (prototype 1 with 5 greater than 45°), the method of the present invention made it possible to prepare hydrogels exhibiting desirable mechanical properties with a molar crosslinking rate of only 0.4% (prototype 6).

[0287] When the crosslinking time is increased, the elastic modulus increases, but the gel has a lower T, in other words, it is potentially more brittle (see prototypes 5 vs. 4 and 7 vs. 6).

[0288] Surprisingly, the gels obtained by the process of the present invention have rheological properties and cohesivenesses at least as interesting, or even superior (the HA concentrations and the manufacturing process being otherwise identical), to the gels obtained conventionally at room temperature and with a significantly higher crosslinking rate (prototype 2).

[0289] Example 2: Process according to the invention of hyaluronic acid crosslinked with DVS

[0290] Prototypes No. X1 and X2 were prepared as follows with the DVS contents (molar TR DVS) described in Table 3.

[0291] 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 for 3 cycles of 15 min at 210 rpm at room temperature. The mixture was placed and maintained at the temperatures and times shown in Table 3. The pH of the mixture was approximately 13.

[0292] The mixture is left to defrost before continuing with the preparation.

[0293] A 1 N HCl solution is added to the sterile bag until a pH of 7 ± 0.5 is obtained. 6- The mixture is diluted to a concentration of 23 mg of hyaluronic acid per gram of product with PBS phosphate buffer.

[0294] The mixture is homogenized for 24 hours using a three-dimensional agitator.

[0295] The mixture is then dialyzed and a fixed amount of sodium hyaluronate (4 MDa) is added as a lubricant.

[0296] An aqueous solution of lidocaine hydrochloride is added to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the resulting product.

[0297] The product thus obtained was sieved and then packaged in a syringe.

[0298] Finally, the product was sterilized in an autoclave (plate temperature between 121°C and 135°C with FO > 15).

[0299] Prototype X0 (comparative) was prepared identically to prototype X1. Only the crosslinking step was changed, namely 0.75 days of crosslinking at 21 °C.

[0300] Table 3: Crosslinking conditions

[0301] After sterilization, the prototypes were analyzed. The elastic modulus G', the phase angle 5 and the stress at the intersection of G' and G”, T, were determined. The results are shown in Table 4.

[0302] Table 4: Results

[0303] The prototypes of DVS crosslinked hyaluronic acid X1 and X2 are gels (phase angles less than 45°C) and have properties suitable for the therapeutic, cosmetic and aesthetic applications targeted by the present invention (stresses at the intersection of G' and G” greater than 50 Pa and G' greater than 20 Pa).

[0304] The X0 prototype produced with the same crosslinking rate as X1 but in a conventional manner (crosslinking at room temperature) does not appear in the form of a gel (5 > 45°).

[0305] The process of the present invention appears well suited to crosslinkers having chemical functions other than epoxides.

[0306] Example 3: Stability study of cryokels according to the invention

[0307] The prototypes presented in Table 5 were placed in an accelerated and forced degradation oven at 40 °C in order to monitor the evolution of the rheological properties over a period of 6 months. These conditions model a storage equivalent to 2 years at room temperature. Each prototype was produced according to the synthesis protocol described in Example 1 with the conditions presented in Table 5.

[0308] Table 5: Crosslinking conditions The evolution of the phase angle was measured over time and is presented in Table 6 as a phase angle loss: % phase angle loss = (phase angle at 6 months at 40 °C - initial phase angle) / initial phase angle * 100

[0309] Table 6: Results

[0310] The prototypes obtained by the process of the present invention, Y1, Y2, and Y3, with low crosslinking rates show a low loss of their viscoelastic properties at the end of an accelerated aging study mimicking 2 years of storage at 25°C in an unfavorable situation. This loss is acceptable and similar to that of a traditional gel (Y4) obtained by a conventional process (crosslinking at room temperature) with a higher molar crosslinking rate.

[0311] Example 4: Analysis of soluble hyaluronic acid in cryogels

[0312] An analysis of soluble hyaluronic acid from the gels in Table 7 (gels prepared according to Example 1) was conducted by HPLC-SEC equipped with MALS and RI detector. The gels after sterilization were diluted under gentle stirring at 37 °C to extract soluble hyaluronic acid. The results are shown in Table 8. The weight-average molecular mass (Mw) and the amount of soluble hyaluronic acid with a Mw less than 50 kDa are listed.

[0313] Table 7: Crosslinking conditions

[0314] Table 8: Results

[0315] All these prototypes are formulated with identical raw materials of hyaluronic acid having a Mw greater than 1 MDa. The production of fragments less than 50 kDa thus denotes a certain degree of degradation of the hyaluronic acid during the process. Thus, it is clear that the 2 prototypes (K1 and K2) obtained conventionally are the only prototypes having traces of soluble hyaluronic acid with a Mw less than 50 kDa. All the prototypes obtained by the process of the invention, all other parameters being equal, present a better preservation of the integrity of the hyaluronic acid chains and therefore no trace of sHA less than 50 kDa.

[0316] Example 5: Comparison of the integrity of the soluble hyaluronic acid chains of cryokels obtained by a process according to the invention or according to a freezing process of the prior art

[0317] The size distribution of hyaluronic acid chains in a hydrogel prepared according to the method described in Oschlaeger C.; Bossier, F; Willenbacher, N. Synthesis, structural and micro-mechanical properties of 3D hyaluronic acid-based cryogel scaffolds Biomacromolecules 2016), and according to the invention was compared in order to evaluate the respective capacity of each method to preserve the hyaluronic acid chains.

[0318] According to the prior art method, a first step of dissolving the hyaluronic acid fibers in soda is carried out over 24 hours at 4°C. The crosslinking process having already started during these 24 hours before cryogelation, a cryogelation step of only 10 days is necessary in order to obtain gels with interesting properties close to those of the present invention.

[0319] In the process of the present invention, this step of dissolving the hyaluronic acid fibers (step a); preparation of the reaction medium) is carried out at room temperature for approximately 1 hour followed by freezing over a much longer period. The conditions of this comparison are summarized in Table 9.

[0320] In order to compare the impact of each process on the integrity of the hyaluronic acid chains, the hyaluronic acid fibers were subjected to both processes without using a crosslinking agent. Thus, it is possible, after each key step of the processes (dilution of the fibers, cryogelation), to monitor the Mw and the distribution of the hyaluronic acid chains by HPLC-SEC. A decrease in Mw and an increase in the presence of small hyaluronic acid fragments are then synonymous with the degradation of hyaluronic acid by the process. For each HPLC-SEC analysis (system described above), the samples collected were neutralized to physiological pH and diluted to approximately 0.5 mg / mL.

[0321] Table 9: Summary of the 2 compared processes Starting from the same raw material with a Mw of 859 kDa and using the same aqueous solutions for dissolution, neutralization and dilution as well as using the same equipment and the same conditions for analyzing hyaluronic acid chains, we were able to observe that the process according to the prior art inflicts a loss of approximately 100 kDa on the Mw of hyaluronic acid compared to the process of the present invention. This is also reflected in the presence of 0.5% of hyaluronic acid with a Mw < 50 kDa while the process of the present invention does not present any trace of these hyaluronic acid fragments.

[0322] It is interesting to note that the freezing steps of the 2 processes do not increase the degradation of hyaluronic acid since the only degradation observed takes place during the fiber dissolution step over 24 h at 4 °C. Thus, only this step has an impact on the Mw of hyaluronic acid. The control sample (dissolution 1 h at 21 °C according to the process of the present invention) showed no degradation compared to the initial raw material. A decrease in Mw will have a negative impact on the final viscoelastic properties of the gels and the presence of small fragments of hyaluronic acid may have an impact on local biological inflammation reactions after implantation of the gel in vivo.

[0323] Example 6: Evaluation of the stringy nature of gels (Tack test)

[0324] The stringiness of the gels presented in Table 10 (prepared according to Example 1) was evaluated by the Tack test by measuring the distance over which the gel is able to maintain a bond / cohesiveness between 2 surfaces.

[0325] Table 10: Crosslinking conditions

[0326] The Tack test data are presented in Table 11.

[0327] Figure 1 illustrates the implementation of the Tack test on prototypes Z2 and Z3. After compression of the gel, the surfaces are separated.

[0328] Prototypes Z1 and Z3 have the highest stringy characteristics. Z1, however, according to its rheological properties, is not a viscoelastic gel (phase angle > 45°) and therefore behaves like a viscous solution.

[0329] Z3, on the other hand, is indeed a gel that has a greater stretch range than the other prototypes.

[0330] Z2 and Z4 do not have a stringy character. Z2 is a gel obtained in a conventional manner (crosslinking at room temperature) while Z4 is a hydrogel obtained by the process of the present invention, with a very high molar crosslinking rate (> 2%), which therefore makes the gel both very elastic, brittle and non-stringy.

[0331] Thus, the Z3 prototype obtained by the method of the present invention with a low modification rate gives the best results in terms of obtaining a viscoelastic gel with desirable properties and a stringy gel (tensile cohesiveness).

[0332] Example 7: Evaluation of the stickiness, affinity of gels with surfaces

[0333] The stickiness of a gel according to the invention J2 (prototype 6 of example 1) and of a conventional gel J1 (prototype 2 of example 1) was observed qualitatively. The gels studied are presented in Table 12. The adhesion or not of the tested gel to a model surface is observed after depositing a bolus of 1 g of gel on said surface. The gel is pushed over 10 cm and the state of the bolus and the behavior of the gel is observed. The four model surfaces are the metal surface (Figure 1A), the glass surface (Figure 1B), the plastic surface (Figure 1C) and a gelatin surface (Figure 1D).

[0334] Table 12: crosslinking conditions The prototype J1 obtained by a conventional crosslinking technique, tends to roll on the model surfaces when one tries to move it. On the other hand, the prototype J2, obtained by cryogelation and having a G' relatively close to J1, tends to adhere systematically to these four model surfaces.

[0335] The effort required to move J2 is also greater on the four model surfaces due to the adhesion of the gel during its movement. It is also noteworthy that despite the adhesion of the prototype J2 during its movement path, it maintains its cohesiveness (a single block of gel) and does not crumble along its movement on the surfaces. This may be an important advantage in vivo because the gel will tend to migrate less when adhering to the surfaces.

Claims

DEMANDS 1. A process for preparing a hydrogel, preferably injectable, comprising the following steps: a) supplying at least one polysaccharide; b) supplying at least one crosslinking agent, said crosslinking agent comprising at least two identical or different Z functional groups selected from the following groups: isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, and an acid anhydride residue; (c) prepare a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total amount of crosslinking agent ranging from 0.001 to less than 0.02 mole per 1 mole of repeating unit of the polysaccharide, the duration of the reaction medium preparation step not exceeding 5 hours;d) place the reaction medium obtained at the end of step c), for a period of 2 weeks to 17 weeks, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P.; 2. The process according to claim 1 wherein the polysaccharide is a glycosaminoglycan, preferably the polysaccharide is hyaluronic acid or a salt thereof.

3. The process according to claim 1 or 2 wherein the Z functional groups of the crosslinking agent are epoxide groups, preferably the crosslinking agent is selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDG), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

4. The process according to any one of the preceding claims wherein the total amount of crosslinking agent varies from 0.001 to 0.01 moles per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.005 moles per 1 mole of repeating unit of the polysaccharide.

5. The process 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 is about -20°C.

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

7. The process according to any one of the preceding claims further comprising a sterilization step.

8. Hydrogel capable of being obtained by the process of one of the preceding claims.

9. Cosmetic or pharmaceutical composition comprising a hydrogel according to claim 8.

10. Hydrogel according to claim 8 or composition according to claim 9 for its use in filling and / or replacing tissues.

11. Cosmetic use of a hydrogel according to claim 8 or of a composition according to claim 9 to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin; to fill volume defects of the skin, in particular to fill wrinkles, fine lines and scars; to reduce nasolabial folds and marionette lines; to increase the volume of the cheekbones, chin or lips; to restore facial volumes, in particular of the cheeks, temples, oval of the face, and around the eyes; to reduce the appearance of wrinkles and fine lines; or to stimulate, regenerate, hydrate, firm or restore the radiance of the skin, in particular by mesotherapy.

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