Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/52—Hydrogels or hydrocolloids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/04—Dispersions; Emulsions
  • A61K8/042—Gels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/73—Polysaccharides
  • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/08—Anti-ageing preparations
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
  • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
  • C08J3/075—Macromolecular gels
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J3/00—Processes of treating or compounding macromolecular substances
  • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
  • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/91—Injection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/95—Involves in-situ formation or cross-linking of polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2400/00—Materials characterised by their function or physical properties
  • A61L2400/06—Flowable or injectable implant compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
  • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
  • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

• TITLE HYDROGELS FOR SOFT TISSUE FILLING
• the present invention relates to a process for preparing a hydrogel comprising a cross-linked polysaccharide, in particular, to a process for preparing an injectable hydrogel comprising cross-linked hyaluronic acid.
• the present invention also relates to a hydrogel, preferably injectable, obtainable by the process, a composition comprising the hydrogel, and the uses of this hydrogel.
• Hydrogels based on polysaccharides are widely used for filling soft tissues, in particular skin, which present, for example, volumetric defects (e.g., wrinkles, scars) or for increasing their volume.
• Hydrogels suitable for filling soft tissues are typically crosslinked hydrogels, i.e., the polysaccharide is crosslinked using one or more crosslinking agent(s). This crosslinking makes it possible to obtain a hydrogel with desirable mechanical properties for filling soft tissues.
• hydrogels based on hyaluronic acid can, with a low incidence, cause adverse effects which develop between 6 and 24 months after application of the filler. In particular, it may happen that the filler migrates to an area far from that of its application in or on the body (Chae SY, Lee KC, Jang YH, Lee SJ, Kim DW, Lee WJ.
• a need remains for the provision of a hydrogel based on crosslinked polysaccharides, in particular a hydrogel based on crosslinked hyaluronic acid. whose hyaluronic acid chains are preserved, having mechanical properties adapted to filling soft tissues and which does not migrate into the tissues after its application.
• the present invention relates to a process for preparing a hydrogel, preferably injectable, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two functional groups Z, identical or different, chosen from isocyanate, amino, epoxy, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl groups, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, and an acid anhydride residue; c) prepare a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total quantity of crosslinking agent ranging from 0.001 to less than 0.02 mole per 1 mole d repetition unit of the polysaccharide
• the present invention also relates to a hydrogel capable of being obtained by a process such as presently described, as well as to a cosmetic or pharmaceutical composition comprising such a hydrogel and their uses in the filling and/or replacement of tissues, to prevent and /or treat the alteration of the viscoelastic or biomechanical properties of the skin; to fill volume defects in the skin, and in particular to fill wrinkles, fine lines and scars; for example, to reduce nasolabial folds and bitter folds, to increase the volume of the cheekbones, chin or lips, to restore the volume of the face, particularly the cheeks, temples, the oval of the face, and around the eye; or to reduce the appearance of fine lines and wrinkles; or to stimulate, regenerate, hydrate, firm or restore radiance to the skin, particularly through mesotherapy.
• Figure 1 illustrates the stickiness of a hydrogel according to the invention compared to a traditional hydrogel on a metal (Fig. 1A), glass (Fig. 1 B), plastic (Fig. 1C) and collagen (Fig. 1 D).
• gel refers to a network of polymers which is expanded throughout its volume by a fluid. This means that a gel is made up of two media, one “solid” and the other “liquid”, dispersed in each other.
• the so-called “solid” medium is made up of long polymer molecules connected together by weak bonds (for example hydrogen bonds) or by covalent bonds (crosslinking).
• the liquid medium consists of a solvent.
• a gel generally corresponds to a product which has a phase angle 5 less than or equal to 45° at 1 Hz for a deformation of 0.1% or a pressure of 1 Pa, advantageously a phase angle 5 ranging from 2° to 45 ° or ranging from 20° to 45°.
• hydrogel designates a gel as defined above in which the solvent constituting the liquid medium is mainly water (for example at least 90%, in particular at least 95%, in particular at least 99% by weight liquid medium).
• the liquid medium comprises, in particular consists of, a buffer solution, advantageously allowing a pH of the liquid medium of between 6.8 and 7.8, in particular a saline phosphate buffer.
• injectable gel designates a gel which can flow and be injected manually using a syringe fitted with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a hypodermic needle of 30 G, 27 G, 26 G, 25 G.
• an “injectable gel” is a gel having an average extrusion force less than or equal to 25N, preferably ranging from 5 to 25 N, more preferably ranging from 8 at 15 N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm/min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle of an external diameter less than or equal to 0.4 mm (27 G) and length 1”, at room temperature.
• the “stringy” nature of a product designates its ability to be stretched between two surfaces to which it has adhered.
• the stringy character can be determined using a texturometer, a sensory analysis carried out by a panel, or even rheological and mechanical measurements including in particular the measurement of the phase angle (5) or traction tests .
• this character can be measured as described by P. Micheels et al. (Micheels et al., Comparison of two swiss-designed hyaluronic acid gels: six-month clinical follow-up, Journal of Drug in Dermatology, 2017, 16:154-161, “Resistance to stretching”) or by carrying out a test of Tack and measure the length of the gel wires in tension.
• the “stickiness” of a product refers to its ability to adhere to a surface. It can be determined qualitatively using a sensory analysis carried out using a panel or by moving a bolus on a surface. It can also be determined quantitatively by measuring the force of adhesion to a surface, traction machine or mechanical analysis.
• polysaccharide designates a polymer composed of monosaccharides (preferably D enantiomers) joined together by glycosidic bonds.
• monosaccharide also called “ose”, designates an unmodified or modified monosaccharide.
• An “unmodified monosaccharide” designates a compound of formula H-(CHOH) that 2 ⁇ x+y ⁇ 5, the monosaccharide being able to be found in a linear form represented by the above-mentioned formula or being able to be found in a cyclized form by reaction of the CO function (aldehyde or ketone) with one of the OH groups for form a hemiacetal or hemiketal group.
• the monosaccharide is in cyclized form.
• Monosaccharides are classified by number of carbons.
• a monosaccharide further comprises x+y asymmetric carbons and therefore 2 (x+y-1) pairs of enantiomers.
• Each pair of enantiomers is designated by a different name and enantiomers of the same pair are referred to as D and L enantiomers, respectively.
• a “modified monosaccharide” designates an unmodified monosaccharide as defined above including, for example:
• an OR group with R representing a group (Ci-Cejalkyl such as methyl or ethyl; hydroxy-(Ci-C6)alkyl such as hydroxyethyl (-CH2CH2OH) or hydroxypropyl (-CH2-CH(OH)- CH3) ; carboxy-(Ci-C6)alkyl such as carboxymethyl (-CH2COOH); or CO-(Ci-C6)alkyl such as acetyl; and/or
• R an NR’R” group with R’ and R” representing, independently of each other, H, (Ci-Ce)alkyl or CO-(Ci-C6)alkyl such as acetyl; and or
• repeating unit of a polysaccharide refers to a structural unit consisting of one or more (usually 1 or 2) monosaccharides, the repetition of which produces the complete polysaccharide chain.
• monosaccharides may be in a modified form.
• Monosaccharides, when modified, can be in different modified forms.
• physiologically acceptable means that which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for human or veterinary cosmetic (i.e. non-therapeutic) or therapeutic use, including for use by injection into the human or animal body or for topical application to the skin.
• salts useful in the context of the present invention are preferably physiologically acceptable salts.
• physiologically acceptable salts designate in particular:
• pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with pharmaceutically acceptable organic acids such as formic acid, acetic acid, benzene sulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethane-sulfonic acid, acid fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, l mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid,
• a metal ion for example an alkali metal ion (eg Na, K), an alkaline metal ion -earth (eg Ca, Mg), a zinc ion, a silver ion or an aluminum ion
• a pharmaceutically acceptable organic base such as diethanolamine, ethanolamine, N-methylglu
• the "modification degree" (MOD) of a polysaccharide corresponds to the molar quantity of cross-linking agent linked to the polysaccharide, by one or more of its ends, expressed per 100 moles of units of repetition of the polysaccharide. It can be determined by methods known to those skilled in the art such as Nuclear Magnetic Resonance spectroscopy (NMR).
• NMR Nuclear Magnetic Resonance spectroscopy
• the “molar crosslinking rate” (TR), expressed in%, designates the molar ratio of the quantity of crosslinking agent relative to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the crosslinking medium.
• therapeutic active ingredient means a substance for curing, relieving symptoms and/or preventing a disease; a substance having curative or preventive properties with regard to human or animal diseases, as well as any substance which can be used in humans or animals or which can be administered to them, with a view to establishing a medical diagnosis or restore, correct or modify their physiological functions by exerting a pharmacological, immunological or metabolic action.
• cosmetic active ingredient designates any non-therapeutic substance, in particular intended to be brought into contact with various superficial parts of the human body, such as the epidermis, the hair and capillary systems, the nails, the lips, the chest and the teeth, with a view, exclusively or mainly, to cleaning them, protecting them, perfuming them, maintaining them in good condition, modifying their appearance or odor.
• spacer group designates a fragment comprising at least one atom aimed at linking two chemical groups together within the same molecule.
• the spacer group contains at least one carbon atom.
• halogen designates an atom of fluorine, chlorine, bromine or iodine.
• An “epoxide” group is an ethylene oxide residue linked to the rest of the molecule by one of its carbon atoms.
• N-succinimidyloxycarbonyl is a group of formula Chem. GR1 below: sulfosuccinimidyloxycarbonyl” is a group with the formula Chem. GR2 above
• a “halogenocarbonyl” group is a group of formula -CO-Hal with Hal representing a halogen, such as Cl or Br.
• an “acid anhydride residue” is a group comprising a -C(O)-OC(O)- motif, and more particularly a monovalent cyclic group comprising the -C(O)-OC(O)- motif, such as a saturated monovalent hydrocarbon monocyclic group comprising 5 to 10, in particular 5 or 6, carbon atoms of which three successive carbon atoms are replaced by C(O)-OC(O) and optionally one or more of which, in particular one, additional carbon atoms, preferably not consecutive to the three carbon atoms substituted by CO-O-CO, are each replaced by a heteroatom such as N, O or S, in particular N.
• the acid anhydride residue can respond in particular to the Chem formula.
• Next GR3 is a group comprising a -C(O)-OC(O)- motif, and more particularly a monovalent cyclic group comprising the -C(O)-OC(O)- motif, such as a saturated monovalent hydrocarbon monocyclic group comprising
• the acid anhydride residue can also be chosen from a maleic anhydride residue or a succinic anhydride residue.
• aliphatic hydrocarbon chain or "aliphatic hydrocarbon group” designates a linear, branched and/or cyclic hydrocarbon group, saturated or unsaturated but non-aromatic, advantageously comprising from 1 to 50, in particular from 1 to 20, for example from 1 to 12 or 1 to 6 carbon atoms. These will in particular be alkyl groups.
• branched aliphatic hydrocarbon chain specifically designates a main aliphatic hydrocarbon chain comprising at least one secondary aliphatic hydrocarbon chain.
• star aliphatic hydrocarbon chain designates a branched aliphatic hydrocarbon chain comprising several secondary aliphatic hydrocarbon chains all starting from a single branching point.
• C1-Cx alkyl or “(Cl-Cx)alkyl” or even “alkyl comprising from 1 to x carbon atoms” designates a saturated monovalent hydrocarbon group, linear or branched, comprising from 1 to carbon, with x an integer, such as for example a methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropyl, cyclohexyl, etc. group.
• (Cl-Cx)alkylene designates a saturated divalent hydrocarbon group, linear or branched, comprising from 1 to x carbon atoms, with x an integer, such as for example a methane-1,1-diyl group, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, pentane- 1,5-diyl, hexane-1,6-diyl, hexane-1,5-diyl, heptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, decane-1, 10- diyl, etc. This is in particular a methane-1,1-diyl or propane-1,3-di
• hydroxy-(C1-Cx)alkyl designates a (Cl-Cx)alkyl group as defined above substituted by a hydroxyl group (OH) such as for example a hydroxyethyl (- CH2CH2OH) or a hydroxypropyl (for example e.g. -CH2-CH(OH)-CH3).
• OH hydroxyl group
• carboxy-(C1-Cx)alkyl designates a (Cl-Cx)alkyl group as defined above substituted by a carboxyl group (COOH) such as for example a carboxymethyl (-CH2COOH).
• aryl designates a monovalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more cycles, such as for example a phenyl or naphthyl group.
• arylene designates a divalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more cycles, such as a phenylene group.
• aryl-(C1-Cx)alkyl designates an aryl group as defined above, linked to the rest of the molecule via a (Cl-Cx)alkyl chain as defined above. with x an integer, such as for example the benzyl or phenylethyl group.
• polyvalent group designates a group capable of forming several covalent bonds with other groups of the same compound or of two different compounds.
• the bonds to the other groups can be formed from the same atom of the polyvalent group or from different atoms of the polyvalent group, and preferably from different atoms of the polyvalent group.
• the versatile group is a divalent group and can therefore form two covalent bonds with two other groups of the same compound or of two different compounds.
• the number of covalent bonds that can be formed refers to the “valency” of the polyvalent group.
• the inventors have developed a process making it possible to prepare a hydrogel meeting the expressed needs.
• the hydrogel obtained has a sticky character allowing the hydrogel to adhere to the tissues and thus not to migrate into the tissues after its application/injection.
• the fillers currently available create volume to fill soft tissues but do not fight against tissue descent.
• chronological aging of the skin is associated with a change in the structure of the skin with the appearance of wrinkles but also ptosis of fatty areas.
• the hydrogel according to the invention can be used to retain tissues and prevent tissue descent linked to age, in particular when it is administered and placed in fillets.
• the hydrogel according to the present invention is therefore particularly useful in filling and/or replacing soft tissues.
• the hydrogel obtained is highly biocompatible. Indeed, its preparation process uses a very small quantity of crosslinking agent (from 0.001 to less than 0.02 moles of crosslinking agent per 1 mole of repeating unit of the polysaccharide). The hydrogel obtained is thus weakly crosslinked.
• the hydrogel obtained is highly mucoadhesive, which allows adhesion to the mucous membranes and thus good persistence of the hydrogel on the site(s) of administration. This is visible by qualitative ex vivo tests or in vitro tests which trace labeled components.
• the process for preparing a hydrogel, preferably injectable, as developed by the inventors comprises the following steps: a) providing at least one polysaccharide; b) providing at least one crosslinking agent, said crosslinking agent comprising at least two functional groups Z, identical or different, chosen from isocyanate, amino, epoxy, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl groups, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, and an acid anhydride residue; c) prepare a crosslinking reaction medium comprising the polysaccharide(s), the crosslinking agent(s) and a solvent, the total quantity of crosslinking agent ranging from 0.001 to less than 0.02 mole per 1 mole d unit of repetition of the polysaccharide(
• the polysaccharide(s) and/or the crosslinking agent(s) may be in salt form.
• step d the crosslinking of the polysaccharide mainly takes place during step d), that is to say when the reaction medium is in a frozen state.
• the properties of the hydrogels obtained by the process of the present invention are particularly surprising since when it came to optimal crosslinking temperature, the prior art advised against going below 10°C and remained silent on the use of temperatures below 0°C (Jaeuk Baek, Yingfang Fan, Seol-Ha Jeong, Ho- Yong Lee, Hyun-Do Jung, Hyoun-Ee Kim, Sukwha Kim, Tae-Sik Jang. Facile strategy involving low-temperature chemical cross- linking to enhance the physical and biological properties of hyaluronic acid hydrogel, Carbohydrate Polymers 202 (2016) 545-553).
• the hydrogels of the present invention are distinguished from hydrogels prepared with the same components but without freezing by their macroporous structure, ie with interconnected macropores, the size of which varies, from less than one micron to several hundred microns, depending on the processing conditions. synthesis, namely according to the nature and quantity of the reagents as well as depending on the temperature and freezing speed. These macropores are visible during morphological analysis by scanning electron microscopy in the frozen state or even by confocal laser scanning microscopy after staining the gel, for example with Rhodamine B.
• Hydrogels which have been brought to room temperature after a crosslinking step in a frozen state can be referred to as “cryogel”.
• cryogelation only generates a few fragments of hyaluronic acid
• the process of the present invention preserves the structure of polysaccharides of high weight average molecular mass, in particular the structure high weight average molecular weight hyaluronic acid.
• the process according to the invention allows the preparation of a crosslinked polysaccharide gel without degradation into fragments of low weight average molecular mass.
• a high weight average molecular mass polysaccharide means a polysaccharide whose weight average molecular mass corresponds to that of naturally (biologically) synthesized polysaccharides.
• a hyaluronic acid polysaccharide of high weight average molecular mass designates a polysaccharide which has a weight average molecular mass greater than or equal to 1 MDa, preferably ranging from 1 MDa to 5 MDa, for example from 1 MDa to 3 MDa or from 1.5 MDa to 3MDa
• a chondroitin sulfate or chondroitin polysaccharide of high molecular weight by weight designates a polysaccharide which has a weight average molecular mass greater than or equal to 30 kDa, preferably ranging from 30 to 150 kDa
• a chitosan polysaccharide of high molecular weight by weight designates a polysaccharide which has an average molecular mass greater than or equal to 100 kD
• a low weight average molecular mass polysaccharide designates a polysaccharide which has an average molecular mass significantly lower than those of biologically synthesized polysaccharides, more particularly a low weight average molecular mass polysaccharide designates a polysaccharide which has a weight average molecular mass less than or equal to one third of the mass of the high molecular weight polysaccharide provided in step a) of the preparation of the hydrogel.
• a hyaluronic acid polysaccharide of low weight average molecular mass designates a polysaccharide which has a weight average molecular mass ranging from 0.04 to 0.3 MDa, preferably from 0.08 to 0.20 MDa, again preferably from 0.08 to 0.15 MDa;
• a chondroitin sulfate or chondroitin polysaccharide of low molecular mass by weight means a polysaccharide which has a weight average molecular mass less than or equal to 50kDa;
• a low molecular weight chitosan polysaccharide means a polysaccharide which has a weight average molecular mass of less than or equal to 30 kDa.
• hyaluronic acid is known to be biocompatible, it has already been reported in the literature that hyaluronic acid of low weight average molecular mass could be the cause of long-term adverse effects after their injection into a patient (Cyphert JM, Trempus CS, Converseziotis S. Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology. International Journal of Cell Biology 2015; 2015:563818).
• the polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds.
• the polysaccharide is chosen from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and their mixtures.
• Pectic substances are polysaccharides composed of a D-galacturonic acid skeleton in acid form possibly esterified with methanol, and L-rhamnose capable of forming branches with other oses.
• Chitosan or “chitosan”, and “chitin” are each a polysaccharide composed of D-glucosamine repeat units linked together in B-(1,4), part of which is N-acetylated. Chitosan more particularly has a degree of acetylation less than 50% while chitin more particularly has a degree of acetylation greater than 50%.
• Cellulose is a polysaccharide composed of a linear chain of D-glucose molecules.
• Cellulose derivatives include methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and carboxymethylcellulose (CMC).
• “Agarose” is a polysaccharide comprising as a repeating unit a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose.
• “Glycosaminoglycans” are linear polysaccharides composed of repeating units of disaccharides, said disaccharides containing a hexosamine (glucosamine (GIcN) or galactosamine (Gal N)) and another ose (glucuronic acid (GIcA), iduronic acid (IdoA) or galactose (Gal)). Hexosamine and the other ose can optionally be sulfated and/or acetylated.
• the glycosaminoglycan may in particular be hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin or chondroitin sulfate.
• Hyaluronic acid is a glycosaminoglycan whose repeating unit is a disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked together by alternating glycosidic bonds
• hyaluronic acid is in the form of a salt, we also speak of “hyaluronate” or “hyaluronan”.
• the hyaluronic acid may have a weight average molecular mass ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, even more preferably greater than 0.05 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa.
• the hyaluronic acid may be in salt form, in particular in the form of a physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, salt of silver, calcium salt and mixtures thereof. More particularly, hyaluronic acid is in its acid form or in the form of sodium salt (NaHA).
• Heparosan or “heparosan” is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid (GIcA) linked by an a-(1,4) bond to an N-acetyl glucosamine (GIcNAc). Each disaccharide repeat unit is connected to the next by a
• GIcA glucuronic acid
• GIcNAc N-acetyl glucosamine
• Chodroitin sulfate or “chondroitin sulfate” is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid linked in
• “Dermatan sulfate” or “dermatan sulfate” is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, that is to say comprising at least one sulfate, L-iduronic acid and N-acetyl substituent. -galactosamine- linked by a(1-3) bonds.
• the disaccharide is sulfated at the C-4 position of N-acetyl-galactosamine, at the C-6 position of N-acetyl-galactosamine, at the C-2 position of L-iduronic acid, or at a combination of these positions.
• Each disaccharide repeat unit is connected to the next by a
• Keatan sulfate or “keratane sulfate” is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, that is to say comprising at least one sulfate substituent, composed of linked D-galactose and N-acetylglucosamine by alternating bonds P(1 -4) and (3(1-3).
• the polysaccharide can be in the form of salt, in particular in the form of physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt and mixtures thereof, more particularly in the form of sodium or potassium salt.
• physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt and mixtures thereof, more particularly in the form of sodium or potassium salt.
• the polysaccharide is a glycosaminoglycan or a salt thereof, preferably hyaluronic acid or a salt thereof, more preferably hyaluronic acid or one of its physiologically acceptable salts such as the sodium salt, the salt thereof potassium, zinc salt, silver salt and mixtures thereof, even more preferably hyaluronic acid or its sodium salt.
• the polysaccharide generally has a weight average molecular mass ranging from 0.03 to 10MDa.
• the polysaccharide is hyaluronic acid, it has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, even more preferably greater than 0.05 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa.
• Mw weight average molecular mass
• crosslinking agent or “crosslinking agent” is a compound comprising at least two functional groups capable of bonding covalently with functional groups present on the polysaccharide, such as OH, CHO, NH2 or COOH groups carried by the polysaccharide, and thus induce connections between the polysaccharide chains (cross-linking) and/or connections on the same polysaccharide chain.
• the functional groups are identical.
• the isocyanate group can react with an OH or NH2 group of the polysaccharide to form a carbamate or urea function.
• the amino group can react with a COOH group of the polysaccharide to form an amide function.
• the epoxide group can react with an OH or COOH group of the polysaccharide to form an ether or ester function.
• the carboxyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function.
• the N-succinimidyloxycarbonyl and N-sulfosuccinimidyloxycarbonyl groups can react with an OH or NH2 group of the polysaccharide to form an ester or amide function.
• the halocarbonyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function.
• the isothiocyanate group can react with an OH or NH2 group of the polysaccharide to form a thiocarbamate or thiourea function.
• the vinyl group can react with an OH group of the polysaccharide to form an ether function.
• the formyl group can react with an OH or NH2 group of the polysaccharide to form a hemiacetal or hemiaminal function.
• the hydroxyl group can react with a COOH group of the polysaccharide to form an ester function.
• the sulfhydryl group can react with a COOH group of the polysaccharide to form a thioester function.
• the hydrazino group (-NH-NH2) can react with a CHO group of the polysaccharide to form a hydrazone function.
• the carbodiimide group can react with a COOH group of the polysaccharide to give a CO-NR a -CO-NH function, and an acid anhydride residue can react with an OH or NH2 group of the polysaccharide to form an ester or amide function .
• the functional groups Z are identical and represent an epoxy or vinyl group, more preferably epoxy.
• the functional groups Z are identical and chosen from amino, vinyl, formyl, and carbodiimide groups, preferably are amino groups.
• the crosslinking agent is chosen from hexamethylene diisocyanate, 4,4'-diphenylmethylene diisocyanate, 4-armed PEG20K-isocyanate, spermine (or 1,12-diamino-5,9-diazadodecane) , spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), poly(ethylene glycol) diamine, ethylenediamine, 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy) ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane
• the crosslinking agent is preferably chosen from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups and mixtures thereof.
• BDDE 1,4-butanediol diglycidyl ether
• PEGDGE poly(ethylene glycol) diglycidyl ether
• EGDGE 1,2-bis(2,3-epoxypropoxy)ethane
• GSDGE 1,3-bis(3-glycidyloxypropyl)tetramethyld
• the crosslinking agent is chosen from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1, 2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.
• BDDE 1,4-butanediol diglycidyl ether
• PEGDGE poly(ethylene glycol) diglycidyl ether
• EGDGE 1, 2-bis(2,3-epoxypropoxy)ethane
• the crosslinking agent is preferably a polyamine chosen from spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5- azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), their salts or a mixture thereof, more preferably the crosslinking agent is a polyamine chosen from spermine, spermidine, their salts and their mixtures.
• the crosslinking agent can be chosen from hydroxyapatite beads modified to carry epoxy groups, a compound of formula Chem. I as described below, and their mixtures.
• the crosslinking agent is a compound of formula Chem. I: Y-(Z) n in which the functional groups Z, identical or different, are as defined above, n is an integer greater than or equal to 2, in particular ranging from 2 to 8, preferably equal to 2 ,
• Y is a versatile hydrocarbon group, in particular aliphatic, having a valence of n and comprising from 1 to 150 carbon atoms:
• R 1 representing a hydrogen atom, an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl
• m an integer between 1 and 20 and the R 2 and the R 3 , identical or different, representing a hydrogen atom; a halogen atom; an -OR 11 group with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl, - said polyvalent group being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, and an aryl-(C1-C6)alkyl, preferably unsubstituted.
• n is an integer ranging from 2 to 8, preferably n represents 2, 3 or 4, even more preferably n is equal to 2.
• R 1 represents a hydrogen atom or a (C1-C6) alkyl group.
• the R 2 and the R 3 represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.
• the polyvalent hydrocarbon group may be an aliphatic or aromatic hydrocarbon polyvalent group, preferably aliphatic and in particular saturated, having a valence of n and comprising from 1 to 150 carbon atoms, preferably from 1 to 50 carbon atoms, more preferably from 1 to 20 carbon atoms, even more preferably from 2 to 20 carbon atoms.
• the hydrocarbon-based polyvalent group is an aliphatic, saturated, in particular linear hydrocarbon-based polyvalent group.
• Y is a polyvalent hydrocarbon group as described above in which one or more CH2 units are optionally replaced by one or more divalent units chosen from -O-, -SO2-, -[SiR 2 R 3 O] m -SiR 2 R 3 - and -NH-, with R 2 , R 3 and m as described above.
• Y is a polyvalent hydrocarbon group as described above, preferably aliphatic and saturated, and in particular linear, branched, or star-shaped, and optionally in which:
• - at least two CH2 motifs are replaced by -O-, particularly between 1 and 50 CH2 motifs, more particularly between 1 and 15 CH2 motifs, or
• Y comprises one or more -CH2-CH2-O- units.
• Y comprises from 1 to 50 -CH2-CH2-O- units, advantageously from 2 to 25 -CH2-CH2-O- units, more advantageously from 2 to 15 -CH2-CH2-O- units.
• Y can only include -CH2-CH2-O- motifs.
• Y is an alkyl group comprising 1 to 150, in particular 1 to 50, in particular 1 to 20, for example 1 to 12, in particular 1 to 6 carbon atoms, preferably linear, in which optionally one or more CH2 units are replaced by one or several divalent units chosen from chosen from -O- and -NH-, more particularly between 1 and 50, in particular between 1 and 15, for example 1 or 2, divalent units chosen from -O- and - NH-.
• the R 2 and the R 3 represent an -OR 11 group with R 11 as described above.
• R 11 represents an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.
• the R 2 and the R 3 represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted (preferably unsubstituted) by one or more groups chosen from a d atom. halogen, an aryl or a hydroxyl, more preferably an unsubstituted (C1-C6) alkyl group such as methyl or ethyl.
• the crosslinking agent is a compound of formula Chem, the following: Z 1 -Y 1 -Z 2 in which the groups Z 1 and Z 2 , identical or different, are chosen from isocyanate, amino, epoxide, carboxyl groups , N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, and an acid anhydride residue, and Y 1 represents a divalent hydrocarbon chain, in particular aliphatic , comprising from 1 to 50 carbon atoms:
• R 1 representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl, m an integer between 2 and 20, and the R 2 and the R 3 , identical or different, representing a hydrogen atom; a halogen atom; an -OR 11 group with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl,
• said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.
• Groups Z 1 and Z 2 have the same definition as group Z defined above.
• Y 1 has the same definition as Y defined above with a valence n being equal to 2.
• Y 1 can only comprise -CH2-CH2-O- motifs, as defined previously.
• the crosslinking agent of formula Chem. I or Chem does not include -[SiR 2 R 3 O] m -SiR 2 R 3 - motifs.
• the additional components may be lubricating agents; cosmetic active ingredients such as antioxidants, co-enzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof.
• Polysaccharides in particular non-crosslinked hyaluronic acid or non-crosslinked heparosan, can be cited as an example of a lubricating agent.
• anesthetics include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, Fomocaine, Guafecainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine,
• antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins , caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, and a mixture thereof.
• amino acids include, but are not limited to, arginine (eg L-arginine), isoleucine (eg L-isoleucine), leucine (eg L-leucine), lysine (eg L-lysine or L- lysine monohydrate), glycine, valine (eg L-valine), threonine (eg L-threonine), proline (eg L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (eg N-acetylated derivatives such as N-acetyl-L-cysteine) and a mixture of these.
• arginine eg L-arginine
• isoleucine eg L-isoleucine
• leucine eg L-leucine
• lysine eg L-lysine or L- lysine monohydrate
• glycine valine (eg L-
• vitamins and their salts include, without limitation, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and better still pyridoxine and its derivatives and/or salts, preferably pyridoxine hydrochloride.
• Examples of minerals include, without limitation, zinc salts (e.g. zinc acetate, particularly dehydrated), magnesium salts, calcium salts (e.g., hydroxyapatite, particularly in ball form), potassium salts , manganese salts, sodium salts, copper salts (e.g. copper sulfate, in particular pentahydrate), optionally in a hydrated form, and mixtures thereof.
• zinc salts e.g. zinc acetate, particularly dehydrated
• magnesium salts e.g., calcium salts (e.g., hydroxyapatite, particularly in ball form)
• potassium salts e.g., manganese salts
• sodium salts e.g. copper sulfate, in particular pentahydrate
• copper salts e.g. copper sulfate, in particular pentahydrate
• nucleic acids examples include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, derivatives thereof, and a mixture thereof.
• coenzyme Q10 coenzyme Q10, CoA, NAD, NADP, and mixtures thereof may be mentioned.
• adrenaline derivatives adrenaline, norepinephrine and a mixture thereof can be cited.
• Provision of at least one polysaccharide or a salt thereof (step a))
• Step a) of the process according to the invention comprises the supply of at least one polysaccharide or a salt thereof, in particular a physiologically acceptable salt thereof.
• the polysaccharide is as described above.
• the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably a sodium salt.
• the polysaccharide may be supplied in hydrated form, completely or partially, or in dry form, such as powder or fiber. More particularly, in step a), the polysaccharide is supplied in dry form such as in the form of powder or fibers. When the polysaccharide is supplied in hydrated form, it is in the form of a non-cross-linked gel or a solution.
• the polysaccharide when it is in hydrated form, it is an aqueous non-crosslinked gel or an aqueous solution. More particularly, the polysaccharide is mixed with water, optionally added with a phosphate buffer or a supplemented phosphate buffer, that is to say possibly comprising additional components as defined in the additional steps. It should therefore be understood that the aqueous non-crosslinked gel or aqueous polysaccharide solution does not include sodium hydroxide. Provision of at least one crosslinking agent or a salt thereof (step b))
• Step b) of the process according to the invention comprises the supply of at least one crosslinking agent or a salt thereof, in particular a physiologically acceptable salt thereof.
• the crosslinking agent is as described above.
• Step c) of the process according to the invention comprises the preparation of a crosslinking reaction medium.
• the reaction medium comprises the polysaccharide(s), the crosslinking agent(s) and a solvent.
• the solvent is typically water or a mixture comprising water and an organic solvent (typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 99% by weight of water relative to the total weight of the solvent).
• an organic solvent such as an alcohol, in particular ethanol, or DMSO, can be used to solubilize the crosslinking agent, for example when it is poly (dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), before its addition to the aqueous reaction medium.
• the reaction medium may further comprise salts, pH adjusters, for example a Bronsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above. and their mixtures.
• a Bronsted base may be particularly necessary when the functional groups Z of the crosslinking agent, such as Z 1 or Z 2 , represent an epoxy group or a vinyl group.
• the crosslinking takes place at a pH greater than or equal to 10, more advantageously greater than or equal to 12, which requires the addition of a Bronsted base to the reaction medium (for example sodium hydroxide). , typically at a concentration between 0.10M and 0.30M.
• the reaction medium is typically prepared from the polysaccharide or polysaccharides in a dry form.
• the reaction medium is prepared from the polysaccharide or polysaccharides in a hydrated form, the aqueous non-crosslinked gel or the aqueous polysaccharide solution used for the preparation of the reaction medium does not include sodium hydroxide.
• the maximum contact time of the polysaccharide with sodium hydroxide before engaging in step d), whether the polysaccharide is supplied in dry or hydrated form is 5 hours, for example from 15 minutes to 4 hours or from 30 min to 2 hours.
• the total quantity of crosslinking agent in the reaction medium varies from 0.001 to less than 0.02 moles per 1 mole of repeating unit of the polysaccharide, for example from 0.001 to 0.015 moles per 1 mole of repeating unit of the polysaccharide, preferably from 0.001 to 0.01 moles per 1 mole of repeating unit of the polysaccharide, even more preferably from 0.001 to 0.008 moles or from 0.001 to 0.005 moles per 1 mole of repeating unit of the polysaccharide.
• the polysaccharide is a glycosaminoglycan such as hyaluronic acid
• the repeating unit is a disaccharide unit.
• the mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg/g of solvent, preferably from 100 to 200 mg/g.
• Step c) of the process according to the invention typically comprises a step of homogenization of the reaction medium. Homogenization is generally achieved by three-dimensional stirring, stirring with a mixer, stirring with paddles or stirring with a spatula. Step c) is typically carried out at a temperature ranging from 4 to 35°C, preferably 15°C to 25°C.
• the duration of the reaction medium preparation step does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 min to 2 hours.
• the reaction medium obtained at the end of step c) is advantageously directly placed in the conditions of step d) according to the invention.
• the reaction medium obtained at the end of step c) is then placed, for a period ranging from 2 weeks to 17 weeks, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the Eutectic point of the reaction medium (i.e. of the mixture comprising the polysaccharide(s), the crosslinking agent(s), the solvent and any salts, pH adjusters and additional components) as measured at pressure P and below the freezing point temperature of the reaction medium as measured at pressure P.
• a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the Eutectic point of the reaction medium (i.e. of the mixture comprising the polysaccharide(s), the crosslinking agent(s), the solvent and any salts, pH adjusters and additional components) as measured at pressure P and below the freezing point temperature of the reaction medium as measured at pressure P.
• the freezing point temperature of the reaction medium designates the temperature at which the mixture of components of the reaction medium, on a macroscopic scale, solidifies, that is to say it becomes non-fluid. Below the freezing point, the mixture is in a frozen state which is characterized by the coexistence of components in solid and liquid form. The freezing state is maintained up to the temperature of the eutectic point of the reaction medium.
• the temperature of the eutectic point of the reaction medium designates the temperature below which the mixture of components of the reaction medium passes from a frozen state (coexistence of liquid and solid phases) to a completely solid state, that is to say a state in which all components of the mixture are in solid form.
• the freezing point and the eutectic point of a mixture depend on the pressure to which the mixture is subjected, therefore the freezing point and the eutectic point are measured at the pressure P.
• the freezing point and the eutectic point can be determined by differential scanning calorimetry. This method makes it possible to determine phase transitions. To do this, the product to be studied is gradually cooled until its phase transitions are observed.
• the temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C. Even more preferably, the temperature T is approximately -20°C.
• the pressure P is preferably atmospheric pressure.
• “Atmospheric pressure” is the pressure exerted by the air constituting the atmosphere on any surface in contact with it. It varies depending on altitude. At an altitude of 0m, the average atmospheric pressure is 101,325 Pa.
• the pressure P is the atmospheric pressure and the temperature T is greater than or equal to -55°C and less than or equal to -5°C, preference T varies from -35°C to -10°C or is around -20°C
• the reaction medium obtained at the end of step c) is placed for a period ranging from 2 weeks to 15 weeks, preferably from 2 weeks to 13 weeks, preferably from 2 weeks to 11 weeks, preferably from 2 weeks to 9 weeks, more preferably from 3 weeks to 6 weeks or even from approximately 4 to 5 weeks or approximately 30 days at said pressure P and said temperature T.
• the crosslinking of the polysaccharide mainly takes place during step d) (it can nevertheless begin as early as step c)). This step therefore makes it possible to cross-link the polysaccharide chains together.
• the functional groups of the crosslinking agent react with functional groups present on the polysaccharides so as to link the polysaccharide chains together and to crosslink them by forming intermolecular bonds.
• the crosslinking agent can also react with functional groups present on the same polysaccharide molecule so as to form intramolecular bonds.
• the functional groups of the crosslinking agent react with the -OH or -COOH, or even -CHO groups, present on polysaccharides such as hyaluronic acid.
• Crosslinked polysaccharides comprising at least one crosslinking link between two polysaccharide chains, said crosslinking link being the residue of the crosslinking agent are thus obtained.
• the crosslinked polysaccharides comprise at least one crosslinking link between two polysaccharide chains, said crosslinking link comprising more particularly the polyvalent group Y as described above, preferably the divalent group Y 1 as described above.
• Some groups functional Z (such as Z 1 and Z 2 ) of the crosslinking agent may however not react with a polysaccharide chain.
• the crosslinking agent comprises two functional groups Z 1 and Z 2
• one of the functional groups Z 1 can react with a polysaccharide while the other functional group Z 2 does not react with any polysaccharide.
• a dangling bond is then formed.
• Crosslinking can be carried out in the presence of several crosslinking agents.
• the crosslinking agents can be added simultaneously or separately over time to the reaction medium.
• Step b) can thus include repeated crosslinking steps.
• the crosslinking is then carried out in the presence of a total quantity of crosslinking agents ranging from 0.1 to less than 2 moles, preferably ranging from 0.1 to 1.5 moles or from 0.1 to 1 mole or from 0 .1 to 0.8 moles or 0.1 to 0.5 moles of crosslinking agents (or their salts) per 100 mole of repeating unit of the polysaccharide.
• the crosslinking conditions in particular the crosslinking agent contents, duration and temperatures as well as the weight average molecular masses (Mw) of the polysaccharide, used are interdependent.
• the higher the temperature of the crosslinking reaction the lower the reaction time can be to obtain the same degree of modification of the polysaccharide by the crosslinking agent.
• crosslinking agent content the longer the reaction time must be to obtain similar mechanical properties of the resulting gel.
• the lower the molar percentage of crosslinking agent the fewer reactive functions there are in the reaction medium and the lower the probability that 2 groups will meet and react together, thus the longer the reaction time must be to allow functions to react with each other and form crosslinking links, and thus obtain a gel with desirable properties.
• the crosslinking reaction with the polysaccharide is advantageously carried out in the presence of at least one activator, and where appropriate associated with at least one coupling auxiliary.
• the activator can be selected from water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-[3-(trimethylamino) propyl]carbodiimide hydrochloride (ETC), 1-cyclohexyl-3-(2-morphilinoethyl)carbodiimide (CMC), their salts and mixtures thereof, preferably is represented by EDC.
• EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
• ETC 1-ethyl-3-[3-(trimethylamino) propyl]carbodiimide hydrochloride
• CMC 1-cyclohexyl-3-(2-morphilinoethyl)
• the coupling auxiliary when present, it can be selected from N-hydroxy succinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4 -oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-azabenzotriazole (HAt) and N-hydroxysylfosuccinimide (sulfo NHS), and mixtures thereof, preferably is represented by HOBt .
• NHS N-hydroxy succinimide
• HOBt N-hydroxybenzotriazole
• HOBt 3,4-dihydro-3-hydroxy-4 -oxo-1,2,3-benzotriazole
• HAt 1-hydroxy-7-azabenzotriazole
• sulfo NHS N-hydroxysylfosuccinimide
• the process according to the invention may comprise one or more additional steps, such as for example steps of addition of one or more additional components, purification, sterilization, sieving, swelling and/or packaging.
• the method according to the invention may include a step of adding at least one additional component.
• the additional component can be chosen from lubricating agents; cosmetic active ingredients such as antioxidants, co-enzymes, amino acids, vitamins, minerals, and nucleic acids; therapeutic active ingredients such as anesthetics, antibiotics, antifungals and adrenaline and its derivatives, and mixtures thereof. Additional components may be as described above.
• the process according to the invention may comprise at least one purification step. Purification can be carried out by dialysis.
• the method according to the invention may include a step of sterilizing the hydrogel.
• Sterilization is preferably carried out by heat.
• Sterilization is generally carried out by increasing the temperature of the sterilization medium to a temperature called “plateau temperature”, which is maintained for a determined period called “plateau duration”.
• Sterilization is preferably carried out at a plate temperature ranging from 121°C to 135°C, preferably with a plate duration ranging from 1 minute to 20 minutes with FO > 15.
• the sterilizing value FO corresponds to the time necessary, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized.
• sterilization can be carried out in particular by gamma, UV or ethylene oxide radiation.
• the process may include a step of sieving the hydrogel, more particularly with a sieve with a porosity of between 50 and 2000 pm. This sieving step makes it possible to obtain a more homogeneous hydrogel with the most constant possible extrusion force, ie, the most regular possible. Those skilled in the art know how to select a sieve with an appropriate pore size depending on the mechanical properties of the hydrogel being prepared.
• the method may include a step of swelling the hydrogel.
• the polysaccharide concentration of the hydrogel is adapted.
• a solvent is added, for example, water, phosphate buffer, water for injection. More particularly, the added solvent has a pH around the physiological pH (6.8-7.8).
• the polysaccharide concentration obtained following the swelling step advantageously varies from 1 mg/g of gel to 50 mg/g of the hydrogel, more advantageously from 5 mg/g to 35 mg/g of the hydrogel, even more advantageously from 10 mg/g to 30 mg/g of hydrogel gel.
• the process may include a step of conditioning the hydrogel.
• the step of adding one or more additional components preferably takes place after the purification step.
• the step of adding one or more additional components preferably takes place before the sterilization step.
• the step of adding one or more additional components may also include the addition of at least one therapeutic active ingredient, or at least one cosmetic active ingredient, or their mixture.
• the step of adding one or more additional components preferably takes place after step d).
• the purification step preferably takes place after the step of adding one or more additional components.
• the purification step preferably takes place before the sterilization step. Where applicable, the purification step preferably takes place before the sieving step.
• the sterilization step is preferably carried out after steps a) to d) and any additional steps.
• the hydrogel is sterilized after having been packaged in its injection device and the conditioning of the gel takes place following all stages of the process and before sterilization.
• the present invention also relates to a hydrogel capable of being obtained by the process of the present invention.
• a hydrogel can also be referred to as “cryogel”.
• the hydrogel comprises one or more polysaccharides as defined above.
• the polysaccharide is crosslinked.
• the molar crosslinking rate of the polysaccharide is greater than 0 and less than 2%, preferably less than or equal to 1.5% or less than or equal to 1%, more preferably less than or equal to 0.8% or 0.5% , in particular between 0.1% and 0.8% or between 0.1 and 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeating unit of the polysaccharide(s).
• crosslinking agent(s) are as defined above.
• the crosslinking agent is preferably a crosslinking agent whose functional groups are epoxy groups.
• the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2 -bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.
• the hydrogel of the present invention has a sticky character. In certain embodiments the hydrogel of the present invention has a stringy character.
• the hydrogel is preferably an injectable hydrogel.
• It is preferably sterile, in particular heat sterilized at a tray temperature of 121°C to 135°C, preferably with a tray duration ranging from 1 minute to 20 minutes with F0 > 15.
• This hydrogel is preferably homogeneous.
• the hydrogel may comprise one or more additional components as described above, in particular one or more additional components chosen from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids and their mixtures.
• a hydrogel according to the present invention acceptable for the therapeutic and/or cosmetic applications targeted by the present invention, has a cross-over stress (or stress at the crossing of the modules G' and G") greater than or equal to 50 Pa , preferably between 50 and 5000 Pa and more preferably between 100 and 1000 Pa, preferably greater than 150 Pa and an elastic modulus G' greater than or equal to 20 Pa, preferably from 100 Pa to 2000 Pa, more preferably from 110 Pa to 1000 Pa.
• a hydrogel according to the present invention has a cohesiveness of 1 N to 30 N, preferably greater than 4 N, for example ranging from 6 to 20N or ranging of 6 to 15N.
• This cohesiveness is measured by mechanical compression using a rheometer. For this, the gel is deposited on a Peltier plane with an initial air gap of 2.60mm; it is then compressed at a constant speed of 100 pm/s to 70% of the initial air gap, at 25°C; Finally, the cohesiveness of the gel is measured at the end of the compression stroke. The more cohesive a gel is, ie has a high cohesive value, the more it is capable of withstanding stresses, such as those it may encounter after its administration to a subject.
• a hydrogel according to the present invention acceptable for the therapeutic and/or cosmetic applications targeted by the present invention, has a Tack test result ranging from 1.5 to 15 cm, preferably from 1.5 to 10 cm, again preferably from 2 to 7.
• a hydrogel according to the present invention acceptable for the therapeutic and/or cosmetic applications targeted by the present invention has a sticky character and will adhere to surfaces such as glass, metal, human tissues, collagen or plastic. Additionally, it will maintain a cohesive structure when moving across these surfaces.
• the present invention also relates to a composition comprising a hydrogel according to the present invention. It is preferably a cosmetic or pharmaceutical composition. It may also include physiologically acceptable excipients.
• the hydrogel according to the invention comprises a crosslinked polysaccharide.
• the composition may further comprise a non-crosslinked polysaccharide.
• the composition according to the present invention can thus comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight of polysaccharide (e.g., hyaluronic acid), relative to the total weight of said composition, the polysaccharide (e.g., hyaluronic acid) being present in crosslinked and optionally non-crosslinked form.
• the content of non-crosslinked polysaccharide (e.g., hyaluronic acid) varies from 0 to 40% by weight, preferably from 1 to 40% by weight, more preferably from 5 to 30% by weight, relative to the total weight of polysaccharide (e.g., hyaluronic acid) present in the composition.
• the polysaccharide of the hydrogel is as defined above.
• the composition according to the present invention is preferably a sterile composition, in particular heat sterilized at a plate temperature of between 121°C and 135°C, preferably with a plate duration of between 1 minute and 20 minutes with F0 > 15. It is preferably an injectable composition.
• the composition according to the invention then preferably comprises a physiologically acceptable medium, preferably a physiologically acceptable aqueous medium.
• the physiologically acceptable aqueous medium may comprise a solvent or a mixture of physiologically acceptable solvents and preferably comprises water.
• the physiologically acceptable medium may also include isotonic agents such as oses, sodium chloride and their mixture.
• the physiologically acceptable medium may further comprise at least one isotonic and physiologically acceptable saline solution.
• said balanced salt solution is a phosphate-buffered saline solution, and particularly a KH2PO4/K2HPO4 saline solution buffer.
• composition according to the invention may further comprise at least one additional compound chosen from lubricants, anesthetics, antioxidants, amino acids, vitamins, minerals, nucleic acids, co-enzymes, adrenaline derivatives. , and mixtures thereof. Additional compounds may be as described above.
• the hydrogel or the composition according to the invention may have therapeutic and/or cosmetic applications.
• the present invention therefore also relates to a hydrogel or a composition according to the invention for its use in the filling and/or replacement of tissues, in particular soft tissues, in particular by injection of the hydrogel or the composition into the tissue.
• the hydrogel or the composition according to the invention is used in oral care and more particularly in the treatment of gingival recession, or to fill periodontal pockets. More particularly, the hydrogel or the composition according to the invention is used to treat defects in the gingival architecture which can occur with tooth loss, with aging, with periodontal diseases and disorders, or after the installation of tooth, crown or bridge implants.
• the hydrogel or the composition according to the invention can also be used in ophthalmology, more particularly to protect the ocular structures during eye surgery such as for example ophthalmic surgery of the anterior or posterior segment, extraction of the cataract possibly with implantation of an intraocular lens, corneal transplant surgery, glaucoma filtering surgery, or even implantation of a secondary lens.
• the hydrogel or the composition according to the invention will be more particularly injected into the eye.
• the hydrogel or the composition according to the invention can also be used in orthopedics or rheumatology, for example by injection into the synovial cavity. The hydrogel or the composition according to the invention is then used as viscosupplementation.
• hydrogel or the composition according to the invention can also be used in the treatment of lipodystrophy.
• the hydrogel or the composition according to the invention can be used in cosmetic surgery, in particular for gynecoplasties and/or penoplasties.
• the hydrogel or the composition according to the invention is administered more particularly by injection.
• the hydrogel or the composition according to the invention can be used for its mucoadhesive properties useful in the treatment of gingival pain.
• the present invention also relates to a method of treating the pathologies indicated above which comprises the administration, to an individual in need, of an effective dose of the hydrogel or the composition.
• the effective dose of the hydrogel or the composition varies depending on numerous parameters such as, for example, the chosen route of administration, weight, age, sex, the state of progress of the pathology to be treated. treat and the sensitivity of the individual to be treated.
• the subject of the present invention is preferably the cosmetic, and therefore non-therapeutic, use of a hydrogel or a composition according to the invention to prevent and/or treat the alteration of the viscoelastic or biomechanical properties of the skin, and in particular to stimulate, regenerate, hydrate, firm or restore the radiance of the skin, in particular by mesotherapy; to fill volume defects in the skin, and in particular to fill wrinkles, fine lines or scars (in particular hollow scars); or to reduce the appearance of fine lines and wrinkles.
• the subject of the present invention is the cosmetic use of a hydrogel or a composition according to the invention to reduce nasolabial folds and bitter folds; to increase the volume of the cheekbones, chin or lips; to restore the volume of the face, particularly the cheeks, temples, the oval of the face, and the rim of the eyes; or to stimulate, regenerate, hydrate, firm or restore radiance to the skin, particularly through mesotherapy.
• the hydrogel or the composition according to the invention is a hydrogel or an anti-aging composition.
• the hydrogel or the composition according to the invention is administered more particularly by injection.
• the present invention also relates to a method of cosmetic treatment, preferably anti-aging, of keratin materials, in particular of the skin, comprising at least a step of administering a hydrogel or a composition according to the invention onto or through said keratin materials, more particularly by injection.
• the administration may be an injection, in particular an intra-epidermal and/or intradermal and/or subcutaneous injection.
• Administration by intra-epidermal and/or intradermal and/or subcutaneous injection according to the invention aims to inject a hydrogel or a composition of the invention into an epidermal, dermo-epidermal and/or dermal region.
• the hydrogel or the composition according to the invention can also be administered by a supraperiosteal injection.
• the hydrogel or the composition according to the invention can be injected using any of the methods known to those skilled in the art.
• a hydrogel or a composition according to the invention can be administered by means of an injection device suitable for intra-epidermal and/or intradermal and/or subcutaneous and/or supra-periosteal injection.
• the injection device may in particular be chosen from a syringe, a set of microsyringes, a wire, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.
• the injection device may comprise any injection means usually used suitable for intraepidermal and/or intradermal and/or subcutaneous and/or supraperiosteal injection.
• such means may be a hypodermic needle or a cannula or a set of microneedles.
• a needle or cannula according to the invention may have a diameter varying from 18 to 34 G, preferably between 25 and 32 G, and a length varying from 4 to 70 mm, and preferably from 4 to 25 mm.
• the needle or cannula is advantageously single-use.
• the needle or cannula is associated with a syringe or any other device making it possible to deliver said hydrogel or said injectable composition through the needle or cannula.
• a catheter can be inserted between the needle/cannula and the syringe.
• the syringe can be operated manually by the practitioner or by a syringe support such as guns.
• the injection device is a syringe.
• the injection device can be adapted to the mesotherapy technique.
• Mesotherapy is a treatment technique by intra-epidermal and/or intradermal and/or subcutaneous injection of a composition or a hydrogel.
• the composition or the hydrogel is administered according to this technique by injection in the form of multiple small droplets at the level of the epidermis, the dermo-epidermal junction and/or the dermis in order, in particular, to produce a subcutaneous coating.
• the mesotherapy technique is notably described in the work “Treatise on mesotherapy” by Jacques LE COZ, published by Masson, 2004.
• Mesotherapy carried out on the face is also called mesolift, or also under the Anglo-Saxon term “mesoglow”.
• Administration can also be topical.
• it is a topical application on the surface of the skin, more particularly on the epidermis, even more particularly on the facial epidermis.
• the present invention thus also relates to an injection device as described above comprising a hydrogel or a composition according to the invention.
• the viscoelastic properties of the hydrogels obtained were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plane geometry and an anodized aluminum peltier plane (42 mm) (air gap 24 p.m.).
• extrusion forces in Newton
• the extrusion force results correspond to the average of the average extrusion forces on at least 2 samples.
• the Tack test is conducted on a DHR-2 rheometer (TA Instruments) equipped with a rough steel plane geometry (40 mm in diameter) and a rough steel peltier plane (40 mm in diameter). 1 g of product is deposited between the two geometries at 25 °C and the upper geometry is quickly brought closer to the peltier plane at a speed of 2 mm/s up to an air gap of 2 mm. The upper geometry then compresses the gel with a closing speed of 0.1 mm/s to an air gap of 0.1 mm. The upper geometry is then raised at 0.1 mm/s and the distance over which the gel maintains its flowing character between the 2 geometries is reported. The stringy character thus determined by a unit of length is expressed in cm.
• the gel is deposited on a Peltier plane with an initial air gap of 2.60mm. The gel is then compressed at a constant speed of 100 pm/s to 70% of the initial air gap, at 25°C. The cohesiveness of the gel is measured at the end of the compression stroke.
• soluble hyaluronic acid or sHA is carried out using high performance liquid chromatography interfaced with a multi-angle light scattering detector and a refractive index detector (HPLC-SEC-MALS-RI, software ASTRA (Wyatt Technology Corp.).
• Samples are diluted approximately 10 times depending on their initial concentration of hyaluronic acid in the SEC mobile phase, a filtered solution of 150 mM sodium nitrate (pH 7.2).
• Said diluted mixture consists of acid compact and insoluble hyaluronic acid and soluble hyaluronic acid.
• the soluble hyaluronic acid portion of the samples was released for 5 days under orbital shaking to avoid any artificial production of soluble hyaluronic acid.
• the soluble part is separated from the insoluble part by a gentle filtration method (syringe equipped with a 0.45 ⁇ m filter) then subjected to SEC analysis.
• Varying injection volumes are tested to achieve a background-free signal of at least five to one to prevent and avoid overloading the SEC columns.
• the HPLC-SEC system uses a column duo for wide hyaluronic acid ranges from 500Da to 20MDa for optimal peak resolution on the chromatograph.
• a value of the refractive increment index or dn/dc refractive index variation / polysaccharide concentration in the analysis solvent
• the chromatograms obtained by SEC are analyzed to quantify the molecular weight, distribution and proportion of soluble hyaluronic acid in each sample.
• the mass average molecular weight of the sample is data provided by the HPLC-SEC software.
• the percentage of soluble hyaluronic acid fractions (%sHA) is also a direct output from the HPLC-SEC software after entering the total hyaluronic acid mass concentration (mg/ml) of the analyzed sample.
• Said percentage may differ from one composition to another depending on its manufacturing technique and the dispersion (p/d) (molar mass by weight / molar mass by number) of the sample.
• %sHAs for several molecular weight limits are also a direct output from the HPLC software. However, this does not take into account the amount of hyaluronic acid actually released from the gel.
• a normalized %sHA for the multiple molecular weight limits was calculated to normalize the values provided by the software taking into account the amount of hyaluronic acid actually released from the analyzed sample.
• Prototypes No. 1 to 9 were prepared as follows with the BDDE contents (BDDE molar TR) described in Table 1.
• BDDE and sodium hyaluronate (1.5 MDa, 120 mg/g) were dissolved in a 0.25 M aqueous sodium hydroxide solution in a sterile bag. The mixture was then homogenized in a paddle mill for 3 cycles of 15 min at 210 rpm at room temperature. The mixture was then maintained at atmospheric pressure and placed at the temperatures and times presented in Table 1. The pH of the mixture was approximately 13.
• a 1N HCI solution was then added to the sterile bag until a pH of 7.3 ⁇ 0.5 was obtained.
• the mixture was diluted to a concentration of 23 mg of hyaluronic acid per gram of product with PBS phosphate buffer.
• the mixture was homogenized for 24 h using a three-dimensional shaker.
• the mixture was dialyzed.
• Sodium hyaluronate (4 MDa) was added as a lubricant.
• lidocaine hydrochloride An aqueous solution of lidocaine hydrochloride was added to obtain 0.3% by weight of lidocaine hydrochloride based on the weight of the resulting product.
• the product thus obtained was sieved then packaged in a syringe.
• the commercial product Restylane Lyft, has a very low cohesiveness (2.2 N) compared to the gels obtained using the process of the present invention.
• the gels obtained by the process of the present invention have rheological properties and cohesivities at least as interesting, or even greater (the HA concentrations and the manufacturing process being identical elsewhere), to the gels. obtained conventionally at room temperature and with a significantly higher crosslinking rate (prototype 2).
• Example 2 Process according to the invention of hyaluronic acid crosslinked with DVS
• Prototypes No. X1 and X2 were prepared as follows with the DVS contents (DVS molar TR) described in Table 3.
• Sodium hyaluronate (1.5 MDa, 120 mg/g) is dissolved in an aqueous solution of DVS and 0.25 M sodium hydroxide in a sterile bag. The mixture is homogenized in a paddle mill for 3 cycles of 15 min at 210 rpm at room temperature. The mixture is placed and maintained at the temperatures and times presented in Table 3. The pH of the mixture was approximately 13.
• the mixture is left to thaw before continuing the preparation.
• a 1 N HCI solution is added to the sterile bag until a pH of 7 ⁇ 0.5 is obtained. 6- The mixture is diluted to a concentration of 23 mg of hyaluronic acid per gram of product with PBS phosphate buffer.
• the mixture is homogenized for 24 hours using a three-dimensional stirrer.
• the mixture is then dialyzed and a fixed amount of sodium hyaluronate (4 MDa) is added as a lubricant.
• lidocaine hydrochloride An aqueous solution of lidocaine hydrochloride is added to obtain 0.3% by weight of lidocaine hydrochloride based on the weight of the resulting product.
• the product thus obtained was sieved then packaged in a syringe.
• the X0 prototype (comparative) was prepared identically to the X1 prototype. Only the crosslinking step was changed, namely 0.75 days of crosslinking at 21°C.
• the prototype X0 produced with the same crosslinking rate as
• phase angle loss (phase angle at 6 months at 40 °C - initial phase angle) / initial phase angle * 100
• the prototypes obtained by the process of the present invention, Y1, Y2, and Y3, with low crosslinking rates present a low loss of their viscoelastic properties following an accelerated aging study mimicking 2 years of storage at 25 °C in unfavorable situation. This loss is acceptable and similar to that of a traditional gel (Y4) obtained by a conventional process (crosslinking at room temperature) with a higher molar crosslinking rate.
• Example 5 Comparison of the integrity of the soluble hyaluronic acid chains of the cryokels obtained by a process according to the invention or according to a freezing process of the prior art
• a first step of dissolving the hyaluronic acid fibers in soda is carried out over 24 hours at 4°C.
• the crosslinking process having already started during these 24 hours before cryogelation, a cryogelation step of only 10 days is necessary in order to obtain gels with interesting properties close to those of the present invention.
• this step of dissolving the hyaluronic acid fibers (step a); preparation of the reaction medium) is carried out at room temperature over approximately 1 hour followed by freezing over a much longer period.
• the conditions of this comparison are summarized in Table 9.
• the hyaluronic acid fibers were subjected to the two processes without using a crosslinking agent.
• the processes fiber dilution, cryogelation
• a decrease in Mw and an increase in the presence of small fragments of hyaluronic acid are then synonymous with the degradation of hyaluronic acid by the process.
• the samples taken were neutralized to physiological pH and diluted to approximately 0.5 mg/mL.
• Table 9 summary of the 2 processes compared Starting from the same raw material having a Mw of 859 kDa and using the same aqueous dissolution, neutralization and dilution solutions as well as using the same equipment and the same conditions for analyzing hyaluronic acid chains, we were able to observe that the process according to the prior art inflicts a loss of approximately 100 kDa on the Mw of hyaluronic acid compared to the process of the present invention. This is also evidenced by the presence of 0.5% of hyaluronic acid with a Mw ⁇ 50 kDa while the process of the present invention does not present any trace of these fragments of hyaluronic acid.
• Figure 1 illustrates the implementation of the Tack test on the Z2 and Z3 prototypes. After compression of the gel, the surfaces are separated.
• Z1 and Z3 prototypes have the highest stringy characteristics.
• Z1 according to its rheological properties, is however not a viscoelastic gel (phase angle > 45°) and therefore behaves like a viscous solution.
• Z2 and Z4 do not have a stringy character.
• Z2 is a gel obtained in a conventional manner (crosslinking at room temperature) while Z4 is a hydrogel obtained by the process of the present invention, with a very high molar crosslinking rate (> 2%), which therefore makes the gel at both very elastic, brittle and non-stringy.
• the Z3 prototype obtained by the process of the present invention with a low modification rate gives the best results in terms of obtaining a viscoelastic gel with desirable properties and a stringy gel (tensile cohesiveness).
• J2 The effort required to move J2 is also greater on the four model surfaces due to the adhesion of the gel to its movement. It should also be noted that despite the adhesion of the J2 prototype on its path of movement, it maintains its cohesiveness (a single block of gel) and does not crumble along its movement on the surfaces. This can be an important advantage in vivo because the gel will tend to migrate less by adhering to surfaces.

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