Method for crosslinking polysaccharide hydrogels by native chemical ligation, the hydrogels obtained by this method, and their uses

Abstract

The invention relates to a method for preparing a crosslinked polymer hydrogel by native chemical ligation, the method using a crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine, and a polymer comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups, the method comprising at least the steps of: a) bringing into contact, in an aqueous solution, at an acidic pH of at least 4.0, the polymer and the crosslinking agent, b) increasing the pH by at least 0.5 units, the pH being at least greater than 5.5 and not exceeding 8.0, and c) obtaining a crosslinked polymer hydrogel. The invention also relates to a hydrogel obtainable by such a method, and the use of such a hydrogel for cosmetic or therapeutic purposes.

Description

Title of the invention: Method for crosslinking polysaccharide hydrogels by native chemical ligation, the hydrogels obtained by this method, and their uses Technical field

[0001] The invention relates to processes for preparing crosslinked polysaccharide hydrogels, as well as the hydrogels obtained by these processes and their cosmetic or medical uses. Prior art

[0002] Crosslinked polysaccharide hydrogels, such as crosslinked glycosaminoglycan hydrogels, such as hyaluronic acid, are widely used in various fields such as cosmetics and reconstructive surgery, as a soft tissue filler, in therapeutics, as a drug delivery agent, in tissue engineering, as a support for the preparation and growth of biological tissues, or in ophthalmology, for example for intravitreal injections, the treatment of dry eye and contact lenses.

[0003] Polysaccharide hydrogels, such as hyaluronic acid hydrogels, can be rapidly degraded after injection into skin tissue, in particular due to enzymatic and / or chemical degradation.

[0004] To slow down the in vivo degradation of polysaccharide hydrogels (and therefore increase their persistence after injection), adjust their viscoelastic properties, improve resistance to sterilization, particularly heat, and increase their stability during storage, it has been proposed to crosslink the polysaccharides.

[0005] Polysaccharide hydrogels are almost exclusively crosslinked by epoxide chemistry, such as 1,4-butanediol diglycidyl ether (BDDE), aldehydes, or polyvinyl sulfones, such as divinyl sulfone (DVS).

[0006] Epoxide chemistry involves the use of alkaline environments and sometimes high temperatures (above 40°C) which can alter the structure and properties of polysaccharides, particularly glycosaminoglycans such as hyaluronic acid. This can result in the production of compounds with reduced molecular weights and which are potentially inflammatory.

[0007] In addition, crosslinking agents, such as BDDE, must be removed by means of specific purification processes (allowing its content to be reduced to 2 ppm or less), which are industrially long, complex and expensive.

[0008] WO 2019 / 238953 A1 describes a crosslinked hyaluronic acid hydrogel obtained by crosslinking a hyaluronic acid, the carboxylic groups of which have been functionalized with thiol groups. The crosslinking carried out in the presence of oxygen leads to the formation of disulfide bridges.

[0009] WO 2021 / 124147 A1 describes a crosslinked hyaluronic acid hydrogel obtained by crosslinking a hyaluronic acid, the carboxylic groups of which have been functionalized with 4-(6-dimethoxy-1,3,5-triazin-2-yl)-4methyl chloride (DMTMM). The crosslinking carried out in the presence of amino acids of the arginine or ornithine type, used as crosslinking agents, leads to the formation of amide bonds between the hyaluronic acid and the crosslinking agents.

[0010] Zhang et al. (Chem Commun, 2015, 51(47): 9662-9665) describes the crosslinking of a hyaluronic acid hydrogel whose carboxylic groups have been functionalized by thiol groups, in the presence of a crosslinking agent carrying thioester groups (PEG-thioester). The crosslinking is carried out by native chemical ligation. The described process involves complex hyaluronic acid modification chemistry requiring up to 9 synthesis steps, which makes this process difficult to industrialize. In addition, the crosslinking step uses BDDE.

[0011] There is therefore a need to have new methods for crosslinking polysaccharide hydrogels, in particular glycosaminoglycan hydrogels, in particular hyaluronic acid, and their biologically acceptable salts.

[0012] There is therefore a need for new methods for crosslinking polysaccharide hydrogels which are gentle and do not denature the polysaccharides to be crosslinked.

[0013] There is still a need for methods for crosslinking polysaccharide hydrogels that do not use crosslinking agents of the epoxide, aldehyde or polyvinylsulfone type.

[0014] There is still a need for a method for crosslinking polysaccharide hydrogels that allows crosslinking by native chemical ligation requiring few steps.

[0015] There is still a need to have a hydrogel of crosslinked polysaccharides whose crosslinking has not been carried out by crosslinking agents of the epoxide, aldehyde or polyvinylsulfone type.

[0016] There is a need for more biocompatible and metabolizable crosslinking agents to facilitate their removal by simple and inexpensive purification processes.

[0017] There is a need for functionalized polysaccharides that can be used in native chemical ligation crosslinking processes.

[0018] There is still a need for a crosslinked polysaccharide hydrogel whose crosslinking has been carried out by native chemical ligation.

[0019] There is still a need for a crosslinked polysaccharide hydrogel having improved biocompatibility with implanted or injected body tissues.

[0020] There is still a need for a crosslinked polysaccharide hydrogel with improved persistence in implanted or injected body tissues.

[0021] The object of the present invention is to satisfy all or part of these needs. Summary of the invention

[0022] According to one of its first objects, the present invention relates to a process for preparing a crosslinked polymer hydrogel by native chemical ligation,

[0023] the process using a non-crosslinked functionalized polymer and a crosslinking agent,

[0024] the crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine, and

[0025] the polymer comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups,

[0026] the method comprising at least the steps of:

[0027] a) bringing into contact, in an aqueous solution, at an acidic pH of at least about 4.0, said non-crosslinked functionalized polymer and said crosslinking agent,

[0028] b) increasing the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than about 5.5 and not exceeding 8.0, and

[0029] c) obtaining a crosslinked polysaccharide polymer hydrogel.

[0030] As illustrated by the examples given below, the inventors have developed a new method for crosslinking polysaccharide hydrogels based on the formation of native chemical ligation (NCL). As demonstrated by the examples, the native chemical ligation crosslinking process is a gentle crosslinking process, involving more biocompatible and metabolizable crosslinking agents, thus reducing the risks of denaturation of the polysaccharides and the formation of potentially toxic or immunogenic side products. In addition, the biocompatible and metabolizable nature of the crosslinking agents used makes it possible to reduce the risks of toxicity, and facilitates their elimination by simple and inexpensive purification processes.

[0031] According to one embodiment, the thiol groups of the crosslinking agent originate from cysteine ​​residues, the cysteine ​​residues being linked together by a spacer.

[0032] According to one embodiment, the spacer is of general formula (I):

[0033] @-AR*-B-@ (I)

[0034] in which

[0035] @- and -@ represent bonds with the acyl groups of the cysteine ​​residues, and

[0036] R1 represents a residue chosen from a saturated CrCio hydrocarbon chain or unsaturated, optionally interrupted by one or more O, NR2, or S, and optionally substituted by one or more groups selected from -OH, -NH 2, -NHR2, -SH, -COOR2 and -CONHR2, in which R2 represents H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched,

[0037] A and B, identical or different, represent NR3, O or S, with R3 representing H or a C1-C6 alkyl.

[0038] According to one embodiment, a crosslinking agent may be represented by the formula (Ia):

[0039] Cys-A-R'-B-Cys (la)

[0040] in which

[0041] Cys represents a cysteine ​​residue, and

[0042] R1 represents a residue chosen from a C1-C10 hydrocarbon chain, saturated or unsaturated, optionally interrupted by one or more O, NR2, or S, and optionally substituted by one or more groups chosen from -OR2, -NHR2, -SR2, -COOR2 and -CONHR2, in which R2 represents H, a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched,

[0043] A and B, identical or different, represent NR3, O or S, with R3 representing H or a CrC6 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0044] According to one embodiment, a crosslinking agent may be represented by the formula (II):

[0045]

[0046] According to one embodiment, at least about 0.5% of the carboxylic groups of the polymer are functionalized by thioester groups.

[0047] According to one embodiment, the proportion of carboxylic groups functionalized with a thioester group varies from approximately 0.5% to approximately 50%.

[0048] According to one embodiment, the carboxylic groups of the polymer are functionalized with a functionalizing compound with a thioester group of general formula (III):

[0049] NH2-R4-C(O)-S-R5 (III)

[0050] in which

[0051] R4 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -OC(O)-NHR6, -S-R6, -SC(O)OR6, -NHR6, -NH-C(O)R6, -NH-C(O)OR6, -C(O)NH-C(O)R6, -NH-C(NHR6)=NR6, -NH-C(NH-C(O)R6)=NH-C(O)R7, -Phe, -Phe-OC(O)R6, and

[0052] R5 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -NHR6, -NH-C(O)R6, -C(O)NH-C(O)R6, -C(O)NH-COOR6, -NHC(O)(CH2)2CH(NHR6)C(O)OR7, -SOR6, S(O)OR6, -Phe, -Phe-OC(O)R6,

[0053] with R6 and R7 representing, indifferently, H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by Phe.

[0054] According to one embodiment, the mixture obtained in step a) is kept stirring for a period varying from approximately 5 minutes to approximately 1 hour.

[0055] According to one embodiment, the mixture obtained in step a) is kept stirring for a period varying from approximately 5 minutes to approximately 20 minutes.

[0056] According to one embodiment, step a) is carried out under an inert atmosphere, for example an atmosphere of nitrogen, argon or CO2.

[0057] According to one embodiment, in step b), the pH is adjusted to a pH of about 6.0 to about 8.0, or about 6.2 to about 7.8, or about 6.4 to about 7.6, or about 6.8 to about 7.4, or is about 7.0.

[0058] According to one embodiment, in step b), the pH is adjusted to a pH of about 6.5 to about 8.0, or about 6.7 to about 7.8, or about 6.8 to about 7.6, or about 6.8 to about 7.4, or is about 7.0

[0059] According to one embodiment, step b) comprises a first sequence b1) and a second sequence b2),

[0060] - sequence bl) comprising an increase in the pH of the mixture obtained in step (a) at least 0.5 units, the pH being at least greater than about 5.5 and not exceeding 8.0, for a period of about 2 min. to about 1 hour, and

[0061] - sequence b2) comprising maintaining the solution obtained at the end of the sequence bl), without agitation, for a duration of approximately 15 min. to approximately 30 h.

[0062] Sequence b2) comprises maintaining the pH at the pH obtained in step b1) (maintaining the pH constant).

[0063] According to one of its objects, the invention relates to a method for preparing a crosslinked polymer hydrogel by native chemical ligation,

[0064] the method using a first polymer and a second non-crosslinked polymer,

[0065] the first polymer comprising at least one carboxylic group, the carboxylic group being functionalized with a thioester group, and

[0066] the second polymer being functionalized with at least one thiol group, the thiol group being beta to a primary amine,

[0067] the method comprising at least the steps of:

[0068] a) contacting the first and second polymers in an aqueous solution, the aqueous solution being at an acidic pH of at least about 4.0,

[0069] b) increasing the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than about 5.5 and not exceeding 8.0, and

[0070] c) obtaining a crosslinked polymer hydrogel.

[0071] According to one of its objects, the invention relates to a method for preparing a crosslinked polymer hydrogel by native chemical ligation,

[0072] the method using a first polymer and a second non-crosslinked polymer,

[0073] the first polymer comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups, and

[0074] the second polymer being functionalized with at least two thiol groups, the thiol groups being beta to a primary amine,

[0075] the method comprising at least the steps of:

[0076] a) contacting the first and second polymers in an aqueous solution, the aqueous solution being at an acidic pH of at least about 4.0,

[0077] b) increasing the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than about 5.5 and not exceeding 8.0, and

[0078] c) obtaining a crosslinked polymer hydrogel.

[0079] The first polymer comprising at least one carboxylic group functionalized with a thioester group may be as described previously.

[0080] In this method, the thiol group of the second polymer reacts with the thioester group of the first polymer, allowing a crosslinking node to be obtained.

[0081] Steps a), b) and c) of this embodiment can be carried out as described previously except that the crosslinking agent is replaced by the second polymer functionalized with at least two thiol groups.

[0082] According to one embodiment, a method as described herein may comprise an additional step of dialyzing the crosslinked polymer hydrogel against a buffer, for example a phosphate buffer.

[0083] According to one embodiment, a method as described herein may comprise an additional step of adding an aqueous solution of non-crosslinked polymer to the crosslinked polymer hydrogel.

[0084] According to one embodiment, the method may comprise an additional step of adding at least one active agent.

[0085] According to one embodiment, the polymer is a polysaccharide or its biologically acceptable salts.

[0086] According to one embodiment, the polysaccharide is hyaluronic acid, or one of its biologically acceptable salts.

[0087] According to one embodiment, the polysaccharide is sodium hyaluronate.

[0088] According to one of its objects, the invention relates to a hydrogel obtainable according to a method as described here.

[0089] According to one of its objects, the invention relates to a sterile injectable composition comprising a hydrogel as described here.

[0090] According to one of its objects, the invention relates to a cosmetic use of a hydrogel or a composition as described herein as a soft tissue filler or for soft tissue augmentation.

[0091] According to one of its objects, the invention relates to a hydrogel or a composition as described herein for use as a medicament or medical device.

[0092] According to one of its objects, the invention relates to a hydrogel or a composition as described herein, said hydrogel or said composition further comprising at least one active agent, for use as a medicament or medical device.

[0093] According to one of its objects, the invention relates to a polymer comprising carboxylic groups in which a part of the carboxylic groups is functionalized with thioester groups, the polymer being represented by the general formula (IV):

[0094] Q (IV) / J x ..CH, \ / O' Y' f ' \ [ CH. L ô ] s " S i \r" : o / \ / T n IYI

[0095] in which n preferably represents the proportion of carboxylic groups of said polymer functionalized with a thioester group and varies from approximately 0.5% to approximately 50% and Y represents a polymer residue comprising carboxylic groups.

[0096] According to one of its objects, the invention relates to a crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine.

[0097] According to one embodiment, a crosslinking agent may be of general formula (Ia):

[0098] Cys-A-R'-B-Cys (la)

[0099] in which

[0100] Cys represents a cysteine ​​residue,

[0101] R1 represents a residue chosen from a saturated or unsaturated CrCio hydrocarbon chain, optionally interrupted by one or more O, NR2, or S, and optionally substituted by one or more groups chosen from -OR2, -NHR2, -SR2, -COOR2 and -CONHR2, in which R2 represents H, a saturated or unsaturated, linear or branched C1-C4 hydrocarbon chain; a polyol residue; an acid residue amino; a peptide residue; an amino saccharide residue; a polyamine residue; and a PEG diamine residue,

[0102] A and B, identical or different, represent NR3, O or S, with R3 representing H or a CrC6 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0103] According to one embodiment, a crosslinking agent may be represented by the formula (II):

[0104]

[0105] According to one of its objects, the invention relates to a use of a crosslinking agent as described here, for crosslinking together two polymers comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups. Brief description of the drawings

[0106] [Fig. 1] is a schematic representation of a crosslinking process described herein. Step 1 represents the functionalization of a polymer comprising carboxylic groups, for example a hyaluronic acid, with thioester groups. Step 2 represents the crosslinking of the functionalized polymer with a crosslinking agent comprising two cysteines linked by a spacer.

[0107] [Fig.2] is a schematic representation of a crosslinking process described herein. Step 1 represents the functionalization of a polymer comprising carboxylic groups, for example a hyaluronic acid, with thioester groups. Step 2 represents the functionalization of a polymer, for example a hyaluronic acid, with thiol groups, provided by cysteine ​​residues. Step 3 represents the crosslinking of the polymers functionalized in steps 1 and 2. Detailed description

[0108] Definitions

[0109] Unless otherwise indicated, scientific and technical terms used in this document have the meaning commonly accepted in the field to which it relates. Exemplary methods and materials are described herein, but similar or equivalent methods and materials may also be used in practicing this disclosure. In the event of a conflict, this description shall prevail.

[0110] Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be implemented separately or in any suitable subcombination.

[0111] The numerical ranges include all numbers defining the range. Each maximum numerical limitation given throughout the description includes any lower numerical limitation, as if such lower numerical limitations were expressly written herein. Each minimum numerical limitation given throughout the description includes any higher numerical limitation, as if such higher numerical limitations were expressly written herein. Each numerical range given throughout the description includes any narrower numerical range that falls within such a broad numerical range, as if such narrower numerical ranges were all expressly written herein.

[0112] All lists of items, such as, for example, lists of ingredients, are intended and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as elements "selected from the group consisting of the list of items" and combinations and mixtures thereof.

[0113] In the specification, embodiments described herein with the terms "having" or "comprising" include embodiments described with the terms "comprising only", "consisting of" and / or "consisting essentially of". The expression "consisting of" implies inclusion of the elements set forth to the exclusion of any other elements. The expression "consisting essentially of" implies inclusion of the elements set forth, and possibly other elements where the other elements do not significantly affect the fundamental characteristic(s) of the disclosure.

[0114] Furthermore, the expression "and / or" is to be considered a specific disclosure of each of the two features with or without the other. Thus, the expression "and / or" used in an expression such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone).

[0115] The terms "about" or "approximately" mean an acceptable measurement error for a particular value of a parameter determined by measurement methods customary in the art and which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within a range of three or more standard deviations, as practiced in the art.

[0116] For the purposes of the invention, the term "polymer" means a macromolecule composed of a linear or branched chain of monomers linked together by covalent bonds. A polymer suitable for the invention is a polymer comprising carboxylic groups. A polymer suitable for the invention may be a protein or a polysaccharide, or their biologically acceptable salts. In particular, a polymer may be a polysaccharide or one of its biologically acceptable salts.

[0117] For the purposes of the invention, the term “functionalized polymer” means a polymer which has specific functional groups, introduced by a chemical modification, and which gives the polymer new properties.

[0118] For the purposes of the invention, the term "crosslinked polymer" means a polymer comprising crosslinking nodes. The expression "crosslinking nodes" is intended to designate the points of chemical bonding between adjacent polymer chains, forming a three-dimensional structure. These crosslinking nodes represent the junctions between the polymer chains which give the hydrogel its ability to retain water. These nodes play a central role in determining the swelling, mechanical strength and permeability properties of the hydrogels.

[0119] For the purposes of the invention, the term “non-crosslinked polymer” means a polymer comprising no, or few, crosslinking nodes.

[0120] For the purposes of the invention, the term “polysaccharide comprising carboxylic groups” means a polymer consisting of a chain of sugar monomers, at least one of which comprises a carboxylic group.

[0121] For the purposes of the invention, the term "hyaluronic acid" means hyaluronic acid, or hyaluronan, and its derivatives. Consequently, the term "hyaluronic acid" also includes hyaluronic acid salts such as sodium hyaluronate and hyaluronic acids chemically modified, for example by oxidation, reduction, deacetylation, sulfation or amidation. Hyaluronic acid is a linear polysaccharide with alternating D-glucuronic acid and N-acetyl-D-glucosamine units, linked by alternating [3-1,3 glycosidic and [3-1,4 glycosidic bonds.

[0122] For the purposes of the invention, the term “protein” means a biological macromolecule composed of a linear chain of amino acids. A protein suitable for the invention comprises amino acids whose side chain comprises a carboxylic group (COOH).

[0123] For the purposes of the invention, the term “hydrogel” means a material composed of a network of interconnected polymers which are surrounded by water capable of absorbing and retaining significant quantities of water.

[0124] For the purposes of the invention, the term “Native Chemical Ligation (NCL)” means a method of chemical synthesis of peptides which uses natural chemical reactions to form covalent bonds between amino acid residues.

[0125] For the purposes of the invention, the term “crosslinking agent” means a compound capable of introducing crosslinking between different polymer chains.

[0126] For the purposes of the invention, the term “cysteine ​​residue” means a cysteine ​​amino acid residue, characterized by the presence of a thiol group (-SH) on the alpha carbon.

[0127] For the purposes of the invention, the term “thiol group” means a functional group composed of a sulfur atom linked to a hydrogen atom.

[0128] For the purposes of the invention, the term “thioester group” means a functional group composed of a sulfur atom linked to an oxygen atom and to an acyl group (R-CO-).

[0129] For the purposes of the invention, the term “spacer” means a segment providing the connection between two molecules or two parts of a molecule.

[0130] For the purposes of the invention, the term “filling agent” for soft tissues means an injectable substance that can be used to increase the volume of soft tissues, fill in wrinkles and fine lines, or reshape the contours of the face.

[0131] For the purposes of the invention, the term "sterile hydrogel" or "sterile composition" is intended to describe a hydrogel or a composition having the required safety for administration in or through the superficial areas of the skin, subcutaneously, i.e. in the upper dermis, the middle dermis, and the hypodermis. In particular, it is essential that the hydrogel or the composition to be administered using an injection technique be free of any contaminating body likely to initiate an undesirable side reaction in the host organism.

[0132] Crosslinking process by native chemical ligation with crosslinking agent

[0133] According to one of its objects, the present invention relates to a method for preparing a crosslinked polymer hydrogel by native chemical ligation.

[0134] According to one embodiment, a method described herein uses a non-crosslinked functionalized polymer and a crosslinking agent, the crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine, and the polymer comprising at least one carboxylic group, the carboxylic group being functionalized with a thioester group.

[0135] According to one embodiment, a method described herein uses a non-crosslinked functionalized polymer and a crosslinking agent, the crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine, and the polymer comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups.

[0136] According to one embodiment, a method described herein comprises at least the steps of:

[0137] a) bringing into contact, in an aqueous solution, at an acidic pH of at least about 4.0, said non-crosslinked functionalized polymer and said crosslinking agent,

[0138] b) increasing the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than about 5.5 and not exceeding about 8.0, and

[0139] c) obtaining a crosslinked polysaccharide hydrogel.

[0140] According to one embodiment, a method described herein implements a first polymer and a second non-crosslinked polymer, the first polymer comprising at least one carboxylic group, said carboxylic group being functionalized with a thioester group, and the second polymer being functionalized with at least one thiol group, the thiol group being beta to a primary amine.

[0141] According to one embodiment, a method described herein implements a first polymer and a second non-crosslinked polymer, the first polymer comprises carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups, and the second polymer being functionalized with at least two thiol groups, the thiol groups being beta to a primary amine.

[0142] In this process, the thiol groups of the second polymer react with the thioester groups of the first polymer, allowing the crosslinking nodes to be obtained.

[0143] According to one embodiment, a method described comprises at least the steps of:

[0144] contacting the first and second polymers in an aqueous solution, the aqueous solution being at an acidic pH of at least 4.0,

[0145] increasing the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than 5.5 and not exceeding 8.0, and

[0146] obtain a crosslinked polymer hydrogel.

[0147] Step a)

[0148] A method of the invention comprises a step a), comprising bringing into contact, in an aqueous solution, at a pH of about 4.0 to about 6.5, or about 4.0 to about 6, a non-crosslinked functionalized polymer and a crosslinking agent or a first non-crosslinked polymer comprising at least one carboxylic group, preferably at least two carboxylic groups, the one or more, or a part of the, carboxylic groups being functionalized with one or more thioester groups, and a second non-crosslinked polymer functionalized with at least one thiol group, preferably with at least two thiol groups, or thiol groups, the thiol group(s) being beta to a primary amine.

[0149] Step a) allows the obtaining of a homogeneous mixture of a non-crosslinked functionalized polymer and a crosslinking agent.

[0150] Alternatively, step a) makes it possible to obtain a homogeneous mixture of a first polymer and a second non-crosslinked polymer.

[0151] In the context of the description, the expressions "homogeneous mixture of a non-crosslinked polymer and a crosslinking agent", "homogeneous mixture of a first polymer and a second non-crosslinked polymer", "homogeneous aqueous gel of a non-crosslinked polymer and a crosslinking agent" or "homogeneous aqueous gel of non-crosslinked polymers" are understood in the context of the present description as a mixture or an aqueous gel of non-crosslinked or weakly crosslinked polymer, that is to say a mixture or a gel whose phase shift angle δ, measured under dynamic rheology conditions at 1 Hz, is greater than 40° when it is subjected to a stress greater than 1 Pa.

[0152] According to one embodiment, step a) is carried out at a pH in a range of about 4.0 to about 6.5, preferably about 4.0 to about 6, or about 4.2 to about 6.2, or about 4.5 to about 5.8, or about 4.8 to about 5.3 or at a pH of about 5.0.

[0153] When bringing a non-crosslinked functionalized polymer and a crosslinking agent into contact, a step of adjusting the pH of the mixture obtained can be carried out.

[0154] Alternatively, when bringing into contact a first and a second non-crosslinked polymer, as defined previously, a step of adjusting the pH of the mixture obtained can be carried out.

[0155] The pH may be adjusted to a value of about 4.0 to about 6.5, or about 4.0 to about 6.0, or about 4.2 to about 6.2, or about 4.5 to about 5.8, or about 4.8 to about 5.3 or to a pH of about 5.0.

[0156] The pH may be adjusted to a value of about 4.0 to about 6.5, or about 4.0 to about 6.0, or about 4.2 to about 6.0, or about 4.5 to about 5.5, or about 5.0.

[0157] According to one embodiment, the pH can be adjusted to a value of about 4.0 to about 6.0.

[0158] The pH adjustment can be carried out by adding a base, for example NaOH, or an acid, for example HCl.

[0159] According to one embodiment, a functionalized polymer is brought into contact with the crosslinking agent by:

[0160] bringing an aqueous solution of functionalized polymer into contact with an aqueous solution of crosslinking agent, or

[0161] bringing an aqueous solution of functionalized polymer into contact with a crosslinking agent in the form of a powder, or

[0162] bringing an aqueous solution of crosslinking agent into contact with a functionalized polymer in the form of fibers.

[0163] According to one embodiment, a functionalized polymer is brought into contact with the crosslinking agent by bringing an aqueous solution of functionalized polymer into contact with a crosslinking agent in the form of a powder.

[0164] According to one embodiment, a first non-crosslinked polymer is brought into contact with a second non-crosslinked polymer by:

[0165] contacting an aqueous solution of the first polymer with an aqueous solution of the second polymer, or

[0166] contacting an aqueous solution of the first polymer with the second polymer in the form of fibers, or

[0167] bringing an aqueous solution of the second polymer into contact with the first polymer in the form of fibers.

[0168] According to one embodiment, a first polymer is brought into contact with a second polymer by bringing an aqueous solution of the first polymer into contact with an aqueous solution of the second polymer.

[0169] The pH of the aqueous solution may be adjusted to be in a range of from about 4.0 to about 6.5, or from about 4.0 to about 6.0, or from about 4.2 to about 6.0, or from about 4.5 to about 5.5, or about 5.0.

[0170] According to one embodiment, the pH of the aqueous solution can be adjusted to be in a range varying from about 4.0 to about 6.0.

[0171] According to an alternative embodiment, in a method using a first non-crosslinked polymer comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups, and a second non-crosslinked polymer functionalized with thiol groups, the thiol groups being beta to a primary amine, the polymers can be solubilized concomitantly in an aqueous solution with a pH in a range of about 6.8 to about 7.8. In such an alternative embodiment, step b) may be present. Alternatively, in such an alternative embodiment, step b) may be absent, the crosslinking of the polymers being carried out concomitantly with the mixture of polymers.

[0172] A polymer and / or a crosslinking agent may be solubilized in an aqueous solution selected from water, a buffer selected from N-carbamoylmethyl taurine (CAS No: 7365-82-4), 3-[N,N-bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid sodium salt (CAS No: 102783-62-0), 3-morpholino-2-hydroxypropanesulfonic acid (CAS No: 68399-77-9), 1,4-piperazinediethanesulfonic acid (CAS No: 5625-37-6), 1,4-piperazine-N,N'-bispropane sulfonic acid) (CAS No: 5625-56-9), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-l-propane sulfonic acid (CAS No: 68399-81-5), 2-[(2-hydroxy-l,l-bis(hydroxymethyl)ethyl)amino]ethane sulfonic acid (CAS No: 7365-44-8), N-tris(hydroxymethyl)methylglycine (CAS No: 5704-04-1), 3-(N-morpholino)propane sulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1), 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (CAS No: 7365-45-9), 1,4-Piperazinediethanesulfonic acid (CAS No: 5625-37-6), 4-(2-hydroxyethyl)piperazine-l-(2-hydroxypropane-3-sulfonic acid) (CAS No: 68399-78-0), phosphate buffers such as PBS with a pH around physiological pH (CAS No: 7647-14-5, 7447-40-7).

[0173] For example, the buffer may be selected from 3-(N-morpholino)propane sulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1), 4-(2-Hydroxyethyl)piperazine-l-ethane sulfonic acid (CAS No: 7365-45-9) and phosphate buffers such as PBS with a pH around physiological pH (CAS No: 7647-14-5,7447-40-7).

[0174] According to one embodiment, the buffer may be a phosphate buffer, particularly a NaH2PO4 / Na2HPO4 or KH2PO4 / K2 hpo4 saline buffer.

[0175] The aqueous solutions can be degassed, before or after solubilization of the polymer and / or the crosslinking agent.

[0176] According to one embodiment, a method of the invention may comprise a step, prior to step a), of preparing an aqueous solution of the polymer. This step may comprise adjusting the pH of the aqueous solution of functionalized polymer to a value of about 4.0 to about 6.5 or about 4.2 to about 6.0, or about 4.5 to about 5.5, or about 5.0.

[0177] According to one embodiment, the step of preparing the aqueous polymer solution may comprise a step of degassing the solution.

[0178] The degassing step can be carried out under magnetic stirring of the solution at 100 - 200 rpm, by inerting / bubbling of an inert gas (nitrogen / argon) or under vacuum.

[0179] The degassing step may be carried out for a period of time from about 5 min to about 30 min, or from about 10 min to about 25 min, or from about 15 min to about 20 min.

[0180] The degassing step can be carried out at room temperature, ranging from about 21°C to about 25°C.

[0181] The step of degassing the aqueous solution advantageously makes it possible to reduce the risks of oxidation of the thiol functions of the crosslinking agent or of the second polymer.

[0182] According to one embodiment, a method of the invention may comprise a step, prior to step a), consisting of preparing an aqueous solution of crosslinking agent.

[0183] According to one embodiment, the mixture obtained in step a) is kept stirring until a homogeneous mixture is obtained.

[0184] When the crosslinking agent or a polymer is added in the form of a powder, the mixture obtained in step a) is kept stirring until the powder dissolves.

[0185] According to one embodiment, the mixture obtained in step a) is kept stirring until a homogeneous mixture is obtained.

[0186] According to one embodiment, the mixture obtained in step a) is kept stirring for a period varying from approximately 5 minutes to approximately 1 hour, or from approximately 10 min. to approximately 30 min. or from approximately 15 min. to approximately 20 min.

[0187] According to one embodiment, the mixture obtained in step a) is kept stirring for a period varying from approximately 5 minutes to approximately 20 minutes.

[0188] The stirring in step a) can be carried out by manual stirring, mechanical stirring, magnetic stirring, or alternating 2 or 3 types of stirring.

[0189] Manual stirring can be achieved with a spatula.

[0190] Mechanical agitation can be achieved with a paddle device, at a speed of about 100 rpm to about 200 rpm. Such devices are known to those skilled in the art.

[0191] Magnetic stirring can be achieved with a magnetic bar, at a speed of about 100 rpm to about 200 rpm.

[0192] Step a) may be carried out at a temperature of about 0°C to about 55°C, or about 5°C to about 40°C, or about 10°C to about 30°C, or about 15°C to about 25°C, or about 21°C to about 25°C (room temperature).

[0193] According to one embodiment, step a) is carried out under an inert atmosphere, for example an atmosphere of nitrogen, argon or CO2.

[0194] Step b)

[0195] A method of the invention comprises a step b), comprising an increase in the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than 5.5 and not exceeding 8.0.

[0196] Increasing the pH in step b) makes it possible to induce crosslinking of the functionalized polymer by formation of native peptide bonds by reaction between the thioester groups of the polymer functionalized with thioester groups and the thiol groups of the crosslinking agent.

[0197] Alternatively, the increase in pH in step b) makes it possible to induce the crosslinking of the first polymer functionalized with thioester groups with the second polymer functionalized with thiol groups by formation of native peptide bonds by reaction between the thioester groups and the thiol groups.

[0198] According to one embodiment, the pH may be adjusted in a range of about 6.0 to about 8.0, or about 6.2 to about 7.8, or about 6.4 to about 7.6, or about 6.8 to about 7.4, or be about 7.0.

[0199] According to one embodiment, in step b), the pH is adjusted to a pH of about 6.5 to about 8.0, or about 6.7 to about 7.8, or about 6.8 to about 7.6, or about 6.8 to about 7.4, or about 7.0 to about 7.4, or is about 7.0.

[0200] According to one embodiment, the pH can be adjusted to a physiological pH. A physiological pH is a pH in the range of about 6.8 to about 7.8.

[0201] The pH can be adjusted by adding a basic aqueous solution. A basic aqueous solution is chosen from a sodium hydroxide solution (NaOH) and a potassium hydroxide solution (KOH). The basic aqueous solution is advantageously of high concentration, for example 1 M, so as not to dilute the mixture obtained in step a).

[0202] According to one embodiment, step b) can be carried out under an inert atmosphere. An inert atmosphere can be chosen from a nitrogen atmosphere or an argon atmosphere.

[0203] According to one embodiment, step b) may comprise a first sequence b1) and a second sequence b2).

[0204] Sequence bl) may comprise an increase in the pH of the mixture obtained in step a) of at least 0.5 units, the pH being at least greater than 5.5 and not exceeding 8.0, for a period of approximately 2 min. to approximately 1 hour.

[0205] Sequence bl) is carried out with stirring.

[0206] The sequence bl) may last from about 2 minutes to about 1 hour, or from about 5 min. to about 30 min. or from about 5 min. to about 15 min.

[0207] Sequence b2) may comprise maintaining the solution obtained at the end of sequence b1), without stirring, for a duration of approximately 15 min. to approximately 30 h.

[0208] Sequence b2) comprises maintaining the pH at the pH obtained in step b1) (maintaining the pH constant).

[0209] The stirring in sequence bl) of step b) can be carried out by manual stirring, mechanical stirring, magnetic stirring, or alternating 2 or 3 types of stirring.

[0210] Manual stirring can be achieved with a spatula.

[0211] Mechanical agitation can be achieved with a paddle device, at a speed of about 100 rpm to about 200 rpm. Such devices are known to those skilled in the art.

[0212] Magnetic stirring can be achieved with a magnetic bar, at a speed of about 100 rpm to about 200 rpm.

[0213] According to one embodiment, the stirring in sequence b1) of step b) can be carried out by mechanical stirring.

[0214] Sequence b2) may comprise maintaining the solution obtained at the end of sequence b1), without stirring, for a duration of approximately 15 min. to approximately 30 h, or approximately 20 min. to approximately 24 h, or approximately 25 min. to approximately 20 h, or approximately 30 min. to approximately 15 h, or approximately 35 min. to approximately 10 h, or approximately 40 min. to approximately 8 h, or approximately 45 min. to approximately 5 h, or approximately 50 min. to approximately 2 h, or be approximately 1 h.

[0215] Sequence b2) may comprise maintaining the solution obtained at the end of sequence b1), without stirring, for a duration of approximately 1 h.

[0216] Step c)

[0217] A method of the invention comprises a step c), comprising obtaining a crosslinked polymer hydrogel.

[0218] At the end of step c), a homogeneous aqueous gel is obtained, containing at least one crosslinked polymer.

[0219] Advantageously, an aqueous gel in accordance with the invention may comprise a total polymer concentration varying from 5 to 40 mg / g, 10 to 35 mg / g, or from 15 to 30 mg / g, or from 20 to 25 mg / g relative to the total mass of the gel.

[0220] The crosslinked polymer hydrogel obtained in step c) may optionally be subjected to various additional steps. The various additional steps may allow the preparation of a sterile injectable composition.

[0221] According to yet another particular embodiment, a method of the invention can be implemented at least in part inside a specific container with a deformable wall, such as, for example, a pocket.

[0222] Indeed, the deformability properties of such a container and its hermeticity make it possible to carry out the different stages of a process of the invention, and in particular the homogenization and crosslinking stages, under optimal conditions which lead to obtaining an even better crosslinked gel, i.e. one having viscoelastic and injectability properties superior to those displayed by a gel obtained according to a process using a conventional container of the pot or tank.

[0223] The deformable wall or pocket has a degree of deformability such that it can be deformed manually, that is to say under the effect of hand pressure, resulting, for example, from simple palpation.

[0224] The container, or even the pocket, for practical reasons but also with a view to limiting contact with the external environment as much as possible, has a hermetic character.

[0225] The container according to the invention can however be advantageously equipped with an opening or port system, preferably resealable, and capable of introducing any compound used for the manufacture of a gel according to the invention.

[0226] A deformable wall container or pouch relating to the invention may be as described, in particular, in WO 2010 / 131175 AL

[0227] Additional steps

[0228] Dialysis, Sieving, Conditioning, Sterilization

[0229] According to one embodiment, a crosslinked polymer hydrogel obtained in step c) can be subjected to an additional dialysis step.

[0230] The dialysis step may be carried out in a buffer solution, for example a buffer solution selected from a phosphate buffer, a Tris-HCl buffer, and a bis-tris buffer. A buffer solution may have a pH in a physiological range, for example from about 6.8 to about 7.8, or from about 7.0 to about 7.4.

[0231] The dialysis step may be carried out at room temperature, for example at a temperature in a range varying from about 21°C to about 25°C.

[0232] Alternatively, the dialysis step can be carried out cold, i.e. at a temperature in a range varying from about 0°C to about 10°C.

[0233] According to one embodiment, a crosslinked polymer hydrogel obtained in step c) can be subjected to an additional sieving step.

[0234] An additional sieving step may consist of a single sieving or a sequence of successive sievings, for example at least 2, 3, 4 or 5 successive sievings.

[0235] The sieving may be carried out with a sieve having a sieve mesh in a range of about 750 and about 30 pm, for example about 710, 500, 250, 150, or 34 pm.

[0236] According to one embodiment, a sieving step may comprise two successive sievings. The first sieving may be carried out with a sieve having a mesh of approximately 710 μm, and the second sieving may be carried out with a sieve having a mesh of approximately 150 μm.

[0237] According to one embodiment, a crosslinked polymer hydrogel obtained in step c) can be subjected to an additional conditioning step.

[0238] Packaging can be carried out in a syringe.

[0239] According to one aspect, the present invention relates to a kit comprising a pre-filled syringe comprising a sterile, injectable composition comprising a crosslinked polymer hydrogel as described herein.

[0240] According to one embodiment, a crosslinked polymer hydrogel obtained in step c) can be subjected to an additional sterilization step.

[0241] The method may comprise a single sterilization step.

[0242] A sterilization step can be carried out by thermal means, for example in an autoclave, in particular at a temperature between 120°C and 140°C.

[0243] A sterilization step can be carried out by autoclave, under humid heat conditions, at a temperature greater than or equal to 121°C so as to obtain an F0 > 15 (sterilizing value).

[0244] Sterilization can be carried out on a hydrogel composition as described herein already packaged in its administration device, for example a syringe.

[0245] Addition of non-crosslinked polymer

[0246] According to one embodiment, a crosslinked polymer hydrogel obtained in step c) may be subjected to an additional step of adding an aqueous solution of non-crosslinked polymer to the crosslinked polymer hydrogel.

[0247] The aqueous solution of non-crosslinked polymer may be added to the crosslinked polymer hydrogel in an amount of about 5% to about 30% w / w, or about 10% to about 25% w / w, or about 15% to about 20% w / w of the crosslinked polymer hydrogel.

[0248] According to one embodiment, the aqueous solution of non-crosslinked polymer can be added to the crosslinked polymer hydrogel in a proportion of about 10% by weight / weight of the crosslinked polymer hydrogel.

[0249] According to one embodiment, the crosslinked polymer and the non-crosslinked polymer are of the same nature or are of different natures.

[0250] According to one embodiment, the crosslinked polymer and the non-crosslinked polymer are of the same nature.

[0251] According to one embodiment, the crosslinked polymer and the non-crosslinked polymer are a polysaccharide.

[0252] According to one embodiment, the crosslinked polymer and the non-crosslinked polymer are a hyaluronic acid or one of its biologically acceptable salts.

[0253] According to one embodiment, the crosslinked polymer and the non-crosslinked polymer are sodium hyaluronate.

[0254] The aqueous solution in which the non-crosslinked polymer is dissolved may be of a different nature or of the same nature as the aqueous solution in which the functionalized polymer intended to prepare a crosslinked polymer hydrogel is prepared. According to one embodiment, the aqueous solutions of non-crosslinked polymer and functionalized polymer to be crosslinked are of the same nature.

[0255] Addition of an active agent

[0256] According to one embodiment, a crosslinked polymer hydrogel obtained in step c) may be subjected to an additional step of adding at least one active agent.

[0257] For the purposes of the present description, the term “active agent” means a substance or composition intended to exert a pharmacological action within biological tissues into which a crosslinked polymer hydrogel as described is injected.

[0258] An active agent may be selected from an anesthetic agent, an antioxidant, an amino acid, a vitamin, minerals, a nucleic acid, a nucleotide, a nucleoside, a coenzyme, an adrenergic derivative, and mixtures thereof.

[0259] An anesthetic agent may be chosen from 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, 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 their salts.

[0260] According to one embodiment, an anesthetic agent may be mepivacaine, lidocaine or one of their salts.

[0261] According to one embodiment, an anesthetic agent may be a salt of mepivacaine hydrochloride or lidocaine.

[0262] According to one embodiment, mepivacaine, lidocaine or one of their salts can be used in amounts varying from approximately 0.1 to approximately 30 mg / mL, for example from approximately 0.5 to approximately 10 mg / mL or more preferably from approximately 2 to approximately 6 mg / mL.

[0263] An antioxidant may be selected from glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, and alpha-lipoic acid.

[0264] An amino acid may be selected from arginine, isoleucine, leucine, lysine, glycine, valine, threonine, proline, methionine, histidine, phenylalanine, tryptophan, and cysteine.

[0265] A vitamin may be chosen from vitamins E, A, C, and B, and in particular vitamins B4, B5, B6, B8, B9, B7, and B12 and preferentially, pyridoxine.

[0266] The minerals can be chosen from zinc, magnesium, calcium, potassium, manganese, sodium and copper salts.

[0267] A coenzyme may be chosen from coenzymes Q10, CoA, NAD, and NADP.

[0268] An adrenergic derivative may be chosen from adrenaline and noradrenaline.

[0269] Crosslinking agents

[0270] A method according to the invention uses a crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine.

[0271] According to one embodiment, the thiol groups of the crosslinking agent come from cysteine ​​residues.

[0272] A crosslinking agent may comprise at least two cysteine ​​residues.

[0273] The two cysteine ​​residues can be linked together by means of a spacer.

[0274] According to one embodiment, a spacer suitable for connecting two cysteines may comprise, before bonding to the cysteine ​​residues, at least two groups each selected from a primary amine, a secondary amine, a hydroxyl, and a thiol.

[0275] According to one embodiment, each cysteine ​​residue can be linked to the spacer by a bond chosen from #-NR3-CO-*, #-O-CO-* and #-S-CO-* in which #- and -* represent, respectively, a covalent bond with the spacer and a covalent bond with the cysteine ​​residue, and R3 representing H or a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0276] For the purposes of the description, the expression “hydrocarbon chain” aims to designate a chain of carbon atoms linked together by covalent bonds.

[0277] For the purposes of the description, the term “saturated” used in connection with the expression “hydrocarbon chain” means that the carbon atoms are linked together by simple covalent bonds.

[0278] For the purposes of the description, the term “unsaturated” used in connection with the expression “hydrocarbon chain” means that the chain comprises at least two carbon atoms linked together by a double or triple covalent bond.

[0279] For the purposes of the description, the term “linear” used in connection with the expression “hydrocarbon chain” means that the chain consists of a single hydrocarbon chain.

[0280] For the purposes of the description, the term “branched” used in connection with the expression “hydrocarbon chain” means that the chain comprises at least one carbon atom forming a secondary chain branched onto a main chain.

[0281] According to one embodiment, R3 represents H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0282] According to one embodiment, R3 represents H, a methyl, an ethyl, a propyl, or a butyl.

[0283] According to one embodiment, R3 represents H.

[0284] According to one embodiment, each cysteine ​​residue is linked to the spacer by a bond selected from an amide bond established between a primary or secondary amine of the spacer and a carboxylic group of a cysteine ​​residue, an ester bond established between a hydroxyl group of the spacer and a carboxylic group of a cysteine ​​residue, and a thioester bond established between a thiol group of the spacer and a carboxylic group of a cysteine ​​residue.

[0285] According to one embodiment, the spacer and the cysteine ​​residues can be linked by amide bonds established between a primary amine or a secondary amine of the spacer and a carboxylic group of a cysteine ​​residue.

[0286] According to one embodiment, the spacer and the cysteine ​​residues can be linked by ester bonds established between a hydroxyl of the spacer and a carboxylic group of a cysteine ​​residue.

[0287] According to one embodiment, the spacer and the cysteine ​​residues can be linked by thioester bonds established between a thiol of the spacer and a carboxylic group of a cysteine ​​residue.

[0288] Spacer

[0289] A spacer may be of general formula (I):

[0290] @-AR*-B-@ (I)

[0291] in which

[0292] @- and -@ represent bonds with the acyl groups of the cysteine ​​residues, and

[0293] R1 represents a residue chosen from a saturated C1-C10 hydrocarbon chain or unsaturated, optionally interrupted by one or more O, NR2, or S, and optionally substituted by one or more groups selected from -OR2, -NHR2, -SR2, -COOR2 and -CONHR2, in which R2 represents H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched,

[0294] A and B, identical or different, represent NR3, O or S, with R3 representing H or a C1-C6 alkyl.

[0295] For the purposes of the description, the expression “interrupted by one or more O, NR2, or S” used in connection with the expression “hydrocarbon chain” means that one or more several oxygen atoms, amino groups and / or sulfur atoms are linked to two carbon atoms by covalent bonds, for example in the form: -COC-, -C-NR2-C-, or -CS C-.

[0296] According to one embodiment, R1 is a hydrocarbon chain, saturated or unsaturated, linear or branched, in C1-C10, or in C2-C8, or in C3-C7, or even in C4-C6.

[0297] According to one embodiment, R1 is a hydrocarbon chain interrupted by one or more O's. According to one embodiment, R1 is a polyethylene glycol chain.

[0298] According to one embodiment, R1 is substituted by one or more groups chosen from -OH, -NH2, -NHR2, -SH, -COOR2 and -CONHR2.

[0299] According to one embodiment, R1 is substituted by one or more -CONHR2 groups.

[0300] According to one embodiment, R2 represents H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0301] According to one embodiment, R2 represents H or a CrC4 hydrocarbon chain

[0302] According to one embodiment, R2 represents a methyl, an ethyl, a propyl, or a butyl.

[0303] According to one embodiment, R2 represents a methyl.

[0304] According to one embodiment, R3 is as defined previously.

[0305] According to one embodiment, -A-R'-B- may be chosen from a polyol residue; an amino acid residue comprising at least two groups each chosen from a primary amine, a secondary amine, a hydroxyl, and a thiol; a residue of a peptide comprising at least two groups each chosen from a primary amine, a secondary amine, a hydroxyl, and a thiol; a residue of an aminated saccharide; a residue of an aminated polysaccharide; a polyamine residue; and a PEG diamine residue.

[0306] A polyol may be chosen from a saccharide, a polysaccharide, ethylene glycol, a polyethylene glycol.

[0307] A saccharide may be trehalose.

[0308] A polysaccharide may be a cyclodextrin.

[0309] An amino acid can be lysine, arginine, asparagine, or glutamine.

[0310] An amino saccharide may be chosen from diamino trehalose

[0311] An amino polysaccharide may be chosen from chitosan and derivatives thereof, a diamino cyclodextrin.

[0312] According to one embodiment, -A-R'-B- may be an amino acid residue chosen from a lysine, arginine, asparagine, or glutamine residue.

[0313] According to one embodiment, -A-R'-B- may be a lysine residue.

[0314] According to one embodiment, a spacer may be represented by the formula (Ib):

[0315] @-NH-(CH2)n-CH(CO2Et)-NH-@ (la),

[0316] in which @- and -@ represent bonds with the acyl groups of the cysteine ​​residues, and n is a natural integer varying from 1 to 10, in particular from 2 to 8, in particular from 3 to 6, and in particular being 4.

[0317] Crosslinking agents

[0318] According to one of its objects, the invention relates to a crosslinking agent of general formula (Ia):

[0319] Cys-A-R'-B-Cys (la)

[0320] in which

[0321] Cys represents a cysteine ​​residue, and

[0322] R1 represents a residue chosen from a C1-C10 hydrocarbon chain, saturated or unsaturated, optionally interrupted by one or more O, NR2, or S, and optionally substituted by one or more groups chosen from -OR2, -NHR2, -SR2, -COOR2 and -CONHR2, in which R2 represents H, a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched,

[0323] A and B, identical or different, represent NR3, O or S, with R3 representing H or a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0324] R1, R2 and R3 are as defined previously.

[0325] According to one embodiment, a crosslinking agent may be represented by the formula (II):

[0326] Nh2 h (II) HS. A,N. XO2Et ï r

[0327] According to one of its objects, the invention relates to the use of a crosslinking agent as described here, for crosslinking together two polymers comprising carboxylic groups, a portion of said polysaccharides being functionalized with a thioester group.

[0328] Functionalized polymers

[0329] A method according to the invention uses a polymer comprising at least one carboxylic group, preferably at least two carboxylic groups.

[0330] According to one embodiment, a method according to the invention uses a polymer comprising at least one carboxylic group, preferably at least two carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups.

[0331] According to one embodiment, at least about 0.5% of said carboxylic groups of said polymer are functionalized with thioester groups.

[0332] A portion of the carboxylic groups functionalized with thioester groups means that at least 0.5% of said carboxylic groups of said polymer are functionalized with thioester groups.

[0333] A portion of the carboxylic groups functionalized with thioester groups means that at most 80% of said carboxylic groups of said polymer are functionalized with thioester groups.

[0334] According to one embodiment, from about 0.5% to about 50%, or from about 5% to about 40%, or from about 6% to about 30%, or from about 8% to about 20%, or about 10%, or about 5% of the carboxylic groups of the polymer may be conjugated to the thioester groups.

[0335] According to one embodiment, from about 5% to about 20% of the carboxylic groups of the polymer may be conjugated to the thioester groups.

[0336] According to one embodiment, approximately 5% of the carboxylic groups of the polymer may be conjugated to the thioester groups.

[0337] A polymer comprising carboxylic groups suitable for the invention may be chosen from polysaccharides and proteins, and their biologically acceptable salts.

[0338] According to one embodiment, a polymer comprising carboxylic groups suitable for the invention is a polysaccharide.

[0339] According to one embodiment, a polysaccharide, or one of its biologically acceptable salts, suitable for the invention may be chosen from alginate, chondroitin, chondroitin sulfate, dermatan sulfate, heparin, xanthan, hyaluronic acid, carboxymethylcellulose, carboxycyclodextrin, their biologically acceptable salts, and mixtures thereof.

[0340] A biologically acceptable salt of polysaccharide may be selected from sodium salts, potassium salts, silver salts, sulfate salts, and mixtures thereof.

[0341] According to one embodiment, a polysaccharide, or one of its biologically acceptable salts, suitable for the invention may be hyaluronic acid, or one of its physiologically acceptable salts.

[0342] According to one embodiment, a polysaccharide salt suitable for the invention may be sodium hyaluronate.

[0343] According to one embodiment, the hyaluronic acid or sodium hyaluronate has a molecular weight ranging from about 100 kDa to about 5 MDa, or from about 500 kDa to about 4 MDa, or from about 1 MDa to about 3 MDa, or is about 1.5 MDa.

[0344] According to one embodiment, a protein suitable for the invention can be chosen among HSA, fibrin, and collagen.

[0345] According to one of its objects, the invention relates to a polymer comprising groups carboxylic in which part of the carboxylic groups is functionalized with thioester groups, the polymer being represented by the general formula (IV):

[0346] (IV)

[0347] in which n represents the proportion of carboxylic groups of said polymer functionalized with a thioester group and varies from approximately 0.5% to approximately 50% and Y represents a polymer residue comprising carboxylic groups.

[0348] According to one of its objects, the invention relates to a polymer comprising carboxylic groups in which a part of the carboxylic groups is functionalized with thioester groups, the polymer being represented by the general formula (IVa): Polymer

[0350] in which n represents the proportion of carboxylic groups of said polymer functionalized with a thioester group and varies from approximately 0.5% to approximately 50%.

[0351] According to one embodiment at least about 0.5% of said carboxylic groups of the polymer are functionalized with a thioester group. According to one embodiment, from about 5% to about 50%, or from about 5% to about 40%, or from about 6% to about 30%, or from about 8% to about 20%, or about 10%, or about 5% of the carboxylic groups of the polymer can be functionalized with a thioester group.

[0352] The proportion of carboxylic groups functionalized by thioester groups can be determined by any method known to those skilled in the art, for example by spectroscopic methods such as NMR.

[0353] According to one embodiment, a polymer may be a polysaccharide as defined previously.

[0354] As indicated previously, the present invention relates to a method for preparing a crosslinked polymer hydrogel by native chemical ligation using a first polymer comprising at least one carboxylic group, preferably at least two carboxylic groups or carboxylic groups, the, or at least one, or part of the carboxylic groups being functionalized with thioester groups, and a second polymer functionalized with at least one thiol group, preferably with at least two thiol groups, or thiol groups, the thiol group(s) being beta to a primary amine.

[0355] The first polymer may be as described previously.

[0356] The second polymer functionalized with at least one thiol function can be prepared by any method known in the art, for example by the methods described in WO 2019 / 238953 A1 or WO 2021 / 123247 A1

[0357] According to one embodiment, a first and a second polymer may be a polysaccharide or one of its biologically acceptable salts, in particular as defined below.

[0358] According to one embodiment, a polysaccharide, or one of its biologically acceptable salts, suitable for the invention may be hyaluronic acid, or one of its physiologically acceptable salts.

[0359] According to one embodiment, a polysaccharide salt suitable for the invention may be sodium hyaluronate.

[0360] A hyaluronic acid, or its biologically acceptable salts, comprising carboxylic groups functionalized with thiol groups can be prepared as described in WO 2019 / 238953 Al or Zhang et al. (Chem Commun, 2015, 51(47):9662-9665).

[0361] Thioester group functionalizing compounds

[0362] According to one embodiment, the carboxylic groups of the polymer can be functionalized with a functionalizing compound comprising a thioester group and a primary amine.

[0363] Before bonding to a carboxylic group of the polymer, a thioester group may be a functionalizing compound comprising a thioester group and a primary amine.

[0364] According to one embodiment, the thioester groups may be linked to the polymer by amide bonds. The amide bonds may be established between a primary amine of the thioester and a carboxylic group of the polymer.

[0365] According to one embodiment, a functionalizing compound with a thioester group may be of general formula (III):

[0366] NH2-R4-C(O)-S-R5 (III)

[0367] in which

[0368] R4 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -OC(O)-NHR6, -S-R6, -SC(O)OR6, -NHR6, -NH-C(O)R6, -NH-C(O)OR6, -C(O)NH-C(O)R6, -NH-C(NHR6)=NR6, -NH-C(NH-C(O)R6)=NH-C(O)R7, -Phe, -Phe-OC(O)R6, and

[0369] R5 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -NHR6, -NH-C(O)R6, -C(O)NH-C(O)R6, -C(O)NH-COOR6, -NHC(O)(CH2)2CH(NHR6)C(O)OR7, -SOR6, S(O)OR6, -Phe, -Phe-OC(O)R6,

[0370] with R6 and R7 representing, indifferently, H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by Phe.

[0371] According to one embodiment, R4 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0372] According to one embodiment, R4 represents a hydrocarbon chain, saturated or unsaturated, linear or branched, in C1-C4, or in C1-C2, or even in C1.

[0373] According to one embodiment, R4 represents a methylene, an ethylene, a propylene, or a butylene.

[0374] One embodiment, R4 may be substituted with one or more groups selected from -OC(O)-R6, -C(O)-O-R6, -OC(O)-NHR6, -S-R6, -SC(O)OR6, -NHR6, -NH-C(O)R6, -NH-C(O)OR6, -C(O)NH-C(O)R6, -NH-C(NHR6)=NR6, -NH-C(NH-C(O)R6 )=NH-C(O)R7, -Phe, -Phe-OC(O)R6.

[0375] According to one embodiment, R4 may be substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -OC(O)-NHR6, -NHR6, -NH-C(O)R6, -NH-C(O)OR6, -C(O)NH-C(O)R6, -Phe, -Phe-OC(O)R6.

[0376] According to one embodiment, R4 may be substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -OC(O)-NHR6, -NHR6, -NH-C(O)R6, -NH-C(O)OR6, -C(O)NH-C(O)R6.

[0377] According to one embodiment, NH2-R4-C(O)- represents an amino acid residue in which the reactive groups of the side chain are protected by a group chosen among an acetyl, a methyl, an ethyl, a tert-butyloxycarbonyl (BOC), or a carbamate.

[0378] According to one embodiment, R5 represents a CrC hydrocarbon chain 6, saturated or unsaturated, linear or branched, optionally substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -NHR6, -NH-C(O)R6, -C(O)NH-C(O)R6, -C(O)NH-COOR6, -NHC(O)(CH2)2CH(NHR6)C(O)OR7, -SOR6, S(O)OR6, -Phe, -Phe-OC(O)R6.

[0379] According to one embodiment, R5 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched.

[0380] According to one embodiment, R5 represents a hydrocarbon chain, saturated or unsaturated, linear or branched, in C1-C4, or in C1-C2, or even in Cp

[0381] According to one embodiment, R5 represents a methylene, an ethylene, a propylene, or a butylene.

[0382] According to one embodiment, R5 may be substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -NHR6, -NH-C(O)R6, -C(O)NH-C(O)R6, -C(O)NH-COOR6, -NHC(O)(CH2)2CH(NHR6)C(O)OR7.

[0383] According to one embodiment, R5 may be substituted by one or more groups chosen from -OC(O)-R6, -C(O)-O-R6, -NHR6, -NH-C(O)R6.

[0384] According to one embodiment, R6 and R7 represent, indifferently, a C1-C4 alkyl residue, optionally substituted by Phe.

[0385] According to one embodiment, R6 and R7 represent, indifferently, H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by Phe.

[0386] According to one embodiment, R6 and R7 represent, indifferently, a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by Phe.

[0387] According to one embodiment, R6 and R7 represent, indifferently, a methyl or an ethyl.

[0388] According to one embodiment, NH2-R4-C(O)- represents a glycine residue.

[0389] According to one embodiment, -S-R5 represents a group chosen from a residue cysteine, a cysteamine residue, or a glutathione residue.

[0390] According to one embodiment, -S-R5 represents a group chosen from a cysteine ​​residue.

[0391] According to one embodiment, a functionalizing compound with a thioester group may be a compound of formula (IIIa):

[0392] (Ilia)

[0393] According to one embodiment, a thioester group may be represented by the general formula (Ilia):

[0394] §-NH-R4-C(O)-S-R5 (Illb)

[0395] in which

[0396] $- represents a bond with an acyl residue of a carboxylic group of the polysaccharide, and

[0397] R4 and R5 are as defined above.

[0398] According to one embodiment, a polymer comprising carboxylic groups functionalized with a thioester group functionalizing compound may be represented by the general formula (IV): (IV) H N CH,. \ O 1st ch3 \ ! ° / \ o<, „ T n Y

[0399] in which n represents the proportion of carboxylic groups of said polymer functionalized with a thioester group and Y represents a polymer residue comprising carboxylic groups.

[0400] According to one embodiment, a polymer comprising carboxylic groups functionalized with a thioester group functionalizing compound may be represented by the general formula (IVa): Polymer

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413] in which n represents the proportion of carboxylic groups of said polymer functionalized with a group. According to one embodiment, n represents a percentage of functionalization varying from about 0.5% to about 50%, or from about 5% to about 40%, or from about 6% to about 30%, or from about 8% to about 20%, or about 10%, or about 5% of the carboxylic groups of the polymer. Alternatively, according to one embodiment, the carboxylic groups of the polymer can be functionalized with a functionalizing compound comprising a thiol group. According to one embodiment, the thioester groups may be linked to the polymer by thioether bonds. The thioether bonds may be established between the sulfur of a compound comprising a thiol function and a carboxylic group of the polymer. According to one embodiment, a functionalizing compound with a thiol group may be of general formula (IIIc): SH-R5 (IIIc) in which R5 is as defined previously. Crosslinked hydrogels, compositions, and uses According to one of its objects, the invention relates to a crosslinked polymer hydrogel obtainable according to the method as described here. According to one of its objects, the invention relates to a composition comprising a crosslinked polymer hydrogel obtained according to the method as described here. According to one of its objects, the invention relates to a sterile injectable composition comprising a crosslinked polymer hydrogel obtained according to the method as described herein. According to one of its objects, the invention relates to a cosmetic use of a hydrogel or a composition as described here as a soft tissue filling agent, in particular the filling of wrinkles.

[0414] According to one of its objects, the invention relates to a hydrogel or a composition as described here for its cosmetic use in the augmentation of soft tissues.

[0415] According to one of its objects, the invention relates to a hydrogel or a composition as described here for its cosmetic use in the prevention and / or treatment of an alteration of the surface appearance of the skin.

[0416] A cosmetic use of a hydrogel or composition as described herein may aim to fill wrinkles, restore lost volume to the face or body, reduce dimples in the case of cellulite, or shape the contours of the face or body.

[0417] A cosmetic use of a hydrogel or composition as described herein may be aimed at the prevention and / or treatment of an alteration of the viscoelastic or biomechanical properties of the skin.

[0418] A cosmetic use of a hydrogel or composition as described herein may be aimed at the prevention and / or treatment of cutaneous signs of chronological aging and / or induced by external factors such as stress, atmospheric pollution, tobacco or prolonged exposure to ultraviolet (UV) rays.

[0419] According to one of its objects, the invention relates to a crosslinked polymer hydrogel obtainable according to the method as described here, or a composition comprising such a hydrogel, for use as a medicament or medical device, or system for delivering active agents, in particular as defined above.

[0420] According to one embodiment, the invention relates to a hydrogel or a composition as described herein, for use in the treatment or prevention of a disease selected from osteoarthritis, glaucoma, cataract, lipodystrophy, to promote healing processes, and lipoatrophy in patients with AIDS. Examples

[0421] The examples described below are given for the purpose of illustrating the claimed invention and should not be construed as limiting it.

[0422] Example 1: Preparation of a thioester to functionalize a polysaccharide: A-acetyl-S-^tert-butoxycarbonyljglycy^methyl cysteinate then A-acetyl-S-(glycyl)cysteinate methyl in the form of its TFA salt cf3co2h*h2n CO2Me NHAc

[0423] Boc-glycine iV-hydroxysuccinimide ester (Boc-Gly-OSu, 10 g, 0.036 mmol, 1.5 eq) and methyl iV-Acetyl-L-cysteinate (Ac-Cys-Ome, 4.3 g, 0.024 mmol, 1 eq) are dissolved in anhydrous dimethyl formamide (DMF) (85 mL) under argon atmosphere in a 500 mL flask. The solution is stirred overnight at room temperature. The reaction medium is diluted with water (40 mL) and then the aqueous phase is extracted (3x80 mL of ethyl acetate (AcOEt)). The organic phases are combined, washed with brine (40 mL). The obtained phase is dried over MgSO4 then filtered and evaporated. A transparent oil is obtained. Purification by silica gel chromatography (Heptane / AcOEt, gradient 50 / 50 to 0 / 100) gives methyl A^-acetyl-5'-[(tert-butoxycarbonyl)glycyl]cysteinate in the form of a white powder.

[0424] Methyl A^-acetyl-5'-[(tert-butoxycarbonyl)glycyl]cysteinate (600 mg, 1.80 mmol, 1 eq) is introduced into a flask and solubilized in dichloromethane (15 mL). The solution is placed under an argon atmosphere. Trifluoroacetic acid (TFA, 3 mL) is added using a syringe and with a rapid dropwise flow. The solution is stirred at room temperature for 1 h 30 min. The reaction medium is diluted with methanol (22.5 mL) in order to co-evaporate the TFA. Successive co-evaporations are carried out with volumes of methanol of approximately 20 mL until the mass is stabilized and a pale yellow oil is obtained which then crystallizes at low temperature (refrigerator, 4°C) from methyl / V-acetyl-5'-(glycyl)cysteinate in the form of its TFA salt.

[0425] Example 2: Preparation of a crosslinking agent comprising two thiol functions: bis-cysteine ​​- A^AU-bisfA-^erLbutoxycarbonylj-S-trityl-L-cysteiny^-L-ethyl lysinate then A^^-bis^-cysteiny^-L-ethyl lysinate H2N^^^^^ / CO2Et । nh2 Lys-OEt + Ph3C-S CO2H ^^NHBoc BocHN

[0426]

[0427] Ethyl lysinate (Lys-Oet, 0.7 g, 2.83 mmol, 1 eq), A-(terLbutoxycarbonyl)-S-trityl-L-cysteine ​​(3.28 g, 7.08 mmol, 2.5 eq), and the coupling agents 1-hydroxybenzotriazole (HOBt, 1 g, 7.08 mmol, 2.5 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1.83 g, 7.08 mmol, 2.5 eq) were placed in a flask under argon atmosphere and anhydrous DMF was added (17.5 mL), followed by MA^-diisopropylcthylaminc (DIEA, 1.83 g, 2.34 mL, 14.2 mmol, 5 eq). The solution is stirred at room temperature (approx. 21°C - 25°C) for 48 hours. The reaction medium is diluted with water (30 mL) and then the aqueous phase is extracted (3x40 mL AcOEt). The organic phases are combined and washed with brine (40 mL), dried over MgSO4. Finally, the solvents are evaporated to obtain a yellow oil.Purification by silica gel chromatography (Heptane / AcOEt, gradient 100 / 0 to 0 / 100) gives A7,ALbis| AA / e / 7-butoxycarbonyl)-5'-trityl-L-cysteinyl]-L-lysinate ethyl in the form of a white powder. Ethyl A“,AE-bis[A-(tert-Lbutoxycarbonyl)-S,-trityl-L-cysteinyl]-L-lysinate (400 mg, 0.375 mmol, 1 eq.) is placed in a flask under an inert argon atmosphere. The product is then dissolved in dichloromethane (5 mL) and then trifluoroacetic acid (5 mL) and triethylsilane (0.24 mL, 1.5 mmol, 4 eq.) are added to the mixture. The solution is stirred at room temperature for 3 h and then the mixture is diluted with water (40 mL). The aqueous phase is washed with diethyl ether (3x25 mL). The aqueous phase is then lyophilized to obtain ethyl A^^-bis^-cysteinyl)-L-lysinate as a white powder.

[0428] Example 3: Functionalization of sodium hyaluronate with thioester functions

[0429] The functionalization of sodium hyaluronate is illustrated by step 1 of [Fig. 1].

[0430] Sodium hyaluronic acid (NaHA or sodium hyaluronate) with a molecular weight of 1.5 MDa was solubilized in water for injection (WFI), at a concentration of 10 mg / g (1% w / v) under magnetic stirring (200 rpm), for 2 to 3 hours, at room temperature (approx. 21°C - 25°C). The pH of the solution was in the range of 5.0 to 5.5. A viscous, homogeneous and transparent solution of NaHA was obtained.

[0431] 0.5 equivalent (Eq.) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl- chloride Morpholinium (DMTMM) was solubilized in 1 mL of water for injection (i.e. 0.5 mole of DMTMM for 1 mole of NaHA), at room temperature (approx. 21°C). A homogeneous and transparent solution with complete dissolution of DMTMM crystals was obtained.

[0432] The solubilized DMTMM was added dropwise to the NaHA solution. The resulting mixture was allowed to react for 30 minutes under magnetic stirring, 200 rpm, at room temperature (approx. 21°C - 25°C). The pH of the reaction mixture was in the range of 5.0 and 5.5.

[0433] 0.5 equivalent (Eq.) (0.5 mole of thioester per 1 mole of NaHA) of the thioester salt of formula (V): G (V> F y ' ' O " ny y S„ . ...JL .OMe AÇ KH {R HAS

[0434] was solubilized in 1 mL of water for injection.

[0435] The solubilized thioester was added dropwise to the reaction mixture of NaHA and DMTMM. The resulting mixture was left to react for 72 hours under magnetic stirring, 200 rpm, at room temperature (approx. 21°C - 25°C), and at room temperature to obtain sodium hyaluronate of which approximately 5%-10% of the carboxylic groups were functionalized with thioester functions (NaHA- thioester). The pH of the reaction mixture was in the range of about 4.0 to about 5.5 during the reaction.

[0436] Example 4: Purification and recovery of sodium hyaluronate functionalized with thioester functions (HA-thioester)

[0437] The purification of the sodium hyaluronate functionalized with thioester functions (NaHA-thioester) obtained in Example 3 was carried out by precipitation in ethanol. The precipitation of the NaHA-thioester makes it possible to eliminate the excess of unreacted DMTMM, any degradation products of DMTMM and the excess thioester. Furthermore, the precipitation makes it possible to recover NaHA-thioester in the form of fibers.

[0438] Adding sodium chloride (NaCl) to the NaHA-thioester solution facilitates the precipitation of NaHA-thioester in ethanol. NaCl was dissolved in the HA-thioester solution to achieve a final concentration of 1 M.

[0439] Precipitation of NaHA-thioester was obtained by dropwise addition of the NaHA-thioester saline solution (obtained after addition of NaCl) into 3 times its volume of cold ethanol (96% EtOH stored between 5°C and 7°C) with stirring to form a vortex.

[0440] The precipitated NaHA-thioester was recovered in the form of fibers. The precipitation step was repeated 3 times to purify the NaHA-thioester.

[0441] After the last precipitation, the NaHA-thioester recovered in the form of fibers was solubilized at the concentration of 5 mg / g of ppi water.

[0442] The purified NaHA-thioester solution was lyophilized. The solution was frozen at -80°C and then placed under vacuum (less than 1 mbar), with a water capture system at -80°C. The lyophilization time is adjusted according to the volume of solution to be lyophilized.

[0443] Example 5: Preparation of a crosslinked sodium hyaluronate hydrogel using a sodium hyaluronate functionalized with thioester functions (NaHA-thioester)

[0444] The crosslinking of sodium hyaluronate functionalized by thioester groups by means of a bi-cysteine ​​is illustrated by step 2 of [Fig.l].

[0445] The lyophilized NaHA-thioester obtained in Example 4 was solubilized, at room temperature (approx. 21°C-25°C), at a concentration of 20 mg / g in a saline phosphate buffer of pH 7.0-7.4, under magnetic stirring (approx. 200 rpm). The final pH of the solution was approximately 5.5.

[0446] Depending on the formulations tested, 0.05 or 0.14 Eq. of bi-cysteine, of formula (VI)

[0447] used as a crosslinking agent, was added in powder form to the NaHA-thioester solution under magnetic stirring (approx. 200 rpm), at room temperature (approx. 21°C-25°C) in order to ensure the solubilization of the bi-cysteine ​​and a homogeneous mixture.

[0448] After dissolving the bi-cysteine, the pH of the NaHA-thioester / bi-cysteine ​​mixture was adjusted to a pH between 6.0 and 7.8, by adding a 0.25 M sodium hydroxide (NaOH) solution, with stirring for approx. 2 min with a spatula, at room temperature (approx. 21°C-25°C).

[0449] The addition of sodium hydroxide allows the crosslinking reaction to be initiated.

[0450] The crosslinking reaction of NaHA-thioester in the presence of bi-cysteine, following the addition of sodium hydroxide, occurs very rapidly and is carried out almost entirely in approximately 5 min.

[0451] The gel obtained was kept at rest, without stirring, for approximately 1 hour.

[0452] Adjusting the pH of the reaction mixture to a pH between 6.0 and 7.8 allows the crosslinking reaction to be initiated and accelerated by the formation of native chemical ligations (amide bonds) between the thioester functions of the functionalized NaHA and the thiol functions of the bi-cysteine. The crosslinking reaction occurs very rapidly, even almost instantly. All of the reagents can be consumed in 5 minutes or less.

[0453] The crosslinking reaction can advantageously be carried out under an inert atmosphere in order to prevent or limit the cyclization reaction of the bi-cysteine, for example as in the case of the preparation of the formulations according to the invention 1 and 2 given below.

[0454] Advantageously, at the end of the crosslinking step, the crosslinked sodium hyaluronate (NaHA) hydrogel obtained can be dialyzed in a saline phosphate buffer bath, with a pH of approximately 7.0 - 7.4, at room temperature (approx. 21°C-25°C).

[0455] Example 6: Preparation of a soft tissue filler

[0456] The crosslinked NaHA hydrogel obtained in Example 5 was added with 10% in mass (mass / mass) of uncrosslinked NaHA at 1.5 MDa, dissolved in a phosphate buffer at a concentration of 23 mg / g (except in the case of the preparation of the formulation according to the invention 1 given below). The addition step is carried out under

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463] manual stirring (spatula) or mechanical stirring (blade robot, at room temperature (approx. 21°C-25°C). The resulting HA mixture was sieved using a sieve with a mesh size of a few hundred microns. The soft tissue filler gel thus obtained was packaged in syringes and then sterilized in an autoclave, under moist heat conditions, at a temperature greater than or equal to 121°C so as to obtain an F0 > 15 (sterilizing value). Example 7: Measurements of elastic moduli (G') Different cross-linked HA gels were prepared according to the protocol described in examples 3-6, but with the following parameters: [Tables 1] Parameters Eq. [HA: D Eq. [HA: thi HA-thioester Eq. [HA-thio Addition of MTMM] ester] mg / g before rest ester: bi-cys HA non-crosslinked (% by gel mass) crosslinking teine] Method 1 0.5 0.5 20 0.14 0 Method 2 0.5 0.5 20 0.14 10 Method 3 0.5 0.5 20 0.05 10 [Tables 2] Parameters Reaction time pH before addition b pH after addition bi HA: DMTMM i-cysteine ​​-cysteine ​​and sodium hydroxide Method 1 1h 4.2 6.4 Method 2 1h 4.2 6.4 Method 3 30 min 4.12 7.3 The elastic modulus G' and the phase shift angle δ of the different formulations were measured before and after sterilization as follows. The measurements were carried out at 25°C, at a frequency of 1 Hz, with stress scanning using a Thermo Haake RS6000 rheometer with a 1 “ / 35 mm diameter cone-plate geometry. G' and r# are recorded at an applied strain stress of 5 Pa, i.e., in the viscoelastic range where G' and r remain stable (i.e., in the linear viscoelastic range). The elastic modulus G', or storage modulus, measures the energy released by the gel when it is subjected to a small deformation. This quantity can be represented by the stiffness of a spring.

[0464] The phase shift angle δ characterizes the degree of viscoelasticity of a material: it varies between 0° for a 100% elastic material (all the deformation energy is restored by the material, i.e. it returns to its initial shape) and 90° for a 100% viscous material (all the deformation energy is lost by the material, i.e. it flows and completely loses its initial shape). A skin filler gel must be predominantly elastic to ensure its filling properties, i.e. r must be <45°.

[0465] The measurement of elastic moduli before and after sterilizations were as follows: [Tables 3] Parameters G' (Pa) ô (°) T (Pa) Process 1 Before sterilization 1876 1.9 226 After sterilization 1786 1.9 137 Process 2 Before sterilization 1340 5.2 132 After sterilization 1102 4.1 96 Process 3 Before sterilization 524 7.7 174 After sterilization 377 4.5 124

[0466] Hydrogels obtained by a process as described herein exhibit elastic moduli, measured after sterilization, compatible with hydrogels suitable for use as soft tissue fillers. Hydrogels obtained by a process as described herein may be adjusted to a pH between 6.8 and 7.8.

Claims

Claims

1. A method for preparing a crosslinked polymer hydrogel by native chemical ligation, the method using a non-crosslinked functionalized polymer and a crosslinking agent, the crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine, and the polymer comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups, the method comprising at least the steps of: a. contacting, in an aqueous solution, at an acidic pH of at least 4.0, said non-crosslinked functionalized polymer and said crosslinking agent, b. increasing the pH of the mixture obtained in step a) by at least 0.5 units, the increased pH being at least greater than 5.5 and not exceeding 8.0, and c. obtaining a crosslinked polymer hydrogel.

2. The method of claim 1, wherein the thiol groups of the crosslinking agent are derived from cysteine ​​residues, the cysteine ​​residues being linked together by a spacer.

3. A method according to claim 1 or 2, wherein the crosslinking agent is of general formula (Ia): Cys-A-R'-B-Cys (Ia) in which Cys represents a cysteine ​​residue, and R1 represents a residue chosen from a saturated or unsaturated C1-C10 hydrocarbon chain, optionally interrupted by one or more 0, NR2, or S, and optionally substituted by one or more groups chosen from -OH, -NH2, -NHR2, -SH, -COOR2 and -CONHR2, in which R2 represents H or a linear or branched, saturated or unsaturated C1-C4 hydrocarbon chain, A and B, identical or different, represent NR3, O or S, with R3 representing H or a linear or branched, saturated or unsaturated C1-C6 hydrocarbon chain.

4. A method according to any one of claims 1 to 3, wherein the crosslinking agent is represented by the formula (II): NH2 h (II) h Et 0 O^.NH A / SH H2N

5. A method for preparing a crosslinked polymer hydrogel by native chemical ligation, the method using a first polymer and a second non-crosslinked polymer, the first polymer comprising at least one carboxylic group, the carboxylic group being functionalized with a thioester group, and, the second polymer being functionalized with at least one thiol group, the thiol group being beta to a primary amine, the method comprising at least the steps of: a. contacting the first and second polymers in an aqueous solution, the aqueous solution being at an acidic pH of at least 4.0, b. increasing the pH of the mixture obtained in step a) by at least 0.5 units, the pH being at least greater than 5.5 and not exceeding 8.0, and c. obtaining a crosslinked polymer hydrogel.

6. A method according to one of claims 1 to 5, wherein at least about 0.5% of said carboxylic groups of the polymer comprising carboxylic groups functionalized with thioester groups are functionalized with thioester groups.

7. Method according to one of claims 1 to 6, in which said carboxylic groups of the polymer comprising carboxylic groups functionalized with thioester groups are functionalized with a thioester group functionalizing compound of general formula (III): NH2-R4-C(O)-S-R5 (III) in which R4 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by one or several groups selected from -OC(O)-R6, -C(O)-O-R6, -OC(O)-NHR6, -S-R6, -SC(O)OR6, -NHR6, -NH-C(O)R6, -NH-C(O)OR6, -C(O)NH-C(O)R6, -NH-C(NHR6)=NR6, -NH-C(NH-C(O)R6)=NH-C(O)R7, -Phe, -Phe-OC(O)R6, and R5 represents a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by one or more groups selected from -OC(O)-R6, -C(O)-O-R6, -NHR6, -NH-C(O)R6, -C(O)NH-C(O)R6, -C(O)NH-COOR6, -NHC(O)(CH2)2 CH(NHR6)C(O)OR7, -SOR6, S(O)OR6, -Phe, -Phe-OC(O)R6, with R6 and R7 representing, indifferently, H or a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched, optionally substituted by Phe.

8. Method according to one of claims 1 to 7, in which the mixture obtained in step a) is kept stirring for a period varying from 5 minutes to 1 hour.

9. A method according to any one of claims 1 to 8, wherein in step b), the pH is adjusted to a pH of 6.0 to 8.0, or 6.2 to 7.8, or 6.4 to 7.6, or 6.8 to 7.4, or is 7.

0.

10. Method according to one of claims 1 to 9, in which step b) comprises a first sequence b1) and a second sequence b2), - sequence b1) comprising an increase in the pH of the mixture obtained in step a) of at least 0.5 units, the pH being at least greater than 5.5 and not exceeding 8.0, for a period of 2 min. to 1 hour, and - sequence b2) comprising maintaining the solution obtained at the end of sequence b1), without stirring, for a period of 15 min. to 30 hours.

11. Method according to one of claims 1 to 10, in which the polymer is a polysaccharide or its biologically acceptable salts, optionally the polysaccharide is hyaluronic acid, or one of its biologically acceptable salts.

12. Crosslinked polymer hydrogel obtainable according to the method as defined according to one of claims 1 to 11.

13. A sterile injectable composition comprising a hydrogel according to claim 12.

14.

15.

16. Cosmetic use of a hydrogel according to claim 12 or a composition according to claim 13 as a soft tissue filler or for soft tissue augmentation. Hydrogel according to claim 13 or composition according to claim 14, said hydrogel or said composition further comprising at least one active agent, for use as a medicament or medical device. A polymer comprising carboxylic groups in which a portion of the carboxylic groups is functionalized with thioester groups, the polymer being represented by the general formula (IV):

17.

18. in which n represents the proportion of carboxylic groups of said polymer functionalized with a thioester group and varies from 0.5% to 50% and Y represents a polymer residue comprising carboxylic groups. A crosslinking agent comprising at least two thiol groups, each of the thiol groups being beta to a primary amine. Crosslinking agent according to claim 17, of general formula (la): Cys-A-R'-B-Cys (la) in which Cys represents a cysteine ​​residue, R1 represents a residue chosen from a C1-C10 hydrocarbon chain, saturated or unsaturated, optionally interrupted by one or more O, NR2, or S, and optionally substituted by one or more groups chosen from -OR2, -NHR2, -SR2, -COOR2 and -CONHR2, in which R2 represents H, a C1-C4 hydrocarbon chain, saturated or unsaturated, linear or branched,

19. A and B, identical or different, represent NR3, O or S, with R3 representing H or a C1-C6 hydrocarbon chain, saturated or unsaturated, linear or branched. Use of a crosslinking agent according to one of claims 17 to 18, for crosslinking two polymers comprising carboxylic groups, a portion of the carboxylic groups being functionalized with thioester groups.

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