INJECTABLE COMPOSITIONS BASED ON FUNCTIONALIZED POLYSACCHARIDES

Abstract

An injectable composition comprising a matrix based on a crosslinked functionalized polysaccharide and a lubricating agent is described. The matrix is ​​capable of being obtained by crosslinking the polysaccharide modified by functional groups capable of reacting with each other and creating intermolecular covalent bonds to form the crosslinked functionalized polysaccharide, the crosslinking being carried out in the presence of a lubricating agent.

Description

Title of the invention: INJECTABLE COMPOSITIONS BASED ON FUNCTIONALIZED POLYSACCHARIDES FIELD OF THE INVENTION

[0001] The present invention relates to injectable compositions based on polysaccharides, in particular hyaluronic acid, and their preparation process. TECHNOLOGICAL BACKGROUND

[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 based on hyaluronic acid. To improve skin quality, compositions prepared from unmodified 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 hyaluronic acid, the hyaluronic acid usually being modified by crosslinking. This crosslinking has the advantage of increasing the in vivo durability and resistance to in vivo degradation of the compositions as well as improving their viscoelastic properties. Crosslinking is typically carried out with one or more crosslinking agent(s). Conventional crosslinking agents have at least two reactive functions allowing them to link polysaccharide molecules together (e.g. 1,4-butanediol diglycidyl ether: BDDE). As a result, these conventional crosslinking agents can also react with biopolymers such as peptides, carbohydrates and DNA, which is not desired.Furthermore, conventional techniques do not allow the addition of other molecules or polymers without fear of their degradation linked to the alkaline pH of the reaction medium, or to unwanted crosslinking, due to the presence of highly reactive epoxide groups. For all these reasons, it is therefore desirable to avoid their use and to employ alternative crosslinking methods when preparing compositions based on polysaccharides.

[0004] Thus, a need remains for the provision of a new injectable composition based on polysaccharide, in particular hyaluronic acid, prepared under mild conditions which allow preservation of the polysaccharide chains and which have rheological properties suitable for applications in the cosmetic and therapeutic field. BRIEF DESCRIPTION OF THE INVENTION

[0005] The present invention relates to an injectable composition comprising a matrix based on a crosslinked functionalized polysaccharide and a lubricating agent, the matrix being capable of being obtained by crosslinking a polysaccharide modified by functional groups capable of reacting with each other and creating intermolecular covalent bonds to form the crosslinked functionalized polysaccharide, the crosslinking being carried out in the presence of a lubricating agent.

[0006] The present invention also relates to a process for preparing such a composition. The process comprises the following steps:

[0007] (a) providing a polysaccharide modified by functional groups capable of reacting with each other and creating covalent intermolecular bonds;

[0008] (b) crosslinking, in the presence of a lubricating agent, the polysaccharide provided in step (a) to form the crosslinked functionalized polysaccharide.

[0009] Finally, the present invention relates to the cosmetic use of such a composition for preventing and / or treating the alteration of the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, in particular for filling wrinkles, fine lines and scars; for reducing nasolabial folds and bitterness folds; for increasing the volume of the cheekbones, chin or lips; for restoring the volumes of the face, in particular the cheeks, temples, oval of the face, and the area around the eyes; for reducing the appearance of wrinkles and fine lines.

[0010] Other aspects of the invention are as described below and in the claims. DEFINITIONS

[0011] The term "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed of two media, one "solid" and the other "liquid", dispersed in each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (for example hydrogen bonds) or by covalent bonds (crosslinking). The liquid medium consists of a solvent. A gel generally corresponds to a viscoelastic product that has a phase angle δ of less than 90°, preferably less than or equal to 70°, preferably less than or equal to 45°, at 1 Hz for a deformation of 0.1% or a pressure of 1 Pa, preferably a phase angle δ ranging from 2° to 45° or ranging from 20° to 45°.

[0012] The term “hydrogel” designates 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%, in particular at least 97%, in particular at least 98% by weight of the liquid medium) and having a pH ranging from 6.8 to 7.8.

[0013] The term “injectable hydrogel” designates a hydrogel that can be injected manually using a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example of a 32G, 30G, 27G, 26G, 25G hypodermic needle. Preferably, an “injectable hydrogel” is a hydrogel having an average extrusion force less than or equal to 25N, preferably ranging from 5 to 25N, or even ranging from 8 to 15N, when measured with a dynamometer, at a fixed speed of approximately 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 (27G) and a length L2”, at room temperature.

[0014] A "superficial application" means the administration, for example by mesotherapy, of a composition superficially into the skin, or onto the skin, for the treatment of the superficial layers of the skin, the epidermis and the most superficial parts of the dermis, to reduce superficial wrinkles and / or improve the quality of the skin (such as its radiance, density or structure) and / or rejuvenate the skin.

[0015] A "midline application" refers to administering a composition into the midline of the skin to treat the midline layers of the skin, as well as to reduce midline wrinkles.

[0016] A "deep application" refers to the administration of a composition into the deepest layers of the skin, the hypodermis and the deepest part of the dermis, and / or under the skin (above the periosteum) to "add volume", such as for filling the deepest wrinkles and / or partially atrophied regions of the contour of the face and / or body. So-called "volumizing" compositions may typically be administered for deep application.

[0017] The term “polysaccharide” designates a polymer composed of monosaccharides (preferably D enantiomers) joined together by glycosidic bonds.

[0018] The term "repeating unit" of a polysaccharide means a structural unit consisting of one or more (usually 1 or 2) monosaccharides whose repetition produces the complete polysaccharide chain.

[0019] A "crosslinked polysaccharide" refers to a polysaccharide modified during a crosslinking reaction. Crosslinking leads to the formation of covalent bonds between the polysaccharide chains.

[0020] Conversely, a “non-crosslinked polysaccharide” designates a polysaccharide which has not undergone a crosslinking reaction.

[0021] A “functionalized polysaccharide” means a polysaccharide modified during a functionalization reaction by the introduction of functional groups. In the context of the present invention, the functionalization aims to chemically modify the polysaccharide in order to make it crosslinkable in a subsequent step.

[0022] A “crosslinked functionalized polysaccharide” designates a polysaccharide modified during a functionalization reaction and then crosslinked by reaction between the functional groups introduced during the functionalization reaction.

[0023] Conversely, a “non-functionalized, non-crosslinked polysaccharide” means a polysaccharide that has not been modified during a functionalization reaction and that has not undergone a crosslinking reaction. A non-functionalized, non-crosslinked polysaccharide may also be called a native polysaccharide.

[0024] The “molar functionalization rate” (TF), expressed in %, designates the molar quantity of functional groups grafted onto the polysaccharide, expressed per 100 moles of repeating units of the polysaccharide. 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.

[0025] By “room temperature” is meant a temperature ranging from 20 to 25°C, more particularly 21°C. DETAILED DESCRIPTION OF THE INVENTION

[0026] Unexpectedly, the inventors discovered that by carrying out the crosslinking of polysaccharide molecules modified by particular functional groups ("functionalized polysaccharides"), in the presence of a lubricating agent, a composition based on crosslinked polysaccharides having mechanical properties

[0027] satisfactory was obtained (suitable for applications in the cosmetic and therapeutic field). More particularly, the compositions obtained have satisfactory viscoelastic properties (elastic modulus G' and phase angle, delta) as well as cohesiveness (represented by the measurement of tau, value of the stress at the intersection of G' and G”).

[0028] In particular, the compositions obtained have improved mechanical properties, for example improved viscoelastic properties (G', delta), compared to those of compositions obtained by a different process (addition of the lubricating agent after crosslinking).

[0029] The particular functional groups are functional groups capable of reacting with each other and forming intermolecular covalent bonds to result in a crosslinked functionalized polysaccharide. Crosslinking carried out in this manner makes it possible to preserve the polysaccharide chains compared to crosslinking carried out using conventional crosslinking agents, for example using BDDE. The molar mass of the polysaccharide is preserved.

[0030] Without wishing to be bound by any theory, the inventors believe that when the lubricating agent is introduced into the crosslinking reaction medium, it becomes integrated into the three-dimensional network formed by the polysaccharides. The lubricating agent is notably trapped in the three-dimensional network. The composition, also called gel or hydrogel, is then formed from a network of polysaccharide chains linked together both by covalent bonds (permanent bonds) and by non-covalent bonds, of the hydrogen type (reversible bonds), contributing to strengthening the viscoelastic properties of the network, as reflected by rheology measurements.

[0031] When the lubricating agent is added after the crosslinking step, it interacts very little with the three-dimensional network, the latter then only plays the role of a lubricating agent between the crosslinked particles, also called grains, of crosslinked polysaccharides.

[0032] The crosslinking carried out under the conditions of the invention forms a matrix based on a crosslinked functionalized polysaccharide and a lubricating agent, i.e. a three-dimensional network formed of polysaccharide chains linked together by covalent (permanent) and non-covalent (reversible) bonds.

[0033] The present invention thus relates to an injectable composition comprising a matrix based on a crosslinked functionalized polysaccharide and a lubricating agent, the matrix being capable of being obtained by crosslinking polysaccharide molecules modified by functional groups capable of reacting with each other and creating covalent intermolecular bonds to form the crosslinked functionalized polysaccharide, the crosslinking being carried out in the presence of a lubricating agent.

[0034] Matrix based on a crosslinked functionalized polysaccharide and a lubricating agent

[0035] The crosslinked functionalized polysaccharide is obtained by crosslinking polysaccharide molecules previously modified by functional groups capable of reacting with each other and forming covalent intermolecular bonds (functionalized polysaccharides).

[0036] The polysaccharide may be any polymer composed of monosaccharides joined together by glycosidic bonds or mixtures thereof. Preferably, the polysaccharide is chosen from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof. Even more preferably, the polysaccharide is chosen from hyaluronic acid, heparosan, chondroitin and mixtures thereof, even more preferably the polysaccharide is hyaluronic acid or one of its salts, in particular a physiologically acceptable salt such as the sodium salt, the potassium salt, the zinc salt, the calcium salt, the magnesium salt, the silver salt, the calcium salt and mixtures thereof.More specifically, hyaluronic acid is in its acid form or in the form of sodium salt (NaHA).

[0037] Preferably, if the polysaccharide is hyaluronic acid or one of its salts, it 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 1 to 5 MDa or ranging from 2 to 4 MDa.

[0038] The polysaccharide molecules have been previously modified by functional groups capable of reacting with each other and forming covalent intermolecular bonds. The polysaccharide molecules are not modified by bifunctional molecules which react with two polysaccharide chains to link them together. In the context of the present invention, two functional groups react with each other in the presence of suitable reaction conditions.

[0039] The modifications can be carried out on the carboxyl, hydroxyl and N-acetyl groups of the polysaccharides, or after oxidation of the polysaccharides, for example with sodium periodate.

[0040] 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, azide, furan, alkynes, aldehydes, ketones, azides, alkynes, imines, oximes, silanes, epoxides, isocyanates, amines, alcohols, hydroxyphenyls, carboxylic acids, diazo, carbodiimides, anhydrides, thioesters, nitriles, boronates, vinylsulfones, triazine, hydrazone, coumarin, oxazolidinones, aziridines and tetrazoles.

[0041] Preferably, the functional groups are chosen from methacrylate, acrylate, aldehyde, thiol, diene, alkyne, oxime, silyl or hydroxyphenyl groups, more preferably the functional groups are chosen from methacrylate, thiol, diene and hydroxyphenyl groups.

[0042] After reaction of the functional groups with each other, the polysaccharide obtained, called “crosslinked functionalized polysaccharide”, has covalent intermolecular bonds which can be represented in the following manner (i.e. it comprises the following structure):

[0044] in which:

[0045] - mlP and m2P are respectively a first and a second molecule of polysaccharides, preferably a first and a second hyaluronic acid molecule;

[0046] - Ar is a functional group capable of reacting with another group functional Ar and form covalent intermolecular bonds, preferably Ar is an aryl group which may be substituted;

[0047] - Y is absent, an oxygen atom, a sulfur atom, a -NR1- group, a group -NR1-(L)n-NR1-, a group -S-(L)nS- or a group -CR'R2-(L)n -CR'R2- with:

[0048] R1 and R2 being independently of one another 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,

[0049] L being a hydrocarbon group comprising from 1 to 3 carbon atoms or a peptide, and

[0050] n being an integer ranging from 0 to 200, preferably from 1 to 100.

[0051] The binding of the Ar group to the polysaccharide can be done via of any functionalized spacer group capable of reacting with a function carried by the polysaccharide, in particular with a carboxyl function carried by the polysaccharide. The spacer group may be a hydrocarbon group comprising from 1 to 20 carbon atoms functionalized by an amine.

[0052] 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 group 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 is a hydroxyl group.

[0053] Preferably, Y is absent, i.e. the two Ar groups are covalently linked by a carbon-carbon bond.

[0054] In some embodiments, when the polysaccharide is functionalized with a group comprising a hydroxyphenyl, the covalent intermolecular bonds formed may be represented as follows (i.e., the crosslinked functionalized polysaccharide comprises the following structure):

[0055] [Chem.2] oa ------ W-MH

[0056] in which:

[0057] - niiP and m2P are respectively a first and a second molecule of polysaccharides, preferably a first and a second hyaluronic acid molecule;

[0058] - 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.

[0059] Preferably, W is a hydrocarbon group with 2 carbon atoms.

[0060] When the polysaccharide is functionalized by a group comprising a hydroxyphenyl, the covalent intermolecular bonds formed may be the following (i.e. the crosslinked functionalized polysaccharide comprises the following structure):

[0061] [Chem.3]

[0062] in which:

[0063] - m । P and m2P are respectively a first and a second molecule of polysaccharides, preferably a first and a second hyaluronic acid molecule;

[0064] - 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.

[0065] Preferably, W is a hydrocarbon group with 2 carbon atoms.

[0066] Typically, when the polysaccharide is functionalized with a group comprising a hydroxyphenyl, the covalent intermolecular bonds formed may comprise a mixture of the following bonds (i.e., the crosslinked functionalized polysaccharide comprises the mixture of the following structures):

[0067]

[0068] [Chem.4]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] with niiP, m2P and W as described previously. Preferably, the polysaccharide molecules, for example hyaluronic acid, have been previously modified by introducing hydroxyphenyl groups. Even more preferably, the polysaccharide molecules, for example hyaluronic acid, have been modified by means of tyramine. In this case, in the above representations of the covalent intermolecular bonds formed, W is a hydrocarbon group with two carbon atoms. Preferably, the crosslinked functionalized polysaccharide is a crosslinked functionalized hyaluronic acid. The matrix is ​​obtained by crosslinking functionalized polymer molecules in the presence of a lubricating agent. The lubricating agent allows the injection forces of the composition to be reduced (compared to a control without lubricating agent), for example by at least 10% or 15% or 20% or 25%. The lubricating agent useful in the context of the invention may be chosen from biocompatible polymers, such as proteins, peptides, polysaccharides or nucleic acids, these biocompatible polymers preferably having a molecular weight ranging from 0.5 to 10 MDa or ranging from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or even from 1.5 to 4 MDa. 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 or a mixture thereof, preferably with a molecular weight ranging from 0.5 to 10 MDa or ranging from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or even from 1.5 to 4 MDa.

[0076] 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 ranging from 0.7 to 10 MDa, more particularly from 1 to 5 MDa or even from 1.5 to 4 MDa. Optional additional components

[0077] The composition may comprise one or more additional components selected from anesthetic agents, antioxidants, amino acids, vitamins, minerals, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof.

[0078] 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, Febuvérine, Fomocaine, Guafecainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine,Proxymetacaine, 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.

[0079] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, and a mixture thereof.

[0080] Examples of amino acids include, but are not limited to, arginine (eg L-arginine), isoleucine (eg L-isoleucine), leucine (eg L-leucine), lysine (eg L-lysine or L-lysine monohydrate), glycine, valine (eg L-valine), threonine (eg L-threonine), proline (eg L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (eg N-acetylated derivatives such as N-acetyl-L-cysteine) and a mixture thereof.

[0081] 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 more preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0082] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated), magnesium salts, calcium salts (e.g., hydroxyapatite, in particular in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, in particular pentahydrate), optionally in a hydrated form, and mixtures thereof.

[0083] Examples of coenzymes include, but are not limited to, coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0084] Examples of adrenergic derivatives include, but are not limited to, adrenaline, noradrenaline, and a mixture thereof. Characteristics of the composition

[0085] The composition of the present invention is an injectable composition, i.e. a composition which can be injected manually by means of a syringe provided with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 32 G, 30 G, 27 G, 26 G, 25 G hypodermic needle.

[0086] The injectable composition has a physiological pH, i.e. ranging from 6.8 to 7.8. The pH of the injectable composition 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.

[0087] The injectable composition advantageously has a phase angle δ less than or equal to 45°, at 1 Hz for a deformation of 0.1% or a pressure of 1 Pa, preferably a phase angle δ ranging from 2° to 45° or ranging from 20° to 45°.

[0088] The composition of the present invention advantageously exhibits mechanical reinforcement of the gel as well as better cohesiveness, which translate rheologically into an elastic modulus G' ranging from 100 to 2000 Pa for a stress of 5 Pa at 1 Hz and 25°C, a stress at the crossing, r, greater than 50 Pa, preferably greater than or equal to 100 Pa or even greater than 150 Pa or 200 Pa.

[0089] The composition of the present invention has mechanical properties suitable for use in filling soft tissues.

[0090] The composition of the present invention may comprise: - from 0.5 to 3% by weight of crosslinked functionalized polysaccharides, and - from 0.001 to 3% by weight, preferably from 0.1 to 2% by weight, of agent lubricant (e.g. a polysaccharide);

[0091] relative to the total weight of the composition.

[0092] The composition of the present invention typically has a functionalized polysaccharide / lubricating agent (e.g. unmodified, uncrosslinked hyaluronic acid) mass ratio ranging from 51 / 49 to 99 / 1, preferably ranging from 70 / 30 to 95 / 5, or else ranging from 70 / 30 to 90 / 10.

[0093] The total concentration of polysaccharides in the composition (the total concentration of polysaccharides includes the concentration of polysaccharides and lubricating agent 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.

[0094] Process for preparing the compositions of the invention

[0095] The composition of the present invention may in particular be prepared by a process comprising the following steps:

[0096] (a) providing polysaccharide molecules modified by groups functional ones capable of reacting with each other and creating covalent intermolecular bonds (functionalized polysaccharides);

[0097] (b) crosslinking, in the presence of a lubricating agent, of the polysaccharide molecules functionalized to form a crosslinked functionalized polysaccharide, more precisely a matrix comprising a crosslinked functionalized polysaccharide and the lubricating agent.

[0098] The polysaccharide 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, in particular thiol, methacrylate, diene and hydroxyphenyl groups.

[0099] In some embodiments, the functionalized polysaccharide molecules have the following structure:

[0100] [Chem.6] p

[0101] in which:

[0102] P is a polysaccharide molecule, the polysaccharide possibly being as described previously;

[0103] 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.

[0104] Preferably, W is a hydrocarbon group with 2 carbon atoms. Thus, the functionalized polysaccharide is a polysaccharide modified by a tyramine.

[0105] It is understood that the polysaccharide is functionalized by several functional groups.

[0106] The functionalized polysaccharide molecules can be prepared according to methods well known to those skilled in the art. In particular, the use of coupling agents such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or 4-(4,6-Dhnethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) makes it possible to graft functional groups having a primary amine onto a polysaccharide containing carboxyl groups (Gürer et al., Carbohydrate Polymers, Volume 267, September 1, 2021, 118226; Tournier et al., Advanced Science 2023).

[0107] Functionalized polysaccharide molecules having the following structure:

[0108] [Chem.7] p

[0109] in which P and W are as described above, can advantageously be prepared at a pH ranging from 4 to 9, preferably ranging from 6.8 to 7.8.

[0110] They are typically obtained by placing a polysaccharide (non-functionalized non-crosslinked polysaccharide) and a reagent of 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 medium aqueous. Preferably, the aqueous medium is deionized water. The coupling agent is introduced with molar ratios of coupling agent / polysaccharide repeating unit varying 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 after the addition of the coupling agent.

[0111] The modified polysaccharide molecules typically have a molar functionalization rate ranging from 0.1 to 10%, preferably ranging from 0.5 to 5%, even more preferably ranging from 2 to 5%.

[0112] Crosslinking (step (b)) involves a step of preparing a reaction medium comprising a lubricating agent and functionalized polysaccharide molecules and a step of reacting the reaction medium to form a crosslinked functionalized polysaccharide, more precisely a matrix comprising a crosslinked functionalized polysaccharide and the lubricating agent.

[0113] The lubricating agent may be as described above.

[0114] 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).

[0115] The reaction medium may further comprise salts, pH adjusters, for example a Bronsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above and mixtures thereof.

[0116] The mass concentration of functionalized polysaccharides or functionalized polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 50 to 200 mg / g.

[0117] Depending on the choice of functional groups, the reaction medium may further comprise a radical initiator and / or a catalyst.

[0118] When the functionalized polysaccharide molecules have the following functional groups:

[0119] [Chem.8] p

[0120] with P and W as described above, the crosslinking reaction is typically carried out in the presence of a peroxide, for example hydrogen peroxide and a peroxidase, for example horseradish peroxidase. Any enzyme or agent capable of generating free radicals may be employed.

[0121] Alternatively, the crosslinking may 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 rays, for example of wavelengths between 200-500 nm, 200-400 nm, 300-500 nm or 400 to 460 nm, particularly the visible UV range. Preferably, the crosslinking is carried out in the presence of riboflavin (vitamin B2), under exposure to visible UV rays of wavelength between 250 and 500 nm, preferably between 400 and 500 nm, even more preferably between 440 and 460 nm. Advantageously, irradiation in the visible range allows better preservation of the polysaccharide chains.

[0122] The riboflavin concentration in the crosslinking reaction medium typically varies from 2 to 2000 ppm, preferably from 25 to 500 ppm or from 50 to 500 ppm.

[0123] The molar ratio “amount of riboflavin / amount 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.

[0124] The amount of functional groups can be determined by UV measurement.

[0125] The preparation of the reaction medium typically comprises a step of homogenization of the latter. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with blades or stirring with a spatula.

[0126] The preparation of the reaction medium is typically carried out at a temperature ranging from 4 to 35°C, preferably from 15°C to 25°C.

[0127] The reaction of the reaction medium (crosslinking) makes it possible to obtain a crosslinked functionalized polysaccharide, more precisely a matrix comprising a crosslinked functionalized polysaccharide and the lubricating agent. The crosslinking is generally carried out at a temperature ranging from -25 to 60°C, preferably from 30 to 60°C or from 0 to 30°C or from -25 to 0°C.

[0128] This step allows the functionalized polysaccharide molecules / chains to be crosslinked with each other. 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.

[0129] The duration of the crosslinking step typically varies from a few seconds to a few days, preferably from 30 minutes to 24 hours, more preferably from 1 hour to 4 hours or from 1 hour to 3 hours, even more preferably from 30 minutes to 2 hours.

[0130] The method of the invention can be carried out at least in part within a specific receptacle with a deformable wall, such as for example a pocket. Indeed, the deformability properties of such a receptacle and its hermetic nature make it possible to carry out the different steps of the method of the invention, and in particular the homogenization and crosslinking steps, under optimal conditions which lead to the production of a further improved crosslinked gel, that is to say having injectability properties superior to those exhibited by a gel obtained according to a method using a conventional receptacle such as a pot or tank.

[0131] The preparation of the injectable composition may further comprise one or more of the following conventional steps: pH adjustment (1); Dilution (2); Purification (3); Addition of at least one additional component (4); Extrusion (5); Packaging (6); Sterilization (7).

[0132] These steps, well known to those skilled in the art, typically carried out after crosslinking, may be as described below. pH adjustment (1)

[0133] The method of preparing the injectable composition may comprise a step of adjusting the pH of the composition to reach the desired pH (pH of 6.8-7.8). Dilution (2)

[0134] The method for preparing the injectable composition may comprise a step of diluting the matrix. The dilution step makes it possible to adapt the concentration of crosslinked functionalized polysaccharide in the prepared composition. In particular, an aqueous solvent is added to the matrix, for example, a physiological saline solution, possibly buffered by the presence of salts, such as phosphate or carbonate or sulfate salts or mixtures thereof. More particularly, the added aqueous solvent has a pH around physiological pH (6.8-7.8). The concentration in crosslinked functionalized polysaccharide obtained following the dilution step 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. Purification (3)

[0135] The process for preparing the injectable composition may comprise at least one purification step. The purification step aims to eliminate any undesirable impurities. This step may also allow a liquid exchange to be carried out, for example a buffer exchange.

[0136] Purification can be carried out by dialysis or by filtration, for example by dynamic tangential filtration (“DCF” for Dynamic Cross-flow Filtration). Addition of additional components (4)

[0137] The method for preparing the injectable composition may comprise a step of adding at least one additional component. The additional component may be chosen from anesthetic agents, antioxidants, amino acids, vitamins, minerals, nucleotides, nucleosides, coenzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof. These components may be as described above.

[0138] The method of the present invention typically does not include steps of adding lubricating agents after crosslinking. Extrusion (5)

[0139] The process for preparing the injectable composition may comprise one or more extrusion steps. This extrusion step makes it possible to obtain a more homogeneous injectable composition, in particular with the most constant extrusion force possible, i.e. the most regular possible. For example, the extrusion step may be carried out using a sieve whose perforations have a diameter of between 30 and 2000 μm. The person skilled in the art knows how to select the perforation diameter according to the desired mechanical properties of the composition. Packaging (6)

[0140] The method for preparing the injectable composition may comprise a step of packaging the composition. The packaging of the composition is typically carried out in an injection device. The packaging is preferably carried out just before the sterilization step. Thus, the composition may be in the form of an injection device pre-filled with the composition, for example a syringe pre-filled with the composition. Sterilization (7)

[0141] The method for preparing the injectable composition may comprise a step of sterilizing the composition. Sterilization is preferably carried out by heat, for example in an autoclave. Sterilization is generally carried out by increasing the temperature of the sterilization medium to a temperature called the "plateau temperature", which is maintained for a determined period of time called the "plateau time". Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, preferably at a plateau time ranging from 1 minute to 20 minutes with FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized. Alternatively, sterilization may be carried out in particular by gamma ray, UV radiation or by means of ethylene oxide.

[0142] The composition obtained at the end of the process typically has a pH ranging from 6.8 to 7.8 (physiological pH). Applications

[0143] The compositions of the invention are particularly useful for filling and / or replacing tissues, in particular soft tissues, in particular by injecting the compositions into the tissue.

[0144] The compositions may be injected using any of the methods known to those skilled in the art. In particular, they may be administered by means of an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supra-periosteal injection. The injection device may in particular be chosen from a syringe, a set of micro-syringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0145] The compositions of the invention are preferably injected subcutaneously.

[0146] They may concern deep applications, medium applications and / or superficial applications.

[0147] They may have therapeutic and / or cosmetic and / or cosmeceutical applications.

[0148] In the cosmetic field, the compositions can be particularly useful for compensating for losses of tissue volume due to aging.

[0149] They can be used in the prevention and / or cosmetic treatment of an alteration of the surface appearance of the skin. For example, the compositions can be used in the cosmetic field 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 bitterness folds; to increase the volume of the cheekbones, chin or lips; to restore the volumes of the face, including cheeks, temples, facial contours, and the area around the eyes; to reduce the appearance of wrinkles and fine lines.

[0150] The present invention also relates to the cosmetic use of a composition as described above for filling tissues, in particular soft tissues, in particular to compensate for losses of tissue volume due to aging. Other embodiments

[0151] Those skilled in the art will recognize that in the proposed method the polysaccharide (functionalized polysaccharide) can be replaced by any polymer. Thus, a matrix based on a crosslinked polymer can be obtained by crosslinking a polymer modified by, or comprising, functional groups capable of reacting with each other and creating intermolecular covalent bonds to form a crosslinked polymer, the crosslinking being carried out in the presence of a lubricating agent.

[0152] The useful polymer may be selected from a group consisting of collagen, elastin, sericin, hyaluronic acid, chondroitin sulfate, dextran, pectin, pullulan, xanthan, carrageenan, lignin, chitosan, alginate, cellulose and its derivatives, chitin, agarose, nucleic acids, rubber, polyethylene glycol (abbreviated as PEG), poly(lactic-co-glycolic acid) (abbreviated as PLGA), polylactic acid (abbreviated as PLLA), polycaprolactone, poly(glycolide-co-trimethylene carbonate) (abbreviated as PGTMC), poly(N-isopropylacrylamide) (abbreviated as polyNIPAM, PNIPAM or PNIPAAm) and polyvinylpyrrolidone (abbreviated as PVP).

[0153] The following examples are given for illustrative purposes, but should in no way be considered as limiting the present invention. EXAMPLES Measurement of viscoelastic properties

[0154] The viscoelastic properties of the compositions (hydrogels) obtained were measured using a cone-plane rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) and an anodized aluminum peltier plane (42 mm) (air gap 24 pm).

[0155] 0.5 g of sterilized hydrogel is deposited between the Peltier plane and the cone. Then a sweep in stresses between 0.1 and 1000 Pa is carried out at 1 Hz and 25°C. The elastic modulus G' (in Pascal), the viscous modulus G” (in Pascal) and the phase angle ô (in degrees) are reported for a stress of 5 Pa.

[0156] The constraint at the intersection of G' and G”, r, is determined at the intersection of the curves of the modules G' and G' ' and is expressed in Pascal. Extrusion force measurement

[0157] The extrusion forces (in Newton) of the gels packaged in syringes were measured using a test bench equipped with a dynamometer at a constant speed of 12.5 mm / min, through a 30G Yi' needle and at room temperature. The extrusion force results correspond to the average of the average extrusion forces on at least 2 samples. Measurement of cohesiveness

[0158] Cohesiveness is measured by mechanical compression using a rheometer. For this, the gel is deposited on a Peltier plane with an initial air gap of 2.60 mm; it is then compressed at a constant speed of 100 qm / s up to 70% of the initial air gap, at 25°C; finally, the cohesiveness of the gel is measured at the end of the compression stroke. Material#:

[0159] - Non-crosslinked sodium hyaluronate 4 MDa (HA-4MDa)

[0160] - Non-crosslinked sodium hyaluronate 1.5 MDa (HA-1.5MDa)

[0161] - Ethanol (Sigma)

[0162] - Water for Injectable Preparation PPI (B.Braun)

[0163] - Tyramine HCl CAS number 60-19-5 (Sigma)

[0164] - DMTMM (Sigma) CAS 3945-69-5

[0165] - NaCl (Sigma)

[0166] - Synthetic peptide Tyramine-Glycine-Glycine-Glycine-Tyramine (YGGGY) mass 515 kDa (Genscript)

[0167] - Phosphate Buffer (BBraun),

[0168] - Three-dimensional agitator

[0169] - DHR-2 Rheometer

[0170] - Dynamometer and test bench

[0171] - Homogenizer Paddle Mill

[0172] - Sterile polyethylene bag

[0173] - Vortex

[0174] - Horseradish Peroxidase (HRP) (Sigma)

[0175] - Hydrogen peroxide (Sigma)

[0176] - Riboflavin 5'-phosphate (Sigma)

[0177] -Light source (Paulmann)

[0178] Preparation of a hyaluronic acid functionalized by tyramine (designated HA-Tyr)

[0179] 20 g of hyaluronic acid with a molar mass of 1.5 MDa (25 mg / mL) and 800 g of water PPI were placed in a reactor. The whole was homogenized until the HA-1.5MDa was completely dissolved.

[0180] 3.56 g of DMTMM (-0.25 eq) was then added into the mixture.

[0181] The mixture obtained was stirred for 15 min and then 4.4 g of Tyramine HCl (~0.5 eq) was added to this mixture.

[0182] The mixture was then left to stir for 72 hours at room temperature. 23.4 g of NaCl (~ 0.5 M) was then added to this mixture and homogenized until the NaCl was completely dissolved.

[0183] 3 successive precipitations were then carried out in ethanol to purify the product, adding NaCl at the same concentration at each resolubilization. The product obtained (HA-Tyr) was placed under vacuum at 37 °C for 24 hours to be dried and stored in powder form. Preparation of the YGGGY peptide

[0184] The YGGGY peptide was solubilized in PPI water at a concentration of 5 mg / ml and stored at -20°C.

[0185] Example 1 - Comparison between a hydrogel according to the invention (hydrogel A) and a hydrogel with the addition of a lubricating agent after crosslinking (hydrogel B)

[0186] A hydrogel A based on HA-Tyr (15 mg / ml) and HA-4MDa (lubricating agent) (5 mg / ml) according to the invention was prepared.

[0187] 300 mg of HA-Tyr prepared according to the previous protocol and 100 mg of HA-4MDa have were homogenized in 20 grams of PBS phosphate buffer for 1.5 hours in a sterile bag using a paddle mill.

[0188] 40 pL of riboflavin (final concentration (FC) = 2 ppm) was added then the mixture obtained homogenized manually.

[0189] The mixture was then irradiated for 2 hours in a black box equipped with a light source (1.5m of LED IP44 18W 8801m / m 6500K 36VA).

[0190] The gel obtained was sieved to the order of a micron and then packaged in a syringe.

[0191] Finally, the gel obtained was sterilized in an autoclave (plate temperature between 121°C and 135°C with F0 > 15).

[0192] A comparative hydrogel B based on HA-Tyr (15 mg / ml) and HA-4MDa (as a lubricating agent) (5 mg / ml) was prepared by adding HA-4MDa after crosslinking HA-Tyr.

[0193] 334 mg of HA-Tyr prepared according to the previous protocol, and 20 grams of buffer phosphate PBS were homogenized for 1h30 in a sterile bag.

[0194] 40 pL of riboflavin (CF = 2 ppm) were added and then the mixture obtained homogenized manually.

[0195] The mixture was then irradiated for 2 hours in a black box equipped with a light source (1.5m of LED IP44 18W 8801m / m 6500K 36VA).

[0196] 250 mg of HA-4MDa was dissolved with PBS to achieve a concentration of 50 mg / g. This solution was added to the cross-linked HA-Tyr gel in a HA-Tyr / HA-4MDa mass proportion of 90 / 10.

[0197] The products obtained were sieved to the order of a micron and then packaged in a syringe.

[0198] Finally, the gel obtained was sterilized in an autoclave (plate temperature between 121°C and 135°C with F0 > 15). Results#:

[0199] [Tables] Hydrogel A Hydrogel B G' (before sterilization) in Pa 312 170 G' (after sterilization) in Pa 220 105 Phase angle (°) before sterilization 13 25 Phase angle (°) after sterilization 19 28 r (Pa) before sterilization 267 175 r (Pa) after sterilization 223 157

[0200] It is observed that hydrogel A prepared from a method according to the invention comprising a step of adding the lubricating agent during crosslinking has a lower elastic modulus (G'), a lower delta and a higher cross-over tau than hydrogel B, prepared under the same conditions except for the addition of the lubricating agent after the crosslinking step. Hydrogel A is therefore more ductile, more cohesive. It has better adaptation to tissue movements.

[0201] Example 2 - Example of production of a gel including a peptide

[0202] 75 mg of HA-Tyr prepared according to the previous protocol, 75 mg HA-4MDa and 12.7 g of PBS buffer were homogenized for 1h30 in a sterile bag.

[0203] 434 pL of the solubilized YGGGY peptide was added to the resulting mixture to reach a final concentration of 0.5 molar eq (relative to the moles of HA-Tyr)) and the solution was homogenized manually.

[0204] 150 pL of HRP (final concentration = 10 ppm) and 1500 pL of hydrogen peroxide (final concentration = 100 ppm) were added quickly and the mixture was homogenized using a vortex.

[0205] The mixture was then left to stand for 2 hours at room temperature without stirring to allow complete crosslinking of the gel.

[0206] The hydrogel C obtained was sieved to the order of a micron and then packaged in a syringe.

[0207] Finally, the obtained hydrogel was sterilized in an autoclave (plate temperature between 121°C and 135°C with F0 > 15). Results#:

[0208] [Tables2] Hydrogel C G' (before sterilization) in Pa 169 G' (after sterilization) in Pa 114 Phase angle (°) before sterilization 12 Phase angle (°) after sterilization 15 r (Pa) before sterilization 75 r (Pa) after sterilization 60

[0209] It is observed that hydrogel C has good rheological properties and forms an injectable gel at 7.35N (30G 1 / 2 TSK® needle).

Claims

Claims

1.

2. Injectable composition comprising a matrix based on a crosslinked functionalized polysaccharide and a lubricating agent, the matrix being capable of being obtained by crosslinking a polysaccharide modified by functional groups capable of reacting with each other and creating intermolecular covalent bonds to form the crosslinked functionalized polysaccharide, the crosslinking being carried out in the presence of a lubricating agent. An injectable composition according to claim 1 wherein the crosslinked functionalized polysaccharide comprises the following structure: in which: - mlP and m2P are respectively a first and a second polysaccharide molecule; - Ar is a functional group capable of reacting with another functional group Ar and forming covalent intermolecular bonds, preferably Ar is an aryl group which may be substituted; - Y is absent, an oxygen atom, a sulfur atom, an -NR1- group, an -NR'-CLjn-NR1- group, an -S-(L)nS- group or a -CR1R2-(L)n-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 n being an integer ranging from 0 to 200, preferably from 1 to 100.

3. Injectable composition according to claim 1 or 2 wherein the crosslinked functionalized polysaccharide comprises one of the following structures, or a mixture thereof: .0 bm?? --.P \ / P? Ç K ? \\ '-Y fl MM La AJ " biH «b T n ûb in which: - m,P and m2P are respectively a first and a second polysaccharide molecule; - W is a hydrocarbon group comprising from 1 to 20 carbon atoms, preferably comprising from 2 to 10 carbon atoms.

4. Injectable composition according to one of the preceding claims in which the crosslinked functionalized polysaccharide is a crosslinked functionalized hyaluronic acid.

5. Injectable composition according to one of the preceding claims in which the polysaccharide modified by functional groups is a hyaluronic acid modified by tyramine.

6. Injectable composition according to one of the preceding claims in which the lubricating agent is chosen from biocompatible polymers, preferably having a molecular weight ranging from 0.5 to 10 MDa.

7. Injectable composition according to one of the preceding claims in which the lubricating agent is a non-functionalized non-crosslinked polysaccharide, preferably having a molecular weight ranging from 0.5 to 10 MDa.

8. Injectable composition according to one of the preceding claims in which the lubricating agent is a non-functionalized and non-crosslinked hyaluronic acid, preferably having a molecular weight ranging from 0.5 to 10 MDa.

9. Injectable composition according to one of the preceding claims further comprising an anesthetic agent.

10. A method of preparing an injectable composition comprising a crosslinked functionalized polysaccharide, the method comprising the following steps: (a) providing a polysaccharide modified by functional groups capable of reacting with each other and creating covalent intermolecular bonds; (b) crosslinking, in the presence of a lubricating agent, the polysaccharide provided in step (a) to form the crosslinked functionalized polysaccharide.

11. A method according to claim 10 wherein the polysaccharide provided in step (a) has the following structure: "rt :O< JJ "YP in which: P is a polysaccharide; W is a hydrocarbon group comprising from 1 to 20 carbon atoms.

12. Method according to claim 11 in which the crosslinking is carried out in the presence of - a peroxide, preferably hydrogen peroxide and a peroxidase, preferably horseradish peroxidase, or - in the presence of a photoinitiator, preferably riboflavin, and UV and / or visible rays.

13. Method according to one of claims 10 to 12 further comprising a sterilization step.

14. Method according to one of claims 10 to 13 further comprising a step of packaging in an injection device.

Images