WO2020157416A1

WO2020157416A1 - Subcutaneous chitosan gel modelling

Info

Publication number: WO2020157416A1
Application number: PCT/FR2020/050105
Authority: WO
Inventors: Caroline Ceccaldi; Alexandre GUERRY; Frédéric BERTAINA
Original assignee: Bioxis Pharmaceuticals
Priority date: 2019-01-30
Filing date: 2020-01-24
Publication date: 2020-08-06
Also published as: FR3091995B1; FR3091995A1; WO2020157416A8; TW202042848A

Abstract

The present invention relates to cutaneous filling processes which involve chitosan. It particularly relates to a cosmetic tissue-filling process comprising a step of modelling an implant which is obtained by injecting a chitosan solution into a tissue having a pH between 6.8 and 7.4. The present invention also relates to a chitosan solution for use in a surgical treatment method, the method comprising injecting, in a patient, a chitosan solution into a tissue having a pH between 6.8 and 7.4 in order to obtain a chitosan implant, then modelling the implant.

Description

Title: Chitosan Gel Modeling

Technical area

The invention relates to the field of cosmetic fillers, biomaterials, physiological skin reconstruction materials, regenerative medicine, reconstructive surgery in particular skin anti-aging, in humans or possibly the animal. The present invention particularly relates to methods of skin fillers involving chitosan. Prior art

[0002] Various injectable fillers, in particular in humans, are already known.

[0003] Hyaluronic acid (HA) is today the most widely used filling product in cosmetic surgery, in particular for filling in wrinkles and fine lines on the face. Direct injection of HA has the advantage of having an immediate filling effect associated with a minor inflammatory reaction, due to its biocompatibility. This product is generally degraded by the body within 6 to 9 months of its injection. Nevertheless, several studies have shown that in some patients, HA could not only be found several years (up to 9 years) after injection, but that it could also migrate to other areas / tissues of the body ( see Chang et al., Ophthalmic plastic and raconstructive surgery, 33.3, 2017: S1 16-S1 18; Chae et al. Annals of dermatology 28.5 (2016): 645-647; or Kopp et al. Dermatologie Surgery 40.1 (2014) : 85-87). This migration and persistence of HA can cause multiple complications, ranging from a bluish coloration of the skin due to the “Tyndall effect” appearing when the HA implant migrates to the surface of the epidermis, to the appearance of protuberances such as lumps or swelling, for example in the periocular area.

As demonstrated by Chae et al. (2016), HA migration can result and / or be accentuated by the modeling applied to the HA implant after injection. This modeling makes it possible to obtain a homogeneous distribution of the product and therefore an optimal filling.

[0005] There is therefore a real need for products making it possible both to obtain optimum filling of wrinkles, fine lines and other roughness of the skin, but which do not present any risks for the patient, in particular linked to a migration of the implant after injection.

[0006] The Applicant has already proposed the use of solutions based on chitosan as fillers. These solutions are the subject of applications published under the references WO2013079646, WO2016 / 170284 and FR3057778.

[0007] Chitosan behaves vis-à-vis the organism like a "lure" of the biological environment (A. Montembault, K. Tahiri, C. Korwin-Zmijowska, X, Chevalier, M. Corvol, A. Domard , Biochimie, 88 (2006), 551-64): on the one hand, it is sufficiently “recognized” not to induce a dangerous inflammatory reaction, and on the other hand sufficiently “unrecognized” not to be degraded too quickly. Chitosan-based products represent a promising development in the field of cosmetic fillers.

Surprisingly, the Applicant has demonstrated that it is possible to obtain a chitosan implant that can be modeled in the hours following the injection, and this without risking any subsequent migration of said implant like that obtained with HA.

[0009] The Applicant has indeed demonstrated that when a chitosan solution is injected into a tissue whose pH is between 6.8 and 7.4, it gels with a gelation kinetics such that the implant thus obtained can be modeled within hours of the injection. This gelation kinetics means that the implant is completely gelled within 12 hours of injection and therefore cannot migrate after this time.

[0010] These properties make it possible to obtain an implant giving an optimal filling effect without presenting any risk of long-term migration. Summary of the invention [0011] Thus, in a first aspect, the present invention relates to a cosmetic method for filling tissues comprising a step of modeling an implant obtained by injecting a solution of chitosan into a tissue having a pH of between 6, 8 and 7.4. [0012] The present invention also relates to a chitosan solution for its use in a method of surgical treatment, said method comprising the injection, into a patient, of a solution of chitosan into a tissue having a pH of between 6, 8 and 7.4 in order to obtain a chitosan implant, then the modeling of said implant. Disclosure of the invention

[0013] The cosmetic method and the use according to the invention implement an implant obtained from an injectable solution of chitosan. Such solutions are disclosed in applications WO2013 / 079646 and WO2016 / 170284 by the applicant. [0014] Chitosan

[0015] Chitosan is an amino-polysaccaharide generally obtained by N-deacetylation of chitin, a polysaccharide widely used in biomass. Chitin is present in particular in the cuticles of arthropods, the endoskeleton of cephalopods, cell walls or else the extracellular matrix of fungi, yeasts or algae.

Advantageously according to the present invention, chitosan is a natural product which comes from an animal source, for example crustaceans of the crab, shrimp or squid type, or from a vegetable source, such as mushrooms or algae. Chitosan and chitin are linear copolymers of 2-acetamido-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-glucopyranose, respectively. We speak more commonly of units N-acetyl-D-glucosamine (GIcNAc) and D-Glucosamine (GIcN)), linked by glycosidic bonds b- (1®4). Chitin and chitosan are differentiated by the molar fraction (expressed in%) of the GIcNAc units present in the copolymer, also called the degree of acetylation (DA). The chemical structures of chitosan and chitin are shown schematically below as a function of the degree of acetylation (DA):

[0018] N-acetyl-D-Glucosamine (GlcNAc) I) -Glucosamine (GlcN)

[0019] Degree of acetylation (DA): nGIcNAc

DA (%) ”x 100

nGlcNAc * nGlcN

[0020]

[0021] with nGIcNAc = number of acetylated units and nGIcN = number of deacetylated units.

[0022] According to the present invention, the chitosan preferably has a degree of acetylation (DA) of less than 20%, for example less than 10%.

Typically, the chitosan according to the invention has a degree of acetylation (DA) of between 0.5 and 20%, for example between 2 and 10%.

The degree of acetylation can be measured according to the method described in the publication "Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium" A. MONTEMBAULT, C. VITON, A. DOMARD Biomaterials, 26 (8) , 933-943, 2005).

The chitosan used in the solutions of the invention preferably has a mass average molecular mass (determined before sterilization according to the method described in "Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium" A. MONTEMBAULT, C. VITON, A. DOMARD Biomaterials, 26 (8), 933-943, 2005) between 100,000 and 1,500,000 g / mol, for example between 300,000 and 600,000 g / mol. Conventionally, a chitosan with a molar mass of between 100,000 to 1,000,000 g. mol ^-1 can also be characterized by its viscosity (conventionally at a concentration of 1% in a 1% aqueous solution of acetic acid at 25 ° C). With this consideration, the molar mass of chitosan can also be defined by a viscosity between 5 Pa.s and 20 Pa.s.

The viscosity of the composition is measured at 25 ° C using a DHR1 rheometer (TA industry) and a plane geometry of 40mm according to a dynamic mode with the shear rate applied from 0.01 to 1 s ^{- 1} .

According to a preferred embodiment, the chitosan used in the solutions according to the invention is not chemically modified, and in particular, is not grafted to promote its solubility in aqueous solution at pH close to neutrality (between 6.2 and 7.2). In this sense, it differs from the chitosans used in application WC03 / 042250 or application JP-H02-69502, in the publication “Synthesis and charactehzation of sugar-bearing chitosan derivatives: aqueous solubility and biodegradability”, Jae Hyung Park and al. , Biomacromolecules 2003, 4, 1087-1091, and in the publication “Water solubility of partially N-acetylated chitosans as a function of pH: effect of Chemical composition and depolymerization” Varum KM et al., Carbohydrate polymer 25 (1994), 65 -70. [0029] According to one particular characteristic, another chitosan of lower average molecular weight, advantageously less than 20,000 g / mol, may be added to the chitosan as defined above.

In this embodiment, this other chitosan may be in the form of particles of crosslinked chitosan. [0031] Solution

[0032] The injectable chitosan solution according to the invention is an aqueous solution of chitosan. It contains, in a physiologically acceptable medium, a chitosan preferably having a degree of acetylation of less than 20% and an average molecular mass by mass of between 100,000 and 1,500,000 g / mol, as described above. By "physiologically acceptable medium" is meant, within the meaning of the present application, a medium which presents no risk of intolerance or toxicity during injection into biological tissues of the injectable solution according to the invention. . The physiologically acceptable medium must therefore in particular be inert and biocompatible with respect to biological tissues such as muscles, joints, eyeballs, and more generally in the soft or hard tissues of the body, for example organs ( digestive or uro-genital system) or fatty tissue, mucous membranes, gums, cartilage, bones ...

The pH of the aqueous solution according to the present invention is between 4 and 7.6. In a particular embodiment according to the present invention, the pH of said solution is between 5.5 and 7.5, preferably between 6 and 6.8, and even more preferably between 6 and 6.2.

In the context of the invention, the chitosan is soluble in aqueous solution, such as water, in the pH ranges mentioned above, advantageously by protonation of the amine groups of the chitosan. Advantageously, the aqueous solution according to the invention is stable, in particular stable (conservation of its rheological characteristics, of its color, of its transparency and / or of its clarity at 25 ° C.) on storage at temperatures ranging from 4 to 25. ° C for at least 1 month, preferably at least 6 months, and more preferably at least 24 months. In other words, even after storage for a prolonged period at temperatures between 4 and 25 ° C, its characteristics at 25 ° C (rheological, color, transparency and / or clarity) of the aqueous solution according to the invention can be preserved.

For the purposes of the present invention, the term “injectable solution” means a composition which is in liquid form, as opposed to a gelled composition. It thus differs from compositions in the form of injectable hydrogels based on solubilized chitosan such as those described in patent application WO 2009/150651. These hydrogels of gel texture exhibit, because of their higher viscosity, poorer injectability properties (or syringeability, that is to say ease of injection due to a more or less satisfactory flow through a needle in a syringe) than the liquid solutions according to the invention. The term “liquid” solution within the meaning of the present invention means a composition which flows under its own weight, in particular after at most 24 hours, preferably at most 10 hours, unlike a gel, and in particular a hydrogel .

[0038] The liquid solutions according to the invention preferably have a viscosity at 25 ° C of less than 1000 Pa.s, preferably between 20 and 800 Pa.s, more preferably between 30 and 600 Pa.s. For comparison, formulations in the form of hydrogels generally have a viscosity of the order of 4000 to 10000 Pa.s.

[0040] The distinction between the solutions according to the invention and formulations in the form of gels can in particular be demonstrated by measuring the rheological properties of these compositions.

The gel / solution distinction is carried out during a rheological study in oscillation from 0.1 to 100 rad.s ^-1 at a constant frequency of 1 Hz at 25 ° C in order to determine the viscous moduli G "and the modulus elastic G '.

In fact, a liquid solution within the meaning of the present invention is characterized in particular by the fact that its viscous modulus G "is greater than its elastic modulus G '. In the case of a gel, on the contrary, the elastic modulus G 'is greater than the viscous modulus G ". The measurements are carried out on a DHR2 rheometer (TA industries) and a 40 mm plane geometry according to a dynamic mode (angular frequency: 100 to 1 rad / s, deformation 1%, 37 ° C.).

The liquid solution according to the present invention is used to form a gel once injected into a tissue having a pH of between 6.8 and 7.4.

By "gel", it may be accepted according to the present invention that it is a body having a modulus of conservation greater than the modulus of loss (cf. HH Winter, F. Chambon Analysis of linear viscoelasticity of a crosslinking polymer at the gel point J. Rheol., 30 (1986), pp. 367-382). The stability and integrity of this body is maintained either by non-covalent interactions (physical gel) or by covalent chemical crosslinking (chemical gel). The solution according to the present invention can be a "homogeneous" solution of chitosan, that is to say a solution in which all the chitosan polymer is dissolved, the solution not containing solid particles in suspension in the phase. liquid. The solution according to the invention can also be transparent. The homogeneity of a chitosan solution can in particular be characterized by measuring the transmittance of light through a sample of solution. In a particular embodiment, the solution according to the present invention can be opalescent or turbid, that is to say it is capable of absorbing white light visible in spectrophotometry with a transmittance of less than 95% compared to the translucent solution of the same composition.

The liquid solutions of chitosan objects of the present invention can be liquid and homogeneous at room temperature, that is to say between 20 and 25 ° C, preferably at 25 ° C. They differ on this point from the intermediate solutions described in the examples in application WO 003/042250, which are liquid at low temperature (4 ° C) but gel and become cloudy when the temperature increases.

The liquid solutions of chitosan which are subjects of the present invention are preferably stable on storage at 25 ° C. over time. In particular, their viscosity at 25 ° C, their injectability and their transmittance remain stable with a variability of less than 30% compared to the initial value for at least 1 hour and up to more than 3 years to allow the solutions to be stored.

According to a particular characteristic of the present invention, the aqueous chitosan solution contains between 0.1 and 4.5%, advantageously between 1 and 3.5%, in particular between 2 and 3.5%, by weight of chitosan, relative to the total weight of the aqueous solution.

In a particular embodiment, the aqueous solution according to the invention is sterilized before injection, for example by autoclave.

After sterilization, the chitosan typically has a mass average molecular mass of between 50,000 and 1,200,000 g / mol, advantageously between 100,000 and 1,000,000 g / mol. After sterilization, the liquid solutions according to the invention preferably have a viscosity of less than 800 Pa.s, preferably between 5 and 800 Pa.s. The viscosity of the solutions after sterilization is measured according to the method described for measuring the viscosity of the solutions before sterilization. In another particular embodiment of the present invention, the aqueous solution contains a chitosan as defined above in a mixture with another chitosan, such as a chitosan oligosaccharide also called ch ito-ol igosaccharide, advantageously having a degree of acetylation less than 20%, advantageously less than 10%, even more advantageously having a degree of acetylation identical to chitosan as defined above, and typically having a very low weight-average molecular weight, for example less than 20,000 g / mol, advantageously less than 17,000 g / mol.

In another particular embodiment of the present invention, the aqueous solution contains, as polymer, a single chitosan advantageously having a degree of acetylation as defined above, advantageously having an average molecular weight as defined above , advantageously in a content of between 0.1 and 4.5%, advantageously between 1 and 3.5%, in particular between 2 and 3.5%, by weight of chitosan, relative to the total weight of the aqueous solution. [0054] Crosslinking agent

In a particular embodiment, the chitosan can be partially crosslinked by ionic interactions induced for example by the addition of sulphate, citrate, metal anions or even anionic molecules, in particular by the formation of polyelectrolyte complexes with polysaccharides exhibiting a COO carboxylic group (alginates, pectin, xanthan, hyaluronic acid), with polysaccharides having a sulfate group, or with polylactic acid (PLA), or by interaction with proteins (collagen), nucleic acids (l 'DNA, RNA, Si RNA, mRNA, etc.) or oxidized polysaccharides. In another particular embodiment, the chitosan can be partially crosslinked using covalent crosslinking agents (eg: genipin), to the exclusion of agents known for their toxicity, such as agents from the epoxy group (for example 1, 4-butanediol diglycidyl ether - BDDE) or bi or polyfunctional esters (for example Ethylene Diamine Tetra-Acetic - EDTA), divinyl sulfone, carbodiimides, and dialdehydes. In a particular embodiment, the aqueous solution according to the invention is composed of the association of an aqueous solution of uncrosslinked chitosan with an aqueous solution of crosslinked chitosan.

[0058] Dispersing agent

In addition to the chitosan, the aqueous solution according to the present invention can comprise at least one agent for dispersing chitosan. This type of dispersing agent is very well known in the art and can for example be chosen from mannitol, glycerol, sorbitol and mixtures thereof.

[0060] Buffer

[0061] The aqueous solution according to the invention can also comprise a pH buffer to be placed at physiological pH. Any pH buffer commonly used for this purpose can be used, for example PBS ("Phosphate Buffer Saline" - phosphate buffered saline solution).

[0062] Salt

In a particular embodiment, the aqueous solution according to the invention comprises a salt such as sodium chloride or potassium chloride, or any other acceptable excipent advantageously for adjusting the osmolarity of the composition. The addition of a salt such as sodium or potassium chloride can be useful in order to obtain an isotonic solution. According to a particular characteristic of the present invention, the solution can also comprise at least one compound having recognized therapeutic activity.

[0064] Additional compound or excipent

The aqueous solution of chitosan according to the present invention may further comprise at least one pharmaceutically, dermatologically and / or cosmetically acceptable compound or excipent. [0066] In addition, the solution according to the present invention may comprise at least one active compound such as an analgesic compound, local anesthetic, such as lidocaine, mepivacaine, bupivacaine or ropivacaine, an angiogenic compound, or else an active compound of the type growth factor or bioactive oligosaccharide.

According to a particular embodiment, the aqueous solution according to the invention can comprise a compound or a substituent of the extracellular matrix.

The "extracellular matrix" typically designates the set of extracellular macromolecules of connective tissue and other tissues. A compound of the extracellular matrix according to the present invention is advantageously an organic polymer, for example of a size greater than 1000 Daltons (Da), or even greater than 5000 Da. Advantageously, the organic polymers of the extracellular matrix according to the present invention can be of protein, glucosidic, proteoglucosic or even glucoprotein nature. Typically the organic polymers of the extracellular matrix can be collagen, hyaluronic acid or fibronectin. A “substituent of the extracellular matrix” is a non-ubiquitous compound (i.e. which is not found naturally in humans for this function) making it possible to fulfill the role of extracellular matrix. This type of compound is known in the art (Jayakumar, R., Menon, D., Manzoor, K., Nair, SV, & Tamura, H. (2010). Biomedical applications of chitin and chitosan based nanomaterials— A short review Polymeric Carbohydrate, 82 (2), 227-232).

The compound of the extracellular matrix is preferably chosen from proteins, glycosaminoglycans and mixtures thereof.

The proteins and their glycosylated derivatives can preferably be chosen from collagen, elastin, fibronectin, laminin or their mixtures.

The glycosaminoglycans can preferably be chosen from hyaluronic acid, chondroitin sulfate, heparan sulfate, keratan sulfate or a mixture of at least two of these glycosaminoglycans. According to a preferred embodiment, the compound of the extracellular matrix is chosen from proteins and their glycosylated derivatives, preferably from collagen, fibronectin, and a mixture of collagen and fibronectin. The substituent of the extracellular matrix can for its part be chosen from carboxymethylcellulose, the chitosan ester, the chitin ester as well as the mixture of at least two substituents of the extracellular matrix.

The aqueous solution according to the present invention preferably contains less than 5% by weight, relative to the total weight of the composition of at least one compound of the extracellular matrix and / or at least one substituent of the extracellular matrix .

Process for preparing the solution

The aqueous chitosan solution according to the invention, as described above, can in particular be prepared by the following successive steps:

[0077] a. dissolving chitosan in water or buffered solution by adding acid such as a weak acid, said weak acid being advantageously chosen from the group consisting of acetic acid, glycolic acid, lactic acid, glutamic acid, or such as a strong acid, said strong acid advantageously being hydrochloric acid, and mixtures thereof,

[0078] b. the addition, where appropriate, of at least one compound and / or a substituent of the extracellular matrix and

[0079] c. the optional readjustment of the pH to obtain an aqueous composition exhibiting a pH between 4 and 7.6, particularly between 5.5 and 7.5, preferably between 6 and 6.8, and even more preferably between 6 and 6.2 .

Preferably, at least one dispersing agent (such as mannitol, glycerol, sorbitol, etc.) is added, where appropriate, to any one of steps (a), (b), or ( c) or after any one of steps (a), (b), or (c) above. In a particular embodiment according to the present invention, the aqueous, gelled chitosan composition can be dispersed by the addition of a dispersion as presented above, optionally with a step of mechanical fragmentation of the gel of said aqueous chitosan composition.

Controlling the pH of the solutions is very important to avoid, on the one hand, the acid necrosis of the tissues after injection, and also to protect the compositions from hydrolysis and degradation of chitosan if sterilization is carried out. (eg by autoclaving at 121 ° C for 15 minutes). The pH is readjusted if necessary with a compound such as sodium bicarbonate or PBS buffer, typically in reduced amounts. The pH value is advantageously controlled using a pH meter. [0082] In the context of the present invention, the adjustment of the pH can preferably be carried out by dialysis. Dialysis is a process of membrane separation of molecules or ions in solution. Thus, in the context of the present application, the aqueous chitosan solution according to the invention can be dialyzed against a buffer solution having a pH equal to the final pH desired for the chitosan solution (target pH), that is to say between 4 and 7.6, particularly between 5.5 and 7.5, preferably between 6 and 6.8, and even more preferably between 6 and 6.2.

The buffer solution can, for example, be a phosphate buffered saline solution (PBS, TBS, PBS-lactic acid), tris (tris (hydroxymethyl) methylamine), 4-2-hydroxyethyl-1 -piperazineethanesulfonic acid ( HEPES), 2 - {[tris (hydroxymethyl) methyl] amino} -ethanesulfonic acid (TES), 3- (N-morphol ino) propanesulfbn ic acid (MOPS), piperazine-N, N '-bis (2- ethanesulfonic acid )), MES (2- (N-morphol ino) ethanesulfbn ic acid (PIPES), sodium chloride (NaCl). [0084] According to a preferred embodiment, the buffer solution is a solution of phosphate buffer PBS (" Phosphate Buffer Saline "- phosphate buffered saline solution) composed of an" acid "salt NaH2P04, a" basic "salt Na2HPO4 and NaCl.

According to a particular embodiment, the buffer is physiologically acceptable, that is to say that it presents no risk of intolerance or toxicity during the injection into the tissues of the injectable solution according to l 'invention. To this As a result, the buffer is preferably different from glycerophosphate, and in particular from b-glycerophosphate which, although not very irritating to the skin, poses problems of calcification when it is injected into the tissues.

According to a particular embodiment of the invention, the dialysis can be carried out statically in a single bath against a buffer solution as described above. When the dialysis is carried out statically in a single bath against a buffer solution, it is preferable that said buffer solution has a pH equal to the final pH desired for the chitosan solution (target pH), for example between 6 and 6.2 . According to a more preferred embodiment, the dialysis can be carried out statically in several successive baths against buffer solutions having different pHs which are increasingly close to the final pH desired for the chitosan solution (target pH).

According to a particularly preferred embodiment, the dialysis is carried out dynamically, that is to say by continuously circulating at least one solution allowing the gradual increase in pH through one or more pockets. dialysis cells consisting of a dialysis membrane containing the aqueous solution of chitosan. This type of dialysis is for example described in patent application WO 2016/170284 of the Applicant. In a particular embodiment according to the invention, the chitosan is dissolved in water using a strong acid of the hydrochloric acid type. In this case, the pH is readjusted with a compound of sodium or ammonium bicarbonate or PBS type, for example, and / or a base of NaOH or KOH type, for example (always by controlling the pH). In a particular embodiment according to the invention, during the dissolution step, the acid is added in an amount necessary for the dissolution of the chitosan. An excess of acid can thus be used for certain chitosans, for example chitosans which are difficult to dissolve with the strictly necessary quantity of acid, then the chitosan is reprecipitated, using ammonia for example. After a series of washes to remove the excess ammonia and the salts, the chitosan can then be freeze-dried to recover the dry matter. This will then be easier to dissolve.

In another particular embodiment according to the invention, during the dissolution step, the acid is added in an amount at least necessary for the dissolution of the chitosan, such as the stoichiometric amount strictly necessary for the desecration. NH2 sites.

Particularly advantageously, the pH of the aqueous composition according to the present invention is between 4 and 7.6, particularly between 5.5 and 7.5, preferably between 6 and 6.8, and even more so more preferred between 6 and 6.2. In the context of the invention, the chitosan is dissolved in aqueous solution, such as water, in an acidic environment in the pH ranges mentioned above, advantageously by desecration of the amine groups of the chitosan. These pH ranges were in particular chosen in order to maximize the stability of the aqueous composition according to the invention.

Applications of the solution according to the present invention

The aqueous solution according to the invention is formulated to be administered to a patient and used in the context of a process for filling tissues, preferably wrinkles and fine lines.

The solution according to the invention is injected into a tissue having a pH of between 6.8 and 7.4. Injection into a tissue having a pH between 6.8 and 7.4 allows the solution to gel with a kinetics such that the implant thus obtained has a gelation gradient starting from the outside towards the inside allowing it to be modeled within hours of the injection. The outer surface of the implant in contact with tissue gels first. Gradually, the implant gels from the outside to the inside, the heart of the implant gels last after a maximum of 12 hours. During this time, the implant can be modeled to give it the desired shape and filling effect.

The present invention therefore relates to a cosmetic process for filling tissues, comprising a step of modeling an implant obtained. by injection of a solution of chitosan into a tissue having a pH between 6.8 and 7.4.

The tissues which can be particularly mentioned in the context of the present invention are soft tissues such as the skin, adipose tissue, mucous membranes, gums, muscles or bone tissue, vitreous humor and intraperitoneal space. In a preferred embodiment, the tissue is the skin, particularly the skin exhibiting wrinkles (“wrinkled” skin).

The term "implant" denotes the aqueous solution according to the present invention once injected into the tissue having a pH of between 6.8 and 7.4. The "modeling" consists in applying an external physical stress (for example through the skin) on the implant in order to ensure its shaping, for example to distribute it homogeneously in the area to be filled . This modeling makes it possible to obtain an optimal filling result. A person skilled in the art knows how to model an implant in order to obtain a filling of the irregularities of the skin: such modeling was in particular known for HA-based implants. The modeling can, for example, be carried out by massage, by rolling palpation, by compression or by pinching.

[0100] The chitosan implant is therefore "deformable", that is to say that its shape is modified under the effect of an external physical constraint, but its volume remains constant.

[0101] The modeling can be done within 12 hours of the injection. In a particular embodiment, the implant modeling step is performed within 5 hours of injecting the solution according to the invention into the patient. Preferably, the modeling is carried out within 2 hours, more preferably within an hour, even more preferably within 30 minutes following the injection of the solution into the patient.

[0102] The “patient” according to the present invention is a mammal, preferably a human.

[0103] A "filling" process aims to correct hollow irregularities or depressions in the skin by restoring volume to it via the use of a product. fluid (filler) which fills said irregularities in order to attenuate them or even make them disappear.

[0104] The hollow irregularities or depressions of the skin which can be corrected by a filling process can be:

[0105] - the furrows including the nasolabial folds (which go from the wing of the nose to the corner of the mouth), the bitterness folds (which start from the corner of the mouth and descend to the chin) or the groove labio-chin (located between the lower lip and the chin);

[0106] wrinkles and fine lines on the forehead (such as frown lines), eyes (crow's feet), cheeks, mouth, chin, neck or even hands;

[0107] - depressions related to a lack of volume in the lips, cheekbones, chin, mandibles, cheeks, nose, or depressions resulting from scarring after acne, chickenpox or any other trauma.

[0108] In a particular embodiment, the method and use according to the present invention are aimed at filling in wrinkles and / or fine lines.

According to the present invention, the chitosan solution can be injected subcutaneously or intradermally. Preferably, the solution is injected subcutaneously. When the method or use according to the present invention is aimed at filling in wrinkles and / or fine lines of the skin, the injection will typically be made subcutaneously. The injection of the solution according to the present invention is made for cosmetic purposes and is therefore defined as not being invasive. Nevertheless, the cosmetic use according to the present invention can be carried out with syringes and needles of small caliber.

[0110] Another object of the invention is therefore a solution of chitosan for its use in a method of surgical treatment, in which said method comprises the injection, into a patient, of a solution of chitosan in a tissue having a pH between 6.8 and 7.4 in order to obtain a chitosan implant, then the modeling of said implant. [0111] The injection can be carried out via all the known injection methods suitable for fillers. The solution can, for example, be injected as a “bolus”, that is to say all at once and at the same injection point; in a “sheet”, that is to say with a single entry point but several passages of the needle; or by retro-injection, that is to say where the solution is injected by withdrawing the needle. An illustration of these different injection modes is provided in Fig 1. According to a preferred embodiment, the solution is injected as a bolus.

[0112] The injection volume depends on the area to be treated. The solution can for example be administered in the following dosages: the concentration of chitosan can be between 0.1 and 5% by weight, advantageously between 1, 5 and 3.5% by weight, more preferably between 2 and 3% by weight even more advantageously between 2.2 and 2.8% by weight, for example at 2.5% ± 0.05% by weight of chitosan relative to the total amount of aqueous composition administered. The amount of aqueous solution administered in a single dose is typically between 0.01 and 20 grams per injection, preferably between 0.05 and 10 grams per injection, more preferably between 0.1 and 5 grams per injection, still more advantageously between 0.4 and 1 gram per injection, for example 0.7 grams ± 0.1 gram per injection. The number of injections can vary from 1 to 20 times in the most extreme cases. [0113] More routinely, this injection number is less than 5 times to effectively fill cavities with a volume of less than 50 mL (therefore at physiological temperature).

The examples which follow illustrate the invention in more detail but should not be interpreted as limiting its scope. Brief description of the drawings

Fig. 1

[0115] [Fig. 1] shows the main injection techniques according to the invention: A) bolus, B) tablecloth and C) feedback;

Fig. 2 [0116] [Fig. 2] shows the macroscopic observation of a bolus resulting from the subcutaneous injection of a solution of chitosan in the rat;

Fig. 3

[0117] [Fig. 3] Shows the macroscopic observation of chitosan solutions injected as a bolus (0.5 ml) and A) explanted one hour after injection and B) two hours after injection;

Fig. 4

[0118] [Fig. 4] Shows a microscopic observation of gelled chitosan solutions injected as a bolus (0.5 ml) and explanted 28, 52 and 78 weeks after injection.

Examples

[0119] Example 1

[0120] The main objective of the study is to evaluate the technique of injection and modeling of a chitosan solution and then to determine the gelation kinetics of this solution in rats.

[0121] For this, the rats are anesthetized by exposure to isoflurane and then receive a bolus of 0.5 ml of a chitosan solution (2.34% w / w in a buffered medium) as a subcutaneous injection. Part of the injections are intended to evaluate the modeling method and the other part is intended to evaluate the gelation kinetics. A photo showing the macroscopic observation of a bolus resulting from the subcutaneous injection of a chitosan solution into a rat is provided in Figure 2.

Ease of injection: The syringe is equipped with a 27G or 30G needle. The operation is performed by a cosmetic surgeon who submits his feelings regarding the ease of injection, in comparison with injections of hyaluronic acid, usually used in the doctor's office.

[0123] During the injection, the practitioner confirms good handling and ease of injection comparable to injections of hyaluronic acid. This is confirmed for injections made with size 27G and 30G needles. [0124] A few minutes after injection, the injection sites are massaged by series of pressures, spreading and by rolling palpation. By this technique, the shape and the external rendering can be modulated. The implant remains easily moldable for at least 20 minutes after injection and then becomes more difficult to mold.

[0125] Gelation kinetics: After 1, 2, 3 and 5 hours, the boluses are excised and the appearance is observed macroscopically to assess the state of gelation.

[0126] The samples taken after one hour after implantation exhibit a gel shell in contact with biological tissues and a still liquid heart. The samples taken two hours after injection show complete gelation with a gelled shell and a gelled core (see Fig. 3). The samples taken at 3 and 5 hours show the same appearance.

[0127] Example 2

[0128] As part of the biocompatibility evaluation tests, volumes of 0.2 to 0.5 ml of chitosan solution (2.37% w / w in a buffered medium) are injected as a bolus subcutaneously at a rate of 3 to 5 implantation sites per rat (38 rats in total). The explants are recovered after 26, 52 (15 sites), 78 (15 sites) weeks (see Fig. 4).

[0129] On this kinetics, macroscopic observation did not show migration over 26 weeks (0 migration over 90 implantation sites). One site (out of 15 implantation sites) and two sites (out of 15 implantation sites) were not found at 52 and 78 weeks respectively, the others not having migrated. The disappearance of these three implantation sites is attributed to the degradation of the chitosan solution.

Claims

[Claim 1] Cosmetic process for filling in tissues comprising a step of modeling an implant obtained by injecting a solution of chitosan into a tissue having a pH between 6.8 and 7.4.

[Claim 2] A solution of chitosan for use in a method of surgical treatment, said method comprising injecting, into a patient, a solution of chitosan into a tissue having a pH between 6.8 and 7.4 in order to obtain a chitosan implant, then the modeling of said implant.

[Claim 3] A method according to claim 1 or a solution for its use according to claim 2, wherein said tissue is skin, preferably with wrinkles.

[Claim 4] A method or solution for its use according to any one of claims 1 to 3, wherein the shaping is performed within 5 hours, preferably within 2 hours of the injection of the chitosan solution.

[Claim 5] A method or solution for its use according to any one of claims 1 to 4, wherein the injection of the chitosan solution is performed as a single injection at the same injection site.

[Claim 6] A method or solution for its use according to any one of claims 1 to 5, wherein the chitosan solution comprises between 0.1 and 4.5% by weight of chitosan.

[Claim 7] A method or solution for its use according to any one of claims 1 to 6, wherein said solution comprises a chitosan having a degree of acetylation of less than 20% and / or a weight average molecular weight of between 100 000 and 1,500,000 g / mol.

[Claim 8] A method or solution for its use according to any one of claims 1 to 7, wherein the pH of said solution is between 4 and 7.6, particularly between 5.5 and 7.5, preferably between 6 and 6.8, even more preferably between 6 and 6.2.

[Claim 9] A method or solution for its use according to any one of claims 1 to 8, wherein the solution comprises an acceptable compound or excipient.

[Claim 10] A method or solution for its use according to claim 9, in which the compound is an active compound such as an analgesic compound, local anesthetic, such as lidocaine, mepivacaine, bupivacaine or ropivacaine, an angiogenic compound, or alternatively an active compound of the growth factor or bioactive oligosaccharide type.