A process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof.

Abstract

The disclosure relates to a process for preparing a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof, the process comprising the following steps: (1) preparing a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof and further comprising at least 1 mM of citrate ions; and (2) sterilizing, preferably by heat, the hydrogel comprising at least 1 mM of citrate ions to obtain a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof.

Description

Title of the invention: Process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof. FIELD OF THE INVENTION

[0001] The present invention relates to a method for preparing a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular comprising a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof. TECHNOLOGICAL BACKGROUND

[0002] 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 unmodified hyaluronic acid are of interest because they have the advantage of being perfectly biocompatible.

[0003] 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 the in vivo durability and resistance to in vivo degradation of the hydrogels. Cross-linked hyaluronic acid-based hydrogels can be obtained by various preparation processes.

[0004] Furthermore, hydrogels based on crosslinked and / or non-crosslinked hyaluronic acid intended for filling soft tissues must be sterile. Therefore, the preparation processes for hydrogels based on crosslinked and / or non-crosslinked hyaluronic acid intended for injection generally include a sterilization step for the previously formed hydrogel. Sterilization is typically achieved by heat, for example in an autoclave. It has been observed that these sterilization conditions tend to degrade crosslinked and / or non-crosslinked hyaluronic acid, leading to degradation of the rheological properties of the hydrogels.

[0005] Thus, a need remains for the provision of a process for preparing sterile hydrogels comprising a crosslinked polysaccharide (e.g., crosslinked hyaluronic acid) and / or a non-crosslinked polysaccharide (e.g., non-crosslinked hyaluronic acid) which is as respectful as possible of the properties of the hydrogels, that is to say, which causes the least possible degradation of the rheological properties of the hydrogels during sterilization, for example by heat, as well as over time. BRIEF DESCRIPTION OF THE INVENTION

[0006] The present invention relates to a process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, the process comprising the following steps:

[0007] (1) preparation of a hydrogel comprising a cross-linked polysaccharide, a poly non-crosslinked saccharide or a mixture thereof, further comprising at least 1 mM of citrate ions; and

[0008] (2) sterilization, preferably by heat, of the hydrogel to obtain a hydrogel sterile comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof.

[0009] The invention also relates to a hydrogel obtained by the process of the invention.

[0010] Finally, the invention relates to the use of citrate ions to protect a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, in particular a cross-linked, non-cross-linked hyaluronic acid or a mixture thereof, and possibly an anesthetic agent, of the degradation of its rheological properties upon sterilization, preferably by heat and on the use of citrate ions to preserve the stability over time of hydrogels comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, in particular a cross-linked, non-cross-linked hyaluronic acid or a mixture thereof, and possibly an anesthetic agent.

[0011] Other aspects of the invention are as described below and in the claims. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0012] The term "gel" refers to a network of polymers that is expanded throughout its volume by a fluid. This means that a gel is formed of two media, one "solid" and the other "liquid," dispersed within each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (for example, hydrogen bonds) or by covalent bonds (cross-linking). The liquid medium consists of a solvent. A gel generally corresponds to a viscoelastic product that has a phase angle θ of less than 90°, preferably less than or equal to 70°, preferably less than or equal to 45°, at 1 Hz for a strain of 0.1% or a pressure of 1 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0013] The term “hydrogel” refers to 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) and having a pH ranging from 6.8 to 7.8.

[0014] The term “injectable hydrogel” refers to a hydrogel that can flow and be manually injected using a syringe fitted with a needle with a diameter of 0.1 to 0.5 mm, for example a 32 G, 30 G, 27 G, 26 G, 25 G hypodermic needle. Preferably, an “injectable hydrogel” is a hydrogel having an average extrusion force 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 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 a length L2”, at room temperature.

[0015] A “superficial application” means 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.

[0016] A “midline application” refers to the administration of a composition into the midline of the skin to treat the midline layers of the skin, as well as to reduce midline wrinkles.

[0017] 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 under the skin (above the periosteum) to “add volume,” such as for filling the deepest wrinkles and / or partially atrophied areas of the facial and / or body contour. So-called “volumizing” hydrogels can typically be administered for deep application.

[0018] A “crosslinked polysaccharide” refers to a polysaccharide modified during a crosslinking reaction.

[0019] Conversely, a "non-crosslinked polysaccharide" refers to a polysaccharide that has not been modified with a crosslinking agent and therefore has not undergone a crosslinking reaction.

[0020] The term “crosslinking agent” refers to any compound capable of introducing crosslinking between different polysaccharide chains.

[0021] The "molar crosslinking ratio" (CR), expressed as a percentage, refers to the molar ratio of the amount of crosslinking agent to the amount of polysaccharide repeat units introduced into the crosslinking reaction medium, expressed per 100 moles of polysaccharide repeat units in the crosslinking medium. For example, a molar crosslinking ratio of 1% means that there is one molecule of crosslinking agent introduced into the reaction medium for every 100 polysaccharide repeat units.

[0022] The expression "repeat unit" of a polysaccharide refers to a structural motif consisting of one or more (usually 1 or 2) monosaccharides whose repetition produces the complete polysaccharide chain.

[0023] The "degree of modification" (MOD) of a polysaccharide, such as hyaluronic acid, corresponds to the molar amount of crosslinking agent bound to the polysaccharide, at one or more of its ends, expressed per 100 moles of polysaccharide repeat units. It can be determined by methods known to those skilled in the art, such as Nuclear Magnetic Resonance (NMR) spectroscopy. For example, a degree of modification of 1% means that there is one molecule of crosslinking agent per 100 polysaccharide repeat units.

[0024] The term “polysaccharide” refers to a polymer composed of monosaccharides (preferably D enantiomers) joined together by glycosidic bonds.

[0025] By "ambient temperature" is meant a temperature ranging from 20 to 25°C, more particularly 21°C.

[0026] The linear viscoelastic region (LVER) corresponds to the range of hydrogel deformations from an initial elastic modulus value G' to a value of the elastic modulus G' reduced by 10% of its initial value. The LVER measurement consists of an oscillatory stress scan measurement in compression mode at a given oscillation frequency to determine the linear viscoelastic region. Process

[0027] Unexpectedly, the inventors discovered that the addition of citrate ions during the preparation of hydrogels comprising a cross-linked and / or non-cross-linked polysaccharide, in particular a cross-linked and / or non-cross-linked 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 modification of their rheological properties compared to hydrogels prepared by an equivalent process without the addition of citrate ions. Hydrogels obtained by the process of the present invention also exhibit better retention of their rheological properties over time.

[0028] The present invention thus relates to a process for preparing a sterile hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide, the process comprising the following steps:

[0029] (1) preparation of a hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide cross-linked and further comprising at least 1 mM of citrate ions; and

[0030] (2) sterilization, preferably by heat, of the hydrogel to obtain a hydrogel sterile comprising a cross-linked and / or non-cross-linked polysaccharide.

[0031] The hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide and further comprising at least 1 mM of citrate ions according to step (1) can be prepared according to two alternative methods:

[0032] - method 1: by adding citrate ions in powder form or in the form of a solution during the preparation of a hydrogel from a previously crosslinked and / or non-crosslinked polysaccharide; or

[0033] - method 2: when the hydrogel comprises a cross-linked polysaccharide, by conducting The cross-linking of the polysaccharide in a reaction medium containing citrate ions, followed by the preparation of the hydrogel from the resulting cross-linked polysaccharide. Method 1

[0034] When the process of the present invention implements method 1, the step (1) of preparing the hydrogel includes a step of adding, to the crosslinked polysaccharide or to the non-crosslinked polysaccharide or to their mixture, a solution comprising citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 1 mM in the hydrogel.

[0035] In one embodiment, when the process of the present invention implements method 1, step (1) of preparing a hydrogel includes a step of adding, to the crosslinked polysaccharide or to the non-crosslinked polysaccharide or to their mixture, citrate ions in powder form in a quantity sufficient to achieve a concentration of citrate ions of at least 1 mM in the hydrogel.

[0036] In some embodiments, the preparation of the hydrogel includes the addition of citrate ions in powder form and in solution form, preferably the powder and the solution being added at different stages of the preparation of the hydrogel. Cross-linked and / or non-cross-linked polysaccharide

[0037] The polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds or mixtures thereof. Preferably, the polysaccharide is selected from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosane, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof. Even more preferably, the polysaccharide is chosen from hyaluronic acid, heparosane, chondroitin and mixtures thereof, even more preferably the polysaccharide is hyaluronic acid or one of its salts, in particular a physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.More specifically, hyaluronic acid is available in its acidic form or as a sodium salt (NaHA). The hydrogel can therefore be a hydrogel based on hyaluronic acid and / or one of its salts.

[0038] Preferably, if the polysaccharide is hyaluronic acid or one of its salts, it has a average molecular mass by weight (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.

[0039] The polysaccharide can be supplied in hydrated form (fully or partially hydrated), or in dry form, such as powder or fiber. When the polysaccharide is supplied in hydrated form, it is typically in the form of a gel.

[0040] A cross-linked polysaccharide can be prepared by any method known to those skilled in the art. For example, the cross-linked polysaccharide can be prepared as described in WO2010131175A1 and WO201277054A1.

[0041] The process of the present invention may thus include, before the hydrogel preparation step, a step of preparing a cross-linked polysaccharide.

[0042] The crosslinked polysaccharide is preferably a crosslinked polysaccharide with a molar crosslinking ratio of 10% or less. Preferably, the crosslinked polysaccharide is a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 6%. Even more preferably, the crosslinked polysaccharide is a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 4%. Even more preferably, the crosslinked polysaccharide is a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 2%, preferably less than or equal to 1%, and 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 repeating unit(s) of the polysaccharide(s)).

[0043] The polysaccharide can be crosslinked by reacting a previously modified polysaccharide. The polysaccharide may have been modified by grafting with a molecule that allows for subsequent crosslinking of the modified polysaccharide. For example, the polysaccharide may have been modified by grafting a silylated molecule, an amino acid, an amino acid derivative, or a protein.

[0044] The polysaccharide is preferably crosslinked using a crosslinking agent selected from bi- or multi-functional epoxy or non-epoxy crosslinking agents, i.e., prepared by reaction of the polysaccharide with a crosslinking agent. Among the epoxy agents, 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 may be mentioned. Among the non-epoxy agents, we can cite endogenous polyamines such as spermine, spermidine and putrescine, aldehydes such as glutaraldehyde, carbodiimides and divinylsulfone, hydrazide derivatives such as adipic acid dihydrazide, bisal-koxyamine, dithiols such as polyethylene glycol dithiol and their mixtures. Non-epoxide agents can be cited as amino acids such as cysteine, lysine; peptides or proteins containing amino acids such as cysteine, lysine; poly(dimethylsiloxane); trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate, or barium trimetaphosphate.

[0045] In some embodiments, the crosslinking agent is an epoxy agent, preferably 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether. Preferably, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0046] In some embodiments, the crosslinking agent is a non-epoxy agent, preferably chosen from endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides and mixtures thereof.

[0047] The cross-linked polysaccharide is preferably a cross-linked polysaccharide having a degree of modification (MOD) of 10% or less, preferably 6% or less, preferably 4% or less, preferably 2% or less, and more preferably 1% or less. Advantageously, the cross-linked polysaccharide is a cross-linked polysaccharide having a degree of modification (MOD) of 1.8% or less, more preferably 1.5% or less, preferably 1.2% or less, and even more preferably less than 1%.

[0048] The cross-linked polysaccharide can in particular be prepared by a process comprising the following steps:

[0049] (al) prepare a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s) and a solvent; and

[0050] (a2) react the reaction medium to obtain a cross-linked polysaccharide.

[0051] The polysaccharide is as described above.

[0052] In step (a1a), the polysaccharide may be supplied in dry form, such as powder or fibers. When the polysaccharide is supplied in hydrated form, it is in the form of a non-crosslinked gel or a solution. In particular, when the polysaccharide is in hydrated form, it is an aqueous non-crosslinked gel or an aqueous solution. More specifically, the polysaccharide is mixed with water, optionally with the addition of an alkaline medium suitable for crosslinking or a phosphate buffer or a supplemented phosphate buffer, i.e., possibly comprising additional components as defined above.

[0053] The crosslinking agent is as described above.

[0054] 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). By For example, an organic solvent such as an alcohol, especially ethanol, or DMSO, can 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.

[0055] The reaction medium may further comprise salts, pH adjusters, for example a Brønsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above, and mixtures thereof. The addition of a Brønsted base may be particularly necessary when the functional groups of the crosslinking agent have an epoxide or vinyl group. In these cases, crosslinking occurs at a pH greater than or equal to 10, more advantageously greater than or equal to 12, which requires the addition of a Brønsted base to the reaction medium, typically at a concentration between 0.10 M and 0.30 M.

[0056] The total amount of crosslinking agent in the reaction medium typically varies from 0.001 to 0.10 moles per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.08 or from 0.001 to 0.06 per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.04 per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.03 per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.02 moles per 1 mole of repeating unit of the polysaccharide, more preferably from 0.001 to 0.01 moles per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.005 per 1 mole of repeating unit of the polysaccharide. When the polysaccharide is a gly-cosaminoglycan such as hyaluronic acid, the repeating unit is a disac-charidic unit.

[0057] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 80 to 200 mg / g.

[0058] Step (al) typically includes a homogenization step of the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with paddles or stirring with a spatula.

[0059] Step (a1a) is typically carried out at a temperature ranging from 4 to 35°C, preferably from 15°C to 25°C. Preferably, the duration of step (1) does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours.

[0060] Step (a2) consists of reacting the reaction medium to obtain a cross-linked polysaccharide. Advantageously, step (a2) is carried out directly after step (a1).

[0061] This step allows the polysaccharide chains to be cross-linked together. The functional groups of the cross-linking agent react with functional groups The functional groups present on polysaccharides link the polysaccharide chains together and cross-link them by forming intermolecular bonds. The cross-linking agent can also react with functional groups present on the same polysaccharide molecule to form intramolecular bonds. In particular, the functional groups of the cross-linking agent react with the -OH, -COOH, or -CHO groups present on polysaccharides such as hyaluronic acid. Cross-linked polysaccharides containing at least one cross-link between two polysaccharide chains, this cross-link being the residue of the cross-linking agent, are thus obtained.

[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 in time to the reaction medium. Step (a2) can thus comprise repeated crosslinking steps; advantageously, step (a2) comprises a single crosslinking step. Crosslinking is then carried out in the presence of a total quantity of crosslinking agents typically ranging from 0.1 to 10 moles, or from 0.1 to 8 moles, or from 0.1 to 6 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 repeating unit of the polysaccharide.The crosslinking conditions, in particular the crosslinking agent content, duration and temperatures, as well as the weight-average molecular weights (Mw) of the polysaccharide used, are interdependent.

[0063] The lower the crosslinking agent content, the longer the reaction time must be to obtain similar mechanical properties of the resulting crosslinked polysaccharide, and ultimately of the prepared hydrogel. In other words, the lower the molar percentage of crosslinking agent, the fewer reactive functional groups there are in the reaction medium and the lower the probability that two groups will meet and react together. Thus, the longer the reaction time must be to allow the functional groups to react with each other and form crosslinking bonds, thereby obtaining a crosslinked polysaccharide, and ultimately a hydrogel with desirable properties.

[0064] In some embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1a) at a temperature of 30°C or lower, preferably 25°C or lower. The temperature is typically above 0°C, above 5°C, or above 10°C. Even more preferably, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1a) at room temperature. When step (a2) is carried out at a temperature of 0°C or higher but 30°C or lower, the crosslinking time is at least 1 minute, preferably at least 10 minutes, and even more preferably at least 10 minutes. minus 1 hour. Preferably, the crosslinking time is at most 5 days.

[0065] In some embodiments, step (a2) can be carried out by placing the reaction medium obtained at the end of step (a1) 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.

[0066] In some embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1) at a pressure P less than or equal to atmospheric pressure and at a temperature T higher than the eutectic point temperature of the reaction medium as measured at pressure P and lower than the freezing point temperature of the reaction medium as measured at pressure P, preferably for a period of at least 1 hour. Crosslinked polysaccharide hydrogels prepared by such a process are highly biocompatible. Indeed, crosslinked polysaccharides can be prepared with smaller amounts of crosslinking agent, for example, amounts ranging from 0.001 to 0.02 moles per mole of polysaccharide repeating unit.

[0067] The freezing point temperature of the reaction medium refers to the temperature at which the mixture of components of the reaction medium, on a macroscopic scale, solidifies, that is, 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. This frozen state is maintained until the eutectic point temperature of the reaction medium is reached.

[0068] The eutectic point temperature of the reaction medium is the temperature below which the mixture of the reaction medium components transitions 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.

[0069] The freezing point and the eutectic point can be determined by differential scanning calorimetry. This method makes it possible to determine phase transitions. For this purpose, the product to be studied is progressively cooled until its phase transitions are observed.

[0070] 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 approximately -20°C.

[0071] 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 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.

[0072] Preferably, during the crosslinking step (a2), when the temperature T is greater than or equal to -55°C and less than or equal to -5°C, the reaction medium obtained at the end of step (1) is placed for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks. Preferably, the crosslinking step (a2) is carried out for a period of 2 to 25 weeks, preferably from 2 to 20 weeks or 2 to 17 weeks, even more preferably from 3 to 8 weeks or 4 to 7 weeks, and at temperature T and pressure P.

[0073] At the end of step (a2), the cross-linked polysaccharide is typically in the form of a gel. This gel is generally directly involved in the continuation of the process of the invention (step 1).

[0074] The crosslinked and / or non-crosslinked polysaccharides described above are useful for carrying out the process of the invention and thus preparing hydrogels comprising a crosslinked and / or non-crosslinked polysaccharide. The crosslinked or non-crosslinked polysaccharide, or a mixture thereof, will constitute the polymer network of the hydrogel. The hydrogel comprising a crosslinked or non-crosslinked polysaccharide, or a mixture thereof, can thus be said to be based on a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof. A hydrogel comprising, as its sole polysaccharide, a non-crosslinked polysaccharide, is prepared from a non-crosslinked polysaccharide. A hydrogel comprising, as its sole polysaccharide, a crosslinked polysaccharide, is prepared from a crosslinked polysaccharide. When the hydrogel comprises a mixture of a cross-linked and a non-cross-linked polysaccharide, the hydrogel is prepared from a cross-linked polysaccharide and a non-cross-linked polysaccharide.The non-crosslinked polysaccharide is typically added to the crosslinked polysaccharide during the preparation of the hydrogel. Preparation of the hydrogel (step (1))

[0075] The process of the present invention according to method 1 comprises the preparation of a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof and further comprising at least 1 mM of citrate ions, preferably from 1 to 12 mM of citrate ions.

[0076] The preparation of the hydrogel includes at least one step of adding citrate ions to the crosslinked and / or non-crosslinked polysaccharide. The amount of citrate ions added in this step makes it possible to achieve a citrate ion concentration in the prepared hydrogel of at least 1 mM, preferably ranging from 1 to 12 mM.

[0077] The preparation of the hydrogel advantageously includes a step of adjusting to physiological pH, in particular between 6.8 and 7.8.

[0078] In one embodiment, citrate ions are added in powder form to the crosslinked and / or non-crosslinked polysaccharide. The amount of citrate ions in powder form added at this step allows a citrate ion concentration in the prepared hydrogel to be achieved of at least 1 mM, preferably ranging from 1 to 12 mM. Typically, when citrate ions are added in powder form, the effect of the citrate ions on the pH of the hydrogel can be neutralized.

[0079] In another embodiment, citrate ions are added as a solution (a solution containing citrate ions) to the crosslinked and / or non-crosslinked polysaccharide. The amount of the solution containing citrate ions added at this step ensures a citrate ion concentration in the prepared hydrogel of at least 1 mM. Preferably, the citrate ion concentration in the hydrogel ranges from 1 to 12 mM.

[0080] In some embodiments, the amount of citrate ions added (in the form of a solution or powder) makes it possible to achieve a citrate ion concentration in the hydrogel of at least 1.5 mM, or at least 2 mM, or at least 2.5 mM, or at least 3 mM, or at least 3.5 mM. The maximum concentration of citrate ions in the hydrogel is generally 12 mM.

[0081] In some embodiments, the quantity of citrate ions added (in the form of a solution or powder) makes it possible to achieve a concentration of citrate ions in the hydrogel ranging from 2 to 12 mM, or from 3 to 11 mM, or from 3 to 9 mM, or from 3 to 8 mM or from 4 to 8 mM or from 3 to 5 mM.

[0082] In some embodiments, the amount of citrate ions added (in the form of a solution or powder) allows a concentration of citrate ions in the hydrogel to be reached from 5 to 12 mM.

[0083] A solution containing citrate ions means a solution whose pH allows citrate ions to be present in solution within that solution, or means a solution capable of releasing citrate ions once added during the preparation of the hydrogel. The solution containing citrate ions is preferably prepared from citric acid.

[0084] The solution containing added citrate ions typically has a pH ranging from 6.8 to 7.8 (physiological pH). If the solution does not have such a pH, the pH is adjusted, if necessary, during the preparation of the hydrogel so that the final hydrogel presents such a pH.

[0085] The citrate ion concentration of the solution is chosen to limit the dilution effect that can be caused by adding the solution during the preparation of the hydrogel, as such dilution of the hydrogel is undesirable. The maximum concentration of citrate ions that can be added to the hydrogel is therefore limited by adjusting the pH. Indeed, adjusting the pH of the solution to reach a physiological pH is more difficult beyond a certain citrate ion concentration. The solution containing citrate ions is typically prepared so that the solution is concentrated in citrate ions, for example, 100 times more concentrated than the final citrate ion concentration in the hydrogel.

[0086] The solution comprising citrate ions is typically prepared in water or in a physiologically acceptable buffer, preferably by adding citric acid 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), and [the following appears to be unrelated and possibly a separate entry:] 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), phosphate buffers such as PBS with a pH around physiological pH (CAS No: 7647-14-5, 7447-40-7). ,

[0087] 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-l-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).

[0088] Preferably the buffer is a phosphate buffer, particularly a saline buffer of NafLPCVNaoHPCL or KH2PO4 / K2HPO4. The pH of the solution containing citrate ions is typically adjusted by adding acid or base.

[0089] Thus, in some embodiments, the solution comprising citrate ions is a solution of citric acid in a phosphate buffer, the pH of which varies from 6.8 to 7.8.

[0090] In preferred embodiments, the citrate ions are added in the form of a solution comprising citrate ions, the solution being as described above. Preferably, the solution is a citric acid solution. The solution is preferably a citric acid solution in a physiologically acceptable buffer, such as a phosphate buffer.

[0091] The preparation of a hydrogel from a cross-linked and / or non-cross-linked polysaccharide can be carried out conventionally, with the difference that citrate ions are added during the preparation of the hydrogel. Thus, the preparation of a hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide can include one or more of the following conventional steps: - pH adjustment (1); - Dilution (2); - Purification (3); - Addition of at least one additional component (4); - Homogenization (5).

[0092] These steps, well known to a person skilled in the art, can be described below.

[0093] These steps, well known to those skilled in the art, can be as described below. The conventional steps can be carried out concurrently. The conventional steps can be carried out sequentially as follows: possible pH adjustment (1), then possible dilution (2), then possible purification (3), then possible addition of an additional component (4), then possible homogenization (5). They can also be carried out in a different order. Advantageously, the homogenization step (5) is carried out last, when at least one of the other conventional steps has been implemented. It can also be carried out repeatedly and inserted between the other conventional steps described.

[0094] For example, conventional steps can be carried out in the following sequential manner: (1), (2), (3), (4), (5); or (2), (1), (3), (4), (5); or (2), (1), (4), (5); or (2), (4), (5); or (1), (4), (5); or (2), (4), (3), (5); or (2), (4), (1), (5); or (2), (4), (1), (5); or (2), (4), (5); or (4), (2), (1); or (4), (1), (2); or (2), (3), (4), (5); or (4), (2), (3), (5); or (2), (4), (1); or (1), (5), (3), (4); or (1), (5), (4); or (2), (4). Steps (2), (3), (4), and (5) can be concurrent. For example, the preparation of the hydrogel, can understand the following sequence: (2) and (4) are carried out simultaneously.

[0095] Citrate ions (in powder form or in solution) can be added at the time of, before or after any of these conventional steps.

[0096] In one embodiment, the citrate ions are added before the homogenization step (5) so as to obtain a homogeneous gel.

[0097] When a purification step (3) is implemented, the citrate ions can be added before or after the purification step (3); advantageously, the citrate ions are added after the purification step (3). Adding the citrate ions after the purification step ensures better control of the citrate ion concentration in the prepared hydrogel.

[0098] Preferably, the citrate ions are added between the purification (3) and homogenization (5) steps.

[0099] The addition of citrate ions can be carried out after the dilution step (2) or during the dilution step (2), for example the citrate ions can be added to the aqueous dilution solvent.

[0100] Preferably, the citrate ions are added during the dilution step (2) and / or during the step of adding at least one additional component (4), preferably during the step of adding at least one additional component (4). In particular, in some embodiments, the addition of the solution containing citrate ions is concurrent with the step of adding at least one additional component (4). In particular, in some embodiments, the addition of the solution containing citrate ions is concurrent with the addition of an anesthetic solution. In particular, in some embodiments, the addition of the solution containing citrate ions is concurrent with the addition of a lubricant.In some embodiments, the solution comprising added citrate ions may include other components, in particular a lubricating agent, for example non-crosslinked hyaluronic acid, non-crosslinked heparosan or a mixture thereof.

[0101] The steps of dilution (2), addition of at least one additional component (4) and addition of citrate ions can be concomitant.

[0102] Citrate ions can be added after the pH adjustment step (1). Citrate ions can be added between the pH adjustment step (1) and the homogenization step (5) when both of these steps are implemented. pH adjustment (1)

[0103] 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). Dilution (2)

[0104] The hydrogel preparation process may include a dilution step of the Crosslinked and / or non-crosslinked polysaccharide. The dilution step allows the polysaccharide concentration in the prepared hydrogel to be adjusted. Specifically, an aqueous solvent is added to the crosslinked and / or non-crosslinked polysaccharide, 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 polysaccharide 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. Purification (3)

[0105] The hydrogel preparation process may include at least one purification step.

[0106] The purification step aims to remove any undesirable impurities. These impurities may result from the crosslinking of the polysaccharide, for example, from step (a2) described above. Such impurities may include, for example, residual crosslinking agent, particularly of the epoxy type, which may not have reacted.

[0107] This step can also be used to perform a liquid exchange, for example a buffer exchange. The purification step can therefore be particularly useful when the hydrogel contains a cross-linked polysaccharide.

[0108] Purification can be carried out by dialysis or by filtration, for example by dynamic cross-flow filtration (“DCF” for Dynamic Cross-flow Filtration). Addition of additional components (4)

[0109] The hydrogel preparation process may include one or more steps of adding at least one additional component. The additional component may be selected from anesthetic agents, antioxidants, lubricating agents, amino acids, peptides, proteins, vitamins, minerals, nucleic acids, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof.

[0110] Non-crosslinked polysaccharides, in particular non-crosslinked hyaluronic acid, non-crosslinked heparosan or mixtures thereof, may be cited as examples of lubricating agents.

[0111] Examples of anesthetics include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betaxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chloro-Robutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryo- Fluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, 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, and any of their salts, in particular a hydrochloride salt, or a mixture thereof.Preferably, the hydrogel according to the invention comprises an anesthetic agent as defined above, and in particular lidocaine, mepivacaine, or one of their salts such as hydrochloride.

[0112] 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 mixtures thereof.

[0113] 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.

[0114] 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.

[0115] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated, or zinc citrate; preferably zinc citrate), 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), possibly in hydrated form, and mixtures thereof. Preferably, zinc citrate is chosen as an additional component.

[0116] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives, and mixtures thereof. As coenzymes, coenzyme Q10, CoA, NAD, NADP, and mixtures of these can be cited.

[0117] Adrenaline, noradrenaline, and mixtures thereof may be cited as derivatives of adrenaline. Homogenization (5)

[0118] The hydrogel preparation process may include one or more homogenization steps. This homogenization step makes it possible to obtain a more homogeneous hydrogel, in particular with the most constant, i.e., the most regular, extrusion force possible. For example, the homogenization step may consist of an extrusion step, more particularly using a sieve with perforations having a diameter between 50 and 2000 µm. Those skilled in the art know how to select the perforation diameter according to the desired mechanical properties of the hydrogel. Method 2

[0119] When the process of the present invention implements method 2, step (1) of hydrogel preparation comprises the following steps:

[0120] (a) preparation of a crosslinked polysaccharide from a reticulated reaction medium culation comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and citrate ions in sufficient quantity to permit the preparation of a hydrogel comprising a crosslinked polysaccharide and further comprising at least 1 mM of citrate ions;

[0121] (b) preparation of a hydrogel from the cross-linked polysaccharide obtained at the end of step (a) and possibly a non-crosslinked polysaccharide.

[0122] The cross-linked polysaccharide can in particular be prepared by a process comprising the following steps:

[0123] (al) prepare a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and citrate ions in sufficient quantity to permit the preparation of a hydrogel based on a crosslinked polysaccharide comprising at least 1 mM of citrate ions; and

[0124] (a2) react the reaction medium to obtain a cross-linked polysaccharide.

[0125] Steps (a), (a1) and (a2) of the process according to method 2 are as described previously in the section "The cross-linked and / or non-cross-linked polysaccharide", with the difference that the reaction medium further comprises citrate ions.

[0126] Citrate ions are typically present in the reaction medium in an amount sufficient to achieve a citrate ion concentration in the hydrogel of at least 1.5 mM, or at least 2 mM, or at least 2.5 mM, or at least 3 mM, or at least 3.5 mM. The maximum concentration of citrate ions in the hydrogel is generally 20 mM or 12 mM.

[0127] In some embodiments, the quantity of citrate ions present in the reaction medium allows a concentration of citrate ions in the hydrogel to be reached ranging from 2 to 20 mM, or 2 to 12 mM, or 3 to 11 mM, or 3 to 9 mM, or 3 to 8 mM, or 4 to 8 mM, or 3 to 5 mM.

[0128] In some embodiments, the quantity of citrate ions present in the reaction medium allows a concentration of citrate ions in the hydrogel to be reached from 5 to 12 mM.

[0129] The citrate ions present in the reaction medium may result from the addition of citric acid or an aqueous solution of citric acid to the reaction medium.

[0130] At the end of step (a2), the cross-linked polysaccharide is typically in the form of a gel comprising citrate ions. This gel is generally directly involved in the continuation of the process of the invention (step (b)).

[0131] The preparation of a hydrogel (step (b)) from the cross-linked polysaccharide obtained at the end of step (a) or (a2) can be carried out conventionally. In particular, the preparation of a hydrogel from the cross-linked polysaccharide obtained at the end of step (a) or (a2) typically comprises one or more of the following conventional steps: pH adjustment (1); Dilution (2); Purification (3); Addition of at least one additional component (4); Homogenization (5).

[0132] These steps, well known to those skilled in the art, can be as described above in relation to method 1. They can be implemented in the sequential ways described above. Sterilization of the hydrogel (step (2))

[0133] The process of the present invention includes a step of sterilizing 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 referred to as the "plateau temperature," which is maintained for a predetermined time referred to 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 minute to 20 minutes with F0 > 15. The sterilizing 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.

[0134] The hydrogel obtained at the end of the process according to the invention typically has a pH ranging from 6.8 to 7.8 (physiological pH). Method 1 or 2: Optional step

[0135] The process of the present invention (method 1 or 2) may further include a step of conditioning the hydrogel. The conditioning of the hydrogel is typically carried out in an injection device. The conditioning is preferably carried out just before the sterilization 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. sterile hydrogel

[0136] The sterile hydrogel obtained by the process of the present invention (method 1 or 2) is a hydrogel based on a cross-linked polysaccharide or a non-cross-linked polysaccharide or a mixture thereof. The sterile hydrogel obtained by the process of the present invention (method 1 or 2) therefore comprises a cross-linked polysaccharide, or a non-cross-linked polysaccharide, or a mixture of a cross-linked polysaccharide and a non-cross-linked polysaccharide. It is understood that the cross-linked polysaccharide may be a mixture of cross-linked polysaccharides.

[0137] The sterile hydrogel obtained by the process of the present invention (method 1 or 2) has a physiological pH, i.e., ranging from 6.8 to 7.8. The pH of the sterile 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.

[0138] The sterile hydrogel obtained by the process of the present invention (method 1 or 2) and comprising a cross-linked polysaccharide, advantageously has a phase angle θ less than or equal to 45°, at 1Hz for a strain of 0.1% or a pressure of 1 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0139] The hydrogel obtained by the process 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 from 0.1 to 0.5 mm, for example a hypodermic needle of 32G, 30G, 27G, 26G, 25G.

[0140] The hydrogel obtained by the process of the present invention may comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of polysaccharide (total weight of polysaccharide, i.e., total weight of cross-linked and / or non-cross-linked polysaccharide, for example, cross-linked and / or non-cross-linked hyaluronic acid), relative to the total weight of the hydrogel. Thus, when the hydrogel comprises, as its sole polysaccharide, a non-cross-linked polysaccharide, the hydrogel obtained by the process of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of non-cross-linked polysaccharide (for example, non-cross-linked hyaluronic acid), relative to the total weight of the hydrogel. When the hydrogel comprises, as its sole polysaccharide, a cross-linked polysaccharide, the hydrogel obtained by the process of the present invention Therefore, it may 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. When the hydrogel comprises a mixture of a cross-linked and non-cross-linked polysaccharide, the hydrogel obtained by the process 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-cross-linked and cross-linked polysaccharide (for example, non-cross-linked and / or cross-linked hyaluronic acid), relative to the total weight of the hydrogel. In particular, the content of non-crosslinked polysaccharide (e.g. hyaluronic acid) can 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 polysaccharide (e.g. hyaluronic acid) present in the hydrogel.

[0141] The total polysaccharide concentration in the hydrogel obtained by the process 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 polysaccharide is hyaluronic acid, and even more preferably, sodium hyaluronate.

[0142] When the hydrogel comprises a crosslinked polysaccharide, the crosslinked polysaccharide preferably has a molar crosslinking ratio of 10% or less. Preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 6%. Even more preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 4%. Even more preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 2%, preferably less than or equal to 1%, and 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 repeating unit(s) of the polysaccharide(s)).

[0143] When the hydrogel comprises a cross-linked polysaccharide, the cross-linked polysaccharide preferably has a degree of modification (MOD) of 10% or less, preferably 6% or less, preferably 4% or less, preferably 2% or less, and more preferably 1% or less. Advantageously, the cross-linked polysaccharide has a degree of modification (MOD) of 1.8% or less, more preferably 1.5% or less, preferably 1.2% or less, and even more preferably less than 1%.

[0144] In some embodiments, the hydrogel comprises an anesthetic agent. The anesthetic agent may be as described above; in particular, the anesthetic agent may be mepivacaine, lidocaine, or one of their salts; more particularly in the form of a hydrochloride salt; preferably in quantities ranging from 0.1 to 30 mg / ml, for example from 0.5 to 10 mg / ml or more preferably from 2 to 6 mg / ml.

[0145] Sterile hydrogels prepared according to the process of the invention are particularly useful for filling and / or replacing tissues, in particular soft tissues, notably by injecting the hydrogel into the tissue.

[0146] They can be injected using any of the methods known to those skilled in the art. In particular, they can be administered by means of an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. The injection device can, in particular, be selected from 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.

[0147] Sterile hydrogels prepared according to the process of the invention are preferably injected subcutaneously.

[0148] They may relate to deep applications, mid-level applications and / or shallow applications.

[0149] They may have therapeutic and / or cosmetic and / or cosmeceutical applications.

[0150] In the cosmetic field, hydrogels can be particularly useful for compensating for tissue volume losses due to aging.

[0151] 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 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 volume, in particular of the cheeks, temples, jawline and around the eyes; to reduce the appearance of wrinkles and fine lines.

[0152] The process for preparing sterile hydrogels of the present invention preserves the properties of the hydrogels, that is to say, it results in less modification of the rheological properties of the hydrogels during sterilization. Indeed, better preservation of the rheological properties of the hydrogels after sterilization (better preservation of the elastic modulus G', better preservation of the phase angle) has been observed compared to hydrogels prepared by a process without the addition of citrate ions.

[0153] The process of the present invention allows the preparation of sterile hydrogels whose reduction in the elastic modulus G' after sterilization does not exceed 50%, 45%, 40%, 35% or 30% of the value of the elastic modulus G' before sterilization.

[0154] Since the step of adding citrate ions according to method 1, particularly in the form of a solution, slightly dilutes the hydrogel, it could be expected that the rheological properties of the hydrogel would be negatively impacted by this addition. Unexpectedly, it was observed that the addition of a solution containing citrate ions has a favorable effect on the properties of the hydrogel during sterilization.

[0155] Furthermore, the addition of citrate ions, particularly in solution form, helps to preserve the properties of the hydrogel over time. Indeed, better preservation of the rheological properties of hydrogels over time (better preservation of the elastic modulus G', better preservation of the phase angle) has been observed compared to hydrogels prepared by a process without the addition of citrate ions, which tend to see their rheological properties decrease more significantly over the months.

[0156] The use of citrate ions, particularly in solution form, in a hydrogel preparation process thus makes it possible to protect a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, particularly one comprising at least one crosslinked polysaccharide, from the degradation of its rheological properties during sterilization, preferably by heat. The use of citrate ions, particularly in solution form, in a hydrogel preparation process comprising a crosslinked and / or non-crosslinked polysaccharide, particularly one comprising at least one crosslinked polysaccharide, also makes it possible to preserve the chain length of the crosslinked and / or non-crosslinked polysaccharide.

[0157] The use of citrate ions, particularly in solution form, in a process for preparing a hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide, particularly comprising at least one cross-linked polysaccharide, also helps to preserve the stability of the hydrogels, especially after sterilization, over time, and in particular to increase the stability of the hydrogels, especially after sterilization, over time compared with identical hydrogels not comprising citrate ions. In other words, the hydrogels obtained according to the invention maintain their rheological properties more effectively over time after sterilization.

[0158] Furthermore, it is known that the additional presence of an anesthetic agent in a hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide leads to increased degradation of the rheological properties of the hydrogels during sterilization, preferably by heat. The addition of citrate ions helps to limit these effects. Hydrogels obtained by the process of the present invention comprising an anesthetic agent exhibit less degradation of their rheological properties after sterilization compared to hydrogels, comprising an anesthetic agent, prepared by an equivalent process without the addition of citrate ions.

[0159] The following examples are given by way of illustration, but shall in no way be considered as limiting the present invention. EXAMPLES 1. Materials

[0160] - Non-crosslinked sodium hyaluronate

[0161] - BDDE (Sigma Aldrich)

[0162] - NaOH 0.25M

[0163] - HCl IM

[0164] - Citric acid (Sigma Aldrich)

[0165] - Phosphate Buffer (BBraun),

[0166] - Lidocaine Hydrochloride

[0167] - Three-dimensional agitator

[0168] - DHR-2 Rheometer

[0169] - Dynamometer and test bench

[0170] - Homogenizer Paddle Mill

[0171] - Sterile polyethylene bag 2. Methods Measurement of viscoelastic properties

[0172] 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).

[0173] 0.5 g of sterilized hydrogel is deposited between the Peltier plane and said cone. Then a Stress scanning is performed at 1 Hz and 25°C. The elastic modulus G', viscous modulus G” and phase angle θ are recorded for a stress of 5 Pa. The measurements are carried out in the linear LVER domain.

[0174] The constraint at the intersection of G' and G”, r, is determined at the intersection of the curves of the modules G' and G'' and is expressed in Pascal. 3. Examples 3.1 Example 1

[0175] Two cross-linked hyaluronic acid hydrogels are prepared from a high molecular weight hyaluronic acid (3 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution (cross-linking for 1 month at -20°C). The cross-linked polysaccharides have a molar cross-linking ratio of 0.2% and either a concentration of 15 mg of hyaluronic acid per gram of product (hydrogel A) or a concentration of 23 mg of hyaluronic acid per gram of product (hydrogel B). Phosphate buffer PBS and a solution of HCl IN are then added to the cross-linked polysaccharides until a pH of 7.3 ± 0.5 is reached. The resulting hydrogels are homogenized using a three-dimensional stirrer. The mixtures are then dialyzed.

[0176] To the hydrogels obtained, a solution of non-crosslinked high molecular weight sodium hyaluronate is then added as a lubricant (same quantity of high molecular weight sodium hyaluronate in the different mixtures) comprising, or not, citrate ions.

[0177] The solution comprising citrate ions and high molecular weight sodium hyaluronate is prepared as follows. Citric acid (in powder form) is dissolved in phosphate buffer, and the pH is then adjusted with 5M NaOH to reach a physiological pH (pH = 6.8–7.8). Finally, high molecular weight sodium hyaluronate is added as a lubricant. The concentration of citrate ions in the solution is adjusted taking into account the dilution effect following the addition of this solution to the cross-linked hyaluronic acid hydrogel. Indeed, the citrate ion concentration indicated in Table 1 corresponds to the final concentration in the hydrogel.

[0178] The prepared solution comprising citric acid and high molecular weight sodium hyaluronate or the solution comprising high molecular weight sodium hyaluronate alone is then mixed with the crosslinked hyaluronic acid-based hydrogel in a stirring tank.

[0179] The products obtained (hydrogels A, B) were sieved and then packaged in syringes.

[0180] Finally, the products were sterilized in an autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0181] Before and after sterilization, the prototypes were analyzed. The elastic modulus G' and the phase angle θ were determined. The results are presented in Table 1 below.

[0182] The prototypes exhibit a molar crosslinking rate of 0.2%.

[0183] [Tables 1] Hydrogels AB Al A2 B1 B2 Molar concentration of citrate ions (mM) in the final hydrogel 0 3.06 0 3.06 Mass percentage of citrate ions relative to the mass of the final hydrogel 0 0.06% 0 0.06% G' (1Hz) before sterilization 261.7 245.0 511.1 499.2 Δ before sterilization 15.4 15.8 13.3 13.4 AG' (%)' -54.5 -33.2 -37.9 -18.8 A Δ (%)2 67.5 39.3 58.9 36.2

[0184] Table 1

[0185] 'AG' (%)= (G' after sterilization - G' before sterilization) / (G' before sterilization) *100

[0186] 2A ô (%)= (ô after sterilization - ô before sterilization) / ( ô before sterilization) *100

[0187] It is observed that hydrogels prepared by a process according to the invention comprising a step of adding a citric acid solution (hydrogels A2, B2) exhibit less degradation of their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of a citric acid solution (hydrogels Al, B1). Indeed, it has been observed that hydrogels A2 and B2 exhibit a higher elastic modulus (G') after sterilization than hydrogels Al and B1 after sterilization. The decrease in the elastic modulus (G') is therefore less after sterilization for hydrogels A2 and B2. It was also observed that hydrogels A2 and B2 exhibit a lower phase angle (θ) after sterilization than hydrogels Al and B1 after sterilization. 3.2 Example 2

[0188] A cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid (4 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution. The cross-linked polysaccharide has a molar cross-linking ratio of 2% and a hyaluronic acid concentration of 15 mg per gram of product. Phosphate buffer (PBS) and HCl (IN) solution are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The mixture is dialyzed.

[0189] To the hydrogels obtained, as appropriate, the following are then added:

[0190] - a high molecular weight sodium hyaluronate solution as a brisant (same quantity in the different mixtures) including, or not including, citrate ions;

[0191] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0192] - a citric acid solution. Hydrogels C

[0193] For hydrogels C, only a non-crosslinked high molecular weight sodium hyaluronate solution comprising, or not comprising, citrate ions is added.

[0194] The solution comprising citrate ions and high molecular weight sodium hyaluronate is prepared as follows. Citric acid (in powder form) is dissolved in phosphate buffer, the pH is then adjusted with 5M NaOH to reach a physiological pH (pH = 6.8–7.6), and finally, high molecular weight sodium hyaluronate is added as a lubricant. The concentration of citrate ions in the solution is adjusted taking into account the dilution effect following the addition of this solution to the mixture comprising the cross-linked hyaluronic acid. Indeed, the citrate ion concentration indicated in Table 2 corresponds to the final concentration in the hydrogel.

[0195] The prepared solution comprising citric acid and high molecular weight sodium hyaluronate is then mixed with the mixture comprising cross-linked hyaluronic acid in a stirring tank. Hydrogels D

[0196] For hydrogels D, a high molecular weight sodium hyaluronate solution, an anesthetic solution and optionally a citric acid solution are added.

[0197] The citric acid solution is added at the same time as the anesthetic solution, the citric acid solution and the anesthetic solution being added after the addition of the high molecular weight sodium hyaluronate solution.

[0198] A citric acid solution is prepared. The citric acid (in powder form) is first dissolved in phosphate buffer, and then 5M NaOH is added to adjust the pH to a physiological level. The aim is to make a solution 100 times more concentrated than the actual concentration desired in the final hydrogel. This prevents excessive dilution of the hydrogel due to the addition of the citric acid solution.

[0199] The products obtained (hydrogels C and D) were sieved on the order of microns and then packaged in syringes.

[0200] Finally, the products were sterilized in an autoclave (temperature at the tray included) between 121°C and 135°C with FO > 15).

[0201] Before and after sterilization, the prototypes were analyzed. The elastic modulus G' and the phase angle θ were determined. The results are presented in Table 2 below.

[0202] [Tables2] Hydrogels CD Cl C2 DI D2 D3 D4 D5 D6 Molar concentration of citrate ions (mM) in the final hydrogel 0 3.06 0 1.53 4.59 7.65 10.71 12.24 Mass percentage of citrate ions relative to the total mass of the final hydrogel 0 0.06% 0 0.03% 0.10% 0.16% 0.23% 0.26% G' (1 Hz) before sterilization 130.1 127.8 124.9 122.7 120.9 122.1 122.3 121.1 0 before sterilization 13 15.3 13.5 13.5 13.5 13.4 13.3 13.4 AG' (%)1 -18.9 -10.7 -33.0 -29.4 -23.4 -18.2 -14.4 -16.6 A ô (%)2 31.6 8.8 45.5 36.3 27.7 21.1 21.1 22.1

[0203] Table 2

[0204] 1 AG' (%)= (G' after sterilization - G' before sterilization) / (G' before sterilization) *100

[0205] 2 A ô (%)= (ô after sterilization - ô before sterilization) / ( ô before sterilization) *100

[0206] It is observed that hydrogels prepared by a process according to the invention comprising a step of adding citrate ions (addition of a non-crosslinked high molecular weight sodium hyaluronate solution comprising citrate ions or addition of a citric acid solution) (hydrogels C2, D2 to D6) exhibit less modification of their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of citrate ions (hydrogels Cl and DI). Indeed, it has been observed that hydrogels C2 and D2 to D6 exhibit a higher elastic modulus (G') after sterilization than Cl and Dl hydrogels. The decrease in elastic modulus (G') is therefore less after sterilization for C2 and D2 to D6 hydrogels. It was also observed that C2 and D2 to D6 hydrogels have a lower phase angle (θ) after sterilization than Cl and Dl hydrogels.

[0207] After 1 month at 40°C, the C2 hydrogel prepared from a process according to the invention comprising a step of adding a solution comprising citrate ions does not show any changes in its rheological properties compared to the Cl hydrogel prepared by an equivalent process without the addition of such a solution. 3.3 Example 3

[0208] A cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid (4 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution. The cross-linked polysaccharide has a cross-linking ratio of 2% and a hyaluronic acid concentration of 15 mg per gram of product. Phosphate buffer (PBS) and HCl (IN) solution are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The mixture is dialyzed.

[0209] The following are then added to the hydrogels obtained:

[0210] - a high molecular weight sodium hyaluronate solution as a brisant (same quantity in the different mixtures);

[0211] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0212] - possibly a citric acid solution.

[0213] For hydrogels E2 and E3, the citric acid solution is added at the same time as the anesthetic solution, the citric acid solution and the anesthetic solution being added after the addition of the high molecular weight sodium hyaluronate solution.

[0214] A citric acid solution is prepared. The citric acid (in powder form) is first dissolved in phosphate buffer, and then 5M NaOH is added to adjust the pH to a physiological level. The aim is to make a solution 100 times more concentrated than the actual concentration desired in the final hydrogel. This prevents excessive dilution of the hydrogel due to the addition of the citric acid solution.

[0215] The products obtained (hydrogels El, E2 and E3) were sieved on the order of micron and then packaged in syringes.

[0216] Finally, the products were sterilized in an autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0217] Before and after sterilization, the E1-E3 hydrogels were analyzed. The module The elastic G' and the phase angle θ were determined. The results are presented in Table 3 below.

[0218] [Tables3] Hydrogels El E2 E3 Molar concentration of citrate ions (mM) in the final hydrogel 0 3.06 7.65 G' (1Hz) before sterilization 127.5 130.2 123.9 G' (1 Hz) at T0 months (after sterilization) 70.3 90.0 103.9 G' (1 Hz) at T2 months (after sterilization) 61.0 86.9 99.8 AG' (%)1 -13.2 -3.4 -3.9

[0219] Table 3

[0220] 1 AG' (%) = (G' T 2 months - G' T0) / (G' T0) * 100

[0221] After 2 months at 40°C, the E2 and E3 hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising citrate ions exhibit a minor modification of their rheological properties compared to the El hydrogel prepared by an equivalent process without the addition of such a solution.

Claims

Demands

1. A process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, the process comprising the following steps: (1) preparing a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, and further comprising at least 1 mM of citrate ions; and (2) sterilizing, preferably by heat, the hydrogel comprising at least 1 mM of citrate ions to obtain a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof.

2. A method according to claim 1 wherein step (1) comprises adding to the crosslinked polysaccharide or noncrosslinked polysaccharide or mixture thereof, a solution comprising citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 1 mM in the hydrogel.

3. A method according to claim 1 wherein step (1) comprises adding, to the crosslinked polysaccharide or to the noncrosslinked polysaccharide or to a mixture thereof, citrate ions in powder form in sufficient quantity to achieve a citrate ion concentration of at least 1 mM in the hydrogel.

4. A method according to claim 2 wherein the solution comprising citrate ions has a pH ranging from 6.8 to 7.

8.

5. A method according to claim 2 or 4 wherein the solution comprising citrate ions is a citric acid solution, preferably a citric acid solution in a physiologically acceptable buffer.

6. A method according to claim 5 wherein the physiologically acceptable buffer is a phosphate buffer.

7. A method according to any one of claims 2 to 6 wherein the amount of citrate ions added to the hydrogel allows a citrate ion concentration of 1 to 12 mM to be achieved in the hydrogel.

8. A method according to any one of claims 2 to 7 wherein step (1) of preparing a hydrogel comprises one or more of the following conventional steps: - pH adjustment; - Dilution; - Purification; - Addition of at least one additional component; - Homogenization.

9. A process according to claim 8 comprising a dilution step and / or an addition step of at least one additional component, wherein the citrate ions are added during the dilution step and / or during the addition step of at least one additional component.

10. A process according to claim 1 wherein step (1) comprises the following steps: (a) preparation of a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and citrate ions in sufficient quantity to permit the preparation of a hydrogel based on a crosslinked polysaccharide comprising at least ImM of citrate ions; (b) preparation of a hydrogel from the crosslinked polysaccharide obtained at the end of step (a) and optionally from a non-crosslinked polysaccharide.

11. A method according to any one of claims 1 to 10 wherein the polysaccharide is hyaluronic acid.

12. A method according to any one of claims 1 to 11 further comprising a step of conditioning the hydrogel, preferably in an injection device, after step (1) and before step (2).

13. A method according to any one of claims 1 to 12 wherein the sterilization is heat sterilization, preferably carried out in an autoclave.

14. Sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof, obtained by the process according to any one of claims 1 to 13.

15. Use of citrate ions to protect a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof, and optionally an anesthetic agent, from the degradation of its rheological properties during sterilization, preferably by heat.

16. Use of citrate ions to preserve the stability over time of a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, in particular a cross-linked hyaluronic acid, a non-cross-linked hyaluronic acid or a mixture thereof, and optionally an anesthetic agent.