WO2014064633A1

WO2014064633A1 - Process for preparing a crosslinked gel

Info

Publication number: WO2014064633A1
Application number: PCT/IB2013/059608
Authority: WO
Inventors: Emeline CHARTON; Stéphane MEUNIER
Original assignee: Teoxane SA
Current assignee: Teoxane SA
Priority date: 2012-10-24
Filing date: 2013-10-24
Publication date: 2014-05-01
Anticipated expiration: 2015-04-24
Also published as: FR2997085B1; FR2997085A1

Abstract

The subject of the present invention is a process for preparing a crosslinked gel of at least one polysaccharide or a salt thereof, comprising at least the steps consisting in: a)providing an aqueous gel comprising at least said polysaccharide(s) in a noncrosslinked form, combined with at least one crosslinking agent; b) crosslinking the mixture obtained at the end of step a) and, where appropriate; c)recovering said crosslinked gel formed, wherein the crosslinking step b) is carried out in a medium supplemented with at least one alkali halide salt present in a content of between 0.5%and 20% by weight relative to the total weight of the aqueous gel of step a).

Description

Processs for preparing a crosslinked gel

The present invention aims to provide a process for preparing hydrogels based on a crosslinked polysaccharide, and preferably on hyaluronic acid or a salt thereof.

Hyaluronic acid, which is naturally present in the skin, is known for its viscoelastic properties and also its very high propensity to absorb water. Its properties contribute to a large extent to the elasticity of the skin.

It is precisely from the viewpoint of these properties that advantage has been taken of this compound for more than 10 years now in numerous applications in the medical and cosmetics fields, or even cosmetic surgery.

Thus, hyaluronic acid is in particular used for filling wrinkles and for reducing, or even eliminating, local weakening of the structure of the dermis represented by a wrinkle, generally via direct injection into the dermis, at the wrinkle under consideration.

In fact, hyaluronic acid is essentially used in the form of a crosslinked gel given the increased resistance to degradation and to heat, and therefore to sterilization, of this particular form.

These crosslinked hyaluronic acid gels can be obtained by various preparation processes. Generally, these processes require two main steps, the first consisting in hydrating the hyaluronic acid so as to convert it into an aqueous gel and the second aimed at crosslinking said aqueous gel in the presence of an agent capable of inducing crosslinking thereof (also referred to as "crosslinking agent").

By way of illustration of these processes, mention may in particular be made of those described in documents US 2006/0105022, WO 2006/056204 or US 2007/0036745.

For obvious reasons, improving the mechanical properties of hydrogels, an objective liable to involve improving the processes for preparing these gels, is a constant objective.

The invention aims precisely to provide a process for obtaining crosslinked gels having particularly advantageous mechanical properties.

Against all expectations, the inventors have noted that carrying out the crosslinking of a polysaccharide, such as in particular hyaluronic acid, in the presence of a particular compound, makes it possible to obtain a crosslinked gel which is particularly advantageous in terms of mechanical properties compared with gels obtained via the conventional processes. Thus, according to a first of these aspects, the present invention relates to a process for preparing a crosslinked gel of at least one polysaccharide or a salt thereof, comprising at least the steps consisting in:

a) providing an aqueous gel comprising at least said polysaccharide(s) in a noncrosslinked form, combined with at least one crosslinking agent; b) crosslinking the mixture obtained at the end of step a) and, where appropriate;

c) recovering said crosslinked gel formed,

wherein the crosslinking step b) is carried out in a medium supplemented with at least one alkali halide salt present in a content of between 0.5% and 20% by weight relative to the total weight of the aqueous gel of step a).

More specifically, and as emerges from the examples hereinafter, the invention results from the unexpected observation by the inventors that the presence of an alkali halide salt during the performing of the crosslinking reaction makes it possible to significantly improve the rheo logical properties of the crosslinked gel.

It follows from this observation that a process in accordance with the invention is also advantageous in that it makes it possible to obtain a gel which is crosslinked using a small amount of crosslinking agent but which, on the other hand, has rheo logical properties equivalent to those shown by a crosslinked gel obtained with a conventional crosslinking process using a larger amount of crosslinking agent.

A process in accordance with the invention thus makes it possible to limit the amount of crosslinking agent to be introduced into the crosslinking reaction, and therefore to limit the amount of residual crosslinking agent, after the crosslinking reaction, which those skilled in the art seek to eliminate as much as possible, in particular by means of subsequent purification steps.

Against all expectations, the alkali halide salt required according to the invention therefore appears to act as an active agent stimulating the crosslinking reaction.

For the purpose of the present invention, the term "skin" encompasses the skin of the face, of the neck, of the neck line, of the hands, of the scalp, of the abdomen and/or of the legs, but also the lips.

According to one particular embodiment, a process of the invention may also comprise a step d) of stopping the crosslinking, which consists in exposing the crosslinked gel to conditions favourable to stopping the crosslinking thereof, it being possible for this step to be carried out before, jointly with or after the recovering step c).

According to one preferred embodiment variant, step d) is carried out before step c).

Advantageously, the crosslinked gel obtained at the end of the implementation of a process in accordance with the invention is a single-phase gel and more particularly a predominantly elastic, viscoelastic gel, i.e. a gel with a decreased capacity, or even devoid of capacity, to flow in the absence of stresses other than its own weight. Polysaccharide

The term "polysaccharide" is intended to mean any polymer consisting of several monosaccharides linked together by O-glycoside bonds and having the general formula: -[C_x(H₂0)_y)]_n-.

For the purpose of the present invention, the term "noncrosslinked" is intended to denote an aqueous gel of polysaccharides which is not crosslinked or not transformed, i.e. a solution of polysaccharides of which the chains of the polymer(s) are not connected to one another by strong or covalent bonds.

A polysaccharide in accordance with the invention is more particularly selected from the viewpoint of the properties that it is desired to see the crosslinked gel obtained according to the invention display. More particularly, such a polysaccharide must have good biocompatibility.

Thus, a physiologically acceptable polysaccharide or polysaccharide salt may be of natural or synthetic origin.

The polysaccharides suitable for the invention may in particular be chosen from chondroitin sulphate, keratan, keratan sulphate, heparin, heparin sulphate, xanthan, carrageenan, hyaluronic acid, chitosan, cellulose and its derivatives, alginate, starch, dextran, pullulan, galactomannan, and biologically acceptable salts thereof.

The polysaccharide salts in accordance with the invention are more particularly chosen from physiologically acceptable salts, such as the sodium salt, the potassium salt, the zinc salt, the silver salt, and a mixture thereof, preferably the sodium salt. Preferably, a polysaccharide or polysaccharide salt according to the invention has a high molecular weight, preferably an average molecular weight greater than or equal to 50 000 Da, or even greater than 3 MDa according to the application under consideration.

Advantageously, the hyaluronic acid used for obtaining a composition according to the invention can have an average molecular weight ranging from 50 000 to 10 000 000 Daltons, preferably from 500 000 to 4 000 000 Daltons.

A particularly preferred polysaccharide is hyaluronic acid (HA) or a salt thereof, preferably sodium hyaluronate (NaHA).

Advantageously, the polysaccharide(s) may be used in a process according to the invention in a content of between 0.5% and 20% by weight, relative to the total weight of the aqueous gel of step a).

Advantageously, in particular in the case where hyaluronic acid is crosslinked with BDDE, the content of hyaluronic acid, preferably in the form of a sodium salt, is between 5% and 15% by weight relative to the total weight of the aqueous gel of step a).

Crosslinking agent

The term "crosslinking agent" is intended to mean any compound capable of inducing crosslinking between the various polysaccharide chains.

The choice of this crosslinking agent from the viewpoint of the polysaccharide to be crosslinked is clearly among the skills of those skilled in the art.

A crosslinking agent in accordance with the invention is chosen from crosslinking agents, in particular bifunctional or multifunctional epoxide crosslinking agents, such as butanediol diglycidyl ether (BDDE), diepoxyoctane or l,2-bis(2,3- epoxypropyl)-2,3-ethylene, and crosslinking agents of polyamine type, such as hexamethylenediamine or l,6-diaminohexane-l ,6-hexanediamine (HMD A), endogenous polyamines such as spermine, spermidine or putrescine, and mixtures thereof.

Preferably, a crosslinking agent in accordance with the invention is butanediol diglycidyl ether (BDDE).

The adjustment of the amount of crosslinking agent for carrying out the crosslinking reaction is also among the skills of those skilled in the art.

Advantageously, the crosslinking agent(s) can be used in a process according to the invention in a content of between 0.1 mol% and 30 mol%, preferably between 0.5 mol% and 20 mol%, relative to the total number of moles of disaccharide units forming the monomer of the hyaluronic acid present in the aqueous gel of step a).

Alkali halide salt

As emerges from the aforementioned, the invention follows from the observation by the inventors that an alkali halide salt can be used during the step of crosslinking a polysaccharide as an active agent stimulating this crosslinking reaction.

For the purpose of the present invention, the term "alkali halide salt" is intended to denote an ionic salt which assembles two types of ions, one being an alkali ion, the other being a non- ion, necessarily representing a halogen atom.

Advantageously, the halogen atom may be chosen from fluorine, chlorine, bromine and iodine, and is preferably represented by chlorine.

Preferably, an alkali halide salt according to the invention can be chosen from the sodium salts, the potassium salts, the lithium salts, the caesium salts, and a mixture thereof, and better still is represented by a sodium salt.

Even more preferably, an alkali halide salt according to the invention may be

NaCl.

The adjustment of the amount of alkali halide salt in a crosslinking process according to the invention is also among the skills of those skilled in the art.

Advantageously, the alkali halide salt(s) may be used in a process according to the invention in a minimum content of greater than or equal to 0.5% by weight, preferably greater than or equal to 1% by weight, relative to the total weight of the aqueous gel of step a).

Advantageously, the alkali halide salt(s) may be used in a process according to the invention in a maximum content of less than or equal to 20% by weight, preferably less than or equal to 15% by weight, relative to the total weight of the aqueous gel of step a).

As previously indicated, the alkali halide salt(s) is (are) used in a process according to the invention in a content of between 0.5% and 20% by weight relative to the total weight of the aqueous gel of step a).

Advantageously, the alkali halide salt(s) may be used in a process according to the invention in a content of between 1% and 15% by weight relative to the total weight of the aqueous gel of step a). Process according to the invention

A process of the invention requires, firstly, providing, prior to the crosslinking step, an aqueous gel comprising at least one polysaccharide in a noncrosslinked form, combined with at least one crosslinking agent and at least one alkali halide salt.

More specifically, the aqueous gel under consideration in step a) can be obtained beforehand by bringing together, in a suitable receptacle:

(i) an aqueous medium;

(ii) at least one polysaccharide, or a salt thereof, in a noncrosslinked form; (iii) at least one crosslinking agent;

(iv) where appropriate, at least one alkali halide salt; and

at least one homogenization of the resulting mixture, the order of addition to the receptacle of said compounds (i), (ii) and (iii), and where appropriate (iv), being unimportant. As a preliminary to its crosslinking, the polysaccharide in the noncrosslinked state is therefore formulated in an aqueous medium in the gel state.

For the purpose of the present invention, the term "aqueous medium" is intended to mean any liquid medium containing water and which has the property of dissolving a polysaccharide or a salt thereof.

The nature of the aqueous medium is more particularly conditioned by the type of crosslinking envisaged, but also by the type of polymer used.

In this respect, an aqueous medium that may be suitable can be acidic or basic. For example, in the case of the use of BDDE as crosslinking agent, the particularly preferred aqueous medium can be an alkaline medium, preferably sodium hydroxide (NaOH), more particularly a solution of sodium hydroxide at a pH above 12.

Whatever the embodiment under consideration, the formation of an aqueous gel as considered in step a) involves at least one homogenization, as indicated above.

The purpose of this operation, optionally carried out in the presence of the crosslinking agent and/or of the alkali halide salt, is more particularly to hydrate and completely homogenize the polysaccharide in the aqueous medium and, where appropriate, the crosslinking agent and/or the alkali halide salt, and thus to contribute to optimizing the qualities of the expected crosslinked gel. The homogenization is considered to be satisfactory when the solution obtained has a homogeneous colouration, without agglomerates, and a uniform viscosity. It can advantageously be carried out under mild operating conditions so as to prevent degradation of the polysaccharide chains.

This step is all the more important when the polysaccharide has a high molecular weight. The hydration of such a compound in fact then has a tendency to generate the formation of a high- viscosity solution within which the appearance of agglomerates is commonly observed.

The duration of this homogenization step depends on the nature of the polysaccharide, and more particularly on its molecular weight, on its concentration, on the operating conditions within the aqueous medium and also on the homogenization device used.

The adjustment of the appropriate homogenization time for obtaining an aqueous polysaccharide gel which is sufficiently homogeneous is part of the general knowledge of those skilled in the art.

Preferably, a homogenization step according to the present invention can be carried out for a period of less than 200 minutes, preferably less than 150 minutes, or even between 5 and 100 minutes. According to a first embodiment variant, this aqueous gel can be formed by introduction, into the receptacle, of the aqueous medium, of the polysaccharide and, where appropriate, of the alkali halide salt, with simultaneous and/or consecutive homogenization of the mixture thus formed, and then addition of the crosslinking agent with simultaneous and/or consecutive homogenization.

According to a second embodiment variant, this aqueous gel can be obtained by introduction, into the receptacle, of the aqueous medium, of the polysaccharide, of the crosslinking agent and, where appropriate, of the alkali halide salt, with simultaneous and/or consecutive homogenization of the mixture thus formed.

This second embodiment variant is advantageous in that a single homogenization step is carried out. Advantageously, this step of forming the aqueous gel can be carried out at a temperature below 35°C, preferably at a temperature ranging from 15 to 25°C, and better still at an ambient temperature.

The aqueous gel of step a) is then subjected to conditions suitable for the crosslinking reaction.

This step is illustrated by step b) of the process of the invention.

The purpose of the crosslinking is to create bridges between the chains of polysaccharides, in particular of hyaluronic acid, making it possible to obtain a solid and dense three-dimensional network from a viscous solution.

According to a first embodiment, and as previously indicated, the alkali halide salt may be present in the aqueous gel of step a).

According to another embodiment, the process according to the invention may also comprise a step e), intermediate between steps a) and b), consisting in adding, to the aqueous gel obtained in step a), said alkali halide salt with simultaneous and/or consecutive homogenization.

Advantageously, this step e) of adding said alkali halide salt can also be carried out at a temperature below 35°C, preferably at a temperature ranging from 15 to 25°C, and better still at an ambient temperature.

According to yet another embodiment, the alkali halide salt can be added to the aqueous gel of step a) concomitantly with the crosslinking step b), with simultaneous and/or consecutive homogenization.

As regards the crosslinking, it requires the presence of at least one crosslinking agent, in particular as defined above.

The particular conditions to be retained for inducing the crosslinking reaction can depend on the nature of the polysaccharide, on its molecular weight, on the aqueous medium and on the nature of the crosslinking agent.

Generally, this stimulation can be acquired by bringing the mixture obtained at the end of step a) into contact with an initiating element, or else a stimulating element, such as, for example, heating, UV exposure, or even bringing said mixture into contact with a material of catalyst type.

The choice of such an initiating element is among the skills of those skilled in the art.

Thus, an initiating element can be carried out by:

- immersion of the receptacle comprising the mixture obtained at the end of step a) in a bath containing a hot fluid;

- exposure thereof to radiation of certain wavelengths of UV type, for example to microwave radiation or else to infrared radiation;

- irradiation by means of ionizing rays, like the process described in document

US 2008/0139796; and

- enzymatic crosslinking; and

- addition of a catalyst and/or of a reaction intermediate. As previously set out, the stopping of the crosslinking (step d)) can occur before, jointly with or after the step of recovering the gel c).

Such a step, according to a process in accordance with the invention, requires exposing the crosslinked gel or the gel undergoing crosslinking, or even the receptacle containing it, to conditions favourable to stopping said crosslinking, or else to conditions capable of stopping the formation of bonds between the various polysaccharide chains.

For example, in the event that the embodiment variant which takes into consideration the application of thermal conditions for stimulating the crosslinking process, the crosslinking can be stopped:

- by simply removing the receptacle from the thermostated bath, and then by cooling until there is a return to ambient temperature;

- by placing the receptacle in a bath of cold water, preferably at a temperature below ambient temperature, until the temperature inside said receptacle is close to ambient temperature; or even

- by extracting the gel from said receptacle. In the case of crosslinking by radiation, said crosslinking is stopped by stopping the exposure of said gel to the radiation.

According to one particularly preferred embodiment, the process according to the invention uses (i) sodium hyaluronate as polysaccharide in an alkaline medium, (ii) butanediol diglycidyl ether (BDDE) as crosslinking agent and (iii) NaCl as alkali halide salt in a content of between 0.5% and 20% by weight relative to the total weight of said aqueous gel of step a).

As indicated above, and according to one particular embodiment, the advantageous effect associated with the presence of the alkali halide salt permits the use of a reduced amount of crosslinking agent, compared with the conventional processes, while at the same time maintaining satisfactory mechanical properties of the crosslinked gels obtained at the end of the implementation of the process according to the invention.

Thus, according to this particular embodiment, a crosslinked gel obtained by implementing a process according to the invention may have a resulting degree of modification of between 0.1% and 15%, preferably between 0.1% and 10%, while at the same time remaining satisfactory in terms of mechanical properties.

For the purpose of the present invention, the term "degree of modification" is intended to denote the ratio between the number of moles of crosslinking agent attached to the hyaluronic acid and the number of moles of hyaluronic acid forming said crosslinked gel. This quantity can in particular be measured by ID 1H NMR analysis of the crosslinked gel.

The expression "number of moles of hyaluronic acid" is intended to mean the number of moles of repeating disaccharide units of the hyaluronic acid, the disaccharide unit being composed of D-glucuronic acid and of D-N-acetylglucosamine linked to one another by alternating beta- 1,4 and beta- 1 ,3 glycosidic linkages.

According to yet another particular embodiment, a process of the invention can be carried out at least partly within a specific receptacle having a deformable wall, such as, for example, a pouch, in particular the one defined in document FR 2 945 293. The crosslinked gel obtained at the end of the process of the invention as previously described cannot be directly injectable, in particular because its polysaccharide concentration is too high and/or because of the possible presence of crosslinking agent residues, or else because of its physiological and/or pH conditions which are incompatible with use in the fields of application considered above.

In addition, the gel obtained at the end of the process of the invention can in particular have too great a stiffness to be injected as such into a patient.

Consequently, several additional steps, known to those skilled in the art, are liable to be carried out in order to obtain an injectable hydrogel.

More particularly, a step of neutralizing and diluting this gel is required in order to give it its implant qualities. The chains of the polysaccharide network are then stretched and hydrated, while the pH is brought to that of the dermis.

A step of adding noncrosslinked hyaluronic acid may also be carried out in order to further improve the qualities of the implant, according to the know-how of those skilled in the art. The gel must be physiologically formulated by virtue of the presence of salts in amounts equivalent to those of the medium injected.

For even higher purity, a purification step can be carried out in order to bring the gel to a physiologically acceptable alkali halide salt concentration and/or to remove the crosslinking agent not attached to the polysaccharide and also any reaction intermediate or auxiliary agent.

Advantageously, this purification step can be carried out by means of one or more dialysis baths.

Finally, the resulting hydrogel can be packed into syringes under controlled atmosphere conditions, it being possible for said syringes to then undergo a sterilization step, preferably heat sterilization.

Throughout the description, including the claims, the expression "comprising a" should be understood to be synonymous with "comprising at least one", unless otherwise specified.

The expressions "between... and ..." and "ranging from ... to ..." should be understood as limits inclusive, unless otherwise specified. The examples which follow are given by way of nonlimiting illustration of the invention.

Example 1: Effect of the NaCl concentration, at constant weight percentage of crosslinking agent

Five crosslinked hyaluronic acid gels A, B, C, D and M, comprising the use of different NaCl concentrations, are prepared according to the following protocol.

For each of the samples, 10 g of NaHA are dissolved in 0.25N sodium hydroxide solution so as to obtain a 12% NaHA solution. NaCl is added to this solution, at the concentration specified in Table 1.

The NaHA is considered to be completely dissolved once all of the NaHA is hydrated and the solution completely homogeneous (absence of agglomerates and uniform colour).

A solution of BDDE diluted to 1/5 in a 1% sodium hydroxide solution is added to each homogeneous NaHA solution obtained at the end of the previous step, in the amount specified in Table 1.

Table 1

Mole percentage of crosslinking agent, defined according to the formula:

τ = (n_BDDE / n_NaHA ) x 100

² Weight concentration of alkali halide salt relative to the total weight of the aqueous gel before carrying out the crosslinking step.

The reaction medium of each sample is then homogenized mechanically and each of the samples is then placed in a water bath for 3 hours, at a temperature of 52°C, in order to carry out the crosslinking. Once the crosslinking reaction has finished, the receptacles are removed from the thermostated bath and placed in a container containing refrigerated sterile water, until the temperature has cooled to 25°C (ambient temperature).

The solids respectively obtained at the end of this incubation step (crosslinked hyaluronic solutions) are then neutralized, and swollen in a solution of phosphate buffer, pH 7.3, so as to obtain a hydrogel at 25 mg/g of hyaluronic acid.

In order to facilitate the extrusion forces for passing the gel through a needle, noncrosslinked hyaluronic acid is then incorporated into and homogenized in the gel obtained. The latter are then purified by dialysis and packaged in syringes.

The sterilization is carried out in an autoclave (wet heat) at T° > 121°C, so as to obtain an F0 > 15 (sterilizing value).

Three crosslinked hyaluronic acid gels B', D' and M' are further prepared according to the above-mentioned protocol, except that the NaCl is added after the step of crosslinking reaction.

The weight concentration of NaCl for these crosslinked hyaluronic acid gels B', D' and M' is similar to, respectively, the weight concentration of NaCl for the crosslinked hyaluronic acid gels B, D and M.

These three crosslinked hyaluronic acid gels B', D' and M' also undergo the same post-crosslinking steps as above-described.

Example 2: Characterization of the gels obtained in Example 1

The measurements of the viscoelastic properties of the gels A, B, C, D, M, B',

D' and M' are carried out using a rheometer (Haake RS6000) equipped with a cone/plate geometry (l°/diameter 35 mm). A stress sweep is performed, and the elastic modulus G'

(in Pa) and the phase angle δ (°) are measured at 1 Hz for a stress of 5 Pa.

Table 2 hereinafter represents the values of the elastic moduli G' (in Pa). By way of indication, the phase angle δ (°) value is also indicated therein. Table 2

In the light of the results represented in Table 2, it emerges that the presence of NaCl in a process according to the invention actually makes it possible to significantly improve the rheological properties of a crosslinked gel for the same weight percentage of crosslinking agent (see gels B, C, D and M).

Also, it emerges that the addition of NaCl after the crosslinking step has no effect on improving the rheological properties of a crosslinked gel of hyaluronic acid (see gels B', D' and M'). Instead, there is even a decrease in G 'with gels B', D' and M'.

Example 3: Effect of the NaCl concentration, with a decrease in weight percentage of crosslinking agent

Four crosslinked hyaluronic acid gels A, E, F and G comprising different NaCl concentrations and for which the crosslinking conditions were varied by means of a modification of the amount of crosslinking agent (see Table 3 hereinafter) are prepared according to the protocol as defined in Example 1.

The mechanical properties inherent in each of the crosslinked gels prepared are given in Table 3 hereinafter. Table 3

¹ and ² as defined in Example 1 It may be noted that the presence of NaCl makes it possible to retain the rheo logical properties of a crosslinked gel with a lower degree of crosslinking.

Example 4: Influence of the nature of the alkali halide salt

Six hyaluronic acid gels C, I, K, A, L and X optionally comprising an alkali halide salt are prepared according to the protocol defined in Example 1 above.

The mechanical properties of each of the crosslinked gels prepared are given in Table 4 hereinafter, which in particular gives an account of mechanical properties of three control gels A, L and X, crosslinked, respectively, in the absence of salt (A) and in the presence of Na₂C0₃ (L) or CaCl₂ (X) by way of salts not in accordance with the invention.

Table 4

as defined in Example 1 It appears from the aforementioned that only the alkali halide salts, namely NaCl, KCl and NaBr, make it possible to significantly improve the rheological properties of a crosslinked gel for the same degree of crosslinking, this improvement being particularly significant with NaCl.

Indeed, Na₂C0₃ reduces the efficiency of the crosslinking reaction. As for CaCl₂, a hyaluronic acid degradation effect is also observed. The crosslinking reaction has not taken place and the gel obtained before sterilization has a viscous, or even runny, stringy appearance, which is generally an indication of hyaluronic acid chain degradation.

Example 5 : Influence of the nature of the crosslinking agent

Protocol for preparing a hyaluronic acid gel crosslinked with HMDA as crosslinking agent

Hyaluronic acid is dissolved at a concentration of 3% (by weight) in water supplemented with NaCl. After a homogeneous solution has been obtained, a solution containing HMDA, EDC as activator (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and HOBt as auxiliary coupling agent (1-hydroxybenzotriazole hydrate) is added to the hyaluronic acid solution. The volume of this solution is defined so as to obtain an overall mixture containing 2.3% of hyaluronic acid.

The pH of this solution is adjusted so as to obtain a pH of the overall mixture of between 5.5 and 6.5 before reaction.

The amounts of reagents are adjusted so as to obtain the following stoichiometries:

0.2 mol/mol of repeating hyaluronic acid units for the polyamine, and 1 mol/mol of repeating hyaluronic acid units for the activator and the auxiliary coupling agent involved in the coupling reaction. For the crosslinking, the aqueous gel is exposed for 15 hours at 25°C.

After crosslinking, the gel obtained is purified by means of several dialysis baths over a period of 48 hours and, if necessary, adjusted to physiological pH.

The aqueous gel obtained after washing comprises a concentration close to

20 mg/g. The sterilization is carried out in an autoclave (wet heat) at T° > 121°C, so as to obtain an F0 > 15 (sterilizing value).

The mechanical properties of each of the crosslinked gels prepared are given in Table 5 hereinafter.

Table 5

⁴ Mole percentage of crosslinking agent, defined according to the formula:

T = (nHMDA n_NaHA ) x 100

² see Example 1.

The improvement in the rheological properties of the gels in the presence of NaCl is also confirmed when HMDA is used as crosslinking agent.

Claims

1. Process for preparing a crosslinked gel of at least one polysaccharide or a salt thereof, comprising at least the steps consisting in:

a) providing an aqueous gel comprising at least said polysaccharide(s) in a noncrosslinked form, combined with at least one crosslinking agent;

b) crosslinking the mixture obtained at the end of step a) and, where appropriate;

c) recovering said crosslinked gel formed,

wherein the crosslinking step b) is carried out in a medium supplemented with at least one alkali halide salt present in a content of between 0.5% and 20% by weight relative to the total weight of the aqueous gel of step a), and the crosslinking agent is chosen from butanediol diglycidyl ether (BDDE), l,2-bis(2,3-epoxypropyl)-2,3-ethylene, crosslinking agents of polyamine type, such as hexamethylenediamine or l ,6-diaminohexane-l,6-hexanediamine (HMD A), endogenous polyamines such as spermine, spermidine or putrescine, and mixtures thereof.

2. Process according to the preceding claim, wherein said process further comprises a step d) of stopping the crosslinking, which consists in exposing the crosslinked gel to conditions favourable to stopping the crosslinking thereof, it being possible for this step to be carried out before, jointly with or after the recovering step c).

3. Process according to any one of the preceding claims, wherein the halogen atom of the alkali halide salt is chosen from fluorine, chlorine, bromine or iodine, and is preferably represented by chlorine.

4. Process according to any one of the preceding claims, wherein the alkali halide salt is chosen from the sodium salts, the potassium salts, the lithium salts, the caesium salts, and a mixture thereof, preferably is represented by a sodium salt, and better still is NaCl.

5. Process according to any one of the preceding claims, wherein the alkali halide salt(s) is(are) present in a content of between 1% and 15% by weight relative to the total weight of the aqueous gel of step a).

6. Process according to any one of the preceding claims, wherein the polysaccharide is hyaluronic acid or a salt thereof.

7. Process according to claim 6, wherein the hyaluronic acid salt is chosen from the sodium salt, the potassium salt, the zinc salt, the silver salt, and a mixture thereof, and is preferably the sodium salt.

8. Process according to any one of the preceding claims, wherein the polysaccharide(s) or a salt thereof is(are) present in a content of between 0.5% and 20% by weight relative to the total weight of the aqueous gel of step a).

9. Process according to any one of the preceding claims, wherein the crosslinking agent is butanediol diglycidyl ether (BDDE).

10. Process according to any one of the claims 6 or 7, wherein crosslinking agent(s) is(are) used in a content of between 0.1 mol%> and 30 mol%>, preferably between 0.5 mol% and 20 mol%, relative to the total number of moles of disaccharide units forming the monomer of the hyaluronic acid present in the aqueous gel of step a).

11. Process according to any one of the preceding claims, wherein it uses (i) sodium hyaluronate as polysaccharide in an alkaline medium, (ii) butanediol diglycidyl ether (BDDE) as crosslinking agent and (iii) NaCl as alkali halide salt in a content of between 0.5%) and 20%> by weight relative to the total weight of the aqueous gel of step a).

12. Process according to any one of the preceding claims, wherein step a) is carried out at a temperature below 35°C, preferably ranging from 15 to 25°C, and better still at ambient temperature.