Sterile cross-linked and / or non-cross-linked hyaluronate hydrogel

Abstract

The present invention relates to the use of a hydrogel in the prevention and / or cosmetic treatment of alterations in the skin's surface appearance, said hydrogel comprising at least one functionalized hyaluronic acid, optionally cross-linked, and at least one thiosulfate salt. The invention also relates to a hydrogel comprising at least one functionalized and cross-linked hyaluronic acid, and at least one thiosulfate salt. Figure for the abstract: None

Description

Title of the invention: Sterile cross-linked and / or non-cross-linked hyaluronate hydrogel. Technical field

[0001] The invention relates to the field of sterile compositions, in the form of a hydrogel, based on hyaluronic acid or hyaluronate, for applications in the prevention and / or cosmetic treatment of alterations in the skin's surface appearance. Prior art

[0002] Polysaccharides, such as glycosaminoglycans, are widely used in the medical and aesthetic fields, particularly for filling soft tissues. In particular, the majority of products marketed for aesthetic applications are hyaluronic acid-based. To improve skin quality, hydrogels prepared from non-crosslinked hyaluronic acid are of interest because they have the advantage of being perfectly biocompatible.

[0003] It is also possible to use compositions based on modified or functionalized hyaluronic acid, the hyaluronic acid usually being modified by cross-linking. This cross-linking has the advantage of increasing in vivo durability, in other words, the resistance to in vivo degradation of the compositions, as well as improving their viscoelastic properties. The cross-linking of non-functionalized hyaluronic acid is typically carried out with one or more cross-linking agents. Conventional cross-linking agents possess at least two reactive functions allowing them to link polysaccharide molecules together (e.g., 1,4-butanediol diglycidyl ether: BDDE). Consequently, these conventional cross-linking agents can also react with biopolymers such as peptides, carbohydrates, and DNA, which is undesirable.

[0004] Furthermore, hydrogels based on cross-linked and / or non-cross-linked hyaluronic acid intended for filling soft tissues must be sterile. Therefore, the preparation processes for hydrogels based on cross-linked and / or non-cross-linked hyaluronic acid intended for injection generally include a sterilization step for the previously formed hydrogel. Sterilization is typically achieved by heat, for example, in an autoclave. Heat sterilization remains the most suitable sterilization method for hyaluronic acid. However, the heat applied during sterilization carries a risk of degradation of the gel's components, particularly hyaluronic acid, which is heat-sensitive, and / or the anesthetic, whose degradation mechanisms, such as oxidation, can be accelerated.

[0005] The present invention aims to overcome the above disadvantages and to provide a cross-linked and / or non-cross-linked functionalized hyaluronic acid-based hydrogel exhibiting reduced loss of rheological properties upon sterilization, and advantageously improved antioxidant properties. Summary of the invention

[0006] More specifically, the present invention relates to the use of a hydrogel in the prevention and / or cosmetic treatment of an alteration in the surface appearance of the skin, said hydrogel comprising at least one functionalized hyaluronic acid, optionally cross-linked, and at least one thiosulfate salt.

[0007] It is understood that the term “hyaluronic acid” also refers to “hyaluronate” or “hyaluronane”.

[0008] According to embodiments, hyaluronic acid and / or thiosulfate salt may have one or more of the characteristics described below: - Thiosulfate salt is sodium thiosulfate, - the thiosulfate salt represents a content strictly less than 10% in mass of thiosulfate salt, preferably sodium thiosulfate, preferably less than or equal to 5% by mass of thiosulfate salt, even more preferably less than or equal to 2% by mass of thiosulfate salt relative to the total mass of the hydrogel, - Functionalized hyaluronic acid is hyaluronic acid modified by at least one functional group chosen from among the following groups: methacrylates, acrylates, aldehydes, thiols, dienes, alkynes, oximes, silyls or hydroxyphenyls, preferably from among methacrylates, thiols, dienes and hydroxyphenyls, - said at least one functionalized hyaluronic acid has a weight-average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or ranging from 1 to 5 MDa, - Functionalized hyaluronic acid is hyaluronic acid modified by at least one functional group corresponding to formula (9):

[0009] H2N - R' 1 - Ar' (9)

[0010] Ar' is an aromatic functional group capable of reacting with another Ar' functional group and forming covalent intermolecular bonds, preferably Ar' is an aryl group comprising at least one -OH function and optionally being substituted by one or more other groups chosen from -OH or hydrocarbon groups comprising from 1 to 10 carbon atoms,

[0011] R' 1 represents a hydrocarbon group possibly comprising one or more oxygen atoms, preferably an alkylene group comprising from 1 to 10 carbon atoms and possibly an -OH or -COOH function, - said at least one functionalized hyaluronic acid is a functionalized and cross-linked hyaluronic acid, preferably having the following structure:

[0013] in which: • mlP and m2P are respectively a first and a second hyaluronic acid molecule; • Ar, identical or different, is a functional group capable of reacting with another functional group Ar and forming covalent intermolecular bonds, preferably Ar is an aryl group that can be substituted; • Y is absent, an oxygen atom, a sulfur atom, an NR1- group, an -NR'-CL^-NR1- group, an -S-(L)PS- group or a -CR'R^O-CR'R2- group with: • R1 and R2 being independently of each other a hydrogen atom or a hydrocarbon group comprising from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 10 carbon atoms, even more preferably from 1 to 5 carbon atoms, • L being a hydrocarbon group comprising 1 to 3 carbon atoms or a peptide, and • p being an integer from 0 to 200, preferably from 1 to 100.

[0014] According to one embodiment, the hydrogel implemented according to the invention further comprises at least one additional additive selected from lubricating agents, anesthetic agents, amino acids, vitamins, minerals, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and one of their mixtures.

[0015] The hydrogel may be in the form of an injectable composition or a composition for topical application.

[0016] Hydrogel is advantageously used to prevent and / or treat alterations in the viscoelastic or biomechanical properties of the skin, to fill volume defects in the skin, in particular to fill wrinkles, fine lines and scars, to reduce nasolabial folds and marionette lines, to increase the volume of the cheekbones, chin or lips, to restore facial volumes, in particular of the cheeks, temples, oval of the face, and around the eye, to reduce the appearance of wrinkles and fine lines.

[0017] The invention also relates to a hydrogel comprising at least one functionalized and cross-linked hyaluronic acid, and at least one thiosulfate salt.

[0018] According to embodiments, the hydrogel according to the invention may have one or more of the characteristics described below: - Thiosulfate salt is sodium thiosulfate, - the thiosulfate salt represents a content strictly less than 10% in mass of thiosulfate salt, preferably sodium thiosulfate, preferably less than or equal to 5% by mass of thiosulfate salt, even more preferably less than or equal to 2% by mass of thiosulfate salt relative to the total mass of the hydrogel, - Functionalized hyaluronic acid is hyaluronic acid modified by at least one functional group chosen from among the following groups: methacrylates, acrylates, aldehydes, thiols, dienes, alkynes, oximes, silyls or hydroxyphenyls, preferably from among methacrylates, thiols, dienes and hydroxyphenyls, - said at least one functionalized hyaluronic acid has a weight-average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or ranging from 1 to 5 MDa, - Functionalized hyaluronic acid is hyaluronic acid modified by at least one functional group corresponding to formula (9):

[0019] H2N - R' 1 - Ar' (9)

[0020] Ar' is an aromatic functional group capable of reacting with another Ar' functional group and forming covalent intermolecular bonds, preferably Ar' is an aryl group comprising at least one -OH function and optionally being substituted by one or more other groups chosen from -OH or hydrocarbon groups comprising from 1 to 10 carbon atoms,

[0021] R' 1 represents a hydrocarbon group possibly comprising one or more oxygen atoms, preferably an alkylene group comprising from 1 to 10 carbon atoms and possibly an -OH or -COOH function, - said at least one functionalized hyaluronic acid is a functionalized and cross-linked hyaluronic acid, preferably having the following structure:

[0022]

[0023] in which: • mlP and m2P are respectively a first and a second hyaluronic acid molecule; • Ar, identical or different, is a functional group capable of reacting with another functional group Ar and forming covalent intermolecular bonds, preferably Ar is an aryl group that can be substituted; • Y is absent, an oxygen atom, a sulfur atom, an NR1- group, an -NR'-CL^-NR1- group, an -S-(L)PS- group or a -CR'R^O-CR'R2- group with: • R1 and R2 being independently of each other a hydrogen atom or a hydrocarbon group comprising from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 10 carbon atoms, even more preferably from 1 to 5 carbon atoms, • L being a hydrocarbon group comprising 1 to 3 carbon atoms or a peptide, and • p being an integer from 0 to 200, preferably from 1 to 100.

[0024] According to one embodiment, the hydrogel according to the invention further comprises at least one lubricating agent, preferably chosen from non-crosslinked and non-functionalized hyaluronic acids advantageously having a molecular mass by weight ranging from 0.5 to 10 MDa, even more preferably ranging from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or even from 1.5 to 4 MDa.

[0025] The invention also relates to a method for preparing a hydrogel according to the invention, comprising the following steps:

[0026] (a) supply of hyaluronic acid modified by functional groups capable of reacting with each other and creating covalent intermolecular bonds;

[0027] (b) crosslinking, possibly in the presence of a lubricating agent, of the acid functionalized hyaluronic acid supplied in step (a) to form the hydrogel,

[0028] (c) Addition of thiosulfate salt to the formulation,

[0029] (d) optionally sterilizing, preferably by heat, the hydrogel to obtain a sterile hydrogel.

[0030] Finally, the invention relates to the use of a thiosulfate salt to reduce the sterilization loss of a hydrogel comprising at least one functionalized hyaluronic acid, crosslinked and / or non-crosslinked. In this application, the thiosulfate salt and / or the functionalized hyaluronic acid advantageously exhibit one or more of the characteristics described in the context of the use of the hydrogel according to the invention.

[0031] Unexpectedly, the inventors discovered that the addition of thiosulfate, in particular sodium thiosulfate pentahydrate, during the preparation of hydrogels comprising functionalized hyaluronic acid, crosslinked and / or non-crosslinked, effectively protects the hydrogel from degradation of its rheological properties during sterilization, particularly heat sterilization. Hydrogels obtained by the process of the present invention thus exhibit less degradation of their rheological properties compared to hydrogels prepared by an equivalent process without the addition of thiosulfate or with the addition of another antioxidant. The hydrogels of the invention, obtained according to the process of the invention, also exhibit better retention of their rheological properties over time.

[0032] More specifically, the inventors discovered that thiosulfate salt significantly improves the sterilization stability of a gel based on functionalized hyaluronic acid, particularly with the aid of hydroxyphenyl compounds such as tyramine or dopamine. In particular, the inventors discovered that even in the presence of a small proportion of thiosulfate salt, the effect on sterilization stability was achieved. Detailed description

[0033] The present invention relates to the use of a hydrogel in the prevention and / or cosmetic treatment of alterations in the skin's surface appearance, said hydrogel comprising at least one functionalized hyaluronic acid, optionally cross-linked, and at least one thiosulfate salt, the functionalization of said hyaluronic acid Functionalized hyaluronic acid is capable of reacting with the functionalization of another functionalized hyaluronic acid molecule. In particular, a functionalized hyaluronic acid molecule is typically capable of reacting with only one other functionalized hyaluronic acid molecule. Advantageously, in this case, the functionalizations of each hyaluronic acid react with each other to obtain a cross-linked functionalized hyaluronic acid.

[0034] For the purposes of the present invention, a "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed from a solid, three-dimensional network that is more or less hydrated. The medium consists of long polymer molecules connected to each other by weak bonds (for example, hydrogen bonds), ionic bonds, or covalent bonds (crosslinking). A gel generally corresponds to a viscoelastic product that has a phase angle θ less than or equal to 45° at 1 Hz for a strain of 0.1% or a stress of 1 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0035] For the purposes of the present invention, a "hydrogel" means a gel as defined above in which the solvent constituting the liquid medium is predominantly water (for example, at least 90%, in particular at least 95%, especially at least 97%, especially at least 98% by weight of the liquid medium). Advantageously, the hydrogel according to the invention is an injectable hydrogel preferably having a pH ranging from 6.8 to 7.8.

[0036] For the purposes of the present invention, an "injectable hydrogel" means a hydrogel that can be manually injected using a syringe fitted with a needle with a diameter of 0.1 to 0.5 mm, for example a hypodermic needle of 32 G, 30 G, 27 G, 26 G, 25 G. Preferably, an "injectable hydrogel" is a hydrogel having an average extrusion force less than or equal to 25 N, preferably from 5 to 25 N, more preferably from 8 to 15 N, when measured with a dynamometer, at a fixed speed of about 12.5 mm / min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle with an external diameter less than or equal to 0.4 mm (27 G) and of length U2, at room temperature.

[0037] A "crosslinked hyaluronic acid" refers to hyaluronic acid or one of its salts, modified during a crosslinking reaction. Crosslinking leads to the formation of covalent bonds between the hyaluronic acid chains.

[0038] Conversely, a "non-crosslinked hyaluronic acid" refers to a hyaluronic acid or one of its salts that has not undergone a crosslinking reaction.

[0039] “Functionalized hyaluronic acid” means hyaluronic acid or one of its salts, modified during a functionalization reaction by the introduction of functional groups. This functionalization does not result in cross-linking. said HA. In the context of the present invention, functionalization aims to chemically modify a hyaluronic acid molecule to enable it to react with another functionalized hyaluronic acid molecule. Thus, the functional group attached to the hyaluronic acid is stable, particularly in solution and over time. In a preferred variant, the functional group introduced onto the hyaluronic acid is heat-stable, particularly at temperatures above or equal to 40°C, typically at 40°C.

[0040] A "crosslinked functionalized hyaluronic acid" refers to a functionalized hyaluronic acid or one of its salts, crosslinked by a reaction between the functional groups introduced during the functionalization reaction. The crosslinking step leads to the formation of covalent bonds between the hyaluronic acid chains.

[0041] Conversely, "non-functionalized, non-crosslinked hyaluronic acid" refers to hyaluronic acid or one of its salts that has not been modified during a functionalization reaction and has not undergone a crosslinking reaction. Non-functionalized, non-crosslinked hyaluronic acid may also be called native hyaluronic acid.

[0042] The "molar functionalization rate" (MF), expressed as a percentage, refers to the molar quantity of functional groups grafted onto the polysaccharide, expressed per 100 moles of repeating units of the polysaccharide (disaccharide for hyaluronic acid). For example, a molar functionalization rate of 1% means that there is one mole of functional groups grafted onto the polysaccharide per 100 moles of repeating units of the polysaccharide.

[0043] By "ambient temperature" is meant a temperature ranging from 20 to 25 °C, more particularly 21 °C.

[0044] The hydrogel according to the invention comprises a thiosulfate salt. Thiosulfate salts that can be used in the invention are chosen from sodium, potassium, ammonium, calcium salts, or mixtures thereof.

[0045] The thiosulfate salt used in the invention is preferably a sodium thiosulfate.

[0046] According to one embodiment, the thiosulfate salt is in hydrate form, preferably in pentahydrate form.

[0047] Thus, for example, the hydrogel according to the invention may comprise a sodium thiosulfate pentahydrate.

[0048] According to a particularly preferred embodiment, the hydrogel according to the invention comprises a content of 1% or less, preferably 0.3% or less by mass of thiosulfate salt, preferably 0.2% or less by mass of thiosulfate salt, preferably even less than or equal to 0.1% by mass of thiosulfate salt, and more preferably less than or equal to 0.08% by mass of thiosulfate salt. mass of thiosulfate salt, more preferably less than or equal to 0.06% by mass of thiosulfate salt, or even less than or equal to 0.05% by mass of thiosulfate salt or less than or equal to 0.04% by mass of thiosulfate salt, relative to the total mass of the hydrogel, the thiosulfate salt preferably being a sodium thiosulfate, possibly in hydrated form, the hydrogel according to the invention preferably being a sterile hydrogel.

[0049] A reduced quantity of thiosulfate salt advantageously limits the by-products from the thiosulfate salt, which are susceptible to being reduced by redox reactions.

[0050] For example, the hydrogel may comprise from 0.01 to 0.3% by mass of thiosulfate salt, preferably sodium thiosulfate, relative to the total mass of the hydrogel, the hydrogel according to the invention preferably being a sterile hydrogel.

[0051] The hydrogel according to the invention comprises hyaluronic acid or one of its salts, functionalized, crosslinked and / or non-crosslinked.

[0052] Hyaluronic acid (HA) can be in the form of its salts.

[0053] The specific functional groups are functional groups capable of reacting with each other and forming intermolecular covalent bonds to produce a cross-linked functionalized polysaccharide. Cross-linking carried out in this way preserves the polysaccharide chains compared to cross-linking achieved using conventional cross-linking agents, for example, BDDE. The molar mass of the HA is preserved.

[0054] The salts are typically physiologically acceptable salts such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.

[0055] The hydrogel can thus be a hydrogel based on hyaluronic acid and / or one of its sodium salts (NaHA).

[0056] According to one embodiment, the HA has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or ranging from 1 to 5 MDa.

[0057] The HA molecules have been previously modified by functional groups capable of reacting with each other and forming covalent intermolecular bonds. The functional groups may be identical or different. The HA molecules are not modified by bifunctional molecules that react with two HA chains to link them together. In the context of the present invention, two functional groups react with each other under suitable reaction conditions.

[0058] The modifications can be made to the carboxyl, hydroxyl and N-acetyl groups of the polysaccharides, or after oxidation of the polysaccharides, for example with sodium periodate.

[0059] Examples of functional groups capable of reacting with each other and forming covalent intermolecular bonds include, but are not limited to, methacrylate, acrylate, vinyl, diene, aldehyde, thiol, thioester, furan, azide, alkyne, aldehyde, ketone, amine, hydrazide, silane, hydroxyphenyl, carboxylic acid, boronate, diol and coumarin groups.

[0060] Preferably, the functional groups are chosen from among the methacrylate, acrylate, thiol, thioester, diene, alkyne, silyl or hydroxyphenyl groups, more preferably the functional groups are chosen from among the methacrylate, thiol, diene and hydroxyphenyl groups, even more preferably the functional groups are chosen from among the hydroxyphenyls.

[0061] According to a particular embodiment, the functionalized hyaluronic acid has been functionalized using one or more hydroxyphenyl molecules.

[0062] Among the hydroxyphenyls, we can mention the molecules of formula (9):

[0063] H2N-R'l -Ar' (9)

[0064] Ar' is an aromatic functional group capable of reacting with another Ar' functional group and forming covalent intermolecular bonds, preferably Ar' is an aryl group comprising at least one -OH function and optionally being substituted by one or more other groups selected from -OH or hydrocarbon groups comprising from 1 to 10 carbon atoms,

[0065] R' 1 represents a hydrocarbon group possibly comprising one or more oxygen atoms, preferably an alkylene group comprising 1 to 10 carbon atoms and possibly an -OH or -COOH function.

[0066] As an example of a functional group for the functionalization of HA, tyramine, tyrosine and dopamine may be cited, preferably tyramine.

[0067] Preferably, the hyaluronic acid molecules have been previously modified by the introduction of one or more functional groups corresponding to the following formula:

[0068] [Chem. 8]

[0069] in which:

[0070] - * represents the point of attachment of the functional group with the acid hyaluronic acid, and

[0071] - R represents one or more substituents, identical or different, independently chosen from a hydrogen atom, an OH group, an -NH2- group, and an -SH group.

[0072] After reaction of the functional groups with each other, the HA obtained, designated "crosslinked functionalized hyaluronic acid", exhibits covalent intermolecular bonds, each of which can be represented in the following way (i.e. it comprises the following structure, repeated as many times as there are reactions between the functional groups on the hyaluronic acid molecule mlP and / or m2P):

[0074] in which:

[0075] - mlP and m2P are respectively a first and second acid molecule hyaluronic acid;

[0076] - Ar, identical or different, is an aromatic functional group capable to react with another functional group Ar and form covalent intermolecular bonds, preferably Ar is an aryl group that can possibly be substituted;

[0077] - Y is absent, an oxygen atom, a sulfur atom, an -NR1- group, a grouping -NR1-(L)P-NR1-, a grouping -S-(L)PS- or a grouping -CR'R2-(L)p-CR'R2- with:

[0078] R1 and R2 being independently of each other a hydrogen atom or a hydrocarbon group comprising from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 10 carbon atoms, even more preferably from 1 to 5 carbon atoms,

[0079] L being a hydrocarbon group comprising 1 to 3 carbon atoms or a peptide, and

[0080] p being an integer from 0 to 200, preferably from 1 to 100.

[0081] The binding of the Ar (or Ar') group to hyaluronic acid can occur by the intermediary of any functionalized spacer grouping capable of reacting with a

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] The function carried by hyaluronic acid, particularly with a carboxyl group, is supported by the hyaluronic acid. The spacer group can be a hydrocarbon group comprising 1 to 20 carbon atoms functionalized with an amine. When the Ar group is an aryl group, it may be substituted by one or more substituents selected from the group consisting of the -NR3R4, -SR3, and -OR3 groups, in which R3 and R4 represent, independently of each other, a hydrogen atom or a hydrocarbon group comprising from 1 to 10 carbon atoms, preferably from 1 to 5 carbon atoms. Preferably, the substituent for the Ar group is a hydroxyl group. Preferably, Y is absent, meaning that the two Ar groups are covalently linked by a carbon-carbon bond. In some embodiments, when hyaluronic acid is functionalized with a group comprising a hydroxyphenyl, each of the covalent intermolecular bonds formed can be represented as follows (i.e., the cross-linked functionalized hyaluronic acid comprises the following structure, repeated as many times as there are reactions between the functional groups on the hyaluronic acid mlP and / or m2P molecule): [Chem. 2] in which: - m,P and m2P are respectively a first and a second molecule of hyaluronic acid; - W is a hydrocarbon group comprising from 1 to 20 carbon atoms, preferably comprising from 2 to 10 carbon atoms, preferably comprising from 2 to 5 carbon atoms. Preferably, W is a hydrocarbon group with 2 carbon atoms. When hyaluronic acid is functionalized with a group including a hydroxyphenyl, each of the covalent intermolecular bonds formed can be the following (i.e., cross-linked functionalized hyaluronic acid comprises the following structure, repeated as many times as there are reactions between the functional groups on the mlP and / or m2P hyaluronic acid molecule):

[0091] [Chem. 3] .0 HlT W

[0092]

[0093]

[0094]

[0095]

[0096] CH in which: - m,P and m2P are respectively a first and a second hyaluronic acid molecule; - W is a hydrocarbon group comprising from 1 to 20 carbon atoms, preferably comprising from 2 to 10 carbon atoms, preferably comprising from 2 to 5 carbon atoms. Preferably, W is a hydrocarbon group with 2 carbon atoms. Typically, when hyaluronic acid is functionalized with a group including a hydroxyphenyl, each of the covalent intermolecular bonds formed can be independently represented by one of the following structures (i.e., cross-linked functionalized hyaluronic acid comprises a mixture of the following structures repeated as many times as there are reactions between the functional groups on the hyaluronic acid mlP and / or m2P molecule):

[0097] [Chem. 4] - ___ / 7 GH KG

[0098]

[0099] And [Chem. 5] * t "0 W-. 'GH 'SJ?'

[0100] Y g ' CH with mæ, m2P and W as described previously.

[0101] Preferably, the hyaluronic acid molecules have been pre-functionalized by the introduction of hydroxyphenyl groups. Even more preferably, the hyaluronic acid molecules have been modified using tyramine, tyrosine, or dopamine. In the case of tyramine, in the preceding representations of the covalent intermolecular bonds formed, W is a two-carbon hydrocarbon group.

[0102] The hydrogel is advantageously obtained by crosslinking functionalized polymer molecules.

[0103] In one embodiment, the hydrogel can be obtained by crosslinking functionalized polymer molecules in the presence of a lubricating agent.

[0104] The lubricating agent makes it possible to reduce the injection forces of the composition (compared to a control without lubricating agent), for example by at least 10% or 15% or 20% or 25%.

[0105] The useful lubricant agent within the framework of the invention can be chosen from biocompatible polymers, such as proteins, peptides, polysaccharides or nucleic acids, these biocompatible polymers preferably having a molecular weight of 0.5 to 10 MDa or of 0.7 to 10 MDa, more particularly of 1 to 5 MDa or even of 1.5 to 4 MDa.

[0106] In particular, the lubricating agent may be a non-functionalized non-crosslinked polysaccharide (native polysaccharide), in particular non-functionalized non-crosslinked hyaluronic acid, non-functionalized non-crosslinked heparosan, non-functionalized non-crosslinked carboxymethylcellulose, or a mixture thereof, preferably with a molecular weight ranging from 0.5 to 10 MDa or from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or from 1.5 to 4 MDa.

[0107] Polyols, in particular mannitol, glycerol, sorbitol, propylene glycol, xylitol, erythritol, maltitol, and lactitol, or mixtures thereof, may be cited as examples of lubricating agents. These polyols may have a synergistic effect on sterilization stability when combined with the thiosulfate salt.

[0108] Preferably, the lubricating agent is non-functionalized non-crosslinked hyaluronic acid or non-functionalized non-crosslinked carboxymethylcellulose, preferably with a molecular weight ranging from 0.5 to 10 MDa, even more preferably from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or from 1.5 to 4 MDa.

[0109] More preferably, the lubricating agent is a non-functionalized, non-crosslinked hyaluronic acid, preferably with a molecular weight ranging from 0.5 to 10 MDa, even more preferably from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or from 1.5 to 4 MDa.

[0110] The hydrogel of the invention may comprise one or more additional components selected from anesthetic agents, amino acids, vitamins, minerals, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and mixtures thereof.

[0111] Examples of anesthetic agents include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, Fomocaine, Guafecaine, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamide, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, PropoxycaineProxymetacaine, Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof. Preferably, the composition according to the invention comprises an anesthetic agent, for example as defined above, and in particular lidocaine, mepivacaine, or one of their salts such as the hydrochloride; preferably in amounts ranging from 0.1 to 30 mg / ml, for example from 0.5 to 10 mg / ml or more preferably from 2 to 6 mg / ml of composition.

[0112] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols such as mannitol, glycerol, sorbitol, propylene glycol, xylitol, erythritol, maltitol or lactitol, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, sulfites, bisulfites, and mixtures thereof.

[0113] Examples of amino acids include, but are not limited to, arginine (e.g., L-arginine), isoleucine (e.g., L-isoleucine), leucine (e.g., L-leucine), lysine (e.g., L-lysine or L-lysine monohydrate), glycine, valine (e.g., L-valine), threonine (e.g., L-threonine), proline (e.g., L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (e.g., N-acetylated derivatives such as N-acetyl-L-cysteine), and mixtures thereof.

[0114] Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and better pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0115] Examples of minerals include, but are not limited to, zinc salts (e.g. zinc acetate, especially dehydrated), magnesium salts, calcium salts (e.g. hydroxyapatite, especially in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g. copper sulfate, especially pentahydrate), possibly in hydrated form, and mixtures thereof.

[0116] Examples of co-enzymes include, but are not limited to, coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0117] Examples of adrenergic derivatives include, but are not limited to, adrenaline, noradrenaline and a mixture thereof.

[0118] Advantageously, the hydrogel according to the invention is free of corticosteroid.

[0119] According to one embodiment, the hydrogel of the present invention is an injectable hydrogel, that is to say a composition which can be injected manually by means of a syringe fitted with a needle of diameter from 0.1 to 0.5 mm, for example a hypodermic needle of 32 G, 30 G, 27 G, 26 G, 25 G.

[0120] The injectable hydrogel has a physiological pH, i.e., ranging from 6.8 to 7.8. The pH of the injectable hydrogel is preferably greater than or equal to 6.9 and less than or equal to 7.4; 7.3; 7.2; 7.1 or 7.

[0121] The injectable hydrogel advantageously has a phase angle θ less than or equal to 45°, at 1Hz for a strain of 0.1% or a shear stress of 5 Pa, preferably a phase angle θ ranging from 0.1° to 45° or from 20° to 45°.

[0122] The hydrogel of the present invention advantageously exhibits good sterilization stability. The hydrogel according to the invention advantageously has an elastic modulus G' ranging from 1 to 2000 Pa for a stress of 5 Pa at 1 Hz and 25°C, preferably greater than or equal to 10 Pa or even greater than 100 Pa or 150 Pa.

[0123] The hydrogel of the present invention has mechanical properties suitable for use in filling soft tissues.

[0124] The hydrogel of the present invention may comprise:

[0125] - 0.5 to 3% by weight of cross-linked functionalized hyaluronic acid, and

[0126] - from 0.001 to 3% by weight, preferably from 0.1 to 2% by weight, of lubricant (per e.g. a polysaccharide, such as native hyaluronic acid);

[0127] in relation to the total weight of the composition.

[0128] The hydrogel of the present invention typically has a mass ratio of functionalized polysaccharide / lubricating agent (e.g. unmodified non-crosslinked hyaluronic acid) ranging from 51 / 49 to 99 / 1, preferably ranging from 70 / 30 to 95 / 5, or even from 70 / 30 to 90 / 10.

[0129] The total concentration of polysaccharides in the composition (the total concentration of polysaccharides includes the concentration of HA and lubricant when the latter is a polysaccharide) advantageously varies from 1 mg / g to 50 mg / g of composition, more advantageously from 5 mg / g to 35 mg / g of composition, even more advantageously from 10 mg / g to 30 mg / g of composition.

[0130] The hydrogel of the present invention can in particular be prepared by a process including the following steps:

[0131] (a) supply of hyaluronic acid molecules modified by groups functionals capable of reacting with each other and creating covalent intermolecular bonds (functionalized HA);

[0132] (b) possibly crosslinking of functionalized HA molecules to form a cross-linked functionalized HA, more precisely a hydrogel comprising a cross-linked functionalized HA and the possible lubricating agent.

[0133] The hyaluronic acid molecules modified by functional groups capable of reacting with each other and creating intermolecular covalent bonds are as described above. The functional groups may be identical or different. In particular, the functional groups may be groups as described above, especially thiol, methacrylate, diene, and hydroxyphenyl groups.

[0134] In certain embodiments, the functionalized hyaluronic acid molecules have one of the following structures:

[0135] [Chem.6] O

[0136] or

[0137] [Chem.7]

[0138] in which:

[0139] P is a hyaluronic acid molecule, hyaluronic acid being able to be as described above;

[0140] W is a hydrocarbon group comprising from 1 to 20 carbon atoms, preferably comprising from 2 to 10 carbon atoms, even more preferably comprising from 2 to 5 carbon atoms; and

[0141] n is the number of functional groups present on the hyaluronic acid, typically n is greater than or equal to 1 or greater than or equal to 2 and may be equal to the number of repeat units of the hyaluronic acid, half of it or a quarter of it, n is for example between 2 and 100, in particular between 2 and 50.

[0142] Preferably, W is a hydrocarbon group with 2 carbon atoms. Thus, the functionalized hyaluronic acid is preferably a hyaluronic acid modified by a tyramine or a dopamine, preferably a tyramine.

[0143] It is understood that hyaluronic acid can be functionalized by one or more functional groups.

[0144] Functionalized hyaluronic acid molecules can be prepared using methods well known to those skilled in the art. In particular, the use of coupling agents such as l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 4-(4,6-Dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) allows functional groups possessing a primary amine to be grafted onto a polysaccharide containing carboxyl groups (Gürer et al., Carbohydrate Polymers, Volume 267, September 1, 2021, 118226; Tournier et al., Advanced Science 2023).

[0145] Functionalized hyaluronic acid molecules having the following structure:

[0146] [Chem.6]

[0147] Or

[0148] [Chem.7]

[0149] in which P, n and W are as described above, can advantageously be prepared at a pH ranging from 4 to 9, preferably ranging from 4 to 5.5 or from 4.5 to 5.5 or from 5 to 5.5 or from 6.8 to 7.8.

[0150] They are typically obtained by placing a polysaccharide (non-functionalized, non-crosslinked polysaccharide) and a reagent of the formula NH2-W-Ph-OH with W as described above in an aqueous reaction medium comprising a coupling agent. Preferably, the polysaccharide is dissolved in the aqueous medium. Preferably, the aqueous medium is deionized water. The coupling agent is introduced with molar ratios of coupling agent / hyaluronic acid (disaccharide) repeat unit ranging from 0.01 to 10, preferably from 0.025 to 1. more preferably from 0.025 to 0.25. The reagent can be introduced concomitantly, before or after the addition of the coupling agent, preferably the reagent is added at least 10 min e.g. from 10 minutes to 24 hours, e.g. from 1 hour to 15 hours, e.g. from 5 hours to 24 hours after the addition of the coupling agent.

[0151] Functionalized hyaluronic acid molecules typically have a molar functionalization rate ranging from 0.1 to 10%, preferably from 0.5 to 5%, even more preferably from 2 to 5% or from 0.8 to 4% or from 0.5 to 2%.

[0152] When implemented, crosslinking (step (b)) involves a step of preparing a reaction medium comprising functionalized hyaluronic acid molecules and optionally a lubricating agent and a step of reacting the reaction medium to form a crosslinked functionalized hyaluronic acid, more specifically a hydrogel comprising a crosslinked functionalized hyaluronic acid and optionally the lubricating agent.

[0153] The lubricating agent may be as described above.

[0154] The reaction medium typically comprises a solvent. The solvent is generally water or a mixture comprising water and an organic solvent (typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 99% by weight of water relative to the total weight of the solvent).

[0155] The mass concentration of functionalized hyaluronic acid or functionalized hyaluronic acid salt in the reaction medium advantageously varies from 5 to 300 mg / g, for example from 10 to 100 mg / g or from 50 to 300 mg / g, preferably from 50 to 200 mg / g or 10 to 50 mg / g.

[0156] The mass concentration of lubricant in the reaction medium advantageously varies from 1 to 50 mg / g, preferably from 1 to 20 mg / g, or from 2 to 8 mg / g, preferably from 2 to 5 mg / g.

[0157] Depending on the choice of functional groups, the reaction medium may also include a radical initiator and / or a catalyst.

[0158] When functionalized hyaluronic acid molecules have the following functional groups:

[0159] [Chem.6]

[0160] Or

[0161] [Chem.7] z\.-' p ; hrhi X « - «U

[0162] With P, n, and W as described above, the crosslinking reaction is typically carried out in the presence of an oxidant such as a peroxide, for example hydrogen peroxide, and a catalyst such as peroxidase, for example horseradish peroxidase. Any oxidant or agent capable of generating free radicals may be used.

[0163] Alternatively, crosslinking can be carried out in the presence of a photoinitiator, for example riboflavin (vitamin B2), Eosin Y, tris(bipyridine)ruthenium(II) (Ru(bpy)3), Irgacure 2959, Irgacure 819, and UV and / or visible light, for example with wavelengths between 200-500 nm, 200-400 nm, 300-500 nm or 400-460 nm, particularly in the UV-visible range. Preferably, crosslinking is carried out in the presence of riboflavin (vitamin B2), under exposure to UV-visible light with wavelengths between 250 and 500 nm, preferably between 400 and 500 nm, and even more preferably between 440 and 460 nm. Advantageously, irradiation in the visible range allows for better preservation of the polysaccharide chains. Preferably, the irradiation dose received by the functionalized cross-linked hyaluronic acid is between 2 and 50 J / cm², for example, an irradiation dose of approximately 10 J / cm².

[0164] The concentration of riboflavin in the crosslinking reaction medium typically varies from 0.5 to 2000 ppm, preferably from 2 to 2000 ppm or from 25 to 500 ppm or from 50 to 500 ppm, typically from 0.5 to 100 ppm or from 0.5 to 5 ppm.

[0165] The molar ratio "quantity of photoinitiator / quantity of functional groups (e.g. hydroxyphenyl groups, such as tyramine groups)" typically varies from 0.001 to 10, preferably from 0.01 to 2, even more preferably from 0.025 to 1.

[0166] The quantity of functional groups can be determined by UV-Vis Spectroscopy and / or NMR measurement.

[0167] The preparation of the reaction medium typically includes a homogenization step. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with paddles, or manual stirring.

[0168] The preparation of the reaction medium is typically carried out at a temperature ranging from 4 to 40°C, preferably from 15°C to 25°C.

[0169] The reaction of the reaction medium (crosslinking) yields a crosslinked functionalized hyaluronic acid, more precisely a hydrogel comprising a crosslinked functionalized hyaluronic acid and the lubricating agent. Crosslinking is generally carried out at a temperature ranging from -25 to 60°C, preferably from 30 to 60°C, or alternatively from 0 to 30°C, or from -25 to 0°C.

[0170] This step crosslinks the functionalized polysaccharide molecules / chains together. The functional groups present on the functionalized polysaccharide molecules react with functional groups present on other modified polysaccharide molecules so as to link the polysaccharide chains together and crosslink them by forming covalent intermolecular bonds. The functional groups can also react with functional groups present on the same polysaccharide molecule so as to form intramolecular bonds. Crosslinked functionalized polysaccharides comprising at least one crosslinking node between two polysaccharide chains are thus obtained.

[0171] The duration of the crosslinking step typically varies from a few seconds to a few days, preferably from 2 seconds to 24 hours, more preferably from 5 seconds to 1 hour, even more preferably from 5 seconds to 10 minutes or from 10 seconds to 5 minutes.

[0172] The process of the invention can be carried out, at least in part, within a specific receptacle with a deformable wall, such as, for example, a pouch. Indeed, the deformability properties of such a receptacle and its airtight nature make it possible to carry out the various steps of the process of the invention, and in particular the homogenization and crosslinking steps, under optimal conditions which lead to obtaining a further improved crosslinked gel, that is to say, one exhibiting injectability properties superior to those exhibited by a gel obtained according to a process using a conventional receptacle such as a pot or vat.

[0173] The preparation of the hydrogel may further include one or more of the following conventional steps: pH adjustment (1); Dilution (2); Purification (3); Addition of at least one component (4); Extrusion (5); Packaging (6); Sterilization (7).

[0174] These steps, well known to those skilled in the art, typically implemented after crosslinking, can be as described below. pH adjustment (1)

[0175] The process for preparing the injectable composition may include a step of adjusting the pH of the composition to achieve the desired pH (pH 6.8-7.8). Dilution (2)

[0176] The hydrogel preparation process may include a hydrogel dilution step. The dilution step allows the concentration of cross-linked functionalized hyaluronic acid in the prepared composition to be adjusted. In particular, an aqueous solvent is added to the hydrogel, for example, a physiological saline solution, possibly buffered by the presence of salts, such as phosphate, carbonate, or sulfate salts, or mixtures thereof. More specifically, the added aqueous solvent has a pH around the physiological pH (6.8–7.8). The concentration of cross-linked functionalized hyaluronic acid obtained following the dilution step advantageously ranges from 1 mg / g to 50 mg / g of composition, more advantageously from 5 mg / g to 35 mg / g of composition, and even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Purification (3)

[0177] The hydrogel preparation process may include at least one purification step. The purification step aims to remove any undesirable impurities. This step may also allow for liquid exchange, for example, buffer exchange.

[0178] Purification can be carried out by dialysis or by filtration, for example by dynamic cross-flow filtration (DCF). Addition of components (4)

[0179] The hydrogel preparation process includes at least one step of adding the thiosulfate salt, said step of adding the thiosulfate salt being advantageously carried out after the crosslinking step of the hyaluronic acid or its salt, when carried out. Indeed, the thiosulfate salt could interfere with the crosslinking of the hyaluronic acid or its salt.

[0180] The hydrogel preparation process may include a step of adding at least one additional component. The additional component may be selected from anesthetic agents, antioxidants, amino acids, vitamins, minerals, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride, and mixtures thereof. These components may be as described above.

[0181] The process of the present invention typically does not include steps of adding lubricating agents after crosslinking. Extrusion (5)

[0182] The hydrogel preparation process may include one or more extrusion steps. This extrusion step makes it possible to obtain a more homogeneous hydrogel, particularly with the most constant, i.e., the most regular, extrusion force possible. For example, the extrusion step may be carried out using a sieve with perforations having a diameter between 30 and 2000 µm. A person skilled in the art knows how to select the perforation diameter according to the desired mechanical properties of the composition. Packaging (6)

[0183] The hydrogel preparation process may include a hydrogel conditioning step. The composition is, for example, conditioned in an injection device. The conditioning is preferably carried out just before the sterilization step. Thus, the hydrogel may be in the form of an injection device pre-filled with the composition, for example, a syringe pre-filled with the hydrogel. Sterilization (7)

[0184] The hydrogel preparation process may include a sterilization step for the composition. Sterilization is preferably carried out by heat, for example in an autoclave. Sterilization is generally performed by increasing the temperature of the sterilization medium to a so-called "plateau temperature," which is maintained for a predetermined time called the "plateau time." Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, and preferably at a plateau time ranging from 1 to 20 minutes with F0 > 15. The sterilization value F0 corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the microorganism population present in the product to be sterilized. Alternatively, sterilization may be carried out, in particular, by gamma radiation, UV radiation, or by means of ethylene oxide.

[0185] The hydrogel obtained at the end of the process typically has a pH ranging from 6.8 to 7.8 (physiological pH).

[0186] The present invention also relates to a hydrogel as such, comprising at least one functionalized and crosslinked hyaluronic acid, and at least one thiosulfate salt, the functionalization of said functionalized hyaluronic acid being capable of reacting with the functional group of another functionalized hyaluronic acid, the crosslinking being implemented by reaction between the functional groups introduced during the functionalization reaction.

[0187] Advantageously, the thiosulfate salt is added after the crosslinking of the functionalized hyaluronic acid.

[0188] The hydrogel according to the invention may have one or more of the characteristics described in the context of the use according to the invention.

[0189] The sterile hydrogels of the invention are particularly useful for filling and / or replacing tissues, especially soft tissues.

[0190] According to a first embodiment, the hydrogel of the invention is applied by injecting the hydrogel into the tissue. In addition to filling soft tissues, it provides biostimulating effects.

[0191] They can be injected using any of the methods known to those skilled in the art. In particular, they can be administered using an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. The injection device can, in particular, be selected from a syringe, a set of microsyringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a microneedle roller.

[0192] The sterile hydrogels according to the invention are preferably injected subcutaneously.

[0193] They may relate to deep applications, mid-level applications and / or shallow applications.

[0194] According to a second embodiment, the hydrogel of the invention is applied topically.

[0195] They may have therapeutic and / or cosmetic and / or cosmeceutical applications.

[0196] In the cosmetic field, hydrogels can be particularly useful for compensating for tissue volume losses due to aging.

[0197] They can be used in the prevention and / or cosmetic treatment of alterations in the skin's surface appearance. For example, hydrogels can be used in cosmetics to prevent and / or treat alterations in the viscoelastic or biomechanical properties of the skin; to fill volume defects in the skin, in particular to fill wrinkles, fine lines and scars; to reduce nasolabial folds and marionette lines; to increase the volume of the cheekbones, chin or lips; to restore facial volume, in particular of the cheeks, temples, jawline and around the eyes; to reduce the appearance of wrinkles and fine lines.

[0198] The present invention also relates to the cosmetic use of a hydrogel according to the invention for filling tissues, in particular soft tissues, in particular to compensate for tissue volume losses due to aging.

[0199] The present invention also relates to the use of a thiosulfate salt to improve the antioxidant properties and / or to reduce the loss during sterilization of a hydrogel comprising at least one hyaluronic acid or one of its salts, functionalized, cross-linked and / or non-cross-linked.

[0200] In the context of the use of the thiosulfate salt according to the invention, preferably the thiosulfate salt has one or more of the characteristics defined in the context of the hydrogel according to the invention.

[0201] In the context of the use of the thiosulfate salt according to the invention, preferably the HA has one or more of the characteristics defined in the context of the hydrogel according to the invention.

[0202] In the context of the use of the thiosulfate salt according to the invention, preferably the hydrogel has one or more of the characteristics defined in the context of the hydrogel according to the invention.

[0203] The invention will now be described by means of the following examples, given of course by way of illustration and not limitation of the invention. Examples 1. Materials

[0204] - Non-crosslinked sodium hyaluronate 4 MDa (HA-4MDa)

[0205] - Non-crosslinked sodium hyaluronate 1.5 MDa (HA-1.5MDa)

[0206] - Ethanol (Sigma)

[0207] - Water for Injection Preparation PPI (B.Braun)

[0208] - Tyramine HCl CAS number 60-19-5 (Sigma)

[0209] - DMTMM (Sigma) CAS number 3945-69-5

[0210] - NaCl (Sigma)

[0211] - Phosphate Buffer (PBS, BBraun),

[0212] - Mepivacaine hydrochloride

[0213] - Sodium thiosulfate pentahydrate (STP) (Sigma)

[0214] - Sodium bisulfite (Sigma)

[0215] - Three-dimensional agitator

[0216] - DHR-2 Rheometer

[0217] - Dynamometer and test bench

[0218] - Homogenizer Paddle Mill

[0219] - Sterile polyethylene bag

[0220] - Vortex

[0221] - Horseradish Peroxidase (HRP) (Sigma)

[0222] - Hydrogen peroxide (Sigma)

[0223] - Riboflavin 5'-phosphate (Sigma)

[0224] -Light source 2. Methods Measurement of viscoelastic properties

[0225] The viscoelastic properties of the hydrogels obtained were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plane geometry and an anodized aluminium peltier plane (42 mm) (air gap 24 pm).

[0226] 0.5 g of sterilized hydrogel is deposited between the Peltier plane and said cone. Then a Stress scanning is performed at 1 Hz and 25°C. The elastic modulus G' and viscous modulus G'' are recorded for a stress of 5 Pa. Measurements are performed in the linear LVER domain.

[0227] The linear viscoelastic region (LVER) corresponds to the range of hydrogel deformations from an initial elastic modulus value G' to a value of the elastic modulus G' reduced by 10% of its initial value. The LVER measurement consists of an oscillatory stress scan measurement in compression mode at a given oscillation frequency to determine the linear viscoelastic region.

[0228] Preparation of a tyramine-functionalized hyaluronic acid (designated HA-Tyr)

[0229] 20 g of sodium hyaluronate with a molar mass of 1.5 MDa (25 mg / mL) and 800 g Water for injection (WFI) was placed in a reactor. The mixture was homogenized until the HA-1.5MDa was completely dissolved.

[0230] 3.56 g of DMTMM (-0.25 eq) were then added to the mixture.

[0231] The resulting mixture was placed under stirring for 15 min and then 4.4 g of Tyramine HCl were added. (-0.5 eq) were added to this mixture.

[0232] The mixture was then left under stirring for 72 hours at room temperature. 23.4 g of NaCl (~0.5 M) were then added to this mixture and homogenized until the NaCl was completely dissolved.

[0233] Three successive precipitations were then carried out in ethanol to purify the product, adding NaCl at the same concentration at each resolubilization. The resulting product (HA-Tyr) was placed under vacuum at 37 °C for 24 hours to dry and be stored in powder form. 3. Examples

[0234] 3.1 Example 1: Effect of STP on the stability of an HA gel in the presence or absence of a anesthetic.

[0235] A cross-linked hyaluronic acid hydrogel is prepared from tyramine-functionalized hyaluronic acid in the presence of unmodified, very high molecular weight sodium hyaluronate (-4 MDa) (10% by mass relative to the weight of tyramine-functionalized hyaluronic acid) in a PBS solution. The cross-linking of the functionalized hyaluronic acid is carried out in an LED irradiation chamber between 300 and 500 nm with an irradiation dose of approximately 10 J / cm² for 5 minutes after the addition of a 217 pL riboflavin solution (initial concentration in the gel (IC) = 2.17 ppm) and manual mixing. The hydrogel has a concentration of 25 mg of hyaluronic acid per gram of product.

[0236] This hydrogel is divided into four equivalent parts.

[0237] To the hydrogels obtained B and D, an aqueous solution of mepivacaine hydrochloride is added to obtain 0.3% by weight of mepivacaine hydrochloride relative to the weight of the final hydrogel. The pH is adjusted to a physiological pH.

[0238] An antioxidant supplementation solution or PBS is added to obtain hydrogels with the following concentrations (Hydrogels A and B: PBS (control), Hydrogels C and D: 4 pmol / mL sodium thiosulfate pentahydrate). The final hyaluronic acid concentration is identical for all conditions (23 mg per gram of product). The antioxidant supplementation solution is prepared in PBS.

[0239] Hydrogels A, B, C and D were sieved and then packaged in syringes.

[0240] The products were sterilized in an autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0241] After sterilization, the hydrogels were analyzed. The elastic modulus G' was determined before and after sterilization. The results are presented in Table 1 below.

[0242] [Tables 1] Hydrogel A Hydrogel B Hydrogel C Hydrogel D Mass percentage of STP in the final hydrogel (%w / w) 0 0 0.1 0.1 Molar concentration of STP in the final hydrogel (pmol / mL) 0 0 4 4 Mass percentage of mepivacaine in the final hydrogel (%w / w) 0 0.3 0 0.3 G' (1 Hz) after sterilization (Pa) 245 211 420 320 G' (%) -53 -57 -16 -31

[0243] AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0244] The results in Table 1 show that thiosulfate prevents the loss of rheological properties upon sterilization of functionalized HA-based hydrogels including or not an anesthetic agent (Hydrogels B compared to D and Hydrogels A compared to C).

[0245] 3.2 Example 2: Effect of STP and bisulfite on the stability of an HA gel in presence of an anesthetic.

[0246] A cross-linked hyaluronic acid hydrogel is prepared from tyramine-functionalized hyaluronic acid and high molecular weight hyaluronic acid in a PBS solution. Cross-linking of the functionalized hyaluronic acid is carried out in an irradiation chamber between 300 and 500 nm with an irradiation dose of approximately 10 J / cm² after the addition of 160 pL of riboflavin solution (initial concentration in the gel (IC) = 2 ppm). The hydrogel has a concentration of 24 mg of hyaluronic acid per gram of product.

[0247] An aqueous solution of mepivacaine hydrochloride is added to this hydrogel to obtain 0.3% by weight of mepivacaine hydrochloride relative to the weight of the final hydrogel. The pH is adjusted to a physiological pH.

[0248] This hydrogel is divided into three equivalent parts. An antioxidant or PBS supplementation solution is added to obtain hydrogels with the following concentrations: (Hydrogel E: PBS (control), Hydrogel F: 4 pmol / mL sodium thiosulfate pentahydrate, Hydrogel G: 4 pmol / mL sodium bisulfite).

[0249] The final concentration of hyaluronic acid is identical for all conditions (23 mg per gram of product).

[0250] The antioxidant supplementation solution is prepared in PBS.

[0251] Finally, the hydrogels were sterilized in the autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0252] After sterilization, the hydrogels were analyzed. The elastic modulus G' was determined before and after sterilization. The results are presented in Table 2 below.

[0253] [Tables2] Hydrogel E Hydrogel F Hydrogel G Antioxidant N / A STP Sodium Bisulfite Mass percentage of antioxidant in the final hydrogel (%w / w) 0 0.1 0.022 Molar concentration of antioxidant in the final hydrogel (pmol / mL) 0 4 4 Mass percentage of mepivacaine in the final hydrogel (%w / w) 0.3 0.3 0.3 G' (1 Hz) after sterilization (Pa) 223 342 125 AG' (%) -53 -28 -53

[0254] AG' (%) = (G' after sterilization - G' before sterilization) / (G' before sterilization) * 100

[0255] The results in Table 2 show that hydrogel F comprising thiosulfate exhibits a lower loss on sterilization than hydrogel G comprising a bisulfite-type antioxidant.

[0256] 3.3 _ Example 3: effect of STP on the thermal and time stability of a HA gel containing an anesthetic

[0257] A cross-linked hyaluronic acid hydrogel is prepared from tyramine-functionalized hyaluronic acid in the presence of a high molecular weight (4 MDa) sodium hyaluronate lubricating agent in a PBS solution. The cross-linking of the functionalized hyaluronic acid is carried out in an irradiation chamber between 300 and 500 nm with an LED irradiation dose of approximately 10 J / cm² for 5 minutes after the addition of 228 pL of riboflavin (initial concentration in the gel (IC) = 3.5 ppm). PBS is then added to the cross-linked HA. The hydrogel is homogenized using a three-dimensional stirrer. The hydrogel has a concentration of 23.5 mg of hyaluronic acid per gram of product.

[0258] To this hydrogel is added an aqueous solution of mepivacaine hydrochloride at 0.3% by weight of mepivacaine hydrochloride relative to the weight of the final hydrogel. The pH is adjusted to a physiological pH.

[0259] This hydrogel is divided into two equivalent parts.

[0260] Sodium thiosulfate pentahydrate is added directly in powder form to hydrogel I.

[0261] The final concentration of hyaluronic acid is identical for all conditions (23 mg per gram of product).

[0262] The antioxidant supplementation solution is prepared in PBS.

[0263] Hydrogels H and I were sieved and then packaged in syringes.

[0264] Finally, the hydrogels were sterilized in the autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0265] After sterilization, the hydrogels were analyzed. The elastic modulus G' was determined before and after sterilization. The sterilized hydrogels were then placed in an oven at 40°C for 3 months. The results are presented in Table 3 below.

[0266] [Tables3] Hydrogel H Hydrogel I Antioxidant N / A STP Mass percentage of antioxidant in the final hydrogel (% w / w) 0 0.3 Mass percentage of mepi-ivacaine in the final hydrogel (% w / w) 0.3 0.3 G' (1 Hz) after sterilization (Pa) at T0 327 438 G' (1 Hz) after sterilization (Pa) T3 months 166 373 G' (%) 3 months -48 -15

[0267] AG' (%)3 monthsS = (G' T3 months after sterilization - G' To after sterilization) / ( G' To after sterilization ) *100

[0268] The results in Table 3 show that the hydrogel according to the invention exhibits very good rheological properties and that these good properties are durable over time.

[0269] The results show that thiosulfate improves the stability over time after sterilization of functionalized HA-based hydrogels.

[0270] The inventors also observed that the hydrogels according to the invention exhibited better stability against aging, since they exhibited reduced oxidation compared to hydrogels free of STP or containing a bisulfite-type antioxidant, particularly in the presence of an anesthetic, since the proportion the oxidation product of mepivacaine (mepivacaine-N-oxide, CAS No. 1346597-75-8) present in these hydrogels has been reduced.

Claims

Demands

1. Use of a hydrogel in the prevention and / or cosmetic treatment of an alteration in the surface appearance of the skin, said hydrogel comprising at least one functionalized hyaluronic acid, optionally cross-linked, and at least one thiosulfate salt.

2. Use according to claim 1, wherein the thiosulfate salt is sodium thiosulfate.

3. Use according to any one of claims 1 to 2, wherein the thiosulfate salt represents a content strictly less than 10% by mass of thiosulfate salt, preferably sodium thiosulfate, preferably less than or equal to 5% by mass of thiosulfate salt, even more preferably less than or equal to 2% by mass of thiosulfate salt relative to the total mass of the hydrogel.

4. Use according to any one of claims 1 to 3, wherein the functionalized hyaluronic acid is hyaluronic acid modified by at least one functional group selected from methacrylates, acrylates, aldehydes, thiols, dienes, alkynes, oximes, silyls or hydroxyphenyls, preferably from methacrylates, thiols, dienes and hydroxyphenyls.

5. Use according to any one of claims 1 to 4, wherein the functionalized hyaluronic acid is a hyaluronic acid modified by at least one functional group corresponding to the formula (9): H2N-R'l -Ar' (9) Ar' is an aromatic functional group capable of reacting with another functional group Ar' and forming covalent intermolecular bonds, preferably Ar' is an aryl group comprising at least one -OH function and optionally substituted by one or more other groups selected from -OH or hydrocarbon groups comprising from 1 to 10 carbon atoms, R'l represents a hydrocarbon group optionally comprising one or more oxygen atoms, preferably an alkylene group comprising from 1 to 10 carbon atoms and optionally an -OH or -COOH function.

6. Use according to any one of claims 1 to 5, wherein said at least one functionalized hyaluronic acid is a functionalized and crosslinked hyaluronic acid, preferably having the following structure: [Chem.l] --Y ---- / K \ \ mlP m2P in which: - mlP and m2P are respectively a first and a second molecule of hyaluronic acid; - Ar, identical or different, is a functional group capable of reacting with another functional group Ar and forming covalent intermolecular bonds, preferably Ar is an aryl group that can be substituted;- Y is absent, an oxygen atom, a sulfur atom, an -NR1- group, an -NR1-(L)P-NR1- group, an -S-(L)PS- group or a -CR1R2-(L)P-CR1R2- group with: R1 and R2 being independently of each other a hydrogen atom or a hydrocarbon group comprising from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 10 carbon atoms, even more preferably from 1 to 5 carbon atoms, L being a hydrocarbon group comprising from 1 to 3 carbon atoms or a peptide, and p being an integer from 0 to 200, preferably from 1 to 100.;

7. Use according to any one of claims 1 to 6, wherein said at least one functionalized hyaluronic acid has a weight-average molecular mass (Mw) of 0.05 to 10 MDa, preferably of 0.5 to 5 MDa, for example of 2 to 4 MDa or of 1 to 5 MDa.

8. Use according to any one of claims 1 to 7, wherein the hydrogel further comprises at least one additive complementary chosen from among lubricating agents, anesthetic agents, amino acids, vitamins, minerals, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and one of their mixtures.

9. Use according to any one of claims 1 to 8, wherein the hydrogel is in the form of an injectable composition or a composition for topical application.

10. Use according to any one of claims 1 to 9, to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin, to fill volume defects of the skin, in particular to fill wrinkles, fine lines and scars, to reduce nasolabial folds and marionette lines, to increase the volume of the cheekbones, chin or lips, to restore facial volumes, in particular of the cheeks, temples, oval of the face, and around the eye, to reduce the appearance of wrinkles and fine lines.

11. Hydrogel as defined in claim 1 comprising at least one functionalized and crosslinked hyaluronic acid, and at least one thiosulfate salt.

12. Hydrogel according to claim 11, wherein the hyaluronic acid has one or more of the characteristics defined in claims 4 to 6 and / or the thiosulfate salt has one or more of the characteristics defined in claims 2 to 3.

13. Hydrogel according to any one of claims 11 to 12, further comprising at least one lubricating agent, preferably selected from non-crosslinked and non-functionalized hyaluronic acids advantageously having a molecular mass by weight from 0.5 to 10 MDa, even more preferably from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or from 1.5 to 4 MDa.

14. A method for preparing a hydrogel according to any one of claims 11 to 13, comprising the following steps: (a) supplying hyaluronic acid modified by functional groups capable of reacting with each other and creating covalent intermolecular bonds; (b) crosslinking, possibly in the presence of a lubricating agent, of the functionalized hyaluronic acid supplied in step (a) to form the hydrogel, (c) addition of the thiosulfate salt to the formulation, (d) optionally sterilization, preferably by heat, of the hydrogel to obtain a sterile hydrogel.

15. Use of a thiosulfate salt as defined in claim 1 to reduce the sterilization loss of a hydrogel comprising at least one functionalized, crosslinked and / or non-crosslinked hyaluronic acid.

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