Sterile hyaluronic acid-based hydrogel having improved antioxidant properties

Abstract

The present invention relates to a hydrogel comprising at least one hyaluronic acid or a salt thereof, crosslinked and / or non-crosslinked, at least one anesthetic and at least one thiosulfate salt. The invention also relates to the use of the hydrogel according to the invention in the prevention and / or cosmetic treatment of an alteration of the surface appearance of the skin.

Description

[0001] Description

[0002] Title: Sterile hyaluronic acid-based hydrogel with enhanced antioxidant properties

[0003] technical field

[0004] The invention relates to the field of sterile compositions, in the form of a hydrogel, based on hyaluronic acid, for applications in the prevention and / or cosmetic treatment of an alteration in the surface appearance of the skin.

[0005] State of the art

[0006] Polysaccharides, such as glycosaminoglycans, are widely used in the medical and aesthetic fields, particularly for filling soft tissues. In particular, the majority of products marketed for aesthetic applications are hyaluronic acid-based. To improve skin quality, hydrogels prepared from non-crosslinked hyaluronic acid are of interest because they offer the advantage of being perfectly biocompatible.

[0007] It is also possible to use hydrogels based on modified hyaluronic acid, the hyaluronic acid usually being modified by cross-linking. This cross-linking has the advantage of increasing in vivo durability, in other words, the hydrogels' resistance to in vivo degradation. Cross-linked hyaluronic acid hydrogels can be obtained by various preparation methods.

[0008] The injection of such a hydrogel may cause pain for the patient. Anesthetic agents may be added to reduce this pain.

[0009] Furthermore, hydrogels based on cross-linked and / or non-cross-linked hyaluronic acid intended for soft tissue filling must be sterile. Therefore, the preparation processes for hydrogels based on cross-linked and / or non-cross-linked hyaluronic acid intended for injection generally include a sterilization step for the previously formed hydrogel. Sterilization is typically achieved using heat, for example, in an autoclave. Indeed, this is the most suitable sterilization method for hyaluronic acid. However, the heat generated during sterilization leads to the degradation of the gel's components, particularly the heat-sensitive hyaluronic acid, and / or the anesthetic, whose degradation mechanisms, such as oxidation, can be accelerated.The present invention aims to overcome the above disadvantages and to provide a hydrogel based on hyaluronic acid and anesthetic, said gel being more stable to oxidation and exhibiting reduced loss of rheological properties upon sterilization.

[0010] Summary of the invention

[0011] More specifically, the present invention relates to a hydrogel comprising at least one hyaluronic acid or one of its salts, crosslinked and / or non-crosslinked, at least one anesthetic and at least one thiosulfate salt.

[0012] It is understood that the term “hyaluronic acid” also refers to “hyaluronate” or “hyaluronane”.

[0013] According to one embodiment, the hydrogel according to the invention has one or more of the following characteristics: the thiosulfate salt is sodium thiosulfate; the thiosulfate salt has a content less than or equal to 0.3% by mass of thiosulfate salt, preferably less than or equal to 0.2% by mass of thiosulfate salt, and preferably even less than or equal to 0.1% by mass of thiosulfate salt, relative to the total mass of the hydrogel; the anesthetic is selected from Ambucaine, Amoxecaine, Amyline, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, Fomocaine, Guafecainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine,Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamide, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propranocaine, Propipocaine, Propoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof, preferably lidocaine, mepivacaine, and one of their salts, in particular a hydrochloride salt; the hydrogel comprises at least one cross-linked hyaluronic acid, optionally in a mixture with at least one non-cross-linked hyaluronic acid, said at least one cross-linked hyaluronic acid being cross-linked using a cross-linking agent,preferably selected from bi- or multifunctional epoxy crosslinking agents, such as 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol) diglycidyl ether (PEGDE), and mixtures thereof, or from non-epoxy bi- or multifunctional crosslinking agents, such as endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides, and mixtures thereof, where the hydrogel comprises at least one crosslinked HA, said crosslinked HA having a degree of modification (MOD) of less than or equal to 8%, preferably less than or equal to 7%, preferably less than or equal to 6%, preferably less than or equal to 5%, preferably less than or equal to 4%, preferably less than or equal to 1%, said at least one hyaluronic acid has a weight-average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa,For example, ranging from 2 to 4 MDa or from 1 to 5 MDa, the hydrogel further comprises (i) citrate ions and / or zinc ions and / or (ii) at least one additional additive selected from vitamins, amino acids, and mixtures thereof, preferably from vitamin C, cysteine, and mixtures thereof. The hydrogel is in the form of an injectable composition or a composition for topical application, preferably in the form of an injectable composition. The invention also relates to a method for preparing a hydrogel according to the invention, said method comprising the following steps:

[0014] (1) preparation of a hydrogel comprising at least one hyaluronic acid or one of its salts, cross-linked and / or non-cross-linked, at least one anesthetic and further comprising at least one thiosulfate salt; and

[0015] (2) optionally sterilization, preferably by heat, of the hydrogel comprising at least one thiosulfate salt to obtain a sterile hydrogel.

[0016] The invention also relates to the use of a thiosulfate salt to improve the antioxidant properties and / or to reduce the loss on sterilization of a hydrogel comprising at least one hyaluronic acid or one of its salts, crosslinked and / or non-crosslinked, and at least one anesthetic.

[0017] According to one embodiment of the use of a thiosulfate salt according to the invention, the hydrogel may have one or more of the following characteristics: the thiosulfate salt is sodium thiosulfate; the thiosulfate salt represents a content less than or equal to 0.3% by mass of thiosulfate salt, preferably less than or equal to 0.2% by mass of thiosulfate salt, preferably even less than or equal to 0.1% by mass of thiosulfate salt, relative to the total mass of the hydrogel; the anesthetic is selected from Ambucaine, Amoxecaine, Amyline, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, the Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, Fomocaine, Guafecainol, Heptacaine, HexylcaineHydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamide, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propranocaine, Propipocaine, Propoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof, preferably from lidocaine, mepivacaine, and one of their salts, in particular a hydrochloride salt; the hydrogel comprises at least one cross-linked hyaluronic acid, optionally in mixture with at least one non-cross-linked hyaluronic acid.said at least one crosslinked hyaluronic acid is crosslinked using a crosslinking agent, preferably selected from epoxy bi- or multifunctional crosslinking agents, such as 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol) diglycidyl ether (PEGDE), and mixtures thereof, or from non-epoxy bi- or multifunctional crosslinking agents, such as endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides, and mixtures thereof, where the hydrogel comprises at least one crosslinked HA, said crosslinked HA has a degree of modification (MOD) of 8% or less, preferably 7% or less, preferably less than or equal to 6%, preferably less than or equal to 5%, preferably less than or equal to 4%, more preferably less than or equal to 1%,said at least one hyaluronic acid having a weight-average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or from 1 to 5 MDa, the hydrogel further comprises (i) citrate ions and / or zinc ions and / or (ii) at least one additional additive selected from vitamins, amino acids, and mixtures thereof, preferably from vitamin C, cysteine, and mixtures thereof, the hydrogel being in the form of an injectable composition or a composition for topical application, preferably in the form of an injectable composition. Finally, the invention relates to the use of a hydrogel according to the invention in the prevention and / or cosmetic treatment of an alteration in the surface appearance of the skin.

[0018] Advantageously, hydrogel is used to prevent and / or treat alterations in the viscoelastic or biomechanical properties of the skin, to fill volume defects in 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.

[0019] Unexpectedly, the inventors discovered that adding thiosulfate, particularly sodium thiosulfate, during the preparation of hydrogels containing crosslinked and / or non-crosslinked hyaluronic acid effectively protects the hydrogel from degradation of its rheological properties during sterilization, especially heat sterilization. Hydrogels obtained by the process of the present invention thus exhibit less alteration of their rheological properties compared to hydrogels prepared by an equivalent process without the addition of thiosulfate or with the addition of another antioxidant. The hydrogels of the invention, obtained according to the process of the invention, also exhibit better retention of their rheological properties over time.

[0020] More specifically, the inventors discovered that thiosulfate salt significantly improves the sterilization stability of a hyaluronic acid-based gel in the presence of an anesthetic agent. In particular, the inventors found that even with a low proportion of thiosulfate salt, this effect on sterilization stability was achieved.

[0021] Also, thiosulfate salt does not have a deleterious effect on the mechanical properties of hyaluronic acid gel, even before any sterilization, unlike some antioxidants, such as bisulfite and cysteine ​​which can negatively impact the mechanical properties of the gel before sterilization.

[0022] The inventors observed in particular that the degradation products, especially the oxidation products, of the anesthetic, particularly of the lidocaine or mepivacaine type, were particularly reduced when the thiosulfate salt is added to the hydrogel comprising an HA, particularly when the hydrogel also further comprises citrate ions.

[0023] Detailed description

[0024] The present invention relates to a hydrogel, in particular sterile, comprising at least one hyaluronic acid or one of its salts, cross-linked and / or non-cross-linked, at least one anesthetic and at least one thiosulfate salt.

[0025] For the purposes of this invention, a "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed from a solid, three-dimensional network that is more or less hydrated. The medium consists of long polymer molecules connected to each other by weak bonds (e.g., hydrogen bonds), ionic bonds, or covalent bonds (crosslinking). A gel generally corresponds to a viscoelastic product that has a phase angle θ less than or equal to 45° at 1 Hz for a strain of 0.1% or a stress of 1 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0026] For the purposes of the present invention, a "hydrogel" means 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%, especially at least 97%, especially at least 98% by weight of the liquid medium). Advantageously, the hydrogel according to the invention is an injectable hydrogel.

[0027] For the purposes of the present invention, an "injectable hydrogel" means a hydrogel that can flow and be manually injected using a syringe fitted with a needle of diameter from 0.1 to 0.5 mm, for example a hypodermic needle of 32 G, 30 G, 27 G, 26 G, 25 G. Preferably, an "injectable hydrogel" is a hydrogel having an average extrusion force less than or equal to 25 N, preferably from 5 to 25 N, more preferably from 8 to 15 N, when measured with a dynamometer, at a fixed speed of about 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 of length U, at room temperature. Advantageously, the hydrogel according to the invention has a pH ranging from 6.8 to

[0028] 7.8.

[0029] By "room temperature" is meant a temperature ranging from 20 to 25 °C, more specifically 21 °C.

[0030] The hydrogel according to the invention comprises a thiosulfate salt. Thiosulfate salts that can be used in the invention are chosen from sodium, potassium, ammonium, calcium salts, or mixtures thereof.

[0031] The thiosulfate salt used in the invention is preferably a sodium thiosulfate.

[0032] According to one embodiment, the thiosulfate salt is in hydrate form, preferably in pentahydrate form.

[0033] Thus, for example, the hydrogel according to the invention may comprise a sodium thiosulfate pentahydrate.

[0034] According to a particularly preferred embodiment, the hydrogel according to the invention comprises a content of less than or equal to 0.3% by mass of thiosulfate salt, preferably less than or equal to 0.2% by mass of thiosulfate salt, more preferably less than or equal to 0.1% by mass of thiosulfate salt, more preferably less than or equal to 0.08% by mass of thiosulfate salt, even more preferably less than or equal to 0.06% by mass of thiosulfate salt, or even less than or equal to 0.05% by mass of thiosulfate salt or less than or equal to 0.04% by mass of thiosulfate salt, relative to the total mass of the hydrogel, the thiosulfate salt preferably being sodium thiosulfate, optionally in hydrated form, the hydrogel according to the invention preferably being a sterile hydrogel.

[0035] A reduced quantity of thiosulfate salt advantageously limits the by-products from the thiosulfate salt, which are susceptible to being reduced by oxy-reduction reactions.

[0036] For example, the hydrogel may comprise from 0.01 to 0.3% by mass of a thiosulfate salt, preferably sodium thiosulfate, relative to the total mass of the hydrogel, the thiosulfate salt preferably being sodium thiosulfate, possibly in hydrated form, the hydrogel according to the invention preferably being a sterile hydrogel. The hydrogel according to the invention comprises an anesthetic.

[0037] According to one embodiment, the anesthetic is chosen from 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, Febuverine, Fomocaine, Guafecaine, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamide, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, ProxymetacainePyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof, preferably lidocaine, mepivacaine, and one of their salts, in particular a hydrochloride salt.

[0038] The hydrogel according to the invention comprises hyaluronic acid or one of its salts, cross-linked and / or non-cross-linked.

[0039] Hyaluronic acid (HA) can be in the form of its salts.

[0040] The salts are typically physiologically acceptable salts such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.

[0041] The hydrogel can therefore be a hydrogel based on hyaluronic acid and / or one of its sodium salts (NaHA).

[0042] According to one embodiment, HA has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or ranging from 1 to 5 MDa.

[0043] The HA implemented according to the invention can be supplied in hydrated form (fully or partially hydrated), or in dry form, such as powder or fibers. When the HA is supplied in hydrated form, it is typically in the form of a gel.

[0044] A cross-linked HA can be prepared by any method known to a person skilled in the art.

[0045] Cross-linked HA can result from the reaction of HA with a cross-linking agent or result from the reaction of HA modified to allow the formation of covalent intermolecular bonds.

[0046] For example, crosslinked HA can be prepared as described in W02010131175A1, WO201277054A1 and WO2023 / 198917.

[0047] Crosslinked HA is preferably crosslinked HA with a molar crosslinking ratio of less than or equal to 20%.

[0048] The "molar crosslinking ratio" (CR), expressed as a percentage, refers to the molar ratio of the amount of crosslinking agent to the amount of HA repeat units (disaccharide units) introduced into the crosslinking reaction medium, expressed per 100 moles of HA repeat units in the crosslinking medium. For example, a molar crosslinking ratio of 1% means that there is one mole of crosslinking agent introduced into the reaction medium for every 100 moles of HA repeat units.

[0049] Preferably, crosslinked HA is crosslinked HA with a molar crosslinking ratio greater than 0 and less than or equal to 20%. More preferably, crosslinked HA is crosslinked HA with a molar crosslinking ratio greater than 0 and less than or equal to 15%, more preferably the ratio is greater than 0 and less than or equal to 14%, more preferably the ratio is greater than 0 and less than or equal to 10%, even more preferably the ratio is greater than 0 and less than or equal to 7%. Even more preferably, crosslinked HA is crosslinked HA with a molar crosslinking ratio greater than 0 and less than or equal to 2%, preferably less than or equal to 1%, even more preferably less than or equal to 0.8%, in particular ranging from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeat unit of HA(s)).

[0050] HA can be crosslinked using a crosslinking agent. Preferably, HA is crosslinked using a crosslinking agent chosen from among bi- or multifunctional epoxy or non-epoxy crosslinking agents, i.e., prepared by reacting the polysaccharide with a crosslinking agent. Examples of epoxy agents include 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol) diglycidyl ether (PEGDE), and mixtures thereof. Examples of non-epoxy agents include endogenous polyamines such as spermine, spermidine and putrescine, aldehydes such as glutaraldehyde, carbodiimides and divinyl sulfone, hydrazide derivatives such as adipic acid dihydrazide, bisalkoxyamines, and mixtures thereof.Non-epoxide agents include amino acids such as cysteine ​​and lysine; peptides or proteins containing amino acids such as cysteine ​​and lysine; and trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate, or barium trimetaphosphate. In some embodiments, the crosslinking agent is an epoxide agent, preferably 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether. Preferably, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0051] In some embodiments, the crosslinking agent is a non-epoxy agent, preferably selected from endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides and mixtures thereof.

[0052] The crosslinking reaction can be carried out under conditions conducive to the coupling of hyaluronic acid and non-epoxy crosslinking agents. For the purposes of this invention, "conducive conditions" means an element that triggers said coupling. The choice of this triggering element falls within the expertise of a person skilled in the art.

[0053] Crosslinked HA is preferably a crosslinked polysaccharide with a degree of modification (MOD) of less than or equal to 8%, preferably less than or equal to 7%, preferably less than or equal to 6%, preferably less than or equal to 5%, or even less than or equal to 4%.

[0054] The "degree of modification" (MOD) of a hyaluronic acid-based (HA) filler corresponds to the molar amount of crosslinking agent bound to the HA at one or more of its ends, expressed per 100 moles of HA repeat units. It can be determined by methods known to those skilled in the art, such as Nuclear Magnetic Resonance (NMR) spectroscopy, as described in the article by Faivre J, Gallet M, Tremblais E, Trévidic P, and Bourdon F. Advanced Concepts in Rheology for the Evaluation of Hyaluronic Acid-Based Soft Tissue Fillers. Dermatol Surg. 2021 May. For example, a degree of modification of 1% means that there is one mole of crosslinking agent per 100 moles of HA repeat units. Also, the thiosulfate salt has a particularly advantageous effect on sterilization losses of hyaluronic acid gel comprising cross-linked HA when the cross-linked HA has a degree of modification (MOD) less than or equal to 8%, preferably less than or equal to 7%.Indeed, the inventors observed that if the HA has a high degree of modification, especially greater than 8%, then the thiosulfate salt has no effect on the stability of the rheological properties of the hydrogel upon sterilization.

[0055] Advantageously, crosslinked HA is crosslinked HA with a degree of modification (MOD) of less than or equal to 8%, preferably less than or equal to 7%, preferably less than or equal to 6%, preferably less than or equal to 5%, or even less than or equal to 4% or less than or equal to 3% or less than or equal to 2%.

[0056] Advantageously, crosslinked HA is crosslinked HA having a degree of modification (MOD) of less than or equal to 1.8%, more preferably less than or equal to 1.5%, preferably less than or equal to 1.2%, even more preferably less than 1%.

[0057] Crosslinked HA can in particular be prepared by a process comprising a step of preparing a crosslinking reaction medium comprising one or more HA(s), one or more crosslinking agent(s) and a solvent to obtain a crosslinked HA.

[0058] The total amount of crosslinking agent in the reaction medium typically ranges from 0.001 to 0.10 moles per 1 mole of HA repeat unit, preferably from 0.001 to 0.08 moles or from 0.001 to 0.07 moles per 1 mole of HA repeat unit, preferably from 0.001 to 0.04 moles per 1 mole of HA repeat unit, preferably from 0.001 to 0.03 moles per 1 mole of HA repeat unit, preferably from 0.001 to 0.02 moles per 1 mole of HA repeat unit, more preferably from 0.001 to 0.01 moles per 1 mole of HA repeat unit, and even more preferably from 0.001 to 0.005 moles per 1 mole of HA repeat unit (DI unit). saccharidic).

[0059] The mass concentration of hyaluronic acid or hyaluronic acid salt in the reaction medium advantageously varies from 15 to 200 mg / g of solvent, preferably from 15 to 50 mg / g.

[0060] The HA crosslinking step is typically carried out at a temperature ranging from 4 to 35 °C, preferably from 15 °C to 25 °C. Preferably, the crosslinking time does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours.

[0061] Crosslinking allows polysaccharide chains to be crosslinked together. The functional groups of the crosslinking agent react with functional groups present on the polysaccharides 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 to form intramolecular bonds. In particular, the functional groups of the crosslinking agent react with the -OH or -COOH groups present on hyaluronic acid. Crosslinked hyaluronic acid (HA) compounds are thus obtained, comprising at least one crosslink between two polysaccharide chains or within the same polysaccharide chain, with this crosslink being the residue of the crosslinking agent.

[0062] 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. Crosslinking can be carried out in the presence of a total amount of crosslinking agents typically ranging from 0.1 to 10 moles, or from 0.1 to 8 moles, or from 0.1 to 7 moles, or from 0.1 to 4 moles, or from 0.1 to 3 moles, or from 0.1 to 2 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 HA repeating units. The crosslinking conditions, in particular the crosslinking agent content, duration and temperatures as well as the weight average molecular weights (Mw) of the HA, used are interdependent.

[0063] In some embodiments, the crosslinking step can be carried out at a temperature of 30°C or lower, preferably 25°C or lower. Typically, the temperature is above 0°C, above 5°C, or above 10°C. Even more preferably, the crosslinking step can be carried out by placing the reaction medium at room temperature. When the crosslinking step is carried out at a temperature of 0°C or higher but below 30°C, the crosslinking time is at least 1 minute, preferably at least 10 minutes, and even more preferably at least 1 hour. Preferably, the crosslinking time is at most 5 days. In some embodiments, the crosslinking step can be carried out by placing the reaction medium at a temperature ranging from 0 to 15°C, from 1 to 10°C, or from 1 to 9°C.When the crosslinking step is carried out at a temperature greater than or equal to 0°C and less than or equal to 10°C, the crosslinking time is at least 1 minute, preferably at least 10 minutes, and even more preferably at least 1 hour.

[0064] In some embodiments, the crosslinking step can be carried out by placing the reaction medium at a temperature above 30°C, or above or equal to 35°C, or above or equal to 40°C, or above or equal to 45°C, or above or equal to 50°C. The temperature is typically below 60°C. When the temperature is above 30°C, the duration of the crosslinking step is at least 1 minute, preferably at least 10 minutes, and even more preferably at least 1 hour, preferably between 1 and 5 hours.

[0065] In some embodiments, the crosslinking step can be carried out by placing the reaction medium at a pressure P less than or equal to atmospheric pressure and at a temperature T above the eutectic point temperature of the reaction medium as measured at pressure P and below the freezing point temperature of the reaction medium as measured at pressure P, preferably for a period of at least 1 hour. Crosslinked HA-based hydrogels prepared by such a process are highly biocompatible. Indeed, crosslinked HAs can be prepared with smaller amounts of crosslinking agent, for example, amounts ranging from 0.001 to 0.02 moles per 1 mole of HA repeating unit.

[0066] The freezing point temperature of a reaction medium refers to the temperature at which the mixture of its components, on a macroscopic scale, solidifies, meaning it becomes non-fluid. Below the freezing point, the mixture is in a frozen state characterized by the coexistence of solid and liquid components. This frozen state is maintained until the eutectic point temperature of the reaction medium is reached.

[0067] The eutectic point temperature of a reaction medium is the temperature below which the mixture of its components transitions from a frozen state (coexistence of liquid and solid phases) to a completely solid state, meaning a state in which all components of the mixture are in solid form. The freezing point and eutectic point of a mixture depend on the pressure to which the mixture is subjected; therefore, the freezing point and eutectic point are measured at pressure P.

[0068] The freezing point and eutectic point can be determined by differential scanning calorimetry. This method allows for the determination of phase transitions. To do this, the product under study is gradually cooled until its phase transitions are observed.

[0069] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably ranging from -35°C to -10°C. Even more preferably, the temperature T is about -20°C.

[0070] 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 with altitude. At an altitude of 0 meters, 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.

[0071] Preferably, during crosslinking, when the temperature T is greater than or equal to -55°C and less than or equal to -5°C, the reaction medium is placed under these conditions for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, and preferably at most 27 weeks. Preferably, the crosslinking step is carried out for a period of 2 to 25 weeks, preferably from 2 to 20 weeks or 2 to 17 weeks, and even more preferably from 3 to 8 weeks or 4 to 7 weeks, at temperature T and pressure P.

[0072] After the crosslinking step, the crosslinked HA is typically in gel form. This gel is generally used directly in the subsequent hydrogel preparation process.

[0073] The crosslinked and / or non-crosslinked HAs described above are useful for implementing the hydrogel preparation process of the invention and thus for preparing hydrogels comprising a crosslinked and / or non-crosslinked HA. The crosslinked or non-crosslinked HA, or a mixture thereof, will constitute the polymer network of the hydrogel. The hydrogel comprising a crosslinked or non-crosslinked HA, or a mixture thereof, can thus be the basis of a crosslinked HA, a non-crosslinked HA, or a mixture thereof. A hydrogel comprising, as its sole HA, a non-crosslinked HA, is prepared from a non-crosslinked HA. A hydrogel comprising, as its sole HA, a crosslinked HA, is prepared from a crosslinked HA. When the hydrogel comprises a mixture of crosslinked and non-crosslinked HA, the hydrogel is prepared from both crosslinked and non-crosslinked HA. The non-crosslinked HA is typically added to the crosslinked HA during the hydrogel preparation.

[0074] The hydrogel according to the invention may optionally further comprise at least one additional additive selected from lubricating agents, amino acids, peptides, proteins, vitamins, minerals, nucleic acids, nucleotides or polynucleotides such as PDRN, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and mixtures thereof, preferably from vitamins, nucleotides or polynucleotides, amino acids, and mixtures thereof. The additional additive(s) are preferably present in an amount ranging from 0.01 to 9.0% by mass relative to the total mass of the hydrogel.

[0075] Non-crosslinked polysaccharides, in particular non-crosslinked hyaluronic acid, non-crosslinked heparosan or mixtures thereof, may be cited as examples of lubricating agents.

[0076] Polyols, particularly mannitol, glycerol, sorbitol, propylene glycol, xylitol, erythritol, maltitol, and lactitol, or mixtures thereof, can be cited as examples of lubricating agents. These polyols can have a synergistic effect on sterilization stability when combined with thiosulfate salts.

[0077] Nucleotides or polynucleotides, particularly PDRN, can be preferentially used in hydrogels. Specifically, PDRN oxidation products could be reduced when thiosulfate salt is added to hydrogels containing HA.

[0078] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives, and mixtures thereof. Examples of coenzymes include coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0079] Examples of adrenaline derivatives include adrenaline, noradrenaline, and mixtures thereof. 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 mixtures thereof.

[0080] 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 preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0081] The inventors discovered that the thiosulfate salt also improved the sterilization stability of a hydrogel based on HA and further containing vitamins, particularly vitamin C. Advantageously, the thiosulfate salt improves the stability of a hydrogel based on HA and further containing an additional component such as vitamins or amino acids.

[0082] In one embodiment, the hydrogel according to the invention further comprises citrate ions and / or zinc ions, preferably at least citrate ions. This embodiment provides a highly biocompatible hydrogel with beneficial effects on skin quality.

[0083] Preferably, when the hydrogel includes citrate ions, the concentration of citrate ions is at least 0.1 mM in the hydrogel.

[0084] Preferably, when the hydrogel includes zinc ions, the concentration of zinc ions is at most 20 mM in the hydrogel.

[0085] According to this embodiment, preferably, the molar ratio [citrate ions] / [zinc ions] ranges from 1 to 20.

[0086] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic and 0.01 to 0.3% by mass of thiosulfate salt, relative to the total mass of the hydrogel.

[0087] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic, citrate ions, optionally zinc ions and 0.01 to 0.3% by mass of thiosulfate salt, relative to the total mass of the hydrogel.

[0088] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic selected from lidocaine, mepivacaine and their mixture, and 0.01 to 0.3% by mass of thiosulfate salt, relative to the total mass of the hydrogel.

[0089] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic selected from lidocaine, mepivacaine and their mixture, citrate ions, optionally zinc ions and 0.01 to 0.3% by mass of thiosulfate salt, relative to the total mass of the hydrogel.

[0090] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic selected from lidocaine, mepivacaine and their mixture, and 0.01 to 0.3% by mass of sodium thiosulfate, relative to the total mass of the hydrogel.

[0091] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic selected from lidocaine, mepivacaine and their mixture, citrate ions, optionally zinc ions and 0.01 to 0.3% by mass of sodium thiosulfate, relative to the total mass of the hydrogel.

[0092] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising a crosslinked and / or non-crosslinked HA, an anesthetic selected from lidocaine, mepivacaine and their mixture, and 0.01 to 0.3% by mass of sodium thiosulfate in pentahydrate form, relative to the total mass of the hydrogel.

[0093] According to a particular embodiment, the hydrogel according to the invention is a sterile hydrogel comprising cross-linked and / or non-cross-linked hydrogel, an anesthetic selected from lidocaine, mepivacaine, citrate ions, optionally zinc ions and a mixture thereof, and 0.01 to 0.3% by mass of sodium thiosulfate in pentahydrate form, relative to the total mass of the hydrogel. Advantageously, the hydrogel according to the invention is free of corticosteroids.

[0094] The hydrogel of the present invention advantageously has a phase angle θ less than or equal to 45°, at 1Hz for a strain of 0.1% or a shear stress of 5 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0095] The injectable hydrogel has a physiological pH, i.e., ranging from 6.8 to 7.8. The pH of the injectable hydrogel is preferably greater than or equal to 6.9 and less than or equal to 7.4; 7.3; 7.2; 7.1 or 7.

[0096] The hydrogel of the present invention is preferably an injectable hydrogel, that is to say, one which can flow and be manually injected by means of a syringe fitted with a needle of diameter ranging from 0.1 to 0.5 mm, for example a hypodermic needle of 32G, 30G, 27G, 26G, 25G.

[0097] The hydrogel of the present invention may comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of HA (total weight of HA, i.e. total weight of crosslinked and / or non-crosslinked HA), relative to the total weight of the hydrogel.

[0098] Thus, when the hydrogel comprises, as the only HA, a non-crosslinked HA, the hydrogel obtained from the present invention can therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of non-crosslinked HA, relative to the total weight of the hydrogel.

[0099] When the hydrogel comprises, as the only HA, a cross-linked HA, the hydrogel obtained by the process of the present invention can therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of cross-linked polysaccharide (for example, cross-linked hyaluronic acid), relative to the total weight of the hydrogel.

[0100] When the hydrogel comprises a mixture of crosslinked and non-crosslinked HA, the hydrogel of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of a mixture of non-crosslinked and crosslinked HA, relative to the total weight of the hydrogel. In particular, the content of non-crosslinked HA may vary from 0.5 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 HA present in the hydrogel.

[0101] The total concentration of HA in the hydrogel of the present invention advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, and even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Preferably, the HA is sodium hyaluronate.

[0102] When the hydrogel comprises a crosslinked HA, the crosslinked HA preferably has a molar crosslinking ratio of 20% or less. Preferably, the hydrogel comprises a crosslinked HA with a molar crosslinking ratio greater than 0 and less than or equal to 15%. Even more preferably, the hydrogel comprises a crosslinked HA with a molar crosslinking ratio greater than 0 and less than or equal to 10%. Even more preferably, the hydrogel comprises a crosslinked HA with a molar crosslinking ratio greater than 0 and less than or equal to 7%, preferably less than or equal to 2%, and even more preferably less than or equal to 1%, in particular ranging from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of HA repeat units).

[0103] When the hydrogel comprises a cross-linked HA, the cross-linked HA preferably has a degree of modification (MOD) of 8% or less, preferably 7% or less, preferably 6% or less, preferably 5% or less, preferably 4% or less, more preferably 2% or less, or even 1% or less. Advantageously, the cross-linked HA has a degree of modification (MOD) of 1.8% or less, more preferably 1.5% or less, preferably 1.2% or less, or even more preferably less than 1%.

[0104] According to a particular embodiment, the hydrogel according to the invention comprises at least one hyaluronic acid or one of its salts, at least one anesthetic and at least one thiosulfate salt, said hyaluronic acid being cross-linked and having a degree of modification less than or equal to 6%, said anesthetic being advantageously chosen from lidocaine and mepivacaine.

[0105] According to a particular embodiment, the hydrogel according to the invention comprises at least one hyaluronic acid or one of its salts, at least one anesthetic and at least one sodium thiosulfate, said hyaluronic acid being cross-linked and having a degree of modification less than or equal to 6%, said anesthetic being advantageously chosen from lidocaine and mepivacaine.

[0106] According to a particular embodiment, the hydrogel according to the invention comprises at least one hyaluronic acid or one of its salts, at least one anesthetic and at least one sodium thiosulfate in pentahydrate form, said hyaluronic acid being cross-linked and having a degree of modification less than or equal to 6%, said anesthetic being advantageously chosen from lidocaine and mepivacaine.

[0107] The invention also relates to a method for preparing a hydrogel according to the invention comprising the following steps:

[0108] (1) preparation of a hydrogel comprising at least one hyaluronic acid or one of its salts, cross-linked and / or non-cross-linked, at least one anesthetic and further comprising at least one thiosulfate salt; and

[0109] (2) optionally sterilization, preferably by heat, of the hydrogel comprising at least one thiosulfate salt to obtain a sterile hydrogel.

[0110] The preparation of the hydrogel typically includes at least one step of adding the thiosulfate salt to the crosslinked and / or non-crosslinked HA.

[0111] The preparation of the hydrogel advantageously includes a step of adjusting to physiological pH, in particular ranging from 6.8 to 7.8.

[0112] In one variation, the thiosulfate salt is added in powder form to the crosslinked and / or non-crosslinked HA. Typically, when thiosulfate is added in powder form, the effect of the thiosulfate salt on the hydrogel's pH is neutralized.

[0113] In another variant, the thiosulfate salt is added as a solution (solution including the thiosulfate salt(s)) to the crosslinked and / or non-crosslinked HA.

[0114] The concentration of thiosulfate salt(s) in the solution is advantageously chosen to limit the dilution effect that can be caused by adding the solution during the preparation of the hydrogel, as such a dilution effect of the hydrogel is undesirable.

[0115] The solution comprising one or more thiosulfate salts is typically prepared in water or in a physiologically acceptable buffer, preferably by adding thiosulfate in powder form to water or a physiologically acceptable buffer. Examples of buffers include, but are not limited to, N-carbamoylmethyl taurine (CAS No: 7365-82-4), the sodium salt of 3-[N,N-bis(hydroxyethyl)amino]-2-hydroxypropane sulfonic acid (CAS No: 102783-62-0), 3-morpholino-2-hydroxypropane sulfonic acid (CAS No: 68399-77-9), 1,4-piperazinediethane sulfonic acid (CAS No: 5625-37-6), 1,4-piperazine-N,N'-bispropane sulfonic acid (CAS No: 5625-56-9), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propane sulfonic acid (CAS No: 68399-81-5), 2- [(2-hydroxy-l,l-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (CAS No: 7365-44-8), N-tris(hydroxymethyl)methylglycine (CAS No: 5704-04-1),3-(N-morpholino)propanesulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1), 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (CAS No: 7365-45-9), 1,4-Piperazinediethanesulfonic acid (CAS No: 5625-37-6), 4-(2-hydroxyethyl)piperazine-l-(2-hydroxypropane-3-sulfonic acid) (CAS No: 68399-78-0), buffers Phosphates such as PBS with a pH around physiological pH (CAS No: 7647-14-5, 7447-40-7).

[0116] Preferably, the buffer is chosen from 3-(N-morpholino)propane sulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1), 4-(2-Hydroxyethyl)piperazine-1-ethane sulfonic acid (CAS No: 7365-45-9) and phosphate buffers such as PBS with a pH around physiological pH (CAS No: 7647-14-5, 7447-40-7).

[0117] Preferably the buffer is a phosphate buffer, particularly a saline buffer of NaH2PO4 / Na2HPO4 or KH2PO4 / K2HPO4.

[0118] The preparation of a hydrogel from crosslinked and / or non-crosslinked HA can be carried out conventionally, with the exception that at least one thiosulfate salt is added during the hydrogel preparation. Thus, the preparation of a hydrogel comprising crosslinked and / or non-crosslinked HA may include, in addition to the crosslinking step when the HA is crosslinked, one or more of the following conventional steps:

[0119] - pH adjustment (1); - Dilution (2);

[0120] - Purification (3);

[0121] - Addition of at least one anesthetic and possibly at least one additional component (4);

[0122] - Extrusion (5).

[0123] These steps, well known to those skilled in the art, can be as described below. They can be at least partially concurrent.

[0124] The conventional steps can be carried out sequentially as follows: optional pH adjustment (1), then optional dilution (2), then optional purification (3), then optional addition of an additional component (4), and finally optional extrusion (5). They can also be carried out in a different order. Advantageously, the extrusion step (5) is performed last, once at least one of the other conventional steps has been completed. It can also be performed repeatedly and inserted between the other conventional steps described.

[0125] The thiosulfate compound(s) (in powder or solution form) can be added at, before, or after any of these conventional steps.

[0126] In one variant, the thiosulfate compound(s) are added before the extrusion step (5) in order to obtain a homogeneous gel.

[0127] When a purification step (3) is implemented, the thiosulfate compound(s) may be added before or after the purification step (3); advantageously, the thiosulfate compound(s) are added after the purification step (3). Adding the thiosulfate compound(s) after the purification step ensures better control of the thiosulfate compound concentration in the prepared hydrogel.

[0128] Preferably, the thiosulfate compound(s) are added between the purification (3) and extrusion (5) steps. The addition of the thiosulfate compound(s) can be carried out after the dilution step (2) or during the dilution step (2), for example, the thiosulfate compound(s) can be added to the aqueous dilution solvent.

[0129] The thiosulfate compound(s) may be added during the dilution step (2) and / or during the step of adding at least one anesthetic and any additional component (4), preferably during the step of adding at least one anesthetic and at least one additional component (4). In particular, in some embodiments, the addition of the solution containing the thiosulfate compound(s) is concurrent with the step of adding at least one anesthetic and any additional component (4). In particular, in some embodiments, the addition of the solution containing the thiosulfate compound(s) is concurrent with the addition of an anesthetic solution.

[0130] The steps of dilution (2), addition of at least one anesthetic and a possible additional component (4) and addition of thiosulfate compound(s) may be concomitant.

[0131] The thiosulfate compound(s) can be added after the pH adjustment step (1). The thiosulfate compound(s) can be added between the pH adjustment step (1) and the extrusion step (5) when both of these steps are implemented.

[0132] Advantageously, the thiosulfate compound and the anesthetic compound are added after the purification step.

[0133] pH adjustment (1)

[0134] The hydrogel preparation process may include a step of adjusting the pH of the hydrogel to achieve the desired pH (pH of 6.8-7.8).

[0135] Dilution (2)

[0136] The hydrogel preparation process may include a dilution step of the crosslinked and / or non-crosslinked HA. This dilution step allows for adjusting the HA concentration in the prepared hydrogel. Specifically, an aqueous solvent is added to the crosslinked and / or non-crosslinked HA, for example, physiological saline, possibly buffered by the presence of salts such as phosphate salts. In particular, the added aqueous solvent has a pH around physiological pH (6.8–7.8). The HA concentration obtained after the dilution step advantageously ranges from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, and even more advantageously from 10 mg / g to 30 mg / g of hydrogel.

[0137] Purification (3)

[0138] The hydrogel preparation process may include at least one purification step.

[0139] The purification step aims to remove any undesirable impurities. These impurities may result from the crosslinking of the HA. Such impurities may include, for example, residual crosslinking agent, particularly of the epoxy type, which may not have reacted.

[0140] This step can also be used to perform a liquid exchange, such as a buffer exchange. The purification step is therefore particularly useful when the hydrogel contains a cross-linked HA.

[0141] Purification can be achieved by dialysis or by filtration, for example by dynamic cross-flow filtration (DCF).

[0142] Addition of anesthetic and possible additional components (4)

[0143] The hydrogel preparation process includes at least one step of adding an anesthetic and may include one or more steps of adding at least one additional component. The additional component may be chosen from among lubricating agents, amino acids, peptides, proteins such as collagen and silk fibroin, vitamins, elements such as silicon (for example via the addition of orthosilicic acid), minerals, nucleic acids, nucleotides or polynucleotides such as PDRN, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof.

[0144] Extrusion (5)

[0145] The hydrogel preparation process may include one or more extrusion steps. This extrusion step allows for a more homogeneous hydrogel, particularly with the most consistent, i.e., the most regular, extrusion force possible. For example, the extrusion step can be carried out using a sieve with perforations ranging from 50 to 2000 µm in diameter. Those skilled in the art know how to select the perforation diameter based on the desired mechanical properties of the hydrogel.

[0146] According to a first embodiment using citrate ions and zinc ions, the hydrogel can be obtained by the following process:

[0147] - contacting cross-linked and / or non-cross-linked HA with a saline physiological solution, preferably buffered, the saline physiological solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof,

[0148] - addition of a thiosulfate salt,

[0149] - addition of an anesthetic,

[0150] - addition of crosslinked and / or non-crosslinked HA citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel,

[0151] - addition of zinc ions to crosslinked and / or non-crosslinked HA in sufficient quantity to achieve a zinc ion concentration of no more than 20 mM in the hydrogel, the addition of citrate ions and zinc ions being carried out in a molar ratio [citrate ions] / [zinc ions] ranging from 1 to 20, and on the condition that the addition of zinc ions is not carried out before the addition of citrate ions when contact with physiological saline solution, preferably buffered, is carried out before the addition of citrate ions.

[0152] According to a second embodiment using zinc ions and citrate ions, the hydrogel can be obtained by the following process:

[0153] (0) preparation of a crosslinked HA from a crosslinking reaction medium comprising one or more HA(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount permitting the preparation of a hydrogel comprising at most 20 mM of zinc ions;

[0154] (1) preparation of a hydrogel from the crosslinked HA obtained at the end of step (0) and optionally from a non-crosslinked HA, the preparation of the hydrogel comprising a step of contacting the crosslinked HA with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof; wherein:

[0155] - the crosslinking reaction medium further comprises citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0156] - step (1) further includes, before the step of contacting the cross-linked HA with physiological saline solution, preferably buffered, a step of adding citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0157] - the physiological saline solution, preferably buffered, further comprises citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

[0158] Typically, the process of the present invention includes a sterilization step for the prepared hydrogel. Sterilization is preferably carried out by heat, for example, in an autoclave. Sterilization is generally performed by increasing the temperature of the sterilization medium to a temperature known as the "plateau temperature," which is maintained for a predetermined time known as the "plateau time." Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, and preferably at a plateau time ranging from 1 to 20 minutes, with F0 > 15. The sterilization value F0 corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the microorganism population present in the product to be sterilized. Alternatively, sterilization can be carried out, in particular, by gamma radiation, UV radiation, or by means of ethylene oxide.

[0159] The hydrogel obtained at the end of the process typically has a pH ranging from 6.8 to 7.8 (physiological pH). The hydrogel preparation process according to the present invention may further include a hydrogel conditioning step. The hydrogel conditioning is typically carried out in an injection device. The conditioning is preferably performed just before the sterilization step (step (2)). Thus, the sterile hydrogel may be in the form of an injection device pre-filled with the hydrogel, for example, a syringe pre-filled with the hydrogel.

[0160] The sterile hydrogels of the invention are particularly useful for filling and / or replacing tissues, especially soft tissues, notably by injecting the hydrogel into the tissue. In addition to filling soft tissues, they also provide biostimulating effects.

[0161] They can be injected using any of the methods known to those skilled in the art. In particular, they can be administered using an injection device suitable for intra-epidermal, intradermal, subcutaneous, and / or supraperiosteal injection. The injection device may include, among other things, a syringe, a set of microsyringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a microneedle roller.

[0162] The sterile hydrogels according to the invention are preferably injected subcutaneously.

[0163] They can relate to deep applications, mid-level applications and / or shallow applications.

[0164] A "superficial application" refers to the administration, for example by mesotherapy, of a composition superficially into or onto the skin, for the treatment of the superficial layers of the skin, the epidermis and the most superficial parts of the dermis, to reduce superficial wrinkles and / or improve skin quality (such as its radiance, density or structure) and / or rejuvenate the skin.

[0165] A "midline application" refers to the administration of a composition into the middle part of the skin to treat the middle layers of the skin, as well as to reduce midline wrinkles.

[0166] A "deep application" refers to the administration of a hydrogel into the deepest layers of the skin—the hypodermis and the deepest part of the dermis—and / or beneath the skin (above the periosteum) to "add volume," such as for filling deep wrinkles and / or partially atrophied areas of the facial and / or body contour. So-called "volumizing" hydrogels are typically used for deep application.

[0167] The hydrogels of the invention may have therapeutic and / or cosmetic and / or cosmeceutical applications.

[0168] In the cosmetic field, hydrogels can be particularly useful to compensate for tissue volume loss due to aging.

[0169] They can be used in the prevention and / or cosmetic treatment of alterations in the skin's surface appearance. For example, hydrogels can be used in cosmetics to prevent and / or treat alterations in the skin's viscoelastic or biomechanical properties; to fill volume defects in the skin, particularly to fill wrinkles, fine lines, and scars; to soften nasolabial folds and marionette lines; to augment the volume of the cheekbones, chin, or lips; to restore facial volume, particularly in the cheeks, temples, jawline, and around the eyes; and to reduce the appearance of wrinkles and fine lines.

[0170] The present invention also relates to the cosmetic use of a hydrogel according to the invention for filling tissues, in particular soft tissues, in particular to compensate for tissue volume losses due to aging.

[0171] The present invention also relates to the use of a thiosulfate salt to improve the antioxidant properties and / or to reduce the loss on sterilization of a hydrogel comprising at least one hyaluronic acid or one of its salts, crosslinked and / or non-crosslinked, and at least one anesthetic.

[0172] In the context of the use of the thiosulfate salt according to the invention, preferably the thiosulfate salt has one or more of the characteristics defined in the context of the hydrogel according to the invention.

[0173] In the context of using the thiosulfate salt according to the invention, preferably the HA exhibits one or more of the characteristics defined in the context of the hydrogel according to the invention. In the context of using the thiosulfate salt according to the invention, preferably the anesthetic exhibits one or more of the characteristics defined in the context of the hydrogel according to the invention.

[0174] In the context of the use of the thiosulfate salt according to the invention, preferably the hydrogel has one or more of the characteristics defined in the context of the hydrogel according to the invention.

[0175] The invention will now be described by means of the following examples, given of course by way of illustration and not limitation of the invention.

[0176] Examples

[0177] 1. Materials

[0178] - Non-crosslinked sodium hyaluronate

[0179] - BDDE (Sigma Aldrich)

[0180] - 0.25M NaOH

[0181] - HCl IM

[0182] - Phosphate buffer (PB S, BBraun),

[0183] - Mepivacaine hydrochloride

[0184] - Sodium thiosulfate pentahydrate (STP) (CAS No. 10102-17-7)

[0185] - L-cysteine ​​hydrochloride monohydrate (CAS No. 7048-04-6)

[0186] - Sodium bisulfite (CAS No. 7631-90-5)

[0187] - Citric acid (Sigma Aldrich)

[0188] - Three-dimensional agitator

[0189] - DHR-2 Rheometer

[0190] - Dynamometer and test bench

[0191] - Homogenizer, Pallet Mill

[0192] - Sterile polyethylene bag

[0193] 2. Methods

[0194] Measurement of viscoelastic properties The viscoelastic properties of the hydrogels obtained were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plane geometry and an anodized aluminum peltier plane (42 mm) (air gap 24 pm).

[0195] 0.5 g of sterilized hydrogel is deposited between the Peltier plate and the cone. A stress scan is then performed at 1 Hz and 25°C. The elastic modulus G' and the phase angle θ are recorded for a stress of 5 Pa. The measurements are carried out in the linear LVER domain.

[0196] The Linear ViscoElastic Region (LVER) corresponds to the range of hydrogel deformations from an initial elastic modulus value G' to a value of G' reduced by 10% of its initial value. LVER measurement consists of an oscillatory stress scan in compression mode at a given oscillation frequency to determine the linear viscoelastic region.

[0197] Measurement of mepivacaine N-oxide

[0198] The mepivacaine N-oxide (oxidized derivative of mepivacaine) content of the hydrogels obtained was measured using ultra-high-performance liquid chromatography (UPLC) on a C18 column (5 µm, 4.6 × 150 mm) at a flow rate of 0.8 mL / min with an injection volume of 10 pL. The instrument was equipped with a diode array detector (DAD), with chromatogram extraction at 230 nm. The mobile phase was prepared by mixing 100 mL of purified water, 540 mL of 50 mM phosphate buffer filtered through a 0.22 µm membrane (pH 7.0), and 360 mL of acetonitrile. Chromatograms were obtained and analyzed using Empower software.

[0199] A standard solution with a concentration of 0.30 mg / mL of mepivacaine HCl was prepared. For the stock solution, 60.0 mg of mepivacaine HCl was dissolved in a 50 mL volumetric flask, which was then filled to the mark with purified water to obtain a concentration of 1.20 mg / mL. This solution was homogenized before being diluted to the 0.30 mg / mL solution by mixing 250 µL of the stock solution with 750 µL of purified water. The diluted solution was also homogenized before analysis.

[0200] Sample preparation involves diluting 100.0 mg of the sample to be analyzed in 100 pL of a 1.0 mg / mL hyaluronidase solution, followed by 800 pL of purified water. The 1.0 mg / mL hyaluronidase solution is prepared as follows: 100.0 mg of hyaluronidase (750–3000 U / mg) is dissolved in a 100 mL volumetric flask filled to the mark with phosphate buffer (50 mM; pH 7.0). The mixture is homogenized and then blended at 37°C for 2 hours at 1100 rpm in a Thermomixer. After tissue incubation, the contents of the tube are homogenized and then transferred to a vial for analysis. The percentage indicated represents the proportion of mepivacaine N-oxide relative to the weight of mepivacaine introduced into the gel.

[0201] 3. Examples

[0202] 3.1 Example the

[0203] A crosslinked hyaluronic acid hydrogel is prepared from high molecular weight hyaluronic acid (1.5 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution (crosslinked for 1 month at -20°C). The hydrogel exhibits a molar crosslinking ratio (TR) of 1%. PBS and IM HCl solution are then added and homogenized using a three-dimensional stirrer. The hydrogel is dialyzed, and a pH of 7.3 ± 0.5 is obtained. The hydrogel has a hyaluronic acid concentration of 20.6 mg per gram of product and a degree of modification (MOD) of less than 1%.

[0204] To this hydrogel, a non-crosslinked high molecular weight sodium hyaluronate solution (4 MDa) and an aqueous solution of mepivacaine hydrochloride are added to obtain 0.3% by weight of mepivacaine hydrochloride relative to the weight of the final hydrogel. The pH is adjusted to physiological pH.

[0205] This hydrogel is divided into four equal parts. An antioxidant or PBS supplementation solution is added to obtain hydrogels with the following concentrations: Hydrogel A: PBS (control), Hydrogel B: 4 pmol / mL sodium thiosulfate pentahydrate, Hydrogel C: 4 pmol / mL sodium bisulfite, and Hydrogel D: 2.85 pmol / mL L-cysteine ​​hydrochloride monohydrate. The final hyaluronic acid concentration is the same for all conditions.

[0206] The solutions containing the antioxidants are prepared as follows. The antioxidants are respectively dissolved in the phosphate buffer, and, if necessary, a 5M NaOH solution is added to reach a physiological pH (pH= 6.8 - 7.8).

[0207] Hydrogels A, B, C, and D were sieved and then packaged in syringes. Finally, the hydrogels were sterilized by autoclave (plate temperature between 121°C and 135°C with FO > 15).

[0208] After sterilization, hydrogels A, B, C, and D were analyzed. The elastic modulus G' was determined before and after sterilization at the same time for each hydrogel. The results are presented in Table 1 below.

[0209] [Table 1]

[0210] AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0211] Hydrogel B according to the invention exhibits a lower loss of G' upon sterilization than hydrogels C and D containing an antioxidant other than a thiosulfate. Hydrogel B even has a lower loss of G' upon sterilization than hydrogel A, which is free of antioxidant.

[0212] The inventors noted that hydrogels C and D began to degrade even before sterilization. The antioxidants in hydrogels C and D thus have detrimental effects on hyaluronic acid gels, unlike thiosulfate, which does not degrade hyaluronic acid gel. Hydrogel D contains a lower molar content of antioxidant compared to hydrogel B, and despite this lower antioxidant content, the rheological properties of gel D are significantly degraded after sterilization.

[0213] 3.2 Example 1b

[0214] A crosslinked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid (1.5 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution (crosslinked for 72 h at 21 °C). The hydrogel exhibits a molar crosslinking ratio (TR) of 6%. PBS and IM HCl solution are then added and homogenized using a three-dimensional stirrer. The hydrogel is dialyzed, and a pH of 7.3 ± 0.5 is obtained. The hydrogel has a hyaluronic acid concentration of 22.2 mg per gram of product and a degree of modification (MOD) of approximately 3.6%.

[0215] To this hydrogel, a solution of high molecular weight non-crosslinked sodium hyaluronate (4MDa) and an aqueous solution of mepivacaine hydrochloride are added to obtain 0.3% by weight of mepivacaine hydrochloride relative to the weight of the final hydrogel. The pH is adjusted to physiological pH.

[0216] This hydrogel is divided into four equal parts. An antioxidant or PBS supplementation solution is added to obtain hydrogels with the following concentrations: Hydrogel E: PBS (control), Hydrogel F: 4 pmol / mL sodium thiosulfate pentahydrate, Hydrogel G: 4 pmol / mL sodium bisulfite, and Hydrogel H: 2.85 pmol / mL L-cysteine ​​hydrochloride monohydrate. The final hyaluronic acid concentration is the same for all conditions.

[0217] The solutions containing the antioxidants are prepared as follows. The antioxidants are respectively dissolved in the phosphate buffer, and, if necessary, a 5M NaOH solution is added to reach a physiological pH (pH= 6.8 - 7.8).

[0218] Hydrogels E, F, G and H were sieved and then packaged in syringes.

[0219] Finally, the hydrogels were sterilized in the autoclave (temperature at the plateau between 121°C and 135°C with FO > 15).

[0220] After sterilization, hydrogels E, F, G, and H were analyzed. The elastic modulus G' was determined before and after sterilization. The results are presented in Table 2 below. [Table 2]

[0221] AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0222] Hydrogel F according to the invention exhibits a lower sterilization loss than hydrogels G and H containing an antioxidant other than a thiosulfate. The inventors noted that hydrogels G and H began to degrade even before sterilization. Hydrogel F has a lower sterilization loss than hydrogel E, which is free of antioxidants.

[0223] Hydrogel F and hydrogel E underwent accelerated aging at 40°C in an oven for 3 months. The concentration of mepivacaine oxidation product (mepivacaine-N-oxide, CAS No. 1346597-75-8) present in these hydrogels was measured. Hydrogel F according to the invention exhibited a lower concentration of mepivacaine-N-oxide than hydrogel E.

[0224] 3.3 Example the

[0225] A crosslinked hyaluronic acid hydrogel is prepared from high molecular weight hyaluronic acid and BDDE in a 0.25 M aqueous sodium hydroxide solution (crosslinked for 1 month at -20°C). The hydrogel has a molar crosslinking ratio (TR) of 0.8%. PBS and IM HCl solution are then added and homogenized using a three-dimensional stirrer. The hydrogel is dialyzed, and a pH of 7.3 ± 0.5 is obtained. The hydrogel has a hyaluronic acid concentration of 20.1 mg per gram of product. It has a degree of modification (MOD) of less than 1%.

[0226] To this hydrogel is added a high molecular weight sodium hyaluronate solution and an aqueous solution comprising mepivacaine hydrochloride, citrate ions and zinc ions to obtain 0.3% by weight of mepivacaine hydrochloride relative to the weight of the final hydrogel and a concentration of 5mM of citrate ions and 0.03 mg / mL of zinc ions in the final hydrogel.

[0227] The solution containing citrate ions, zinc ions, and high molecular weight sodium hyaluronate is prepared as follows. Citric acid (in powder form) is dissolved in PBS, and the pH is adjusted with 5M NaOH to physiological pH (pH 6.8–7.8). High molecular weight sodium hyaluronate is then added. The pH of the gels is adjusted to physiological pH.

[0228] This hydrogel is divided into three equal parts. An antioxidant or PBS supplementation solution is added to obtain hydrogels with the following concentrations: Hydrogel I: PBS (control), Hydrogel J: 1.2 pmol / mL sodium thiosulfate pentahydrate, and Hydrogel K: 2.85 pmol / mL L-cysteine ​​hydrochloride monohydrate. The final hyaluronic acid concentration is the same for all conditions.

[0229] The solutions containing the antioxidants are prepared as follows. The antioxidants are respectively dissolved in PBS, and, if necessary, a 5M NaOH solution is added to reach a physiological pH (pH= 6.8 - 7.8).

[0230] Hydrogels I, J and K were sieved and then packaged in syringes.

[0231] Finally, the hydrogels were sterilized in an autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0232] After sterilization, hydrogels I, J, and K were analyzed. The elastic modulus G' was determined before and after sterilization. The sterilized hydrogels I and J were placed in an oven at 40°C for 1 month. The results are presented in Table 3 below. [Table 3]

[0233] AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0234] AG' (%)i months = (G' Ti months after sterilization - G' To after sterilization) / (G' To after sterilization) * 100 The hydrogel J according to the invention exhibits a lower loss upon sterilization than the hydrogel K comprising an antioxidant other than a thiosulfate. The hydrogel J has a lower loss of G' upon sterilization than the antioxidant-free hydrogel I, both immediately after sterilization and 1 month after sterilization.

[0235] Hydrogel I and hydrogel J underwent accelerated aging at 40°C in an oven for 3 months. The concentration of mepivacaine oxidation product (mepivacaine-N-oxide, CAS No. 1346597-75-8) present in these hydrogels was measured. Hydrogel J according to the invention exhibits a lower concentration of mepivacaine-N-oxide than hydrogel I.

[0236] Hydrogel J according to the invention exhibits a lower level of mepivacaine N-oxide after 3 months at 40°C compared to the level found in hydrogel I. 3,4 Example Id

[0237] Two cross-linked hyaluronic acid hydrogels were prepared from a high molecular weight hyaluronic acid (1.5 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution (cross-linked for 3 h at 52 °C). The cross-linked polysaccharides exhibited a molar cross-linking of 14%. PBS and an IM HCl solution containing non-cross-linked high molecular weight hyaluronic acid were then added to the cross-linked polysaccharides, and the resulting hydrogels were homogenized using a three-dimensional shaker. The mixtures were dialyzed, yielding hydrogels with a pH of 7.3 ± 0.5. The resulting hydrogels had a hyaluronic acid concentration of 25 mg per gram of product and a degree of modification (MOD) of approximately 7%.

[0238] An aqueous solution of mepivacaine hydrochloride is added to the resulting hydrogels to obtain 0.3% mepivacaine hydrochloride by weight of the final hydrogel. The pH of the gels is adjusted with a 0.25 M NaOH solution.

[0239] For each hydrogel, the same volume of an antioxidant supplementation solution is added.

[0240] Antioxidant solutions are finally added to obtain hydrogels with the following concentrations: Hydrogel N: PBS (control: Ctrl), Hydrogel O: 1.20 pmol / mL sodium thiosulfate pentahydrate.

[0241] The solution containing sodium thiosulfate is prepared as follows. The sodium thiosulfate is dissolved in PBS, and, if necessary, a 5M NaOH solution is added to achieve a physiological pH (pH= 6.8 - 7.8).

[0242] The prepared solution containing the antioxidant is then mixed with the cross-linked hyaluronic acid-based hydrogel in a stirring tank.

[0243] The products obtained (N, O hydrogels) were sieved and then packaged in syringes.

[0244] Finally, the products were sterilized in an autoclave (temperature at the tray between 121°C and 135°C with FO > 15).

[0245] After sterilization, the N and O hydrogels were analyzed. The elastic modulus G' was determined before and after sterilization. The mepivacaine n-oxide concentration was measured after sterilization. The sterilized N and O hydrogels were placed in an oven at 40°C for 3 months. The results are presented in Table 5 below.

[0246] [Table 5]

[0247] AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0248] The O hydrogel according to the invention exhibits a lower loss on sterilization than the N hydrogel, both immediately after sterilization and 1 month after sterilization.

[0249] The O hydrogel according to the invention has a lower level of mepivacaine N-oxide whether after sterilization, after 1 month at 40°C or after 3 months at 40°C compared to the level found in the N hydrogel.

[0250] In summary of all the preceding examples, the results show that thiosulfate salt improves the sterilization stability of hydrogels based on HA and an anesthetic.

[0251] On the contrary, examples show that antioxidants such as bisulfite or cysteine ​​do not provide such an improvement in sterilization stability. Furthermore, antioxidants like bisulfite or cysteine ​​have a detrimental effect on the rheological properties of the gel, unlike thiosulfate salts, which do not have this detrimental effect.

[0252] The results obtained show that the addition of thiosulfate salt helps to improve the protection of the anesthetic against degradation by oxidation.

Claims

Demands 1. Hydrogel comprising at least one hyaluronic acid or one of its salts, cross-linked and / or non-cross-linked, at least one anesthetic and at least one thiosulfate salt.

2. Hydrogel according to claim 1, wherein the thiosulfate salt is sodium thiosulfate.

3. Hydrogel according to any one of claims 1 to 2, wherein the thiosulfate salt represents a content less than or equal to 0.3% by mass of thiosulfate salt, preferably less than or equal to 0.2% by mass of thiosulfate salt, preferably even less than or equal to 0.1% by mass of thiosulfate salt, relative to the total mass of the hydrogel.

4. Hydrogel according to any one of claims 1 to 3, wherein the anesthetic is selected from 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, Febuverine, Fomocaine, Guafecaine, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamide, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propranocaine, PropipocainePropoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof, preferably lidocaine, mepivacaine, and one of their salts, in particular a hydrochloride salt.

5. Hydrogel according to any one of claims 1 to 4, comprising at least one cross-linked hyaluronic acid, optionally mixed with at least one non-cross-linked hyaluronic acid.

6. Hydrogel according to claim 5, wherein said at least one crosslinked hyaluronic acid is crosslinked using a crosslinking agent, preferably selected from bi- or multi-functional epoxy crosslinking agents, such as 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol)-diglycidyl ether (PEGDE), and mixtures thereof, or non-epoxy bi- or multi-functional crosslinking agents, such as endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides, and mixtures thereof.

7. Hydrogel according to any one of claims 1 to 6, wherein if said hydrogel comprises cross-linked hyaluronic acid then said cross-linked hyaluronic acid has a degree of modification (MOD) less than or equal to 8, preferably less than or equal to 7%, preferably less than or equal to 6%, preferably less than or equal to 5%, more preferably less than or equal to 4%, more preferably less than or equal to 1%.

8. Hydrogel according to any one of claims 1 to 7, wherein said at least one hyaluronic acid has a weight average molecular mass (Mw) of 0.05 to 10 MDa, preferably of 0.5 to 5 MDa, for example of 2 to 4 MDa or of 1 to 5 MDa.

9. Hydrogel according to any one of claims 1 to 8, further comprising (i) citrate ions and / or zinc ions and / or (ii) at least one additional additive selected from vitamins, amino acids, and mixtures thereof, preferably from vitamin C, cysteine, and mixtures thereof.

10. Hydrogel according to any one of claims 1 to 9, in the form of an injectable composition or a composition for topical application.

11. A method for preparing a hydrogel according to any one of the preceding claims, comprising the following steps: (1) preparation of a hydrogel comprising at least one hyaluronic acid or one of its salts, cross-linked and / or non-cross-linked, at least one anesthetic and further comprising at least one thiosulfate salt; and (2) optionally sterilization, preferably by heat, of the hydrogel comprising at least one thiosulfate salt to obtain a sterile hydrogel.

12. Use of a thiosulfate salt to enhance antioxidant properties and / or to reduce sterilization loss of a hydrogel comprising at least one hyaluronic acid or one of its salts, crosslinked and / or non-crosslinked, and at least one anesthetic.

13. Use according to claim 12, wherein the hydrogel has one or more of the characteristics described in claims 2 to 10.

14. Use of a hydrogel according to any one of claims 1 to 10, in the prevention and / or cosmetic treatment of an alteration in the surface appearance of the skin.

15. Use according to claim 14, 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 eye, to reduce the appearance of wrinkles and fine lines.

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