EP2094736A1

EP2094736A1 - Cross-linked hyaluronic acid and method for producing the same

Info

Publication number: EP2094736A1
Application number: EP07866489A
Authority: EP
Inventors: Jérôme ASIUS; Nicolas Riviere; Bénédicte ASIUS
Original assignee: Stiefel Laboratories Inc
Current assignee: Sinclair Pharmaceuticals Ltd
Priority date: 2006-11-10
Filing date: 2007-10-25
Publication date: 2009-09-02
Also published as: CN101611063B; CN101611063A; AU2007316520A1; BRPI0718577A2; KR20090109084A; AU2007316520B2; FR2908415A1; RU2456299C2; JP2010509425A; KR101478849B1; US20090263447A1; FR2908415B1; MX2009004969A; CA2668650A1; JP5389661B2; RU2009120214A; CA2668650C; WO2008056069A1

Abstract

The invention relates to a cross-linked hyaluronic acid that can be produced by a method comprising: (a) activating a hyaluronic acid; (b) reacting the activated hyaluronic acid with a cross-linking agent containing an oligo- or polypeptide in a reaction medium at a pH of between 8 and 12 in order to obtain a cross-linked hyaluronic acid; (c) adjusting the pH of the reaction medium to a value between 5 and 7; and (d) precipitating the cross-linked hyaluronic acid in an organic solvent. The invention also relates to the above method, to a hydrogel obtained from said cross-linked hyaluronic acid, and to the use of the cross-linked hyaluronic acid for the production of implants that can essentially be used in plastic surgery.

Description

Crosslinked hyaluronic acid and process for its preparation

The present invention relates to a novel crosslinked hyaluronic acid and its method of preparation and uses, especially cosmetics.

Hyaluronic acid is a polysaccharide consisting of D-glucuronic acid units and N-acetyl-D-glucosamine units, which is particularly known for use in reconstructive or ocular surgery or in the aesthetic field as a filling product for wrinkles. In this latter application in particular, hyaluronic acid is preferred over other fillers because of its biocompatibility and physicochemical properties. However, it has the disadvantage of degrading quickly, thus requiring repeated injections. To overcome this disadvantage, it has been proposed various methods of crosslinking hyaluronic acid to make it less sensitive to various degradation factors such as enzymatic and / or bacterial attacks, temperature and free radicals and to improve its resistance to degradation in vivo and therefore its duration of action. These processes involve, in particular, etherification, esterification or amidification of the hydroxyl and / or acid functions of the native hyaluronic acid.

However, the methods for crosslinking hyaluronic acid, in particular by amidification, of the prior art have the disadvantage that they lead to hyaluronic acid derivatives which are difficult to formulate and syringes in an aqueous medium and / or insufficiently resistant to degradation factors, in particular after sterilization of the product.

This is the case of water-insoluble hyaluronic acid prepared according to application US 2001/0393369 by reaction in an acid medium of hyaluronic acid with an activating agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and a nucleophile which may be a polylysine.

It is believed that at pH values of less than or equal to 7, the expected amidification reaction is in competition with an intramolecular esterification reaction, which leads to a self-crosslinking of the primary alcohol carried by hyaluronic acid. on the activated hyaluronic acid ester. This parasitic reaction results in particular in a significant increase in the viscosity (caking) and an opacification of the reaction mixture which is thus in the form of a heterogeneous mixture of water and insoluble polymer. It becomes impossible to formulate the hyaluronic acid obtained.

In addition, the application EP-I 535 952 discloses a coating consisting of crosslinked hyaluronic acid formed in situ by reaction of a polylysine with hyaluronic acid in the presence of EDC and NHS at a pH of 2 to 9 and preferably from 4 to 7.5. The article provided with this coating may in particular be a prosthesis that can be used in cosmetic surgery. This document does not disclose crosslinked hyaluronic acid precipitated in an organic solvent in order to be available in dry form and susceptible thus to be formulated as a hydrogel extemporaneously.

Furthermore, US Pat. No. 6,632,457 discloses a modified hyaluronic acid prepared by reacting a primary amine with a carbodiimide-activated hyaluronic acid such as EDC and an N-hydroxysulfosuccinimide derivative such as NHS. at a pH of 7.0 to 8.5. The compound obtained can be cross-linked under physiological conditions, for example with glutaraldehyde, to obtain a hydrogel which remains sensitive to glycosidases and degrades substantially completely in less than 50 hours. This kinetics of degradation is compatible with the intended use as a vector of cells and growth factors but is not suitable for use as a filler in cosmetic surgery, for example.

Finally, the application WO 2006/021644 describes a process for preparing cross-linked hyaluronic acid by activating hyaluronic acid with a coupling agent such as EDC and a catalyst such as NHS, followed by a reaction with a polypeptide such as dilysine, at a pH of 4 to 10, for example from 4 to 6. The pH may optionally be raised, at the end of the reaction, to a value of 6 to 7 to increase the extraction yield during the reaction. precipitation phase. Thus, either the crosslinking is carried out in an acidic medium which is then optionally neutralized, or it is carried out in basic medium without subsequent modification of pH.

The Applicant has discovered that the use of a The acidic pH during the reaction phase was not always favorable to the amidification reaction and could lead, as indicated previously, to parasitic reactions, in particular of intramolecular esterification, which may affect the physicochemical properties of the product obtained. .

There remains therefore the need to provide a crosslinked hyaluronic acid that can be obtained in dry form and then reformulated easily in aqueous medium to form a hydrogel having good physico-chemical properties, resulting in particular by an elastic modulus G and a loss angle delta less than 30, which hydrogel is itself capable of being subjected to a heat treatment, in particular of sterilization, in order to be used for the manufacture of an implant which is itself sufficiently stable with respect to the different degradative factors such as enzymatic and / or bacterial attacks, temperature and free radicals to not fully resorb in vivo in less than 4 months.

However, the Applicant has discovered quite fortuitously that the pH of precipitation in an organic solvent of hyaluronic acid crosslinked with a polypeptide determined its rheological properties and its sensitivity to degradation factors such as temperature, the free radicals and enzymes such as hyaluronidases. Following multiple experiments, the Applicant then identified the optimal precipitation conditions for obtaining a crosslinked hyaluronic acid insensitive to thermal degradation, that is to say, retaining its properties. rheological properties after redissolution of the precipitated compound and sterilization. Everything happens as if reticulated hyaluronic acid once reformulated a "memory" of its molecular organization at the time of precipitation. It has furthermore been demonstrated that this molecular arrangement furthermore influences the ability of the polymer to resolubilize.

Without wishing to be bound by this theory, it is believed that the aforementioned method makes it possible to densify and solidify the macromolecular network of hyaluronic acid not only by means of covalent bonds with the crosslinking agent, but also by means of ionic interactions and / or or hydrogen bonds developing at the moment of precipitation.

The present invention therefore relates to a crosslinked hyaluronic acid, obtainable by a process comprising:

activation of a hyaluronic acid using a coupling agent and a coupling aid, to obtain an activated hyaluronic acid,

reacting the activated hyaluronic acid with a crosslinking agent comprising at least 50% by weight of oligonucleotide or polypeptide, in a reaction medium adjusted to a pH of 8 to 12, to obtain a crosslinked hyaluronic acid,

adjusting the pH of the reaction medium to a value ranging from 5 to 7,

precipitation of the crosslinked hyaluronic acid in an organic solvent to obtain reticulated hyaluronic acid fibers, and optionally, drying the crosslinked hyaluronic acid fibers obtained.

The crosslinked hyaluronic acid obtained according to the invention is soluble in water. By this expression, it is meant that 1 g of the dehydrated fibers obtained as described above are disintegrated in a few minutes and solubilize completely in one liter of saline solution after a few hours, without stirring.

The hyaluronic acid used in the above process is generally used in the native state, that is to say as naturally occurring in a living organism or excreted by the bacteria during its production by bacterial fermentation. It thus generally has a molecular weight ranging from 500,000 to 7,000,000 Daltons and is usually used in the form of sodium salt.

Hyaluronic acid is activated before crosslinking, using a coupling agent and a coupling aid.

Examples of coupling agents are water-soluble carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide (ETC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). cyclohexyl-3- (2-morphilinoethyl) carbodiimide (CMC) as well as their salts and mixtures thereof. EDC is preferred for use in the present invention.

Examples of coupling aids are N- hydroxy succinimide (NHS), N-hydroxy benzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole

(HAt) and N-hydroxysylfosuccinimide (sulfo NHS) and mixtures thereof. Without being limited to the choice of NHS, this is preferred for use in the present invention.

The role of the agent and the coupling aid is illustrated in Example 1 below.

According to the invention, it is preferred that the molar ratio of the coupling agent to the carboxylic acid units of hyaluronic acid be between 2% and 200%, more preferably between 5% and 100%.

In addition, the molar ratio of the coupling aid to the coupling agent is advantageously between 1: 1 and 3: 1, preferably between 1.5: 1 and 2.5: 1 inclusive, and more preferentially equal to 2.

The activation reaction of the hyaluronic acid by the coupling agent can be carried out at a pH ranging for example from 3 to 6, preferably from 4 to 5.

The concentration of hyaluronic acid in the reaction medium is for example between 0.1 and 5% by weight, for example between 0.1 and 1% by weight, inclusive.

The crosslinking agent comprises at least 50% by weight, and is advantageously constituted of an oligo- or polypeptide which can be a homo- or copolypeptide statistic, block, segmented, grafted or star. The crosslinking agent is generally in salt form and in particular hydrochloride or optionally hydrobromide or trifluoroacetate, in particular.

Examples of polypeptides that can be used in the present invention are homo- and copolymers of lysine, histidine and / or arginine, in particular polylysines having at least two or even at least five lysine units, such as dilysine, polyhistidines and polyarginines, without this list being exhaustive. These amino acids may be in D form and / or L form. Dilysin and its salts and derivatives are preferred for use in the present invention.

According to the invention, it is preferred that the number of amine functions of the polypeptide involved represents from (1 to 100%), preferably from 10 to 50%, of the number of carboxylic acid functions of the hyaluronic acid involved.

In a first preferred variant of the invention, the coupling agent is used in stoichiometric amount relative to the amine functions of the crosslinking agent. In this way, at the end of the first step of the process according to the invention, the amount of carboxylic acid functions of hyaluronic acid that are activated is equal to the amount of amine functions that will be added to the second step.

In a second variant of the invention, the agent of coupling is carried out in stoichiometric amount with respect to the carboxylic acid functions of hyaluronic acid. In this case, at the end of the first step of the process according to the invention, all the carboxylic acid functions of the hyaluronic acid are activated and the quantity of crosslinking agent used in the second step may, for example, be less than 30%, better still, less than 10%, or even about 5% (in number of moles of crosslinking agent relative to the number of moles of carboxylic acid functions).

The crosslinking reaction is generally carried out under temperature conditions and for a time entirely conventional for those skilled in the art, for example at a temperature of 0-45 ⁰ C, preferably 5- 25 ° C for 1 to 1Oh, preferably 1 to 6h. To promote the formation of amide bonds, the pH of the reaction is between 8 and 12 and preferably between 8 and 10 (limits included). This pH can be adjusted using any base, preferably a weakly nucleophilic base such as an amine such as diisopropylethylamine (DIEA).

This reaction is usually carried out in a solvent such as an aqueous solution of sodium chloride.

The concentration of hyaluronic acid in the reaction medium is, for example, between 0.01 and 5% by weight, for example between 0.1 and 1% by weight, limits included.

After reaction, the pH of the reaction medium is adjusted to a value ranging from 5 to 7 and preferably from 5.5 to 7 with the aid of any acid such as hydrochloric acid, before the crosslinked hyaluronic acid obtained is precipitated. The precipitation step is carried out in an organic solvent such as ethanol, isopropanol, ether or acetone, or mixtures thereof, for example, ethanol being preferred in this invention. The solvent is advantageously used in an amount representing from 5 to 20 times, for example approximately 10 times, the volume of reaction medium.

A possible drying step is then preferably carried out, so as to obtain a dehydrated form of crosslinked hyaluronic acid which is easier to handle and is better preserved. The preservation can in particular be carried out in negative cold.

The invention also relates to the process for producing a crosslinked hyaluronic acid, as described above.

This process may also comprise other steps than those explicitly mentioned and in particular a step of mixing said dehydrated crosslinked hyaluronic acid with an aqueous solvent, such as a sodium chloride solution, a physiological saline solution or an injectable buffered solution ( especially a saline solution of phosphate buffer), to form a hydrogel. The concentration of hyaluronic acid in said hydrogel may range from 1 to 4% and preferably from 1.5 to 3% w / v. The invention therefore also relates to such a hydrogel, containing a crosslinked hyaluronic acid as described above, in an aqueous solvent.

The hydrogel thus obtained has, after sterilization, for example at 118 ^° -10 ^° C. for 2 to 30 minutes, according to the invention, an elastic modulus G 'of at least 100 and for example between 200 and 600 Pa, terminals included, and a variation of its elastic modulus of less than 30%, and preferably less than 20%, after steaming at 93 ° C for 1 hour. It also advantageously has a viscous modulus G '' ranging from 50 to 200 Pa; a loss angle δ [= Inv tan (G '' / G ')] ranging from 15 to 35 ° and a viscosity η ranging from 1000 to 3000 Pa. s. The measurement of the elastic modulus, the viscous modulus and the loss angle can be carried out in the following manner: the hydrogel is treated with a 4cm, 4 ° cone-plane geometry at a temperature of 25 ° C. It undergoes a non-destructive viscoelastic test at IHz, with an imposed deformation of 1%. The measurement of the elastic modulus is carried out using a rheometer type AR 1000 from TA Instruments. The same apparatus can be used to measure the viscosity using a shear rate of 50 ° ^{~ 2} sec ^{~ x} .

The invention therefore also relates to a sterilized hydrogel containing hyaluronic acid crosslinked by a crosslinking agent containing at least 50% by weight of oligo- or polypeptide, characterized in that it has a variation of its elastic modulus of less than 30% after stoving at 93 ° C for 1 hour. This hydrogel is advantageously used for the manufacture of implants.

These implants can in particular be injected subcutaneously (hypodermic) or intradermally into the fibrous tissue.

They may contain, in addition to the aforementioned hydrogel, a carrier fluid comprising at least one polysaccharide, for example at least one cellulose derivative such as carboxymethylcellulose and / or at least one glycosaminoglycan such as sodium hyaluronate and / or particles of a biocompatible bioresorbable material such as polylactic acid (PLLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), tricalcium phosphate (TCP), hydroxyapatite (PAHs), and mixtures thereof.

Examples of such implant minerals containing them are described in particular in WO 2004/069090.

The implants according to the invention are bioabsorbable, in that they are capable of degrading in the body in 6 to 18 months.

They can in particular be used for: the supplementation of a hyaluronic acid-deficient cavity or organ (typically in dermatology, aesthetic medicine or in orthopedic treatments); the reconstitution of an effused volume during surgical procedures (typically in ocular surgery), or

topical application to the healthy or injured dermis (typically in cosmetology and dermatology).

The aforementioned implant is particularly well suited for use in filling facial wrinkles and fine lines and / or scars of the human body. The present invention therefore also relates to the use of crosslinked hyaluronic acid as described above for the manufacture of injectable implants intended for use in cosmetic and / or restorative surgery, for the manufacture of fillers, especially wrinkles, fine lines, scars or skin depressions such as lipodystrophies.

The invention will now be illustrated by the following nonlimiting examples.

EXAMPLES

Example 1 Synthesis of Hyaluronic Acid Crosslinked with a Polypeptide According to the Invention

1. Reaction scheme

The reaction scheme followed can be illustrated in the following manner (taking the dilysine as an example):

Hyaluromic acid reticle

The crosslinking reaction (scheme 1) consists of a double peptide coupling between the carboxylic acid functions of two hyaluronic acid chains and the amine functions of dilysine. The coupling reagents used are 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

The mechanism of the coupling reaction can be illustrated as follows:

The first step consists of a nucleophilic attack of the carboxylic acid function of hyaluronic acid on the carbodiimide function of the coupling agent EDC. The resulting O-acylurea is then substituted with NHS to form a more stable activated ester (production of 1-ethyl-3- (3-dimethylaminopropyl) urea). Indeed, O-acylurea can rearrange into inert N-acylurea in a slightly acidic aqueous medium and during a long reaction time. The last step consists finally in the nucleophilic attack of one of the amine functions of the dilysine (preferably terminal, sterically favored) on the activated ester in order to form an amide bond with NHS release.

2. Protocol

^1st stage: swelling phase

In a 500 ml glass reactor, 3 g of sodium chloride are successively introduced into 300 ml of milliQ water. After dissolving the sodium chloride with the sonicator, 2 g of hyaluronic acid (HTL Sari, batch) are introduced into the reactor containing the saline solution.

No. PH 1016, Mw = 2.6 × 10 ⁶ daltons, hereinafter HA), taking care to fray the HA fibers as much as possible by hand. After stirring the heterogeneous medium with a spatula for 1 minute, the reactor is placed at 4 ° C. for 15 hours without stirring and covered with aluminum foil to protect the reaction medium.

2 ^nd step: cure phase

The reaction mixture is removed from the refrigerator and stirred at room temperature (18-25 ° C) for 10 minutes, (visually the solution should be perfectly clear and homogeneous having a certain viscosity, such as fluid honey).

The stirring used is of mechanical type with a stirrer in the form of a half-moon teflon. The rotation speed is 60 rpm.

A solution of 464 mg (4.03 mmol) of N-hydroxysuccinimide (ACROS, purity 98%, hereinafter NHS) is then prepared in 5 ml of milliQ water in a hemolysis tube and then vortexed to dissolve the totality of the NHS. This solution is added to the reaction medium dropwise at 5 ml / mm.

The mixture is left stirring for 5 minutes, and then a solution of N- (3-dimethylaminopropyl) -N-ethylcarbodumide hydrochloride (313 mg, 2.02 mmol) (Sigma-Aldrich, ref 03450-5G, cf. after EDC) in 4 ml of milliQ water. The solution is vortexed and then added dropwise at a rate of 5 ml / mm.

The mixture is left stirring for thirty minutes and then the aqueous solution of dilysme is added to the reaction medium at the rate of 1 ml / mm. This solution is prepared by vortex solubilizing in 1 ml of water rruiliQ, 233 mg (0.67 mmol) of dilysme hydrochloride (BACHEM supplier, ref G2675), then 1302 μl f 10, 08 mmol) of propylethylamine (supplier ACROS ref 115225000, hereinafter DIEA), all in a hemolysis tube. This mixture has two distinct phases forming a reversible emulsion after vigorous stirring. It is attempted to mix the emulsion as much as possible while it is added to the reaction medium. The pH of the reaction medium must be between 8.5 and 10.5.

The whole is left stirring for 3h.

^3rd step: purification phase

After stopping the stirring, the pH of the solution is adjusted before precipitation with 1M HCl to reduce it to a pH of 5.7.

Then a reactor of a liter capacity, equipped with a mechanical stirrer and a stirring rod in the form of a rake is prepared. 420 ml of ethanol at 95 ° are poured into this reactor and the mechanical stirring is started at a very high speed (approximately 1000 rpm).

42 ml of reaction mixture containing the crosslinked hyaluronate is then aspirated with a 50 ml syringe and then fed continuously into the reactor. The solution should be clear, colorless and fairly viscous.

When the addition is complete, stirring is continued for two more minutes. The stirring rod is then removed from the reactor and the resulting polymer is unrolled on a porosity II frit using a pair of pliers. The polymer is rapidly dried on a vacuum flask for up to 15 seconds and then allowed to dry in a desiccator under vacuum for a minimum of 12 hours. The final product must be perfectly white.

^4th stage: stage reformulation

To prepare 10 ml of 2.4% gel, 240 mg of crosslinked and dried polymer are introduced into a standard polypropylene syringe equipped with a stopper (at the outlet of the syringe). 10 ml of buffered solution ** are then added to the solid, and the whole is allowed to swell at 4 ° C. for 12 to 15 hours.

After removing the syringe from the refrigerator, the product is rapidly stirred with mechanical agitation at a speed of 1000 rpm. The stirring rod used is a laboratory spatula shaped like a stainless steel spoon. The stirring time is about 5 minutes for this product, but varies according to the viscosity. The final gel must be colorless and perfectly homogeneous.

Example 2: Degradation or persistence test

Principle: The skilled person is accustomed to practice accelerated degradation tests predictive of the resistance of a polymer to the various degradation factors in vivo (see in particular FR 2861 734).

In this example, one of these tests, which consists in measuring the rheological characteristics of previously sterilized crosslinked products then having undergone a heating phase at 93 ° C. for one hour, was implemented. The percentage of loss of the module is then calculated elastic (G ') during heating. The lower this percentage, the better the product is heat resistant and is considered to be able to withstand other degradation factors as well. This test is therefore predictive of the degradation rate in vivo hyaluronic acid reticle and therefore the duration of filling wrinkles that can be obtained.

Tested products: All tested products are sterile products.

Several commercial products have been tested, as well as:

the product 1 which was a hyaluronic acid obtained as described in Example 1, and the product 2 which was a hyaluronic acid obtained as described in Example 1, except that 45% mol of EDC was used; 90 mol% of NHS; and 15% mol of dilysme, based on the number of moles of COOH units of hyaluronic acid, and a DIEA / NHS ratio of 2.22.

Results:

Table 1 below summarizes the results obtained for the various tested reticulated hyaluronic acids.

Table 1 Degradation test of crosslinked hyaluronic acids

It can be seen from this table that the modified hyaluronic acids according to the invention exhibit a lower drop in their elastic modulus than the commercially available crosslinked hyaluronic acids, which shows that they are more resistant to degradation factors.

Example 3 Influence of the pH of precipitation

The physico-chemical properties of synthesized crosslinked hyaluronic acids were compared substantially as described in Example 1 and precipitated at different pH in ethanol. The parameters of the methods of synthesis of these compounds are collated in Table 2 below:

Table 2 Synthetic parameters of crosslinked hyaluronic acids

* relative to the number of moles of carboxylic acid functions of hyaluronic acid

The physico-chemical properties of the above products, once reformulated as described in Example 1, were evaluated before and after passage in an oven for one hour at 90 ^° C. The viscosity of the product was more precisely evaluated. hydrogel and measured its elastic modulus. The results obtained are summarized in Table 3 below:

Classes from 1 to 5 were used, which represent a synthetic note taking into account the elasticity and the viscosity of the gel. The more the gel is considered elastic, the higher the score. On the other hand, a non-homogeneous and / or fluid gel has a low score.

Table 3

Physico-chemical properties of crosslinked hyaluronic acids

It can be seen from this table that the cross-linked hyaluronic acids precipitated at basic pH, although they have been reformulated in the form of hydrogels, do not give satisfactory hydrogels for an application of wrinkle filler. This phenomenon is thought to be due insufficient development of ionic bonds during precipitation.

In addition, the cross-linked hyaluronic acids precipitated at a pH that is too acidic give hydrogels having good viscoelasticity (provided that they can be reformulated, which is not always possible), but which deteriorate markedly during the transition to and will be sensitive to endogenous degradation factors.

It appears in fact that only a pH of precipitation ranging from 5 to 7 makes it possible to easily formulate a homogeneous hydrogel having a very satisfactory viscoelasticity and which is not substantially reduced after a degradation test. This confirms that at this pH range, the macromolecular network formed by the electrostatic and covalent bonds is optimal for application as a filler.

Claims

A crosslinked hyaluronic acid obtainable by a process comprising:

1 / activation of a hyaluronic acid using a coupling agent and a coupling aid, to obtain an activated hyaluronic acid,

reacting the activated hyaluronic acid with a crosslinking agent comprising at least 50% by weight of oligo- or polypeptide, in a reaction medium adjusted to a pH of 8 to 12, to obtain a crosslinked hyaluronic acid,

adjusting the pH of the reaction medium to a value ranging from 5 to 7,

precipitation of the crosslinked hyaluronic acid in an organic solvent to obtain crosslinked hyaluronic acid fibers, and

optionally, drying the crosslinked hyaluronic acid fibers obtained.

2. Hyaluronic acid according to claim 1, characterized in that the coupling agent is chosen from: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide (ETC) and 1-cyclohexyl-3- (2-morphilinoethyl) carbodiimide (CMC) as well as their salts and mixtures thereof.

3. Hyaluronic acid according to claim 1 or 2, characterized in that the coupling aid is selected from: N-hydroxy succinimide (NHS), N-hydroxy benzotriazole (HOBt), 3,4-dihydro-3 -hydroxy-4-oxo- 1, 2, 3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt) and N-hydroxysylfosuccinimide (sulfo NHS) and mixtures thereof.

4. Hyaluronic acid according to any one of claims 1 to 3, characterized in that the molar ratio of the coupling agent to the carboxylic acid units of hyaluronic acid is between 5 and 100% inclusive.

5. Hyaluronic acid according to any one of claims 1 to 4, characterized in that the molar ratio of the coupling aid to the coupling agent is between 1: 1 and 3: 1 inclusive.

6. Hyaluronic acid according to any one of claims 1 to 5, characterized in that the reaction of activation of hyaluronic acid by the coupling agent is carried out at a pH ranging from 3 to 6.

7. Hyaluronic acid according to any one of claims 1 to 6, characterized in that the polypeptide is a homo- or copolymer of lysine.

8. hyaluronic acid according to claim I ₁ characterized in that the homopolymer of lysine is dilysine.

9. Hyaluronic acid according to any one of claims 1 to 8, characterized in that the coupling agent is implemented in a stoichiometric amount relative to the amine functions of the crosslinking agent.

10. Hyaluronic acid according to any one of claims 1 to 8, characterized in that the coupling agent is implemented in stoichiometric amount relative to the carboxylic acid functions of hyaluronic acid.

11. Hyaluronic acid according to claim 10, characterized in that the amount of crosslinking agent used in the second step is less than 30% by number of moles of crosslinking agent relative to the number of moles of acidic functions. carboxylic.

12. Hyaluronic acid according to any one of claims 1 to 11, characterized in that the crosslinking reaction is carried out at a pH of 8 to 10.

13. Hyaluronic acid according to any one of claims 1 to 12, characterized in that the precipitation pH is from 5 to 7.

14. Hyaluronic acid according to claim 1, wherein the organic solvent is ethanol or isopropanol.

15. A process for producing a crosslinked hyaluronic acid, characterized in that it is as described in any one of claims 1 to 14.

16. Hydrogel characterized in that it contains a crosslinked hyaluronic acid as described in any one of claims 1 to 14, in an aqueous solvent.

17. Sterilized hydrogel containing hyaluronic acid crosslinked by a crosslinking agent containing at least 50% by weight of oligo- or polypeptide, characterized in that it has a variation of its elastic modulus of less than 30% after a parboiling at 93 ° C for 1 hour.

18. Use of crosslinked hyaluronic acid according to any one of claims 1 to 14 for the manufacture of injectable implants intended for use in cosmetic and / or reconstructive surgery, for the manufacture of fillers, in particular wrinkles, fine lines, scars or skin depressions.