EP3624764A1

EP3624764A1 - Method for preparing an aqueous hyaluronic acid gel

Info

Publication number: EP3624764A1
Application number: EP18724248.2A
Authority: EP
Inventors: Frédéric BERTAINA; Alexandre GUERRY; Caroline Ceccaldi
Original assignee: Bioxis Pharmaceuticals
Current assignee: Bioxis Pharmaceuticals
Priority date: 2017-05-18
Filing date: 2018-05-17
Publication date: 2020-03-25
Also published as: BR112019024106A2; FR3066386A1; KR20200008560A; FR3066386B1; WO2018210999A1; CN110621294A; CA3060431A1; US20200060959A1

Abstract

The present invention concerns, according to a first aspect, a method for preparing an aqueous hyaluronic acid gel, comprising the following steps: a) preparing a crosslinked aqueous hyaluronic acid gel, b) homogenising the aqueous gel formed in step (a) by rolling, c) neutralising the aqueous gel homogenised in step (b). The invention also concerns, according to a second aspect, an aqueous hyaluronic acid gel able to be obtained by such a method. The invention further concerns, according to a third aspect, the cosmetic use of such an aqueous gel in tissue repair or reconstruction, in particular for filling lines and wrinkles, or the medical use of said aqueous gel in tissue repair or reconstruction.

Description

Process for the preparation of an aqueous gel of hyaluronic acid

The subject of the present invention is a process for preparing an aqueous gel of homogeneous hyaluronic acid, the aqueous gel thus obtained and its uses, in particular for filling wrinkles and fine lines.

Collagen has long been the filling product of choice for the face, especially for filling wrinkles and fine lines or for re-lipging. However, since the placing on the market of hyaluronic acids, they are increasingly used. In fact, to the biodegradability of collagen considered too fast, are added the safety problems related to the animal origin (bovine or porcine) of it. The injection of hyaluronic acid has two advantages: an immediate mechanical filling effect and the absence of inflammatory phenomena, because of its biocompatibility. When administered in a linear (non-crosslinked) form, hyaluronic acid has excellent biocompatibility but is rapidly degraded by the body (in about a week). The life of injected products based on hyaluronic acid could be significantly prolonged until approximately 12 months by the use of cross-linked hyaluronic acid. Indeed, the crosslinked hyaluronic acid is in the form of a cohesive gel having viscoelastic properties of particular interest for wrinkle filler products.

However, during crosslinking, particles of "hard zones" are formed within the hyaluronic acid gel, rendering it inhomogeneous. These hard zones affect the injectability of the product and are likely to pose tolerance problems for the patient. It is therefore essential to eliminate these hard zones crosslinked hyaluronic acid gels in order to have a perfectly homogeneous product for its administration. Conventionally, the crosslinked hyaluronic acid gels are homogenized by sieving or by extrusion. These methods only allow a partial elimination of hard zones. In addition, the shear stress exerted on the gels causes an alteration of its structure and its viscoelastic properties. The gel subjected to sieving, filtration or extrusion is therefore not perfectly homogeneous and its viscosity is reduced. Once injected it may migrate into tissue and degrade more rapidly. Its filling properties are degraded.

It is therefore desirable to have a method for effectively removing hard areas present in crosslinked hyaluronic acid gels, so as to obtain a homogeneous gel, without altering its viscoelastic properties.

The present invention therefore proposes a process for preparing a homogeneous gel of cross-linked hyaluronic acid, in which the homogenization of the gel is obtained by rolling.

In particular, the subject of the present invention is, according to a first aspect, a process for preparing an aqueous gel of hyaluronic acid comprising the following steps:

a) the preparation of an aqueous gel of cross-linked hyaluronic acid,

b) homogenizing the aqueous gel formed in step (a) by rolling,

c) the neutralization of the aqueous gel homogenized in step (b).

FIRE I LLE OF REM PLACEM ENT (RULE 26) The subject of the invention is also, according to a second aspect, an aqueous gel of hyaluronic acid that can be obtained by such a method.

The invention further relates, in a third aspect, to the cosmetic use of such an aqueous gel in the repair or reconstruction of tissues, in particular for filling wrinkles and fine lines, or said aqueous gel for its medical use. for repair or reconstruction of tissue.

figures

Figure 1 illustrates a rolling of the gel made between two cylinders rotating at the same tangential speed.

Figure 2 illustrates a rolling of the gel made between three cylinders whose tangential speed increases to allow the entrainment of the gel on the surface of the adjacent cylinder.

Figures 3, 4 and 5 illustrate the ejection forces of compositions 3, 4 and 5 of aqueous gels prepared in the exemplary embodiments.

Aqueous gel

The present invention provides a novel process for preparing aqueous gels of hyaluronic acid.

For the purposes of the present application, the term "gel" means a cohesive composition which does not flow under its own weight and which has viscoelastic properties giving it a certain deformability. The gel, if sheared, does not reform, unlike viscous fluids.

The hyaluronic acid gels, according to the invention, are thus distinguished from hyaluronic acid solutions. The distinction gel / solution can be observed by a rheological study in deformation and constant frequency at 25 ° C. in order to determine the viscous modulus G "and the elastic modulus G 'in the zone of linear viscoelasticity. the present invention is characterized in particular by the fact that its elastic modulus G 'is greater than the viscous modulus G "according to the definition of Winter and Chambon (1986). In the case of a viscous solution on the contrary, the viscous modulus G '' is greater than its elastic modulus G '.

The measurements are carried out on a Discovery HR1 rheometer (TA industries) and a 40 mm plan / plane geometry in a continuous mode (strain stress 10%, frequency 1 Hz, at 25 ° C, for 120s). Samples consisting of about 1.2 ml are deposited in a gap of ΙΟΟΟμιη.

The hyaluronic acid gels according to the invention are preferably homogeneous. For the purposes of the present invention, the term "homogeneous gel of hyaluronic acid" is intended to mean that the crosslinked hyaluronic acid is dispersed uniformly within the gel. The homogeneity of the hyaluronic acid gel can in particular be characterized by measuring the variation of the force of ejection of the gel through a syringe whose needle has an internal diameter of 300μιη (27G TSK UTW). The measurement of the ejection force (or extrusion force) is carried out using an EZ-Test SX shimadzu force bench equipped with a 50N cell. The extrusion is carried out at 10 mm.min ^-1 and the sampling is set at 100 points ^"1 . The tests are made with long needles BD LML equipped with TSK needle 27G 1/2 ". The acquisition is treated between the ^20th and the ^140th second extrusion to ignore the constraints of contacting At the end of the acquisition, the sequence of point N = f (t) (extrusion force as a function of time) is linearized A margin of ± 10% to the linearity is materialized. Each intersection of the curve N = f (t) with linear lines N = f (t) linearized N ₊ io% = f (t) and N-io% = f (t) corresponds to the extrusion of a heterogeneous part .

Thus, according to a preferred embodiment, the homogeneous hyaluronic acid gel does not exhibit a variation of the extrusion force of more than ± 10% with respect to the linearized extrusion force. Hyaluronic acid

The aqueous gels according to the invention comprise at least one hyaluronic acid.

Hyaluronic acid is a linear glycosaminoglycan (GAG) composed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine linked together by alternating beta-1,4 and beta-1,3 glycosidic linkages. .

Hyaluronic acid has the following structure:

Preferably, the hyaluronic acid used in the preparation of the aqueous gel according to the invention has a molar mass of between 1,000,000 Da and 5,000,000 Da, preferably between 1,500,000 Da and 3,500,000 Da. The molecular weight can in particular be determined by Waters GPCV Alliance 2000 steric exclusion chromatography, eluent NaN0 ₃ 0.1M in water coupled in line with three Wyatt detectors: a refractometer, a viscometer and a measurement of the light diffusion.

The hyaluronic acid is present in the aqueous hyaluronic acid gel obtained in step a) in a content of between 1 mg / ml and 300 mg / ml, preferably between 75 and 200 mg / ml, more preferably between 100 and 100 mg / ml. and 175 mg / mL. Aqueous phase

In addition to hyaluronic acid, the aqueous gels according to the invention also comprise an aqueous phase.

The gel may comprise water in a content ranging from 60% to 99% by weight, relative to the total weight of the composition, preferably ranging from 70% to 99% by weight, and preferably ranging from 80% to 99% by weight. % in weight.

The gel may also comprise a water-miscible polyol at ambient temperature (25 ° C.), chosen in particular from polyols having in particular from 2 to 20 carbon atoms, preferably having from 2 to 10 carbon atoms, and preferentially having from 2 to 6 carbon atoms, such as glycerine, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, dipropylene glycol, diethylene glycol; glycol ethers (having in particular from 3 to 16 carbon atoms), such as the mono-, di- or tripropylene glycol (C1-C4) alkyl ethers, the mono, di or triethylene glycol (C1-C4) alkyl ethers; ; and their mixtures.

The water-miscible polyol may be present in the gel according to the invention in a content ranging from 0.1% to 20% by weight, relative to the total weight of the composition, and preferably ranging from 3% to 15% by weight. weight.

Preparation of an aqueous gel of crosslinked hyaluronic acid

The method according to the invention implements a first step a) of preparing an aqueous gel of cross-linked hyaluronic acid.

This step a) comprises, preferably at least the crosslinking in acidic or basic medium of said hyaluronic acid in the presence of at least one crosslinking agent.

According to one particular embodiment, the crosslinking is carried out in a basic medium and comprises at least the following stages:

dissolving at least one hyaluronic acid and / or one of its salts in a basic solution having a pH greater than 7.5, preferably greater than or equal to 10, more preferably between 10 and 14,

the crosslinking in basic solution of said hyaluronic acid in the presence of at least one crosslinking agent.

Crosslinking in a basic medium promotes the formation of ether bonds between the hyaluronic acid and the crosslinking agent, which are slowly degraded.

According to another particular embodiment, the crosslinking is carried out in an acid medium and comprises at least the following stages:

the dissolution of at least one hyaluronic acid and / or one of its salts, in an acidic solution having a pH of less than 6.5, preferably less than or equal to 5, more preferably between 4.5 and 2. the crosslinking in acid solution of said hyaluronic acid in the presence of at least one crosslinking agent.

The crosslinking in an acidic medium favors the formation of ester bonds between the hyaluronic acid and the crosslinking agent, which degrade more rapidly than the ether bonds.

According to a preferred embodiment, the step of crosslinking hyaluronic acid comprises at least a crosslinking in basic medium of hyaluronic acid and an acid crosslinking of hyaluronic acid, so as to control the ether and ester formed, and thus the rate of degradation of the crosslinked hyaluronic acid gel thus formed.

More preferably, the crosslinking step of hyaluronic acid comprises a first crosslinking in basic medium of hyaluronic acid followed by crosslinking in acidic medium of hyaluronic acid.

Before dissolution, the hyaluronic acid used in the process according to the invention is typically in dry form, preferably in the form of powder or flakes.

When it is used in salt form, the hyaluronic acid may preferably be a sodium salt, a calcium salt, a zinc salt or a potassium salt of hyaluronic acid, and preferably a salt sodium.

The content of linear hyaluronic acid dissolved in the aqueous solution (corresponding to the content of hyaluronic acid in step a)) is between 50 mg / ml and 300 mg / ml, preferably between 100 and 200 mg / ml. .

The crosslinking of the linear hyaluronic acid dissolved in the aqueous solution is carried out in the presence of at least one crosslinking agent.

The crosslinking agent is preferably chosen from di-functional epoxides, multifunctional epoxides, bi or polyfunctional esters, divinylsulfones, carbodiimides, formaldehyde, dialdehydes and mixtures thereof, and preferably the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE, also known under the name 1,4-Diglycidyloxybutane, Tetramethylene Glycol Diglycidyl Ether and under the name IUPAC 2- [4- (oxiran-2-ylmethoxy) butoxymethyl] oxirane.

The crosslinking agent is in particular introduced in an amount of between 10 mg and 250 mg per gram of linear hyaluronic acid introduced at the crosslinking step.

The crosslinking step is preferably carried out at a temperature of between 30 and 70 ° C, preferably between 45 and 55 ° C, which makes it possible to catalyze the crosslinking of hyaluronic acid. Homogenization by rolling

In the context of the process of the invention, the aqueous gel of crosslinked hyaluronic acid prepared in step a) is then homogenized by rolling to eliminate the hard zones (aggregates formed during the crosslinking) without altering the mechanical and visco properties. -elastic gel. In particular, the rolling consists of a continuous compression between at least two counter-rotating rolls, preferably three counter-rotating rolls.

The inlet cylinder may for example rotate at a tangential speed between 0.1 ms ^"1 and 5 ^{ms" 1,} preferably between 0.5ms and 3m.s ^_1 ^_1.

When the rolling is carried out between two rolls, they preferably rotate at the same tangential speed, and the gel is introduced between the two rolls, as illustrated in FIG.

When the rolling is carried out between three rolls, the tangential speed of the various rolls should increase to allow the entrainment of the gel on the surface of the second roll to conduct a second rolling between the 2 ^nd and the 3 ^rd roll. By way of example, as illustrated in FIG. 2, if the first cylinder rotates at a tangential speed x 1, the second could rotate at this tangential speed x 2, and the third at this tangential speed x 3 to allow double rolling of the gel.

According to a preferred embodiment, the spacing between the counter-rotating cylinders (also called air gap) is between 20 μιη and 1 mm, preferably between 20 μιη and 100 μιη. The rolls may preferably be made of stainless steel, so that they can be easily cleaned, and optionally provided with a microporous or ceramic coating, capable of promoting adhesion of the gel to the surface of the rolls.

According to a preferred embodiment, the rolling homogenization step b) is carried out for 1 minute to 2 hours, preferably between 15 minutes and 45 minutes. These relatively short rolling times can be implemented insofar as, in the context of the present invention, step b) of homogenizing the aqueous gel is carried out before the step c) of neutralization, which leads to a swelling of said aqueous gel. Indeed, before swelling, the volume of aqueous gel to be homogenized by rolling is significantly less important than after swelling.

Purification

According to a particular embodiment, and in particular in the case where the neutralization is not carried out by dialysis, the aqueous gel of crosslinked hyaluronic acid can be purified before or after step c) of neutralizing the aqueous gel in order to remove traces of residual crosslinking agent.

According to a preferred embodiment, the purification is preferably carried out by dialysis under the conditions described above. The dialysis purification makes it possible, in addition to removing the residual crosslinking agent, to further refine the pH obtained after neutralization and to control the osmolarity of the gel.

Purification may result in further dilution of hyaluronic acid. The content of crosslinked hyaluronic acid present in the gel after purification is between 1 mg / ml and 60 mg / ml, preferably between 5 and 50 mg / ml.

Neutralization

After homogenization by rolling, the aqueous gel of hyaluronic acid is neutralized in a step c).

This neutralization is carried out by adjusting the pH to a pH between 6.5 and 7.5. The neutralization can be carried out by adding an acid or a base depending on whether the crosslinking has been carried out in a basic or acidic medium.

Neutralization causes the dilution of hyaluronic acid. The content of crosslinked hyaluronic acid present in the gel after neutralization is between 10 mg / ml and 100 mg / ml, preferably between 20 and 80 mg / ml.

For example, when the crosslinking has been carried out in an acid medium, the pH adjustment can be carried out by adding a compound such as ammonia, sodium hydroxide, sodium hydrogencarbonate, sodium bicarbonate, sodium carbonate or their derivatives or a solution of phosphate buffer (PBS "Phosphate Buffer Saline" - phosphate buffer saline solution).

When the crosslinking has been carried out in a basic medium, the adjustment of the pH for the neutralization can be carried out by adding a compound such as hydrochloric acid, acetic acid, phosphoric acid and sodium dihydrogenphosphate or their derivatives.

Alternatively, the neutralization can be performed by dialysis. Neutralization by dialysis makes it possible to adjust the pH in a very gradual manner, which makes it possible to best preserve the mechanical and viscoelastic properties of the hyaluronic acid gel formed.

Dialysis is a process of membrane separation of molecules or ions in solution. Thus, in the context of the present application, the hyaluronic acid gel according to the invention can be dialyzed against a buffer solution having a pH equal to or close to the desired final pH for the hyaluronic acid gel (target pH), c. is between 6.5 and 7.5, preferably between 6.75 and 7.2.

The buffer solution may, for example, be a saline solution of phosphate buffer (PBS, PBS-lactic acid), tris (hydroxymethyl) methylamine (TRIS), TRIS saline solution (TBS), 4-2- hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2- {[tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES), 3- (N-morpholino) propanesulfonic acid (MOPS), piperazine-N acid , N'-bis (2-ethanesulfonic acid), MES (2- (N-morpholino) ethanesulfonic acid (PIPES), and sodium chloride (NaCl). According to a preferred embodiment, the buffer solution is a solution of phosphate buffer PBS ("Phosphate Buffer Saline" - salt solution of phosphate buffer) composed of an "acid" salt NaH ₂ PO 4, a "basic" salt Na ₂ HPO ₄ and NaCl.

According to one particular embodiment, the buffer is physiologically acceptable, that is to say that it presents no risk of intolerance or toxicity during the injection of the hyaluronic acid gel according to the invention or of its contact with the tissues.

According to a particular embodiment of the invention, the dialysis can be carried out in one or more baths against a buffer solution as described above.

According to a more preferred embodiment, the dialysis can be carried out in several successive baths against buffer solutions having different pHs closer and closer to the desired final pH for the hyaluronic acid solution (target pH). It is thus possible to increase the pH more gradually according to the number of buffer baths implemented.

According to a preferred embodiment, to simultaneously control the osmolarity of the hyaluronic acid gel, the buffer used for the dialysis can be associated with a so-called neutral salt, that is to say that does not interact with the buffer, in particular one of sodium salt (NaCl) or potassium salt (KC1) in a salt concentration to reach the osmolarity of the tissues between 280 mOsmol.L ^{~ 1} and 380 mOsmol.L ¹ .

In particular, the buffer solution may have an osmolarity of between 250 and 350 mOsm / L, preferably between 280 and 330 mOsm / L.

In the context of the invention, step c) of neutralization causes swelling of the crosslinked hyaluronic acid gel. In general, the swelling in fact leads to an increase in the volume of hyaluronic acid gel between 2 and 4 times relative to the volume of the aqueous gel of crosslinked hyaluronic acid obtained in step a).

In the context of the present invention, it is important that this swelling (and therefore the neutralization) is not or very little initiated before the rolling step b) in order to allow the use of a sufficiently narrow air gap (between 20 and 20 μm). and 1 mm) so as to effectively remove the hard areas (aggregates formed during the crosslinking) without altering the mechanical and viscoelastic properties of the gel.

Indeed, if the rolling is carried out simultaneously (as for example described in document US2013 / 0217872) or after the neutralization and therefore the swelling of the gel, the spacing between the rollers of the rolling mill must be significantly increased to allow the passage inflated gel, occupying a larger volume. Such spacing greater than 1 mm between the rollers no longer makes it possible to effectively eliminate the hard zones present in the gel to guarantee its excellent homogeneity and injectability. The document US2013 / 0217872 in this case does not describe injectable hyaluronic acid gels, and does not seek, by rolling, to eliminate the hard zones present in the gel. In this document, rolling allows the neutralized gel to be mixed for a very long period of 18 to 24 hours to reach a swelling equilibrium. To allow a good injectability of the gels obtained by means of the process according to the invention, it is preferable that the gel be free of hard zones having a diameter greater than 1 mm, preferably greater than 20 μιη.

Linear hyaluronic acid

According to a particular embodiment, a linear hyaluronic acid can be added after the step (a) for preparing the aqueous gel of cross-linked hyaluronic acid so as to reduce the viscosity of the gel and thus to adjust its mechanical properties, in particular to reduce the force of ejection of the gel and to facilitate the filling of the syringes.

The introduction of linear hyaluronic acid can be carried out before or after step b) of homogenization by rolling, the step c) of neutralization (dilution) or the purification step previously described. According to a preferred embodiment, the introduction of linear hyaluronic acid can be carried out before or after the previously described purification step.

The amount of linear hyaluronic acid introduced into the crosslinked hyaluronic acid gel is preferably less than or equal to the amount of crosslinked hyaluronic acid present in the gel after neutralization and optionally purification, so as not to further dilute the acid. hyaluronic.

In particular, the content of crosslinked hyaluronic acid present in the gel after purification is between 0.1 mg / ml and 100 mg / ml, preferably between 1 and 50 mg / ml.

Additional polymer

According to one particular embodiment, the aqueous gel of hyaluronic acid also comprises at least one additional polymer other than hyaluronic acid, such as chondroitin, cellulose, alginate, polycaprolactone, polylactic acid, polyglycolic acid, collagen, silk, PTFE and their derivatives.

The additional polymer may be introduced during step a), before the crosslinking of the hyaluronic acid to lead to a co-crosslinking of the hyaluronic acid with the additional polymer, or after the step a) of preparing the aqueous gel crosslinked hyaluronic acid, and in particular before step b) homogenization by rolling.

The additional polymer may for example be introduced in a content ranging from 0.1% to 5%, preferably from 0.5% to 4%.

Injectable compositions

According to a preferred embodiment, the aqueous gel prepared according to the process of the invention is injectable.

For injectable gel is meant in the sense of the present invention a composition in the form of gel having injectability properties (or syringuability, that is to say ease of injection due to a more or less satisfactory flow through a needle in a syringe) satisfactory, and in particular capable of being injected by means of a syringe having a needle of internal diameter approximately equal to 300 μιη. For the purposes of the present application, gels preferably having a viscosity of less than or equal to 10,000 Pa.s and a loss factor (Tan6) of between 0.01 and 5 are considered to be rheologically injectable.

The rheological measurements (G ', G "and Tanô) are carried out on a Discovery HR-1 rheometer (TA industries) and a 40 mm plan / plane geometry in a continuous mode (strain stress 10%, frequency 1 Hz, at 25 ° C). ° C, for 120s) The samples consist of about 1.2 ml are deposited in a gap of ΙΟΟΟμιη.

The maximum viscosity measurements are carried out in dynamic mode (angular frequency from 0.1 to 100 rad.s ^-1 ) The samples consisting of about 1.2 ml are deposited in a gap of ΙΟΟΟμιη.

The aqueous gel prepared according to the process of the invention can therefore be packaged in syringes in order to be injected into the tissues.

The invention thus provides, in another aspect, a syringe containing the gel prepared according to the method of the invention, as described above. Such a syringe is particularly intended for filling wrinkles or fine lines.

According to this embodiment, degassing can be performed before filling the syringes to remove any air bubbles.

uses

In a particular embodiment, the aqueous gel obtained according to the present invention is intended for use in the repair or reconstruction of tissues.

In particular, the aqueous gel according to the present invention can be used for the constitution or the substitution of biological tissues, for example as an implant, or the filling of biological tissues, for example the injection into the bone cartilages or in the joints or for the filling of the cavities of the body or face, such as wrinkles or fine lines, for the creation or increase of volumes of the face or the human body, or for the healing of the skin.

According to other particular embodiments, the aqueous gel according to the present invention can be used:

- in surgery, particularly in the repair of organs, or in medicine or cosmetic surgery,

- in urology, especially for the treatment of urinary incontinence,

- in infectiology, especially as a carrier fluid for vaccines, - in ophthalmology, particularly for corneal scarring,

- in odontology, in particular for the installation of a dental implant or for the bone repair,

- in orthopedics, especially in the periosteum for volume creation,

- for cell therapy or tissue engineering, as part of the vectorization of therapeutic cells or biactive factors, or in angiology.

The aqueous gel according to the present invention can also be used in rheumatology.

Advantageously, the aqueous gel according to the present invention can also be used as a vector of active principle, especially a therapeutic substance, such as cells, a vaccine or an insulin or estrogen-type hormone, and more generally for all the principles. assets whose controlled and / or prolonged release or release has an advantage.

The present invention also relates to the cosmetic use of an aqueous gel according to the invention for treating or combating aging of the skin.

The following example is intended to illustrate the invention without in any way limiting its scope.

Example:

An aqueous gel of hyaluronic acid according to the invention (Composition 1) was prepared according to the following method:

Hyaluronic acid (HTL, France) was completely dissolved in an alkaline solution of phosphate buffer (SOOmOsmol ^1, pH = 12.9, Merck, France) to get a final concentration of hyaluronic acid 150mg.mL ^-1.

A solution of BDDE at 20% by weight (SA, France) is slowly added. The mixture is then heated to 50 ° C until the texture no longer changes and the mixture turns yellow.

The resulting gel is then rolled using an EXAKT 50i G line tricylindrical rolling mill (Exakt, Germany).

When the rolling is complete, an acid solution of phosphate buffer (472mOsmol.L ^-1 , pH = 1.59) is added to neutralize the reaction mixture and to dilute the gel at a concentration of 32.5mg.mL- ¹ to hyaluronic acid.

The gel is then dialyzed against phosphate buffer (300mOsmol.L ^_1, pH 7.4, Merck, France). Dialysis is stopped when neutrality is reached. Finally, 4% (w / w) of a solution of hyaluronic acid 25mg.mL ^_1 (HTL, France) are then added. The acid gel is then placed in syringes of 1 mL (BD, 1 mL long) and sterilized by autoclaving (121 ° C. for 15 minutes). An aqueous gel of comparative hyaluronic acid (Composition 2) was also prepared according to the same process, except for the rolling step which was not performed.

The effect of rolling on the homogeneity of the gel was demonstrated by measuring the ejection force. The more the ejection force is stable during the expulsion of the product through the syringe and the needle, the more the gel is homogeneous.

The ejection forces of the previously prepared aqueous gel compositions 1 and 2 were measured. The results of these measurements are presented in Figures 3 and 4.

Excellent stability of the ejection force is observed for Composition 1 according to the invention (FIG. 3). On the other hand, for Composition 2 (comparative), non-laminated, non-sieved and unmilled, large variations in the ejection force exceeding several times the margin of ± 10% compared to the extrusion force are observed N = F (t) linearized (Figure 4).

The ejection force of a Teosyal Ultradeep crosslinked hyaluronic acid aqueous gel composition (Composition 3) was also measured, the production method of which involves a sieving / milling step as described in US patent application 2013/0237615. The result of this measurement is shown in FIG. 5. A wide variation in the ejection force during ejection is observed which demonstrates a heterogeneity of the gel.

Claims

A process for preparing an aqueous gel of hyaluronic acid comprising the following steps:

a) the preparation of an aqueous gel of cross-linked hyaluronic acid,

b) homogenizing the aqueous gel formed in step (a) by rolling,

c) the neutralization of the aqueous gel homogenized in step (b).

2. Method according to claim 1, characterized in that the rolling consists of a continuous compression between at least two counter-rotating cylinders, preferably three counter-rotating cylinders.

3. Method according to any one of claims 1 or 2, characterized in that the spacing between the counter-rotating cylinders is between 20 μιη and 1 mm, preferably between 20 μιη and 100 μιη.

4. Method according to any one of claims 1 to 3, characterized in that step a) for preparing an aqueous gel of crosslinked hyaluronic acid comprises at least the crosslinking in acidic or basic medium of said hyaluronic acid in the presence at least one crosslinking agent.

5. Method according to claim 4, characterized in that the crosslinking in basic medium of hyaluronic acid comprises at least the following steps:

dissolving at least one hyaluronic acid and / or one of its salts in a basic solution having a pH greater than 7.5, preferably greater than or equal to

10, more preferably between 10 and 14,

6. Method according to claim 4, characterized in that the crosslinking in acidic medium of hyaluronic acid comprises at least the following steps:

the dissolution of at least one hyaluronic acid and / or one of its salts, in an acidic solution having a pH of less than 6.5, preferably less than or equal to 5, more preferably of between 4.5 and 2; .

the crosslinking in acid solution of said hyaluronic acid in the presence of at least one crosslinking agent.

7. Method according to any one of claims 4 to 6, characterized in that the crosslinking step of hyaluronic acid comprises at least a crosslinking in basic medium of hyaluronic acid and a crosslinking in an acid medium of the hyaluronic acid, and preferably cross-linking in a basic medium of hyaluronic acid followed by cross-linking in an acid medium of hyaluronic acid

8. Process according to any one of claims 1 to 7, characterized in that step c) of neutralizing the hyaluronic acid solution is carried out by adjusting the pH to a pH between 6.5 and 7. 5.

9. Process according to any one of claims 1 to 8, characterized in that the hyaluronic acid used in the preparation of the aqueous gel in step a) has a molar mass of between 1,000,000 Da and 5,000. 000 Da, preferably between 1,500,000 Da and 3,500,000 Da.

10. Process according to any one of claims 1 to 9, characterized in that the content of hyaluronic acid in the aqueous gel of hyaluronic acid obtained in step a) is between 1 mg / ml and 300 mg / ml. preferably between 75 and 200 mg / mL, more preferably between 100 and 175 mg / mL.

11. Process according to any one of claims 4 to 10, characterized in that the crosslinking agent is chosen from di-functional epoxides, multifunctional epoxides, bi or polyfunctional esters, divinylsulfones, carbodiimides and formaldehyde. dialdehydes and mixtures thereof, and preferably the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

12. Method according to any one of claims 4 to 11, characterized in that the crosslinking agent is introduced in an amount of between 10 mg and 250 mg per gram of linear hyaluronic acid introduced in step i.

13. Process according to any one of claims 5 to 12, characterized in that the hyaluronic acid salt is chosen from a sodium salt, a calcium salt, a zinc salt and a potassium salt, preferably a sodium salt.

14. Method according to any one of claims 1 to 13, characterized in that the aqueous gel is purified before or after step b) homogenization by rolling, said purification being preferably carried out by dialysis.

15. Method according to any one of claims 1 to 14, characterized in that linear hyaluronic acid is added before or after step b) homogenization by rolling, step c) of neutralization (dilution) or the purification step previously described.

16. Method according to any one of claims 1 to 15, characterized in that the aqueous gel prepared is injectable.

17. An aqueous gel of hyaluronic acid obtainable by the method according to any one of claims 1 to 16.

18. Aqueous gel according to claim 15 for its medical use for the repair or reconstruction of tissues.

19. Cosmetic use of an aqueous gel according to claim 17 in the repair or reconstruction of tissues, in particular for filling wrinkles and fine lines.