WO2006021644A1

WO2006021644A1 - Water-soluble crosslinked hyaluronic acid, a method for the preparation thereof, implant containing said crosslinked hyaluronic acid and the use thereof

Info

Publication number: WO2006021644A1
Application number: PCT/FR2005/001808
Authority: WO
Inventors: Jérôme ASIUS; Bénédicte ASIUS; Olivia Giani; Nicolas Riviere; Jean-Jacques Robin; Koumalen Vayaboury
Original assignee: Asius Jerome; Asius Benedicte; Nicolas Riviere; Olivia Giani; Jean-Jacques Robin; Koumalen Vayaboury
Priority date: 2004-07-23
Filing date: 2005-07-12
Publication date: 2006-03-02
Also published as: FR2873379B1; AR050010A1; FR2873379A1

Abstract

The invention relates to a crosslinked hyaluronic acid exhibiting a water-solubility and obtainable by a preparation method consisting in bringing into contact at least one type of hyaluronic acid or the salts thereof and at least one type of cross-linking agent containing at least one type of oligopeptide or polypeptide having at least two, preferably at least five lysine units, in an aqueous medium, wherein said contact operation is carried out in the presence of at least one type of hydrosoluble coupling agent and at least one catalyst. An implant containing the inventive crosslinked hyaluronic acid and the use thereof for human medicine and veterinary science, in particular for repair or aesthetic surgery is also disclosed.

Description

RETICULATED HYALURONIC ACID SOLUBLE IN WATER, SOUND

PREPARATION METHOD, IMPLANT CONTAINING SAID ACID

RETICULATED HYALURONIC AND USE THEREOF

The invention relates to a crosslinked hyaluronic acid, generally soluble in water, a process for preparing said crosslinked hyaluronic acid, and an implant containing said crosslinked hyaluronic acid. Said cross-linked hyaluronic acid is intended for use in human or veterinary medicine, and in particular in mainly reconstructive and / or aesthetic surgery.

Hyaluronic acid is a polysaccharide which has the advantage of being in the same chemical form whatever its source. It consists of an alternation of monosaccharide units of N-acetyl-D-glucosamide and D-glucuronic acids linked by β 1-3 glycolide linkages. Hyaluronic acid is usually found in the form of a sodium hyaluronate gel. The molecular weight of _; Hyaluronic acid is generally in the range of 500,000 to 7 million g / mol and even beyond. The structural formula of hyaluronic acid is given below, where n is generally from 1,200 to 18,000. This corresponds to a range of hyaluronic acid of 500,000 to 7,000,000 g / mol. The end groups are as known to those skilled in the art.

Hyaluronic acid

Hyaluronic acid is present in the human body, in the vitreous humor in high concentration / in the soft tissues where it forms one of the major components of the extracellular matrix, in the hyaline cartilage and in the synovial fluid where it plays. the role of lubricant and damper against shocks, as well as in the dermal and epidermal tissue where it plays a role of hydration and contributes to the elasticity of the tissues.

Hyaluronic acid can also be obtained by several methods, among which mention can be made of extraction from cock crest or production by bacterial fermentation. These two origins each have advantages and disadvantages. Indeed, hyaluronic acid extracted from the rooster crest has very high molecular weights but contains animal proteins responsible for allergenic phenomena. In contrast, hyaluronic acid obtained by bacterial fermentation (streptococcus) has lower molecular weight even if it is devoid of animal protein.

The applications of hyaluronic acid in several fields have been widely referenced, for example in the medical field, for example in ocular surgery, in rheumatology, or in pharmacology, or in the surgical and dermatological field (restorative and / or aesthetic surgery). ), or in the field of cosmetics. Indeed, its ease of injection and its safety of use, but especially its biocompatibility and its lack of toxicity, as well as its physicochemical properties, make it a compound of choice in various biomedical applications. Sodium hyaluronate gels are particularly popular and widely used in eye surgery. Such gels remarkably exhibit viscoelasticity of interest in cataract surgery procedures. These gels are used in dermatology and plastic surgery in the ^'treatment of aging skin as product filling, but - the drawback to deteriorate rapidly, hence the need to address repeated injections.

Hyaluronic acid concentrations and quality in various tissues of the body have been shown to decrease over the course of life, either naturally or through external factors such as sun exposure. or an inflammatory pathology. For example, in the problems of knee osteoarthritis (inflammatory pathology of the knee), it has been observed a decrease in the concentration and the molecular weight of the hyaluronic acid present in the synovial fluid. This phenomenon is explained partly by a fall in the endogenous synthesis of hyaluronic acid and secondly by the inflammation which generates free radicals responsible for the degradation of hyaluronic acid. ^• The hyaluronic acid supplementation gives excellent results, in terms of tolerance and efficiency, but remains very ephemeral. Indeed, hyaluronic acid is very sensitive to degradation by various factors such as thermal, enzymatic, radical and / or bacterial factors. This is why the various chemical modifications of hyaluronic acid known today, which are mainly cross-links consolidating its three-dimensional network with respect to enzymatic attacks, are intended to make available to the patient. medical body a modified hyaluronic acid that retains its original properties by only improving its resistance to degradation in vivo. 1

Thus, it has been widely described in the literature different methods for synthesizing crosslinked derivatives of hyaluronic acid, most often either by etherification or by amidification. If the crosslinking is done by etherification, it is possible to use at least one crosslinking agent such as divinylsulfone, formaldehyde, a bis-epoxide, a bis-halogenated compound or an alcohol. If the crosslinking is by amidation, at least one crosslinking agent such as a functional amino acid or an oligomer or polymer having two or more amino groups can be used. In all cases, the state of the art refers to methods of preparation giving crosslinked products insoluble in water. the

Whatever the method used, the degree of crosslinking and the choice of the crosslinking agent (more or less hydrophilic) determine the insolubility of the gel in water. Thus, patent application WO 01 / 58,961 'describes a process for crosslinking hyaluronic acid with a bifunctional mono-amino acid, including L-Lysine. But the bonds thus established, by crosslinking between hyaluronic acid and amino acid, mainly by amidation of the amine side chain linkage, are too weak to withstand for a long time the enzymatic hydrolysis phenomena that occur in vivo. In addition, crosslinking agents such as L-Lysine have only a limited number of reactive amino groups, which limits the formation possibilities of the desired network. •

The application ^{^'WO'} 00/46 252 discloses a hyaluronic acid cross-linking process and at least one other polymer. This crosslinking process involves at least two different functional groups of said polymer and the hydroxyl and / or carboxyl functions of hyaluronic acid. Among a numerous list of said polymers, the polypeptides are generally mentioned without identifying the precise nature of these polypeptides. However, with the exception of poly-L-lysine which is not mentioned in said patent application, the polypeptides generally have no side chain amine group, and are not likely to cross-link. hyaluronic acid by amidification.

The patent application WO 02/30990 describes a method of crosslinking hyaluronic acid and of the

minus another polymer having two or more amino groups giving insoluble products in water. This crosslinking process involves said two amino groups of said polymer and the carboxyl function of hyaluronic acid. Among said polymers, the peptides are generally mentioned, without indicating the possibility that said polymer is oligo-L-lysine. Of the polymers mentioned, only chitosan is exemplified. Finally, the crosslinked hyaluronic acids of these three patent applications WO 01 / 58.961, WO 00 / 46.9252 and WO 02/30990 have the disadvantage of being insoluble in water and very sensitive to degradation by the bacterial route. The object of the present invention is to overcome the drawbacks of known products based on crosslinked hyaluronic acid. ^'In particular, these drawbacks are too short life, difficulty of resetting said crosslinked hyaluronic acid aqueous solution or a high bacterial sensitivity. The invention remedies mainly by the development of an original preparation process which leads to original crosslinked hyaluronic acids, both in terms of homogeneity in aqueous solution and possible resistance to bacterial growth.

The invention therefore relates to a crosslinked hyaluronic acid having a solubility in water and obtainable by a preparation process comprising bringing into contact with the aqueous medium at least one hyaluronic acid or a salt thereof, and at least one crosslinking agent comprising at least one oligopeptide or polypeptide having at least two, preferably at least one at least five, more preferably at least six lysine units, said contacting being carried out in the presence of at least one water-soluble coupling agent, and at least one catalyst. The contacting reaction medium is generally and preferably regularly stirred.

In a preferred embodiment, the water-soluble coupling agent is selected from water-soluble carbodiimides, preferably from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3 - (3-trimethylaminopropyl) carbodiimide (ETC) and 1-cyclohexyl-3- (2-morphilinoethyl) carbodiimide (CMC), their derivative salts and mixtures thereof. In a preferred embodiment, independent or not of the previous embodiment, the catalyst is chosen from amidation catalysts for the formation of activated esters, preferably in the group formed by N-Hydroxy Succinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzzo triazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt), and N-hydroxysulfosuccinimide (Sulfo-NHS), and mixtures thereof.

The lysine units of the crosslinking agent are generally and most often in salt form (such as hydrobromide, hydrochloride, trifluoroacetate, etc.) so that they can be substantially completely soluble in water.

The hyaluronic acid or one of its salts used for this crosslinking phase is generally and preferably in the native state (that is to say, as it is present in the body in the state physiological and ^' or that it is excreted by the bacteria during its production by bacterial fermentation), that is to say unmodified. Said hyaluronic acid used in the process of the present invention generally has a molecular weight of 500,000 to 7,000,000 daltons, preferably 1,000,000 to 5,000,000 daltons. IT 'is most often used in the form of sodium hyaluronate. concentration of 0.1 to 5% by weight, preferably 0.5 to 3% by weight. Several hyaluronic acids of different source or concentration can be used for contact with the crosslinking agent, although a single source of hyaluronic acid is generally preferred. By "one of its salts" is meant according to the invention a salt of hyaluronic acid such as sodium hyaluronate whose acid function has been protected by a sodium chloride.

The crosslinking agent comprising at least one oligopeptide or polypeptide having at least two, preferably at least five, lysine units is generally and preferably polylysine, preferably in the form of a polylysine salt, such as a trifluoroacetate. polylysine, a polylysine hydrobromide, or a polylysine hydrochloride. Advantageously, such a salt is water-soluble. The lysine units of polylysine put

^• in game may be in the form of levorotatory (L-Lysine or Poly-L-Lysine) or in the form of dextrorotatory (D-Lysine or Poly-D-Lysine), the levorotary form being the one most often considered because of its presence in nature. The formula of polylysine is given below, where m is generally from 2 to 50. The formula of a lysine unit, it corresponds to m = 1. The terminal groups of this polylysine are: -COOH at one end and -NH ₂ at the other end or in the form of their salts.

The formula of cross-linked hyaluronic acid

^• according to the invention is partially given below, where n is generally from 1,200 to 18,000 and m is generally from 2 to 50 and preferably from 2 to 15 Polylysine is, by another of its groups - NH _2, connected 'to a carboxylic function of a unit of hyaluronic acid / giving an amide bond (not shown' in the formula ¹ below). At the same number of moles used, the more the number of lysine units increases, the more the final product is crosslinked. If a polylysine having (n) lysine units is selected, it has (n + 1) amine groups capable of reacting. In general, it is one of the pendant amino functions (see that located in epsilon, on the pendant chain) which has a higher reactivity than that located on the alpha carbon of the function polylysine acid (see diagram below). To simpify, we speak here and only exemplifies polylysine but this applies more generally for a crosslinking agent according to the invention such as a polymer comprising lysine units.

Crosslinked hyaluronic acid

Another representation of said crosslinked hyaluronic acid may be:

HA - [poiyLys] - HA

I I

[HA] _χ *

Where *: hyaluronic acid molecules capable of reacting with one or more amino functions remaining free of polylysine; HA: hyaluronic acid unit; and polylys = polylysine.

Preferably, said crosslinking agent comprises at least 50% by weight of said oligopeptide or polypeptide.

The polypeptides may be random, block, segmented, grafted, star-shaped lysine homopolypeptides or copolypeptides; or block copolymers of which at least one of the polymers is of peptide structure based on lysine, i.e. is an oligopeptide or polypeptide comprising at least 50% by weight of lysine units.

According to a preferred embodiment of the invention, said oligopeptide or polypeptide is based on polylysine. It is thus understood according to the invention that said oligopeptide or polypeptide comprises at least 50% by weight of polylysine.

Advantageously according to the invention, the crosslinking agent has biocompatibility, biodegradability and optionally bacteriostatic properties.

Poly-cationic oligopeptides are recognized for their bacteriostatic power on Grand¬ and Gram- germs. For example, polylysine is bacteriostatic at concentrations below 5 μg / ml (Liang, JF & Kim, SC. Not only does the nature of the peptide but also the characteristics of the cell membrane determine the antimicrobial mechanism of a peptide. J. Peptide Res. , 1999, 53, 518-522; Nicholas Delihas Lee W.Riley Winnie Loo _Dr. Jonathan Berkowitz _Dr. Natalia Poltoratzskaia. High sensitivity of mycobacterium species to the bactericidal activity by Polylysine. Fems Microbiology Letters 132 (1995) 233-237). These biocompatibility properties and these advantageous bacteriostatic properties can be found advantageously during the synthesis of. hyaluronic acid crosslinked with these agents. Such bacteriostatic properties are generally verified by implementing the ^'test test, known in the art and ^briefly explained below.

The general principle of the "challenge test" is to contaminate voluntarily with a determined rate, a matrix (raw material, an implant, a hydrogel, etc.) using a previously selected inoculum (homogeneity and choice of strains). ). The microorganism inoculated is investigated and monitored over time.

Preferably, the crosslinking agent has bacteriostatic properties. In particular, it generally has NH ₃ ⁺ functions (from groups

NH ₂ ) responsible for the bacteriostatic activity vis-à-

^• screws Gram + and Gram- germs.

In particular, the polylysine advantageously has bacteriostatic properties (at the concentrations and at the pH used in the reaction medium) which may be of interest during the process for the preparation of crosslinked hyaluronic acid according to the invention, which makes it possible to limit any bacterial contamination of the reaction medium.

In addition, polylysine has excellent tolerance to the body compared to commonly used agents. Indeed, the oral LD50 in rats according to the "material safety data sheet" gives the following elements for various potential cross-linking agents of hyaluronic acid. The LD 50 (Lethal dose 50) corresponds to the quantity of a given substance administered which will cause death in 50% of animals tested.

The contacting according to the invention is generally primarily an amidation reaction, which is the intended reaction. By "bringing into contact" is meant according to the invention, the creation of ionic and / or hydrogen bonds. By "reacting" is meant according to the invention the creation of covalent bonds.

Advantageously according to the invention, the crosslinking agent comprises several cationic units which intervene to establish covalent bonds with hyaluronic acid by amidification and non-covalent bonds (hydrogen, ionic) with the same acid

- hyaluronic. A network is then formed by chemical and physical crosslinking reactions. It is the presence of at least three amine groups in each of the polylysine-based polymers capable of generating these amidification reactions, which makes these compounds quite unique within this family of polypeptides. Thus, the crosslinking with an oligomer or a polymer (oligopeptide or polypeptide), containing at least two lysine units, and preferably based on polylysine, involves only one type of functional group, the amine functions. In addition, at least 50% more covalent or ionic reactions occur than with lysine, which makes the thus created network much more resistant to enzymatic hydrolysis phenomena that occur in vivo. Finally, the number of reactions is maximized so as to create a true three-dimensional network while maintaining solubility properties in water, virtually identical to those of native hyaluronic acid.

The different work by the Applicant Company on the cross-linked hyaluronic acid of the invention preferably have determined firstly an ideal ratio between the number of amino ^functions' polylysine involved and the number of carboxylic functions hyaluronic acid capable of reacting; and on the other hand a ratio between the number of equivalents involved with polylysine and the number of catalyst equivalents.

Thus, the number of amino functions involved in the oligo or polypeptide must generally represent from 2 to 7% of the number of carboxylic functions of the hyaluronic acid likely to react. In addition, the number of polylysine equivalents involved must generally represent from 1 to 8 times and preferably from 3 to 7 times the equivalent number of catalyst.

Whatever the size of the polylysine involved, it is generally important to obtain the crosslinked hyaluronic acid according to the invention to maintain the ratio between the number of amino function and carboxyl function number and the ratio between the number of catalyst equivalent and the carboxyl function number of hyaluronic acid.

By "cross-linked hyaluronic acid" is meant according to the invention a molecular network consisting of hyaluronic acid molecules ^• linked together by covalent and / or ionic bonds. This three-dimensional network thus formed is advantageously dense according to the invention, and therefore resistant to various degradation factors. Indeed, the network formed by the crosslinked hyaluronic acid is made particularly dense, which consequently advantageously makes it less sensitive to various in vivo degradation factors such as thermal factor, enzymatic factor, bacterial factor and. oxidation, and different non-specific enzymatic factors of hyaluronic acid such as peptidases, transglutaminase, etc.

Indeed the steric hindrance of the network thus formed does not allow these various factors to easily access the internal structure of the network. This chemical modification between the hyaluronic acid, generally in the native state, starting material and the crosslinked hyaluronic acid according to the invention can be quantified at equivalent concentration by comparative rheological measurements between the native state of hyaluronic acid and the same crosslinked hyaluronic acid. Thus, the viscous modules, the elastic modules, the angle of loss (giving the strength of the gel thus obtained) and its viscosity are measured at a given shear rate. This is done according to the following protocol:

The cross-linked hyaluronic acid is dissolved at a concentration of 2.4% w / v in a liquid such as physiological saline (the skilled person is generally able to choose said liquid adequately). It is treated with a cone-plane geometry 4cm, 4 °, at a temperature of about 25 ° C. It first undergoes a non-destructive viscoelastic test at IHz, with a deformation imposed of 1%. The elastic module

(G '), the viscous modulus (G''), as well as _t the loss angle (δ) [= Inv Tan (G''/G')] can thus be measured.

After this viscoelastic test, the sample immediately undergoes at about 25 ⁰ C a shear gradient which allows to observe the evolution of the viscosity (η) as a function of shear. The measurements were all carried out using an AR 2000 type rheometer from TA Instruments. The values obtained for the crosslinked hyaluronic acid according to the invention are generally and preferably as follows: Elastic Module Viscous Module δ (Viscosity Angle η Pa.s G '(Pa) G''(Pa) loss) in ° (0.05 s ^"1 )

• 550 to 1000 100 to 200 10 to 20 2600 to 7000

Thus the crosslinked hyaluronic acid according to the invention has rheological characteristics, once dissolved, very different from hyaluronic acid (for which, by way of example, it is possible to give, under the same conditions of measurement, elastic modulus of 382, viscous modulus of 153, loss angle δ of 22 and viscosity η of 894). Nevertheless, the crosslinked hyaluronic acid according to the invention advantageously has numerous properties of the hyaluronic acid from which it is derived, namely, among other things, biocompatibility, solubility in water, and preservation in dehydrated form.

The catalyst ¹ is generally soluble in water.

The invention also relates to the method of preparation as described above and whose conditions are detailed below, which makes it possible to obtain the crosslinked hyaluronic acid according to the invention.

The contacting is generally carried out at a temperature of 0 to 45 ⁰ C, preferably 5 to 25 ⁰ C, for a period of 0.5 to 10 hours, preferably 1 to 6 hours, and a pH 4 to 10, preferably 4 to 8, more preferably 5 to 8. A pH range of 4 to 6 is also preferred.

Preferably, the pH of the reaction medium is generally maintained during the reaction of from 4 to 6 and more preferably from 5 to 6 in order to obtain advantageously the best compromise between the yield of the crosslinking and the less degradation of hyaluronic acid by acid hydrolysis. In fact, the lower the pH, the greater the kinetics of degradation. At the end of the reaction only, the pH of the reaction medium is optionally raised to a value of 6 'to 7 in order to increase the extraction yield during the precipitation phase.

The preparation process is generally carried out in the presence of a solvent, for example consisting of an aqueous solution of NaCl (typically 1% by weight). The crosslinking agent is generally used in a proportion of 0.02 to 0.2 equivalent of amine unit per equivalent of hyaluronic acid monomer unit and, preferably, from 0.02 to 0.07. equivalent of amine unit per equivalent of hyaluronic acid monomer unit. The catalyst is generally used in an amount of from 0.003 to 0.025, preferably from 0.015 to 0.025, equivalent per equivalent of hyaluronic acid monomer unit. PH is usually established and maintained at its value

^• (substantially the same or a value substantially in the

(the) range (s) indicated), using for example a dilute hydrochloric acid solution. During the contacting, the reaction medium is generally stirred.

After the time required for the reaction, the crosslinked hyaluronic acid obtained being generally and practically completely soluble in water, the reaction mixture is generally precipitated in an organic solvent such as ethanol, isopropanol, acetone, ether or

other, then generally dried under vacuum and optionally lyophilized. The organic solvent allows advantageously to eliminate, on the one hand, any trace of catalyst that has not reacted during the chemical crosslinking reaction, any trace of unreacted crosslinking agent and, on the other hand, any type of reaction product such as urea. This makes it possible to guarantee a final product that is practically pure, biocompatible and sanitized by the action of the organic solvent of ethanol, isopropanol, acetone, ether or other type. Finally, a product is obtained, which is crosslinked hyaluronic acid according to the invention, in dehydrated form, soluble in water, which advantageously makes it more manipulable for a subsequent formulation, and which improves its shelf life. conservation. Such a dehydrated form thus makes it possible to optimize the handling and the storage with a view to its use.

The reproducible nature of the preparation process has been demonstrated by the determination of the rheological measurements (G ', G' ', δ and η) or; by determining the molecular weight of the crosslinked hyaluronic acid obtained, molecular weight generally obtained by serum exclusion chromatography, as will be explained later. In fact, it has been possible to obtain, in a regular manner, soluble fractions of said crosslinked hyaluronic acid polymer.

The invention therefore also relates to any cross-linked hyaluronic acid obtainable according to the invention, generally and most often exhibiting a solubility in water generally revealed in that all the fiber Ig of the dehydrated polymer. obtained according to the invention are disintegrated in a few minutes and solubilize completely in one liter of physiological saline solution after a few hours, without stirring. The solubility in water can also be shown by the ability of calculating the ^one molecular weight by gel permeation chromatography serum exclusion which generally shows a soluble fraction of said crosslinked hyaluronic acid from 100 to 5%, preferably from 100 to

^• 30%. A crosslinked hyaluronic acid having such a solubility in water is qualified indifferently as "soluble in water" or "water-soluble". Such a crosslinked hyaluronic acid has not been described so far. Indeed, in the patent applications WO 00 / 46.252 and WO 02/30990 previously cited, it is stated that, in any case, the product obtained is not soluble in water (see page 20 lines 1 to 5 of said patent application WO 00 / 46.252 and see all the text of the patent application WO 02/30990). The molecular weight determination of the crosslinked hyaluronic acid according to the invention is

• generally obtained by serum exclusion chromatography. This method of molecular weight analysis generally determines the average molecular weight of cross-linked hyaluronic acid, ranging from the smallest fractions to the largest fractions. The soluble fractions of said polymer crosslinked hyaluronic acid thus measured are always from 100% to 5%, preferably from 100% to 30%. Advantageously, such a solubility property of the crosslinked hyaluronic acid according to the invention makes it possible to formulate a hydrogel. the

The invention also relates to a hydrogel comprising at least said crosslinked hyaluronic acid according to the invention and at least one aqueous solvent. Preferably, such an aqueous solvent is a solution of sodium chloride, physiological saline, an injectable buffer solution such as a phosphate buffer solution or Water for Injection Preparation (or EPP I), generally containing a salt. .

According to the invention, the term "hydrogel" means a crosslinked hyaluronic acid gel obtained by solubilization in water (for example via an aqueous solvent) of said acid.

^• cross-linked hyaluronic. The concentration of cross-linked hyaluronic acid in such a hydrogel is generally and preferably from 1 to 4%, preferably from 1.8 to 3%, w / v.

The invention further relates to an implant comprising at least one crosslinked hyaluronic acid according to the invention. In particular, the invention relates to an implant comprising at least one hydrogel according to the invention, as specified above. Thus the invention relates to any implant comprising at least one crosslinked hyaluronic acid according to the invention, and optionally at least one aqueous solvent. ^'Such an implant undergoes generally at least a sterilization cycle as known in the art before being used as an implant. The crosslinked hyaluronic acid according to the invention has particularly interesting physicochemical characteristics, especially with respect to its sensitivity to temperature in the context of the at least one sterilization cycle recommended by the European Pharmacopoeia. Indeed its parameters The rheological methods according to the invention (namely: elastic modulus G ', viscous modulus G'', loss angle δ and viscosity η) are less affected than a native hyaluronic acid by such a cycle, ie the loss angle generally varies. during such a cycle of less than 50% and the viscosity generally varies during such a cycle by less than 30%. Typically, such a cycle is a heat sterilization cycle, from 118 ^° C. to 130 ^° C. for a duration of two to thirty minutes. This is particularly advantageous.

The concentration of crosslinked hyaluronic acid in such an implant is generally and preferably from about 1 to about 4%, preferably from about 1.8 to about 3%, weight / volume. Generally, for a concentration of less than 1%, ^'the efficiency of the implant over time is not _sufficient, and in excess of 4%, the values of rheological parameters measured i stagnate while one is faced with a concern for injectability through fine gauge needle (> 26G). ^{'Advantageously} and according to the invention, said implant is injectable, water-soluble, and generally has a sufficient inherent viscosity to be injected through a needle gauge of 25 to 30, for example from 800 to 4000 m ³ / kg at 25 ° C. The invention finally relates to the use of said implant. in human or veterinary medicine, in particular in reconstructive and / or aesthetic surgery. In the context of its use in cosmetic surgery and / or restorative, preferably aesthetic, said ^implant according to the invention is used as filling material. These sites may have the purpose of: supplementation of a cavity or organ deficient in hyaluronic acid (typically in dermatology, aesthetic medicine, or in orthopedic treatments); ^'The reconstruction of a volume effused during surgery (typically in eye surgery);

topical application to the healthy or injured dermis (typically in cosmetology and dermatology); or the participation, as a vector fluid, in the implantation of a material or a pharmaceutical active ingredient.

In cosmetic dermatology, for example, such an implant may be used for filling purposes. Such filling comprises filling wrinkles, fine lines, skin depressions and scarring of the human or animal body, including filling of ^defects' cutaneous side to the outlet of a treatment may cause lipodystrophy characterized most sourvent by facial lipoatrophy . Such use is therefore mainly in the field of reconstructive or plastic surgery, or in the field of aesthetic dermatology.

The implant is generally injectable subcutaneously or intradermally into the fibrous tissue. Said implant can also be injected directly intra-articular synovial way to restore the physiological functions of the synovial fluid in the orthopedic field, and more specifically in the treatment of knee osteoarthritis.

Advantageously, the implant according to the invention overcomes the disadvantages of the prior art, since said implant is advantageously a product that is soluble in water and substantially totally biocompatible with the human or animal body. It allows, for example, to fill in wrinkles, fine lines, citations or skin depressions with a simple and effective product, virtually total bioabsorbability, without releasing toxic side products. In fact, the degradation products of such an implant in vivo are on the one hand the hyaluronic acid monomers and on the other hand the lysine, hydrosolouble amino acid, biocompatible and non-toxic units.

By "implant" is meant according to the invention both a composition intended to be implanted a composition that has been implanted in the human or animal body.

The implant according to the invention may further comprise at least one carrier fluid, different from the hydrogel according to the invention. In one embodiment of the invention, the implant thus also comprises at least one element chosen from the group formed by cellulose derivatives such as Carboxy Methyl Cellulose, HPMC (Hydroxy Propyl Methyl Cellulose), HPC (Hydroxy Propyl Cellulose) and glycosaminoglycans such as sodium hyaluronate or branched polysaccharides xanthan type. Preferably in such a case, the implant is a gel comprising at least one element selected from the family of glycosaminoglycans such as hyaluronic acid or a salt thereof or a derivative of one of its salts, more or less viscous, crosslinked or not, in addition to the crosslinked hyaluronic acid according to the invention.

The term "fluid vector" according to the invention a compound which coexists with the crosslinked hyaluronic acid of the invention and which can convey another possible compound, for example in the form of a solid powder, and which is in fluid form. The term "fluid" here also includes a gel for example viscoelastic. The term "gel" according to the invention means a three-dimensional physical structure having interesting viscosifying and thixotropic properties. By "fibrous tissue" is meant according to the invention a subcutaneous space of essentially fibrous nature, and capable of being filled by fillers. By "subcutaneous" is meant according to the hypodermic invention, therefore under the dermis. By "intradermal" is meant according to the invention in the thickness of the dermis. • For "intra-articular" means according to the invention in the space between two joints in synovial ^fluid. By

"Biocompatible" means according to the invention compatible with the human or animal body. In part ilier a biocompatible material is, according to the invention, a material meeting the criteria given by the ISO 10993 standard for medical devices.

The establishment (or implantation) of the implant in the body is essentially intended to overcome a deficit in hyaluronic acid, especially in the treatment of osteoarthritis of the knee, but also to fill a skin defect of origin natural in the case of skin aging or iatrogenic origin and / or in the case of facial lipoatrophy in patients infected with HIV The approach followed is to supplement the body with a fully biocompatible chemically modified implant respecting the properties physicochemical properties of endogenous hyaluronic acid by having a life in the body sufficient to overcome this deficit. The choice of an implant comprising at least one crosslinked hyaluronic acid according to the invention advantageously allows to combine the maximum efficiency with a minimum of risk.

Indeed, no non-absorbable implant does not seem generally desirable. Thus, advantageously according to the invention, the crosslinked hyaluronic acid according to the invention is degraded or solubilized almost completely after subcutaneous or intradermal or intra-articular injection, and is then virtually completely eliminated from the body by natural processes. such as enzymatic hydrolysis. An implant according to the invention in the form of crosslinked hyaluronic acid hydrogel according to the invention, generally should not degrade in less than 6 months and must generally be degraded in less than 18 months. I

Further, the implant according to the invention, ^'when it is injection, advantageously combines the convenience of use, the syringability of the product, the resorbability in a controlled time.

The implant according to the invention is particularly preferably a biocompatible hydrogel, soluble in water for injection with added sodium chloride or directly isotonic sodium chloride solution or in injectable-grade buffer solution.

The following examples illustrate the invention without limiting its scope. Examples

Twenty-six examples (from 1 to 16) of crosslinked hyaluronic acids according to the invention and twelve examples of implants (from 27 to 38) are given below.

The examples given below made it possible to obtain a crosslinked hyaluronic acid polymer which, when formulated at 2.4% in an isotonic sodium chloride solution, has rheological values comprised as follows:

Elastic Module Viscous Module δ (Viscosity Angle η Pa.s G '(Pa) G''(Pa) loss) in ° (0.05 s ^"1 )

550 to 1000 100 to 200 10 to 20 2600 to 7000

All the examples given below are given with polylysines available in the SIGMA ALDRICH catalog.

Example 1

1 g (2.49 mmol disaccharide units of sodium hyaluronate (M _w = 2500000-3000000 g / mol) was dissolved in 150 mL -Of aqueous solution of 1% NaCl by weight. To this solution 50 mg of N-hydroxysuccinimide (M = 115 g / mol), 66.38 mg (274.30 μmol) of poly-L-trifluoroacetate were added successively at 5 ^° C. in succession to 7. 17 mg (62.35 μmol). lysine (M _w = 2700 g / mol, comprises 11 lysine units) and 0.44 mL ^'EDC (l-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (M = 155 g / mol) (2.49 At 50 ^° C., the pH of the solution was then adjusted to 5 with a 1N hydrochloric acid solution After 15 minutes stirring at 5 ⁰ C, the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P ₂ O ₅ for 24 hours to give 1 g of crosslinked hyaluronic acid.

This product was then placed in solution at a concentration of 2.4% w / v in physiological saline.

This solution was treated with a 4cm cone-planar geometry, 4 ° at about 25 ° C. It first underwent a non-destructive viscoelastic test at IHz, with an imposed deformation of 1%. The elastic modules (G ') and viscous

(G ''), as well as the loss angle (δ) could thus be measured. These measurements truly account for the performance of a gel, for example a high G 'value coupled with a low G' value reflect a strong gel.

After the viscoelastic test, the solution! was immediately subjected to a shear rate at 25 ° C., which shows the evolution of the viscosity as a function of shear.

The same measurements were carried out for the native hyaluronic acid starting material.

The measurements, carried out using an AR 2000 type Rheometer of the company TA Instruments, gave the following results:

the

We can see that the cross-linked hyaluronic acid according to the invention has a lower loss angle (δ) and a higher viscosity (η) than the native hyaluronic acid starting material. This demonstrates that the crosslinking according to the process of the invention has made the gel denser and stronger, which advantageously allows it to better withstand the degradation factors in vivo, and therefore to be more effective when used in an implant.

The cross-linked hyaluronic acids of Examples 2 to 27 below all have rheological measurement parameter values substantially similar to those of the cross-linked hyaluronic acid of Example 1.

Example 2

The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 7.17 mg

(62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol),

16.6 mg (68.6 μmol) of poly-L-lysine trifluoroacetate

(M _w = 2700 g / mol) and 0.44 mL of EDC (M = 155 g / mol) (2.49 mmol). After filtration and drying, 1 g of cross-linked hyaluronic acid in the form of a white solid was obtained.

Example 3

The same procedure as in Example 1 was used ^'with 1 g (2.49 mmol disaccharide unit) of sodium hyaluronate (M _w = 2.5 to 3 million g / mol), 7.17 mg (62.35 micromol) of N-hydroxysuccinimide ^(M = 115 g / mol), 33.19 mg (137.15 micromol) trifluoroacetate poly-L-lysine (M _w = 2700 g / mol) and 0.44 mL of EDC (M = 155 g / mol) (2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 4 The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 7.17 mg ( 35 μmol) of N-hydroxysuccinimide (M = 115 g / mol),

Mg-49.79 (205.74 micromol) trifluoroacetate poly-L-lysine (M _w = 2700 g / mol) and 0.44 ml of EDC (M = 155 g / mol)

(2.49 mmol). After filtration and drying, 1 g of acid Crosslinked hyaluronic acid in the form of a white solid was obtained.

EXAMPLE 5 The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 7.17 mg (62, 35 μmol) of N-hydroxysuccinimide (M = 115 g / mol), 165.96 mg (685.79 μmol) of poly-L-lysine trifluoroacetate (M _w = 2700 g / mol) and 0.44 ml of EDC (M = 155 g / mol) (2.49 mmol). 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 6 The same procedure as in Example 1 was used with 1 g (2.49 mmol disaccharide unit) of ^sodium hyaluronate (M _w = 250 000 ^'0-3000000 g / ^mol)', ^'' 4 ^' , 3 ^' mg

(37.41 μmol) of N-hydroxysuccinimide (M = 115 g / mol),

66.38 mg (274.3 micromol) trifluoroacetate poly-L-lysine (M _w = 2700 g / mol) and 0.44 ml of EDC (M = 155 g / mol)

(2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 7

The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 4.3 mg (37.41. μmol) of N-hydroxysuccinimide (M = 115 g / mol), 16.6 mg (68.6 μmol) of poly-L-lysine trifluoroacetate

(M _w = 2700 g / mol) and 0.44 mL of EDC ^' (M = 155 g / mol) (2.49 mmol). After filtration and drying, 1 g of acid Crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 8 The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 28.68 mg (249, 38 μmol) of N-hydroxysuccinimide (M = 115 g / mol),

16.6 mg (68.6 μmol) of poly-L-lysine trifluoroacetate (M _w = 2700 g / mol) and 0.44 mL of EDC (M = 155 g / mol) (2.49

• mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 9

The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharidic units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 114.71 mg (1 mmol) of N-hydroxysuccinimide (M = 115 g / mφl), 16.6 mg (68.6 μmol) of poly-l-lysine trifluoroacetate

(M _w = 2700 g / mol) and 0.44 mL of EDC (M = 155 g / mol) (2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 10

The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 16.6 mg (68.6 μmol ) of poly-L-lysine trifluoroacetate (M _w = 2700 g / mol), 0.44 ml of EDC (M = 155 g / mol) (2.49 mmol) and without addition of N-hydroxysuccinimide (M = 115 g / mol). After After filtration and drying, 1 g of cross-linked hyaluronic acid in the form of a white solid was obtained.

Example 11 The same procedure as in Example 1 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 66.38 mg ( 274.30 μmol) of poly-L-lysine trifluoroacetate

(M _w = 2700 g / mol), 0.44 ml of EDC (M = 155 g / mol) (2.49 mmol) and without addition of N-hydroxysuccinimide (M - = 115 g / mol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 12

1 g (2.49 mmol disaccharide unit) of sodium hyaluronate (M _w = 2.5 to 3 million g / mol) ^'was dissolved ⁱⁿ years and 150 mL of an aqueous solution of 1% NaCl by weight. To this solution, 7.17 mg (62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol), 66.38 mg (274.30 μmol) of poly (trifluoroacetate), were added successively at 50 ^° C. L-lysine (M _w = 2700 g / mol) and 478 mg EDC.HCl (M = 191.5 g / mol) (2.49 mmol). During the reaction, the pH was stable at around 5. After stirring for 4 hours at room temperature, the pH of the reaction mixture was adjusted to 6.5 and this reaction mixture was precipitated in 1.5 L of ethanol. . The precipitate obtained was then filtered and then dried under reduced pressure in the presence of

• P ₂ O ₅ for 24 hours to give 1 g of cross-linked hyaluronic acid. The solubility of the crosslinked polymer obtained according to the invention was confirmed by a rapid and complete dissolution without stirring of the Ig of fibers obtained in 1 liter of physiological saline solution.

Example 13 The same procedure as in Example 12 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 66.38 mg (274, 30 μmol) of poly-L-lysine trifluoroacetate

(M _w = 2700 g / mol), 478 mg of EDC.HCl (M = 191.5 g / mol) (2.49 mmol) and without addition of N-hydroxysuccinimide (M = 115 g / mol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 14

The same procedure as in Example 12 was used with 1- g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 16.6 mg

(68.6 micromol) trifluoroacetate poly-L-lysine (M _w = 2700 g / mol), 478 mg EDC.HCl (M = 191.5 g / mol) (2.49 mmol) and without addition N-hydroxysuccinimide (M = 115 g / mol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 15

The same procedure as in Example 12 was used with 1 g (2.49 mmol disaccharide unit) of sodium hyaluronate (M _w = 2.5 to 3 million g / mol), 7.17 mg

(62.35 μmol) N-hydroxysuccinimide (M = 115 g / mol), 16.6 mg. (68.6 μmol) poly-L-lysine trifluoroacetate

(M _w = 2700 g / mol) and 478 mg of EDC.HCl (2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 16 The same procedure as in Example 12 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 7.17 mg

(62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol),

33.19 mg (137.15 micromol) trifluoroacetate poly-L-lysine (M _w = 2700 g / mol) and 478 mg EDC.HCl (M = 191.5 g / mol) (2.49 mmol ). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 17

The same procedure as in Example 12 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol) L 4.3 mg (37.41 μmol ) of N-hydroxysuccinimide (M = 115 | g / mol), 66.38 mg (274.3 micromol) trifluoroacetate poly-L-lysine (M _w = 2700 g / mol) and 478 mg EDC.HCl (M = 191.5 g / mol) ^(2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 18

The same procedure as in Example 12 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 4.3 mg (37.41 μmol ) of N-hydroxysuccinimide (M = 115 g / mol), 16.6 mg (68.6 μmol) of poly-L-lysine trifluoroacetate (M _w = 2700 g / mol) and 478 mg of EDC.HCl ( M = 191.5 g / mol) (2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 19

The same procedure as in Example 12 was used with 1 g (2.49 mmol of disaccharide units) of sodium hyaluronate (M _w = 2500000-3000000 g / mol), 7.17 mg (62.35). μmol) of N-hydroxysuccinimide (M = 115 g / mol), 3.32 mg (13.72 μmol) of poly-L-lysine trifluoroacetate (M _w = 2700 g / mol) and 478 mg of EDC.HCl. (M = 191.5 g / mol) (2.49 mmol). After filtration and drying, 1 g of crosslinked hyaluronic acid in the form of a white solid was obtained.

Example 20

2.49 mmol of disaccharide units of sodium hyaluronate (Mw = 2500000-3000000 g / mol) were dissolved in

150 ml of a 1% by weight aqueous solution of NaCl. To this solution, were added at 5 ⁰ C in succession

(62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol,

(274.30 μmol) of poly-L-lysine hydrochloride

(347.28 g / mol, ie 2 lysine units) and 0.44 ml of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) (M = 155 g / mol) (2.49 mmol). At 5 ^° C., the pH of the solution was then adjusted to 5 with a 1N hydrochloric acid solution. After stirring for 15 minutes at 5 ° C., the reaction mixture was precipitated in 1 ml. 5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P ₂ O ₅

24 hours to give 1 g of crosslinked hyaluronic acid. Example 21

2.49 mmol of the disaccharide units of sodium hyaluronate (Mw = 2500000-3000000 g / mol) were dissolved in 150 ml ^'of an aqueous solution of 1% NaCl by weight. To this solution were added at 25 ^° C. in succession (62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol), (274.30 μmol) of poly-L-lysine hydrochloride (347.28 g). mol (2 units lysine) and EDC.HCL (M = 191.5 g / mol) (2.49 mmol). The pH should be from 4 to 6. After stirring for 4 hours at 25 ⁰ C, the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P2O5 for 24 hours to give 1 g of crosslinked hyaluronic acid.

Example 22

2.49 mmol of disaccharide units of sodium hyaluronate (Mw = 2500000-3000000 g / mol) were dissolved in 150 ml of a 1% by weight aqueous solution of NaCl. To this solution were added successively at 25 ^° C. (62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol), (411.45 μmol) of poly-L-lysine hydrochloride (347.28 g / mol). mol is 2 lysine units) and EDC.HCL (M = 191.5 g / mol) (2.49 mmol). The pH should be from 4 to 6. After stirring for 4 hours at 25 ⁰ C, the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P2O5 for 24 hours to give 1 g of crosslinked hyaluronic acid. Example 23

2.49 mmol of disaccharide units of sodium hyaluronate (Mw = 2500000-3000000 g / mol) were dissolved in 150 ml of a 1% by weight aqueous solution of NaCl. To this solution were added at 25 ° C successively (62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol), (548.60 μmol) of poly-L-lysine hydrochloride (347.28 g / mol). mol, ie 2 lysine units) and EDC.HCL (M = 191.5 g / mol) (2.49 mmol). The pH should be from 4 to 6. After stirring for 4 hours at 25 ⁰ C, the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P2O5 for 24 hours to give 1 g of crosslinked hyaluronic acid.

Example 24

2.49 mmol of disaccharide units of sodium hyaluronate (Mw = 2500000-3000000 g / mol) were dissolved in 150 ml of a 1% aqueous NaCl solution in Iweight. To this solution were added at 25 ° C successively (62.35 μmol) N-hydroxysuccinimide (M =

115 g / mol), (274.30 μmol) of poly-L-lysine hydrobromide

(500-10 ^' OOg / mol, between 3. and 8 lysine units) and EDC.HCL (M = 191.5 g / mol) (2.49 mmol). The pH should be from 4 to 6. After stirring for 4 hours at 25 ⁰ C, the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P2O5 for 24 hours to give 1 g of crosslinked hyaluronic acid. Example 25

2.49 mmol of disaccharide units of sodium hyaluronate (Mw = 2500000-3000000 g / mol) were dissolved in 150 ml of a 1% by weight aqueous solution of NaCl. To this solution were added at 25 ⁰ C successively (62.35 μmol) of N-hydroxysuccinimide (M = 115 g / mol), (411.45 μmol) of poly-L-lysine hydrobromide (500-1000g). mol / between 3 and 8 lysine units) and EDC.HCL (M = 191.5 g / mol) (2.49 mmol). The pH should be from 4 to 6. After stirring for 4 hours at 25 ⁰ C, the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P2O5 for 24 hours to give 1 g of crosslinked hyaluronic acid.

Example 26

2.49 mmol of disaccharide units of hyaluronate

^• sodium (Mw = 2500000-3000000 g / mol) were dissolved in

150 ml of 1% aqueous NaCl solution in weight. To this solution were added at 25 ° C successively (62.35 μmol) N-hydroxysuccinimide (M = 115 g / mol), (548.60 μmol) of hydrobromide.

(500-1000g / mol, between 3. and 8 lysine units) and EDC.HCL (M = 191.5 g / mol) (2.49 mmol). The pH should be from 4 to 6. After stirring for 4 hours at 25 ° C., the reaction mixture was precipitated in 1.5 L of ethanol. The precipitate obtained was then filtered and then dried under reduced pressure in the presence of P2O5 for 24 hours to give 1 g of crosslinked hyaluronic acid.

. Some examples of formulation of crosslinked hydrogel implants are given below. Example 27

0.2 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were dissolved with stirring in 10 ml of EPPI, and then 90 mg of NaCl was dissolved. It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C (representing a sterilization cycle).

Example 28

240 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were dissolved with stirring in 10 ml of EPPI, then 76 mg of Na ₂ HPO ₄ and 18 mg of KH ₂ PO were dissolved. ₄ . It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C.

Example 29

6 mg of cross-linked hyaluronic acid, 240 mg of crosslinked acid according to the invention, were dissolved with stirring in 6 ml of EPPI, and then 76 mg of Na ₂ HPO ₄ and 18 mg of KH ₂ were dissolved. PO ₄ in 4 ml of EPPI. The 4 ml of buffer solution thus prepared were added with stirring in the 6 ml of hydrogel prepared previously. It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C.

Example 30

10 mg of EPPI was dissolved with stirring in 76 ml of

Na ₂ HPO ₄ and 18 mg of KH ₂ PO ₄ and then 240 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were gradually dissolved. distributed in syringes, corked and autoclaved for 15 minutes at 121 ° C.

EXAMPLE 31 180 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were dissolved with stirring in 10 ml of EPPI and then 90 mg of NaCl was dissolved. It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C.

Example 32

10 mg of crosslinked hyaluronic acid according to the invention were dissolved with stirring in 10 ml of EPPI,

• that of Example 1, then 76 mg of Na ₂ HPO ₄ and 18 mg of KH ₂ PO ₄ were dissolved. It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ^° C.

Example 33

6 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were dissolved with stirring in 6 ml of EPPI, and then 7 μg of Na ₂ HPO ₄ and 18 mg of KH ₂ were dissolved. PO ₄ in 4 ml of EPPI. The 4 ml of buffer solution thus prepared were added with stirring in the 6 ml of hydrogel prepared previously. It was syringed, capped and autoclaved for 15 minutes at 1-21 ° C.

Example 34

10 micrograms of Na ₂ HPO ₄ and 18 mg of KH ₂ PO ₄ were dissolved with stirring in 10 ml of EPPI and then 180 mg of crosslinked hyaluronic acid according to the invention, such as that of the example, was gradually dissolved. 1. We have divided into syringes, capped and autoclaved for 15 minutes at 121 ° C.

EXAMPLE 35 300 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were dissolved with stirring in 10 ml of EPPI, and then 90 mg of NaCl was dissolved. It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C.

Example 36

300 mg of crosslinked hyaluronic acid according to the invention, such as that of Example 1, were dissolved with stirring in 10 ml of EPPI, then 76 mg of Na ₂ HPO ₄ and 18 mg of KH ₂ PO were dissolved. ₄ . It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C.

Example 37 ^• is dissolved with stirring in 6 ml of EPPll 300 mg of cross-linked hyaluronic acid of the invention; as that of Example 1, then 7βmg of Na ₂ HPO ₄ and 18 mg of KH ₂ PO ₄ were dissolved in 4 ml of EPPI. The 4 ml of buffer solution thus prepared were added with stirring in the 6 ml of hydrogel prepared previously. It was divided into syringes, capped and then autoclaved for 15 minutes at 121 ° C.

Example 38

10 micrograms of Na ₂ HPO ₄ and 18 mg of KH ₂ PO ₄ were dissolved with stirring in 10 ml of EPPI, then 300 mg of cross-linked hyaluronic acid were progressively dissolved.

. according to the invention such as that of Example 1. We have distributed in syringes, it was capped and then autoclaved for 15 minutes at 121 ^° C.

All the implants above (Examples 27 to 38) can be formulated in the same way but sterilized with different sterilization cycles, for example:

• 115 ⁰ C, 60 minutes,

• 118 ⁰ C, 30 minutes,

• 125 ⁰ C, 6 minutes, • 128 ⁰ C, 3 minutes, or

• 130 ⁰ C, 2 minutes.

Claims

42REVENDICATIONS

A crosslinked hyaluronic acid having a solubility in water and obtainable by a preparation process comprising contacting an aqueous medium with at least one hyaluronic acid or at least one of its salts, and at least one crosslinking agent comprising at least one oligopeptide or polypeptide having at least two, preferably at least five, even more preferably at least six lysine units, said contacting being carried out in the presence of at least one agent of water - soluble coupling and at least one catalyst.

2. Cross-linked hyaluronic acid according to the preceding claim, such that the water-soluble coupling agent is chosen from water-soluble carbodiimides, preferably from the group formed by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3- (3-trimethylaminopropyl) carbodiimide ETC) and 1-cyclohexyl-3- (2-morphilinoethyl) carbodiimide (CMC), their derivative salts and mixtures thereof; and such that the catalyst is selected from amidation catalysts for the formation of activated esters, preferably in the group formed by N-Hydroxy Succinimide (NHS), N-hydroxybenzotriazole (HOBt), 3, 4-dihydro-3-hydroxy-4-oxo-2, 3-benzo triazole ^(HOOBt), l-hydroxy-7-azabenzotriazole (HAT) and N- hydroxysulfosuccinimide (Sulfo-NHS) and their mixtures.

3. crosslinked hyaluronic acid according to one of the preceding claims, such that said oligopeptide or polypeptide is based on polylysine. 43

4. Cross-linked hyaluronic acid according to one of the preceding claims, such that the contacting is carried out at a temperature of 0 to 45 ° C, preferably 5 to 25 ° C, for a duration of 0.5 to 10 minutes. hours, preferably for a period of 1 to 6 hours, and at a pH of 4 to 10, preferably from 4 to 8, more preferably from 5 to 8.

Crosslinked hyaluronic acid according to the preceding claim, such that the crosslinking agent has bacteriostatic properties.

6. The crosslinked hyaluronic acid according to one of the preceding claims, such that it has rheological measurement parameters of the following values:

Elastic Module Viscous Module Angle of Loss δ Viscosity η Pa.s G '(Pa) G''(Pa) in O (0.05 s ^"1 )

550 to 1000 100 to 200 10 to 20 2600 to 7000

said values having been measured according to the following protocol:

The cross-linked hyaluronic acid is dissolved at a concentration of 2.4% w / v in liquid uri, such as physiological saline. It is treated with a 4cm, 4 ° cone-plane geometry at a temperature of approximately 25 ^° C. It first undergoes a non-destructive viscoelastic test at 1Hz, with a deformation imposed of 1%. The elastic module

(G '), the viscous modulus (G''), as well as the loss angle (δ) can thus be measured. After this viscoelastic test, the sample immediately undergoes at around 25 ^° C. a shear gradient which allows to observe the evolution of the viscosity (η) as a function of shear. The measurements being all 44

performed using an AR 2000 rheometer of TA Instruments.

7. A process for preparing a crosslinked hyaluronic acid comprising contacting an aqueous medium with at least one hyaluronic acid or at least one of its salts, and at least one crosslinking agent comprising at least one oligopeptide or polypeptide having at least two, preferably at least five, even more preferably at least six lysine units, said contacting being carried out in the presence of at least one water-soluble coupling agent generally selected from the group consisting of

^{Water-soluble} carbodiimides, preferably from the group consisting of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), l-ethyl-3- (3-trimethylaminopropyl) carbodiimide hydrochloride (ETC) and l-cyclohexyl- 3- (2-morphilinoethyl) carbodiimide (CMC), their derivative salts and mixtures thereof, and at least one catalyst selected from amidation catalysts for the formation of activated esters, preferably in the group consisting of N-Hydroxy Succinimide (NHS), N-hydroxy benzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzo triazole (HOOBt), 1-hydroxy-7 azabenzotriazole (HAt), and N-hydroxysulfosuccinimide (Sulfo-NHS), and mixtures thereof. 8. Hydrogel comprising at least one crosslinked hyaluronic acid according to one of claims 1 to 6 and at least one aqueous solvent.

9. Implant comprising at least one crosslinked hyaluronic acid according to one of claims 1 to 6, and optionally at least one aqueous solvent.

10. Use of at least one implant according to the preceding claim in human medicine or 45

veterinarian, in particular in reconstructive and / or aesthetic surgery, preferably in cosmetic surgery.