THERMOGELIFIABLE COMPOSITION

专利摘要:
The invention relates to a thermogelifiable composition comprising a chitosan having N-acetylglucosamine units, glucosamine units, and substituted glucosamine units other than N-acetylglucosamine units, said substituted chitosan having preferably a degree of glucosamine units ranging from 10 to 50%, expressed as the number of moles of the substituent relative to the number of moles of total units, its preparation and its applications.
公开号:FR3024363A1
申请号:FR1457547
申请日:2014-08-01
公开日:2016-02-05
发明作者:Mickael Chausson；Renaud Lecler
申请人:Synolyne Pharma SA；
IPC主号:

专利说明:

[0001] The present invention relates to a thermogelifiable composition. This composition is particularly suitable for its injection, for example in a human being or an animal, and more particularly in a human being. The present invention also relates to a method of prevention or therapeutic treatment and a method of aesthetic care, in particular comprising the injection of a thermogelifiable composition of the invention. It is well known to inject compositions based on biopolymers, for example hyaluronic acid, because of its viscoelastic properties, for example for a therapeutic treatment. This type of composition is particularly useful in the treatment of joint pain, in particular osteoarthritis. These compositions are also known for filling tissue, such as the dermis. This filling can intervene for an aesthetic purpose or as part of a repair surgery treatment. It is also known to use chitosan-based compositions as a biopolymer for tissue repair, particularly the repair of cartilage defects mixed with blood. It is also known to prepare and use chitosan-based solutions associated with agents that cause a sol-gel transition of the solution at physiological temperature, for injection into tissues and joints. Such compositions may be used for example in intra- tissue injection (for example subcutaneous, intradermal), intra-articular, or intraocular injection. It may be for example to protect or repair a joint that shows signs of osteoarthritis and cartilage damage by injection of a viscous fluid. It may still be a question of filling a tissue or an organ to favor its repair or increase its volume. For the supply of such compositions, there is generally the technical problem of being able to provide a composition whose physicochemical properties are close to those of the tissues or fluids of the injection zone. However, the problem of injecting such a viscous fluid arises particularly because, when it has physicochemical properties, close to those desired, for example in terms of viscosity, these compositions are very difficult to inject, or it is impossible to inject them.
[0002] Among the sought solutions, mention may in particular be made of US Pat. No. 6,344,488, which teaches the preparation of a thermogelifiable composition comprising chitosan. This composition is also very sensitive to pH, and may, depending on the pH value, have or not a reversibility of the sol-gel transition. The reversibility of the sol-gel transition is important because it allows sterilization by temperature rise. The composition also comprises glycerol phosphate in a rather high concentration. However, it is noted from Table 2 of this patent that chitosan at a concentration of less than 1.5% (w / v) is not gelled at 37 ° C. This is the average body temperature of a nonfevery human being. The compositions used have a concentration of 2% (w / v). It is advantageous to provide a composition comprising chitosan in concentration which can be lower. The compositions described in this patent have a pH of at most 7.05. It is therefore advantageous to provide a composition comprising chitosan having a pH closer to physiological pH for the intended applications. Furthermore, the thermogel described in this patent does not seem to be sterilized by steam. This is therefore a major drawback.
[0003] There is therefore the need to provide a thermogelifiable composition at physiological temperature of a human having good viscoelastic properties for therapeutic or aesthetic applications. The invention therefore aims to solve these technical problems. The object of the invention is in particular to provide a thermogelifiable composition which can be easily injected while being gelled at physiological temperature of a human being. Such a composition must have good physicochemical properties for therapeutic or aesthetic applications, in particular at the physiological temperature of the human or animal body, for example 37 ° C. for uses after implantation. More particularly, such a composition should be able to have physico-chemical properties that can be modulated according to the intended application so as to be as close as possible to the properties necessary for the intended therapeutic or aesthetic purpose. The invention also aims to provide a thermogelifiable composition whose pH is acceptable for therapeutic or aesthetic applications. More particularly, such compositions should be able to have a modulable pH according to the intended application, in particular to be as close as possible to the physiological pH. The present invention also aims to provide a thermogelifiable composition whose osmolarity is controlled and for example close to the physiological for the intended therapeutic or aesthetic application, in particular an osmolarity ranging from 200 to 500 mosm / kg.
[0004] The object of the present invention is also to provide a composition whose sol-gel transition is reversible, in particular so that the storage and / or transport conditions do not require a drastic temperature control, and / or undergo a sterilization by a rise. in temperature.
[0005] The invention also aims, according to a variant, to provide a composition whose biopolymer content, and more precisely in chitosan, is low. Such a content should be less than 5%, preferably less than 2%, by mass of chitosan relative to the total mass of the composition. It has surprisingly been found that such a thermogelifiable composition could be prepared when this composition comprises a chitosan having N-acetylglucosamine units, glucosamine units, and substituted glucosamine units other than N-acetylglucosamine units, said substituted chitosan having preferably a degree of substitution of glucosamine units ranging from 10 to 50%, expressed as the number of moles of the substituent relative to the number of moles of total units. According to a particular embodiment, the glucosamine units are D-glucosamine units (D-glucosamine units, substituted D-glucosamine units, N-acetyl-Dglucosamine units and optionally substituted N-acetyl-D-glucosamine units). Alternatively, chitosan has N-acetyl-D-glucosamine units, D-glucosamine units, and substituted D-glucosamine units other than N-acetyl-D-glucosamine units, and optionally N-acetyl units. D-glucosamine substituted. By "substituted D-glucosamine other than N-acetyl-D-glucosamine units" it is meant that the substituted D-glucosamine units do not form N-acetyl D-glucosamine units after substitution.
[0006] According to one variant, a substituted chitosan has a substitution of D-glucosamine units only. According to another variant, a substituted chitosan exhibits a substitution of the D-glucosamine and N-acetyl-D-glucosamine units simultaneously, and in which the substitution group is covalently linked, alternatively to the amine groups of chitosan only, or according to another variant to the amine and hydroxyl groups of chitosan simultaneously. According to one embodiment, the degree of substitution of the D-glucosamine units expressed as the number of moles of D-glucosamine units relative to the number of moles of total units (D-glucosamine and N-acetyl-D-glucosamine units, substituted or unsubstituted) of the substituted chitosan, ranges from 0.1 to 0.5%.
[0007] It has been discovered according to a specific variant that a degree of substitution of the glucosamine units, expressed as the number of moles of glucosamine units on which the substituent group is attached by a covalent bond, relative to the number of total moles of units. glucosamine and N-acetyl-glucosamine of the substituted chitosan, from 0.1 to 0.5 advantageously allowed to solubilize the substituted chitosan, optionally in the presence of a gelling agent, for example a polyol or sugar salt, and more particularly a glycerol phosphate salt at a pH and at a temperature in a range particularly adapted to the targeted applications. It may be simpler to express the degree of substitution by the molar ratio or the mass ratio of the starting reagents with respect to chitosan, as explained below. According to one variant, the substituted chitosan is soluble by its degree of substitution in a buffered water solution at pH = 7, for example with a phosphate buffered saline (PBS), the solution comprising a concentration of substituted chitosan of 1% by weight. mass relative to the mass of the total solution, the pH being measured at 8 ° C.
[0008] According to one variant, especially when the substituted chitosan has a zwitterionic character, there is a narrow pH range in which the substituted chitosan is insoluble. This feature is circumvented by the addition of a gelling agent, for example polyol salt or sugar, and more particularly glycerol phosphate salt, so that the substituted chitosan is solubilized at the pH considered of interest for the invention.
[0009] By substituting chitosan, it has been possible to prepare a solution of a substituted chitosan soluble in an aqueous solution whose pH varies over a wide range, while the unsubstituted chitosan is only soluble at pH below 5. , 5 to 6.5. The aqueous solution of substituted chitosan may in general have a pH ranging from 6.5 to 8.5. This solution according to the invention can be gelled. The gelation or sol-gel transition can therefore be carried out at neutral pH or at physiological pH, for example at a pH of between 7 and 8.5, whereas chitosan is not soluble at pH = 7, the pKa of amine groups being about 5.5 (and therefore not loaded at pH 7 at which the amine groups are not protonated). The substituted chitosan thus has the ability to be solubilized at different pH levels by virtue of the presence of substituent groups which modify its solubilization profile. Surprisingly, it has been possible to prepare thermogelifiable compositions with a substituted chitosan, and in particular at a low concentration of chitosan, in particular with a chitosan of very low, low or medium molecular weight, while having a sol-gel transition. reversible. Such a composition comprising a substituted chitosan has a sol-gel transition as the temperature increases. Thus, very advantageously, the sol-gel transition, that is to say the transition from the solution to the fluid state to the gel state, can be modulated according to the degree of substitution for this ground transition. gel occurs in particular under conditions of pH, osmolarity, and desired temperature. The invention thus advantageously makes it possible, according to one variant, to prepare a fluid thermogelifiable composition at a temperature lower than that of use, typically at a temperature below the physiological temperature, for example 37 ° C., but which is in the form of gel at the temperature of use, typically at physiological temperature, for example 37 ° C, at neutral pH (pH = 7) or at physiological pH, and for example from 7 to 8.5, with a suitable osmolarity for intended use. This is for example a physiological osmolarity.
[0010] The term "thermogelifiable composition" or "thermo-gel" means a fluid, and especially a solution which has the property of undergoing a sol-gel transition by temperature modification, preferably by increasing the temperature, and more preferably by increasing the temperature to the physiological temperature of the human or animal body, and thus be in the form of a gel at physiological temperature after injection or implantation. The thermogelifiable composition of the invention may be in the form of a hydrogel, that is to say an aqueous gel. According to the present invention, the term "gel" means a composition that does not flow under its own weight, and more specifically does not exhibit any flow in the absence of external stimuli by inverting a container containing the composition, for example for 30 seconds, and characterized by an elastic modulus G 'greater than the loss modulus G ", the modules being measured by rheology using a" Couette "geometry rheometer, the difference between the gel modules G' and G" being characteristic gel cohesion.
[0011] According to the present invention, the term "fluid" means a composition which flows under its own weight by inverting a container containing the composition for example for 30 seconds and characterized by an elastic modulus G 'less than the loss module G " , the modules being measured by rheology using a Couette shear rotation rheometer (shearing of the fluid between two coaxial cylinders). Advantageously, it has been discovered that the degree of substitution of the Dglucosamine units makes it possible to modulate the transition. sol-gel Thus, the invention advantageously makes it possible to provide a thermogelifiable composition in which the minimum degree of substitution of chitosan is chosen so that the substituted chitosan is soluble in the thermogelifiable composition, it is also possible to check the solubility in different buffers. By "water-soluble" it is meant that the substituted chitosan does not present a disorder visible to the naked eye More specifically, the absence of haze can be confirmed by an optical density of less than 0.5, and preferably less than 0.2, measured by UV-visible spectrometry at the wavelength of 600 nm of a sample comprising water optionally buffered with a buffer of the pH concerned, for example 0.5M sodium acetate, with reference to a reference tank comprising only the aqueous solvent used for the sample measured, but in the absence of substituted chitosan. When the chitosan is not sufficiently substituted, the composition is not soluble in a satisfactory pH range, for example ranging from pH = 7 and pH = 8.5, at room temperature, and therefore is not more thermogelifiable when increasing the temperature. The invention makes it possible to provide a thermogelifiable composition in which the maximum degree of substitution of chitosan is chosen so that a sol-gel transition takes place at a temperature below the temperature of use, preferably below the physiological temperature. for example at the temperature of 37 ° C. The sol-gel transition can be seen by the crossing of the G 'and G "modules according to the method defined in the invention Advantageously, the degree of substitution is determined by magnetic resonance spectrometry (NMR) of the proton in solution in an aqueous medium. with the aid of a magnetic resonance spectrometer, for example a Bruker spectrometer of frequency 400 MHz The samples are prepared in the following manner: 5 to 6 mg of substituted chitosan are dissolved in 1 ml of deuterated water. Deuterium hydrochloric acid 12M concentration is added to the solution of the substituted chitosan to reach a pH range suitable for analysis.The appropriate pH range depends on the nature of the substituent.The spectrum is recorded at a temperature at 70 ° C, with a scan count ranging from 64 to 256 and a relaxation time of 1 to 8 seconds.The resulting spectrum is deconvolved to determine the value of the integral of the areas of the signals of interest to be able to calculate the degree of substitution of the substituted chitosan. The sample preparation method, the recording conditions of the NMR spectrum, and the formula used to calculate the degree of substitution should be adapted to each type of substituted chitosan, as they depend on the nature and position of the substituent . An example of calculation of the degree of substitution (DS) is given below for the case of chitosan substituted with a succinyl group bonded to the amine group of D-glucosamine units (chitosan succinamide, Formula 1). The abbreviations are as follows: 4/2 is equal to the integral of the proton signal area of the D-glucosamine units at the 2-position; / cH2 succi is equal to the integral of the proton signal area of the two -CH 2 groups of the succinyl substituents linked to the D-glucosamine units (on the alpha and beta carbon atoms of the amide function); ICH3 acetyl is equal to the integral of the proton signal area of the acetyl groups of the N-acetyl-D-glucosamine units.
[0012] An example of the NMR spectrum of chitosan succinamide and the structural formula of chitosan succinamide are illustrated in Figure 1. Formula 1 - Calculation of the degree of substitution of succinyl-substituted chitosan by proton NMR If another NMR method and more advantageous for to estimate the degree of substitution reliably, such a method should be used. The above formula must be adapted by those skilled in the art as regards the preparation of the sample and the signals to be integrated, in particular as a function of the resolution, the robustness and the position of the protons of the signals to be used. for the calculation of the degree of substitution. Advantageously, the composition has a thermoreversible sol-gel transition.
[0013] Advantageously, the present invention makes it possible to provide a composition with a low concentration of substituted chitosan. Advantageously, the concentration of chitosan is less than 4%, for example less than 3%, or even less than 2% by weight relative to the total mass of the composition (m / m).
[0014] According to a specific variant, the concentration of substituted chitosan is less than 1.9% (w / w), expressed by weight relative to the weight of the final composition. Advantageously, the concentration of substituted chitosan is between 0.5 and 1.5% (w / w), expressed by weight relative to the weight of the final composition. According to a particular variant, the concentration of substituted chitosan is about 1.2% (w / w), expressed by weight relative to the mass of the final composition. According to one particular variant, the concentration of substituted chitosan is approximately 1.1% (w / w), expressed by weight relative to the weight of the final composition. According to one particular variant, the concentration of substituted chitosan is about 1.0% (w / w), expressed by weight relative to the mass of the final composition.
[0015] According to a particular variant, the concentration of substituted chitosan is about 0.9% (w / w), expressed by weight relative to the weight of the final composition. Moreover, the degree of substitution of the D-glucosamine units advantageously makes it possible not to use a solution with an acidic pH (typically 5.0 to 5.5) to solubilize the chitosan then to necessarily have to add a sugar or polyol salt, as the glycerol phosphate, to increase the pH up to about 7. On the contrary, the present invention advantageously allows to directly prepare a solution at neutral pH or at physiological pH, for example from 6.2 to 8.5, in which the substituted chitosan is soluble and therefore also has the advantage of, for example, not necessarily having to add an acidic solution to solubilize chitosan. This has the advantage of providing a much greater freedom over the pH range that can be used for the thermogelifiable composition, and thus preparing thermogelifiable compositions at neutral or physiological pH. According to one variant, the composition comprises a gelling agent, preferably an agent gelling agent inducing a sol-gel transition of the composition, for example a glycerol phosphate salt, for example in the sodium or calcium form, for example in its pentahydrate form, said gelling agent preferably being present in the composition at a concentration of between 1 and 20%, preferably 3 and 9%, by weight relative to the total mass of the final composition (m / m).
[0016] The gelling agent is advantageously at least one polyol salt or sugar, including any of their mixtures. Among the polyol or sugar salts, mention may be made especially of the phosphate salts, and more particularly the dibasic salts of polyol monohydrogen or sugar. Mention may also be made of sulfate salts, for example polyol monoalphate or sugar salts. Among the phosphate salts, there may be mentioned dibasic mono-phosphates of glycerol, including glycerol-2-phosphate, snglycerol-3-phosphate and 1-glycerol-3-phosphate. According to one variant, it is betaglycerol phosphate. Among the polyols and sugars for such salts, mention may be made of the following polyols and sugars: histidinol, acetol, diethylstilbestrol, indoleglycerol, sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol, palmitoyl-glycerol, linoleoyl-glycerol , oleoyl-glycerol, arachidonoyl-glycerol, fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fuculose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose , threose, xylulose, hexose, methylthio-ribose or methylthio-deoxy-ribulose, or any of their mixtures.
[0017] According to a specific variant, the concentration of polyol salt or sugar, and preferably glycerol, is between 1 and 10%. Advantageously, the concentration of polyol salt or sugar, and preferably glycerol, is between 1 and 7%. Advantageously, the concentration of polyol salt or sugar is from 2% to 5%. The values are expressed in mass relative to the total mass of the composition. According to one variant, the glycerol salt is a glycerol phosphate, and more specifically a sodium salt, for example disodium glycerol phosphate. The polyol or sugar salt, and preferably glycerol phosphate, is used to bring the pH to a basic pH, then the pH of the composition is adjusted by the addition of an acid, which has the advantage of provide a thermogelifiable composition whose pH is adjustable very easily and accurately. Thus, the composition according to the invention has a pH greater than or equal to 7, for example greater than or equal to 7.1, and for example a pH of 7.2 to 8.5. The pH is measured on the final thermogelifiable composition in the form of a solution, that is to say before the sol-gel transition. The pH is determined by following the method described in the European Pharmacopoeia (EP 2.2.3). The pH meter used is a pH meter from the Sartorius range with a combined glass electrode (PY-P11). PH measurements are made between 20 and 25 ° C. Advantageously, the pH is adjustable in the wide range from 6.5 to 8.0.
[0018] According to one variant, the pH is greater than 7.40. According to a specific variant, the pH is 7.50 +/- 0.05. According to another specific variant, the pH is 7.20 +/- 0.05. According to another specific variant, the pH is 7.00 +/- 0.05. According to a preferred variant, the composition of the invention has a sol-gel transition a temperature greater than 30 ° C, preferably at a temperature between 30 and 50 ° C, and preferably between 32 and 45 ° C, and even preferably between 35 and 40 ° C. Advantageously, the thermogelifiable composition of the invention is in a fluid form at room temperature, that is to say between 20 and 25 ° C.
[0019] According to a preferred variant, the composition of the invention is fluid at a temperature below 37 ° C, preferably at a temperature below 35 ° C, and more preferably at a temperature between 2 ° C and 20 ° C. According to one variant, the composition of the invention is fluid at the storage temperature, for example from 2 to 8 ° C., or at extended ambient temperature, for example between 15 and 30 ° C.
[0020] According to one embodiment, the composition has an osmolarity of 100 to 700mosm / kg, preferably 200 to 500mosm / kg.
[0021] According to one variant, the thermogelifiable composition according to the invention is isosomolar. According to a variant, when the composition of the invention is intended to be injected into the plasma of a human being, it is preferable that the osmolarity be between 250 and 400, and more specifically between 280 and 350mosm / kg. According to another variant, when the composition of the invention is intended to be injected into the synovial fluid of a human being, it is preferable that the osmolarity be between 300 and 490, and preferably between 360 and 470mosm / kg. . The determination of the osmolarity of the solutions is carried out with an automatic micro-osmometer (Osmometer Type 15M from Leiser Messtechnik). The equipment is calibrated beforehand with a solution of 300mosm / kg. The sample is placed in a container provided for this purpose, and is put to the standard temperature of the measurement. Advantageously, the thermogelifiable composition of the invention has in the fluid state a dynamic apparent viscosity of between 20 and 800mPa.s, for example from 40 to 500mPa.s. The dynamic apparent viscosity is measured using a rotational mobile viscometer, for example a rotational mobile viscometer of the Brookfield brand, for example equipped with a S18 type spindle at a speed of 5. turn / min and at a temperature of 8 ° C. According to one variant, the composition of the invention has an apparent viscosity allowing easy injection into an injection device such as, for example, a syringe, during its filling. According to one variant, the composition of the invention has an apparent viscosity allowing easy injection through a fine needle, for example a 22 Gauge syringe, at ambient temperature. By "easy" injection, it is preferably meant that the force to be exerted on such a syringe is less than 50 Newton to make the thermogelifiable composition flow through a 22-gauge needle, preferably a force of less than 20 Newton. The thermogelifiable composition according to the invention may be diluted, for example in water, optionally buffered. For example, the composition of the invention may be diluted in a buffer which makes it possible to adjust the pH to a physiological pH. More particularly, for example, the composition of the invention can be diluted in an acetate buffer (for example a 10mM sodium acetate trihydrate buffer) to adjust the pH to about 7.5.
[0022] For a gel, the module G 'is greater than G "For a solution, the module G' is less than G". The sol-gel transition is characterized by the crossing of the modules G 'and G ". When the solution has a" thermogelifiable "character, the module G' becomes greater than the modulus G" beyond a certain temperature, and in particular at a temperature below the physiological temperature (i.e., the temperature at which the product is injected or implanted) or after a certain time after implantation or injection into the body at physiological temperature. The module G 'and the module G "intersect when the temperature increases.
[0023] The modules G 'and G "are measured, for example, using a Couette shear rotation rheometer which shears the fluid between two coaxial cylinders, for example an ARES rheometer of the Rheometrics brand, for example with a frequency of 1 Hz and a deformation of 5% It is possible to measure the modules G 'and G "at a certain temperature. The measurement of the modules given here is carried out starting from the product at a certain temperature, for example the storage temperature of the product of 4 ° C. The product is allowed to reach a certain temperature naturally, for example the physiological temperature, for example 37 ° C., without controlling the rate at which the temperature increases. Advantageously, the storage modulus G 'is between 0.001 and 1000, the loss modulus G "is between 0.001 and 1000, G' is less than G" at storage temperature and / or at ambient temperature, and G 'is greater than G "at physiological temperature, for example 37 ° C., after gelation Advantageously, the preservation modules G 'and loss G" intersect when the product passes from a storage temperature in a refrigerator, for example 4 ° C or an ambient temperature, for example 18 ° C to 25 ° C, at a physiological temperature, for example 37 ° C, reflecting the sol-gel transition and the thermogelifiable character of the system, with a transition time sol- gel adapted to the intended application. Gelation can be achieved by maintaining at a sufficient temperature and for a time sufficient to gel the chitosan solution. This gelation is carried out for example in an oven, for example maintained at 40 ° C. According to the present invention gelation can occur in situ, that is to say for example after injection into the human body or animal (warm blood). Gelation notably allows the gel to be positioned in a localized manner.
[0024] More particularly, the time required for the sol-gel transition is generally between 1 second and 48 hours after passing from the temperature of 4 ° C. (storage temperature) to 37 ° C. (physiological temperature), that is to say by example in situ after injection of the solution into a human or animal body. According to one variant, the sol-gel transition is complete after a duration that varies between 5 seconds and 24 hours, preferably less than 4 hours, more preferably less than 2 hours after the passage of the temperature from 4 ° C. to 37 ° C. vs. According to another variant, the gelation occurs instantaneously (gelation occurred as soon as the temperature increases (and before the measurement) .The gel must not be gelled in the injection syringe but gels in situ at the desired location. It is possible to carry out several sol-gel transition cycles since the composition according to the invention advantageously has a reversible sol-gel transition.This advantageously makes it possible to sterilize the composition according to the invention by raising the temperature, that is to say This advantageously makes it possible to provide a thermogelifiable composition whose ability to obtain gelling is not affected by the temperature variations, in particular during storage, transport, or sterilization. invention being intended in particular to be injected, the gelling properties are adapted to its injection via a e syringe provided with a needle of variable size according to the intended application, for example from 19 to 25 gauge, for example 22 gauge. Usually, the person performing the injection must manually press the plunger of the syringe. It is therefore necessary that the compressive strength of the ungelled fluid allows an injection as easy as possible. Once injected, the composition must gel to exhibit the required viscoelastic properties. According to one variant, the force required for the flow of the fluid composition in the orifice of a 22-gauge needle is between 1 and 20 Newton. Preferably the strength of the gel is between 2 and 15 Newton. According to one variant, the strength of the gel is between 2 and 8 Newton. Injectability is measured using a test bench for the measurement of mechanical properties, for example the Instron Bluehill brand, equipped with a 500N force cell. The injection system is specifically designed to measure the force required to inject the solution by pushing the plunger of the syringe, provided with the needle with the desired diameter. The system has a metal cylinder with a height of 15cm provided with a vertical slot of 4cm wide. The cylinder is surmounted by a square metal plate of 10cm side. This plate is provided with a hole in the middle, 0.5 cm radius. The syringe, equipped with a 22 gauge needle, is put at a temperature of 4 ° C. A descent rate of the plunger of the syringe is applied constant of 1 mm per second.
[0025] According to one variant, the composition comprises a buffer, for example an acetate buffer. Buffering agents are known to those skilled in the art. Preferably, the composition comprises a reducing sugar, for example mannitol. According to one variant, the present invention relates to a thermogelifiable composition comprising 0.1 to 5% by weight of substituted chitosan, and preferably of 1 to 7% of polyol salt or of sugar, for example of glycerol phosphate, and optionally of 0.1 10% reducing sugar. Advantageously, the composition of the present invention comprises from 0.1 to 1.9% by weight of substituted chitosan and from 2 to 6% by weight of glycerol, preferably glycerol phosphate, and optionally from 0.1 to 2.5% by weight of reducing sugar, relative to the total mass of the composition It is advantageous to use at least one polyol in the composition of the invention (in addition to the polyol salt such as glycerol phosphate).
[0026] Such polyols may for example be selected from the group consisting of: isopropanol, sorbitol, mannitol, alkylene glycol such as propylene glycol, poly (alkyl glycol), for example poly (ethylene glycol), fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxyribose, ketose, mannose, arabinose, fuculose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose, methylthio-deoxy-ribulose, and any of their mixtures. According to a specific variant, the reagent that allows the substitution is succinic anhydride. According to this specific variant, the substituted chitosan is a chitosan succinamide, that is to say a chitosan with "succinyl" groups covalently bonded to the nitrogen atom of the amine groups of the D-glucosamine units. The chitosan is modified by substitution by a reaction involving a chitosan and a substitution agent in which the chitosan and the substitution agent are reacted thereon to obtain the substituted chitosan on the Dglucosamine moieties.
[0027] The invention also relates to a process for the preparation of substituted chitosan. This process comprises in particular: a step of dissolving the chitosan in an aqueous solution, preferably water, preferably by adjusting the pH to an acidic pH at which the chitosan is soluble; a substitution reaction step of chitosan with a substitution agent; a step of stopping the substitution reaction preferably at a degree of substitution of chitosan ranging from 10 to 50%, expressed as the number of moles of the substituent relative to the number of moles of total units, for example by modification of the pH of the reaction medium, or by precipitation of chitosan in a "non-solvent" in which the substituted chitosan is not soluble; purification of the substituted chitosan.
[0028] The term "non-solvent" a solvent in which the chitosan is not soluble, that is to say that a disorder is observable to the naked eye. Such solvents are, for example, polar solvents, for example ethanol, methanol, acetone, etc. The step of dissolving chitosan in an aqueous solution can be carried out in water by adding a suitable acid.
[0029] In the reaction step, the amount of substitution agent can be adjusted depending on the degree of substitution desired for the substituted chitosan. In the reaction step, the reaction time may be adapted depending on the degree of substitution desired for the substituted chitosan. Advantageously, the reaction is stopped to terminate the substitution reaction of chitosan according to the degree of substitution desired for the substituted chitosan. Different reactions are known to substitute a chitosan. One can refer for example to the application WO 2010/142507 in particular relating to the preparation of chitosan derivatives. The substituted chitosan may comprise D-glucosamine and N-acetylglucosamine units, wherein at least some of the units are grafted (or coupled) with one or more functional groups that may be the same or different. According to one variant, the substituted chitosan comprises glucosamine and N-acetyl-glucosamine units, said units comprising one or more functional groups. For example, chemical modifications are carried out by coupling one or more functions of the N-acetylglucosamine units, that is to say the hydroxyl functions and / or amine functions, and / or glucosamine units. that is, hydroxy functions and / or amine functions. In a particular embodiment, the functional groups present on the substituted chitosan are selected from the group consisting of, but not limited to, hydrophobic functional groups, hydrophilic functional groups, ionic functional groups, and any combination thereof. According to a particular variant, the hydrophobic functional groups are chosen from the group consisting of alkyl, alkenyl, araalkyl and alkaryl groups, and any one of their combinations. These groups generally comprise between 1 and 100 carbon atoms, and more generally 1 between 50 carbon atoms, for example between 1 and 25, or else 1 and 10 carbon atoms, one or more of the carbon atoms being able to be replaced by by a heteroatom and / or an atom chosen from boron, nitrogen, oxygen, sulfur, silicon, germanium, an ester function, amide, urea, urethane, and any combination thereof, and wherein one or more hydrogen atoms may be replaced for example by a heteroatom and / or an atom selected from the group consisting of halogen atoms, alkoxy groups, amide, and any combination thereof. According to another embodiment, the hydrophobic functional groups are chosen from the group consisting of carboxylic acids, organosulfonic acids, polyethers, amine polyethers, polyesters, sterols, porphyrins and one of their combinations. . According to another variant, the hydrophilic functional groups are chosen from the group consisting of diamines, polyamines, diols, polyols, diacids, polyacids, crown ethers, glymes, polyalkenyl ethers, polyalkenylamines, polyalkenyletheramines, polyacrylic acids, polyvinylacools, and any of their combinations. According to another particular variant, the ionic functional groups are chosen from the group consisting of metal salts, ammonium salts, phosphonium salts, sulphate salts, carboxylic acid salts, phosphate salts, dicarboxylic acids, polycarboxylic acids, a carboxylic acid function being used to form a covalent bond with chitosan, diamines, polyamines, an amine function being used to form a covalent bond with chitosan, and any of their combinations . According to a particular variant, the functional group substituting chitosan is an aminoalkyl group. For example, the functional group substituting chitosan is an aminoethyl group. According to one variant, the substituted chitosan is: an amino-alkyl chitosan, such as, for example, an aminoethyl chitosan, etc. and their equivalents; a hydrophobicized N-alkyl or O-alkyl chitosan, such as for example a chitosan ethanoyl, propanoyol, butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonaoyl, decanoyl, or dodecanoyl and their equivalents; positively charged chitosane, such as, for example, a trialkyl ammonium chitosan (eg a trimethylchitosan or TMC) and their equivalents; an N- (2-hydroxy) propyl-3-trimethylammonium chitosan and its counterion as a chloride and their equivalents; a negatively charged chitosan, such as a succinamide chitosan (succinyl chitosan), an N, O-carboxyalkyl chitosan, an N, O-sulfoalkyl chitosan, and their equivalents; a neutral chitosan such as, for example, N, O-acetyl chitosan, N, O-alkyl chitosan, ethanoyl, propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, nonaoyl, decanoyl, dodecanoyl, and their equivalents; a zwitterionic chitosan, such as for example a chitosan comprising hydrophobic polymers, such as for example an aliphatic polyester, such as the homopolymers and copolymers of lactic acid, glycolic acid, epsilon-caprolactone, p-dioxanone; aliphatic and / or aromatic polyesters; aliphatic polyamides; ethylenic polymers and their copolymers; propylene polymers and copolymers; polycarbonates; polyacrylates, and their equivalents. According to a particular embodiment, other derivatives may be used in the context of the present invention and include 0-substituted chitosan whose substitution is carried out on the hydroxyl groups of the glucosamine and / or N-acetyl-g lucosamine units. . According to one variant, the chitosan has, as substituent, hydrophobic polymers, preferably chosen from the group consisting of aliphatic polyesters, and aliphatic polyamides, homopolymers or copolymers of alkene, such as, for example, polymers of ethylene or of propylene, polycarbonates, polyacrylates, and any combination thereof. According to one variant, chitosan has as substituent an aliphatic polyester, and in particular a polylactide. Another method of substituting chitosan is the reaction described in US Pat. No. 7,838,643, and the resulting chitosans. US Pat. No. 3,953,608 may also be mentioned. According to one variant, an N-alkylating agent, or an alkyl anhydride, optionally comprising one or more unsaturations, optionally comprising one or more heteroatoms, may be used as substitution agent. and / or functions (such as, for example, ether, thiether, carboxy, ester, amide, etc.) optionally comprising one or more substituents (such as alkyl, amine, hydroxyl, carboxyl, carboxylic acid, etc.). According to one variant, mention may be made, by way of example, of succinic anhydride, glutamic anhydride, acetoxy succinic anhydride, methylsuccinic anhydride, diacetyl tartaric anhydride, diglycolic anhydride and maleic anhydride. itaconic anhydride, citraconic anhydride, and any of their mixtures.
[0030] In theory, any substitution agent capable of providing a soluble chitosan in the final composition at neutral or physiological pH would be suitable. When the step of stopping the reaction is carried out by precipitation, the purification may consist of the separation of the insoluble substituted chitosan and the non-solvent.
[0031] After purification, the process of the invention may comprise a step of drying the substituted chitosan, and optionally grinding it to obtain a powder. According to a specific variant, the substitution agent is succinic anhydride. The substituted chitosan is a chitosan succinamide.
[0032] Chitosan succinamide can be obtained as follows: Dissolution of chitosan in an aqueous solution at pH below 6.5; The solution is maintained at a temperature between 0 ° C and 100 ° C, preferably between 20 ° C and 50 ° C and more preferably between 25 ° C and 35 ° C. After dissolving the chitosan, adding the succinic anhydride, for example in powder form, to the chitosan solution. The amount of succinic anhydride and the number of succinic anhydride additions are adapted according to the degree of substitution of the desired substituted chitosan. After a time sufficient for a minimum degree of substitution to be reached, for example after a minimum of 15 minutes, the reaction is stopped. The reaction can be stopped, for example by modifying the pH of the reaction medium or by a precipitation step in a non-solvent such as ethanol, methanol, acetone, etc. The substituted chitosan is then advantageously purified by for example by a dialysis technique, or by cycles of precipitation / solubilization in a non-solvent, or by a tangential filtration technique. The purified product is then preferably dried. The product can be dried, for example by spray-drying, or in an oven (under vacuum or at atmospheric pressure), or by lyophilization. The pH of the aqueous chitosan solution can be adjusted, for example with a solution. weak acid, such as acetic acid, lactic acid, etc., to dissolve chitosan. The degree of substitution of chitosan succinamide is correlated with the mass ratio of the reactants relative to chitosan at the start of the reaction. According to one variant, it is preferred to use a mass ratio of the reagents greater than 0.13 in the case of a chitosan succinamide. According to one variant, it is preferred to use a mass ratio of reagents of less than 0.2 in the case of a chitosan succinamide.
[0033] According to a specific variant, the substitution agent is an alkylating agent for the amine groups of the D-glucosamine units. The substituted chitosan is advantageously an alkylated chitosan on the D-glucosamine units. As alkylating agents, mention may be made of haloalkyls such as halomethyls, such as methyl iodide, methyl bromide, acyl chloride, such as for example those bearing one or more carboxymethyl, aminoethyl and / or trimethyl groups, etc. . According to this variant, the substituted chitosan is an N-alkylated chitosan. According to a specific variant, the substituted chitosan is chitosan bearing a tri-methyl substitution group (N, N, N-trimethyl chitosan, abbreviated "TMC").
[0034] According to one variant, the alkylated chitosan, preferably TMC, is obtained with a molar ratio of the reactants ranging from 0.1 to 0.35, and preferably from 0.15 to 0.30, expressed as number of moles of agent. alkylating agent relative to the number of moles of amine groups initially present in chitosan. Chitosan is for example referenced under the CAS number 9012-76-4.
[0035] The chitosan used for the invention is advantageously of fungal origin, and preferably derived from the mycelium of a fungus of the Ascomycete type, and in particular Aspergillus piger, and / or a Basidiomycete fungus, and in particular Lentinula edodes. (shiitake) and / or Agaricus bisporus (Paris mushroom). Preferably, chitosan is derived from Agaricus bisporus. The chitosan is preferably very pure, that is to say containing few impurities from its fungal origin, and a microbiological quality compatible with its use as an implant or pharmaceutical composition. A method for preparing chitosan is that described in WO 03/068824 (EP 1483299, US 7,556,946). The chitosan prepared may be of different molecular weights, and generally ranging from 10,000 to 300,000. According to one variant, the average molecular mass is between 20,000 and 60,000. According to another variant, the average molecular mass is between 60,000 and 100,000.
[0036] According to another variant, the average molecular mass of between 120 000 and 150 000. According to another variant, the average molecular mass is between 150 000 and 220 000. It is possible to hydrolyze chitosan to reduce its molecular weight.
[0037] Preferably, the average molecular weight is the viscosity average molecular weight (Mv), calculated from the intrinsic viscosity according to the Mark-Houwink equation. The intrinsic viscosity is measured by capillary viscosimetry, with a Ubbelohde capillary viscometer, according to the method of the European Pharmacopoeia monograph EP2.2.9. The flow time of the solution is measured through a suitable capillary tube (Lauda, for example the capillary tube Ubbelohde 510 01 with a diameter of 0.53 mm) using a Lauda Visc automatic viscometer, first at the concentration initially in chitosan, then for several dilutions, for example according to the recommendations of method EP2.2.9. The reduced intrinsic viscosity is deduced for each of the concentrations. The reduced viscosity is plotted as a function of temperature, and the value is extrapolated to the concentration 0 to deduce the intrinsic viscosity. For example, the reduced viscosity (llred in ml / g) of the dilutions as a function of the concentration C of the dilutions (g / ml) according to the formula 5 should be taken. Formula 2 [11recl] = (t1-t0) - (1 C). To calculate the average viscometric mass, we apply the MarkHouwink equation with the k and alpha constants recommended by Rinaudo et al. (J. Biol.
[0038] Macromol, 1993, 15, 281-285), according to the DA of chitosan, according to one of the following three formulas. Formula 3. Mv = (VO / 0.082) (110'76), for a DA of 2%; Formula 4. Mv = (W0,076) (110'76), for a DA of 10% (for example 11.5%); Formula 5. Mv = (W0.074) (110'76), for a DA of 20% (for example 21%).
[0039] For the intermediate DA values, a linear interpolation is performed to calculate the average viscometric mass (Mv). Preferably, the chitosan used has an average molecular weight of between 120,000 and 150,000 or between 150,000 and 220,000.
[0040] According to a specific variant, the substituted chitosan has, preferably, an average molecular weight of 150 000 to 220 000 and a degree of substitution ranging from 10 to 50%, the molecular mass being preferably expressed before substitution. According to another specific variant, the substituted chitosan has an average molecular weight of 120,000 to 150,000 and a degree of substitution ranging from 12 to 40%, preferably from 12 to 30%, or even from 20 to 30%, the molecular weight. preferably being expressed before substitution. Advantageously, chitosan has a degree of acetylation of between 5 and 50%. The degree of acetylation is expressed as the number of N-acetyl-D-glucosamine units relative to the number of total N-acetyl-D-glucosamine and D-glucosamine units present.
[0041] According to one variant, the degree of acetylation is between 20 and 45%. According to one variant, the degree of acetylation is between 5 and 20%.
[0042] According to one variant, the degree of acetylation is between 15 and 25%. According to one variant, the degree of acetylation is between 20 and 30%. According to one variant, the degree of acetylation is between 25 and 40%. The degree of acetylation is determined by potentiometry. Chitosan is dissolved in a solution of hydrochloric acid. The excess of unreacted hydrochloric acid with the amine functions of chitosan is determined by a standard solution of sodium hydroxide. This gives the number of moles of D-glusamine unit present in the chitosan and thus by subtraction the degree of acetylation. Chitosan advantageously has a controlled degree of acetylation. By the term "chitosan having a controlled degree of acetylation" is meant a product whose degree of acetylation, i.e. the proportion of N-acetyl-glucosamine units, can be adjusted in a controlled manner. The invention also relates to a process for the preparation of a thermogelifiable composition according to the invention.
[0043] This process typically comprises: dissolving a substituted chitosan in an aqueous solution, preferably water, optionally buffered, preferably at a pH of between 6.2 and 8.5 and preferably between 6.5 and 7; , 5; the eventual adjustment of the pH to a physiological pH; - mixing with a solution of sugar salt or polyol, such as a glycerol phosphate salt solution; the eventual adjustment of the pH to a physiological pH, for example by addition of a buffering agent; the possible adjustment of the osmolarity of the composition; the possible adjustment of the viscosity of the composition. According to one variant, the dissolution of chitosan is carried out in water comprising a reducing sugar, such as, for example, mannitol. According to one variant, the buffering agent is an acetate buffer. Advantageously, the method also comprises a subsequent step of filling an injection device, such as for example a syringe, with the composition according to the invention. Advantageously, the injection device, such as for example a syringe, can then undergo steam sterilization. This device, for example a syringe, can then be packaged, preferably in a sterile manner. It is advantageous to use a chitosan having a degree of purity sufficient for the intended application.
[0044] The present invention relates more particularly to an injectable composition comprising or consisting of a thermogelifiable composition according to the invention. According to one variant, the composition according to the invention is used as an injectable pharmaceutical composition or an injectable or implantable medical device.
[0045] The present invention relates more particularly to a composition according to the invention for use for a therapeutic treatment, for example comprising subcutaneous, intradermal, intraocular or intra-articular injection of said composition, for example for repair or filling at least one body tissue requiring repair or filling.
[0046] According to one variant, the body tissue is chosen from the tissues belonging to the vocal cords, muscles, ligaments, tendons, cartilages, sexual organs, bones, joints, eyes, dermis, or any of their combinations, and more particularly to the joints. joint. The present invention relates more particularly to a composition according to the invention for the treatment of osteoarthritis, or the repair of a cartilage defect, for example by injection into the synovial fluid or after mixing with the blood and implantation in the cartilage. The present invention relates more particularly to a medical device, for example a medical implant, characterized in that it comprises or consists of a composition according to the invention. The present invention may also include one or more additives or excipients for modulating its properties. According to a specific variant, the composition of the invention comprises a suspension of particles, for example solid or gel particles.
[0047] The present invention also relates to a composition according to the invention for use in a method of treatment or cosmetic care by filling the dermis ("dermal filling"). This involves, for example, injecting a composition according to the invention subcutaneously or intradermally. The present invention also relates to a composition according to the invention for use in a method of treatment or aesthetic care in which the composition is a viscosupplement agent. This is for example to inject intra-articularly the composition of the invention especially to limit the friction of the synovial membrane located on either side of the joint. The present invention also relates to a composition according to the invention for use as a cellular vector, of one or more cell type, and / or one or more of active agents. It can be active agents from a pharmaceutical or biological point of view. The composition of the invention may indeed be compatible with the presence of cells, preferably living cells. Among the living cells of interest, there may be mentioned, for example: chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscle), mesenchymal stem cells, red blood cells (blood) and keratinocytes (skin). The composition of the invention may also be intended as a therapeutic vector for the targeted delivery and / or controlled release of at least one therapeutic agent. According to a variant, blood, or plasma, or platelet lysate, or platelet-rich plasma, or any biological fluid with the composition of the invention is added, for example to increase the performance of the product. According to one variant, the composition according to the invention is formulated in a solid form (for example a film or a porous foam), which solubilizes once implanted. According to one variant, the composition is formulated in a form of a nebulizable composition (spray). The present invention also relates to a composition according to the invention for use in a method of treatment or aesthetic care of one or more tissues or organs affected by excessive temperature, as in the case of a burn.
[0048] The present invention also relates to a composition as an artificial synovial fluid comprising or consisting of the thermogelifiable composition according to the invention. The composition according to the present invention makes it possible to mimic the synovial fluid while seeking to mimic its shock absorption properties (identifiable by the modulus of elasticity G '), while being easily injectable to fill a syringe for example or to be injected into the human or animal body. For indication, the elastic modulus G 'of the healthy synovial fluid is between 40 and 100 Pa, and its loss modulus G "is between 1 and 10 Pa.In general, the ranges of osmolarity and pH values sought in biomedical applications are close to the following values: -Osmolarity: Iso-osmolar plasma: 285-295mosm / kg, Iso-osmolar synovial fluid: 404 +/- 57 mosml / kg, according to "Clin Orthop Relat Res, 1988, 235, 289 And physiological pH is in general above 6.8, in particular above 7.0, and in particular 7, 4 (as for plasma or synovial fluid) The pH of the plasma is generally 7.4 The pH of the synovial fluid is in general 7.768 +/- 0.044 according to J Bone Joint Surg Br, 1959, 41- B (2), 388-400 ", or 7.3 according to" Acta Orthop Scand, 1969, 40, 634-641 ", or" Clin Rheumatol 2006, 25, 886-888. "Synovial pH in the case of oste oarthritis is considered to be generally lower than that of the healthy synolvial fluid. Advantageously, the composition according to the present invention is sterile. Very advantageously, the composition according to the present invention is sterilized by temperature rise, preferably under an autoclave. According to one variant, the compositions of the invention are transparent or translucent. By "translucent" we mean that we can distinguish an object when placing its composition between the eye of the observer and the object. By "transparent" it is meant that alphanumeric characters can be distinguished when the composition is placed between the eye of the observer and the characters observed. In general, this evaluation is carried out with a thickness of approximately 1 cm. According to one variant, the composition of the invention is colorless, that is to say in particular that an observer with the naked eye does not attribute a specific color to the composition. The invention relates in particular to articles or packaging, preferably sterile, comprising one or more injection devices pre-filled with a thermogelifiable composition according to the invention. These are typically pre-filled syringes.
[0049] The composition of the invention may advantageously be sterilized. According to one variant, the composition of the invention is sterilized by steam, for example by raising the temperature to a temperature greater than 100 ° C., and preferably greater than 120 ° C., for example between 121 and 138 ° C. under an autoclave, for a sufficient period of sterilization, for example in general from 15 to 20 minutes.
[0050] The invention also covers a composition of the invention in a dry form, especially in a freeze-dried form. In particular, this freeze-dried composition can be (re) dispersed before use. The present invention also covers a therapeutic treatment method comprising the injection of a composition according to the invention.
[0051] The present invention also covers a method of aesthetic care, in other words non-therapeutic, comprising the injection of a composition according to the invention. This is for example the filling of wrinkles or the filling of one or more visible tissue areas damaged, for example following an accident or surgery, for aesthetic purposes. A tissue is a set of similar cells of the same origin, grouped into a functional whole, that is to say, contributing to the same function. Tissues include: epithelial tissue, connective tissue, muscle tissue, and nerve tissue. The term "composition according to the invention" or equivalent terms means a composition defined as in the present invention, including according to any one of the variants, particular or specific embodiments, independently or according to any one of their combinations, including according to the preferred characteristics. In the figures: Figure 1 shows a nuclear magnetic resonance spectrum of the proton of a chitosan succinamide of the invention.
[0052] Other objects, features and advantages of the invention will become apparent to those skilled in the art following the reading of the explanatory description which refers to examples which are given by way of illustration only and which can not in in no way limit the scope of the invention. The examples are an integral part of the present invention and any features appearing novel from any prior art from the description taken as a whole, including the examples, form an integral part of the invention in its function and its generality. Thus, each example has a general scope. On the other hand, in the examples, all percentages are given by weight, unless otherwise indicated, and the temperature is expressed in degrees Celsius unless otherwise indicated, and the pressure is atmospheric pressure unless otherwise indicated.
[0053] Examples The chitosan precursors of the substituted chitosans according to the invention have average molecular masses in viscosity (Mv, determined by capillary viscosimetry) and the degrees of acetylation (DA, proportion of N-acetyl-glucosamine unit, determined by potentiometric titration) included in the ranges of Table 1. Table 1. Characteristics of precursor chitosan substituted chitosan Range of Mv range DA range molecular weight of chitosan (mol%) ultra low 20,000 - 60,000 5 - 20% low 60,000 - 120 000 15 - 25% medium 120 000 - 150 000 20 - 30% high 150 000 - 220 000 25 - 40% Example 1 - Preparation of a substituted chitosan (chitosan succinamide, CSS) To obtain the chitosan succinamide reference CSS5 Table 2, 10 g of chitosan of known molecular weight and DA are dissolved in 266 ml of a 1% (v / v) aqueous solution of acetic acid. The solution is maintained at a temperature of 30 ° C. 6.75 g of succinic anhydride (AS) are added, which corresponds to a mass ratio of succinic anhydride on chitosan (AS / chitosan) of 0.675, and to a molar ratio of succinic anhydride on the NH 2 groups chitosan (AS / NH 2) of 1; 7. After 15 minutes, the pH of the reaction medium is adjusted to 7.5 by addition of 30% sodium hydroxide. The solution is then precipitated in 2.5 liters of ethanol. The precipitate is recovered and again solubilized in water. The substituted chitosan undergoes these steps of precipitation in ethanol then solubilization in water 3 times. At the last precipitation, the precipitate is recovered, pressed and dried in an oven at 60 ° C at atmospheric pressure. Once the chitosan succinamide is dry, it is ground to obtain a powder. The degree of substitution of chitosan is determined by proton NMR according to the method described above. An NMR spectrum is shown in Figure 1.
[0054] The batches of chitosan succinamide used in the examples were prepared in the same manner, with the parameters of Table 2.
[0055] Table 2. Parameters and batch characteristics of chitosan succinamide N ° CSS Range Ratio Ratio DS pH range of Mv molar mass (% mol) for AS / chitosan AS / NH2 insolubility in water CSS1 medium 0.119 0.3 12 5, 4 - 6.7 CSS2 medium 0.159 0.4 18 6.3 - 7.3 CSS3 medium 0.199 0.5 6.1 - 7.0 CSS4 medium 0.262 0.6 42 CSS5 medium 0.675 1.7 52 <5.5 CSS6 medium 0.178 0.45 5.8 - 6.9 CSS7 high 0.19 0.5 48 CSS8 high 0.258 0.6 4.2 - 7.0 CSS9 high 0.68 1.7 <5.2 CSS10 medium 0.133 0.3 6.4 - 6.9 CCS11 medium 0.133 0.3 5.9 - 7.1 CSS12 medium 0.133 0.3 6.6 - 7.1 CSS13 medium 0.133 0.3 6.5 - 7.2 CSS14 medium 0.133 0.23 6.1 - 7.6 CSS15 medium 0.133 0.23 4.8 - 6.3 CSS16 medium 0.178 0.4 15 5.8 - 6.9 CSS17 medium 0.222 0.5 CSS18 medium 0.172 0, 4 5.5 - 7 CSS19 high 0.2 0.5 5.2 - 6.3 Note: The addition of beta-glycerol sodium phosphate (65%, Salfic-Alcan, abbreviated to GP) makes the CSS soluble beyond the limit of solubility in water. CSS has a zwiterionic character. This presents no problem because we can add a low concentration of GP to find the soluble nature of the CSS.
[0056] Example 2 - Preparation of a substituted chitosan (trimethyl chitosan, TMC) A suspension of 50 g of chitosan in a volume of 800 ml of water is prepared. The suspension is heated to 95 ° C. A volume of 26.27 ml of the reagent 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (abbreviation CHTAC) is added dropwise to the medium, a mass and molar CHTAC / chitosan ratio of 0.31 and 0.5, respectively. After 4 hours of reaction, the reaction medium is diluted with 400 ml of water and then precipitated in 8.4 liters of ethanol. The precipitate is recovered and again solubilized in water. The substituted chitosan undergoes this step of solubilization / precipitation in ethanol 3 times. At the last precipitation, the precipitate is recovered, pressed and dried in an oven at 60 ° C at atmospheric pressure. Once the trimethylated chitosan is dry, it is milled to obtain a powder. The batches of trimethyl chitosan used in the examples were prepared in the same way, with the parameters of Table 3. Table 3. Parameters and characteristics of trimethyl chitosan batches No. TMC Range of Mv Ratio Ratio DS of the molar mass chitosan (% mol) CHTAC / chitosan CHTAC / NH2 TMC1 medium 0.37 0.5 16 TMC2 medium 0.79 1.05 17 TMC3 medium 0.37 0.5 22 TMC4 medium 0.37 0.5 26 TMC5 medium 0.37 0 , 5 28 TMC6 medium 0.61 0.8 36 TMC7 medium 1.21 1.5 75 Note: All these trimethylated chitosans are perfectly soluble in water in the DS range of 16 to 75%. Example 3 - Example for preparing a thermogelifiable composition A chitosan succinamide (CSS2) obtained according to Example 1 is solubilized in water in the following proportions: 0.195 g of chitosan succinamide CSS2 in 14.809 g of water containing 0.5% (m / ml) m) mannitol. A solution of sodium beta-glycerol phosphate (65%, Salfic-Alcan, abbreviated GP) at 39.7% (w / w) is prepared. With magnetic or mechanical stirring, 1.49 ml of the GP solution are added to the chitosan succinamide solution. 0.325 ml of 0.5M acetate buffer (pH 3) is added to the solution of chitosan succinamide / GP until a given pH is obtained, for example 7.5 in the case of solution No. 2 of Table 4. The osmolarity of the final solution is measured. Example 4 - Capacity to gel solutions of chitosan succinamide at a final concentration of 1.14% (m / m) The solutions of Table 4 are prepared according to the method described in Example 3. The ability to gel the thermogelifiable solution is dependent in particular on the degree of substitution. The degree of substitution is directly related to the molar ratio between the amine functions present on the chitosan and the amount of succinic anhydride used during the substitution reaction. If the degree of substitution is too high, Table 4. CSS solutions of Mv "medium" and "high" at a concentration of 1.14% (m / m), GP at a concentration of 4.11 % (m / m) and mannitol at a concentration of 0.5% (w / w); the pH is adjusted to 7.5 Mv range N ° Ratio Ratio Ratio DS Ease Transition solid mass CSS solution (% mol) of sol-gel injection to AS / chitosan AS / NH2 37 ° C medium CSS1 0.12 0 , 3 12 Yes No 2 CSS2 0.16 0.4 18 Yes Yes 3 CSS3 0.20 0.5 Yes Yes 4 CSS4 0.26 0.6 42 Yes No 5 CSS5 0.68 1.7 52 Yes No high 6 CSS6 0.18 0.4 Yes Yes 7 CSS7 0.19 0.5 Yes Yes 8 CSS8 0.26 0.6 Yes No 9 CSS9 0.68 1.7 Yes No gelation does not occur (case of solution No 5). Gelation does not take place if the degree of substitution is too low (case of solution No. 1). Table 4 shows that for chitosan succinamide, the optimal AS / chitosan mass ratio leading to the thermogelifiable character is between 0.12 and 0.26, which translates into a DS range of 10 to 45%. Moreover, the thermogelifiable solutions of Table 5 are prepared starting from different lots of chitosan succinamide, to demonstrate the reproducibility of the preparation of the hydrogels with a mass ratio of 0.13, which makes it possible to target a DS between 14 and 18 mol%. 0. It can be seen that the osmolarity of the final solution is between 363 and 447mosm / kg, and that all the solutions are easy to inject and thermogelifiable. The presence of mannitol (which is optional for the thermo-gelling effect) increases the osmolarity.
[0057] Example 5 - Capacity of the TMC chitosan substitute to be gelled at a final concentration of 0.9% (m / m) In the case of the trimethyl chitosan of "medium" molecular weight, the optimum degree of substitution for the thermogelling gel formulation, for example at a low concentration of 0.9% (w / w) is between 10 and 36% (Table 6). Table 5. Solutions of chitosan succinamide of Mv "medium" at a concentration of 1.14%, of GP at a concentration of 4.11% and mannitol at a concentration of 0.5% Range of Mv N ° N ° Osmolarity Facility Solution gel CSS (mosm / kg) injection at 37 ° C medium 10 CSS10 363 Yes OK 11 CSS11 382 Yes OK 12 CSS12 447 Yes OK 13 CSS13 405 Yes OK 14 CSS14 Yes OK 15 CSS15 Yes OK Table 6. Solutions based on TMC at a concentration of 0.9% (w / w) and GP at a concentration of 3.0% (w / w) (no mannitol); the pH is adjusted to 7.5 N ° N ° DA DS Gel solution TMC (mol%) (mol%) at 37 ° C TMC 1 TMC1 16 16 Yes 2 TMC2 16 17 Yes 3 TMC3 15 22 Yes 4 TMC4 18 26 Yes TMC5 18 28 Yes 6 TMC6 17 36 No 7 TMC7 19 75 No 5 Example 6 - Capacity of chitosan succinamide solutions to gel at low concentration, at osmolarity and physiological pH values We compare the solubility, the injectable and thermal character gelatable solutions of unsubstituted chitosan chitosan (CS) with those of solutions based on substituted chitosan (chitosan succinamide CSS), prepared according to the method of Example 2 (Table 7).
[0058] Table 7. Solutions based on chitosan (CS) or chitosan succinamide (CSS) CS CS or GP range Final pH Ability Gel or CSS of Mv CSS (%, m / m) (adjusted) injection at (% m / m) 37 ° C CS high 1.5% 6% 6.8-7.0 Yes No CS high 1.5% 6% 6.7-7.0 Yes No CS high 2% 6% 6.7-7 , 0 Yes Yes CS high 2% 4.1% 6.7-7.0 Yes No CS medium 2% 4.1% 7.4 No No (precipitates) CS low 2% 4.1% 7.4 No No (precipitates) CS medium 1.5% 4.1% 7.4 No No (precipitates) CS low 1.5% 4.1% 7.4 No No (precipitates) CSS medium 1.5% 4.1% 7 , 4 Yes Yes With the unsubstituted chitosan of the prior art, the osmolarity, pH, solubility in water, injectability and gelation at 37 ° C can not be met at the same time.
[0059] With succinyl-substituted chitosan, solubility, injectability and sol-gel character can be achieved even at lower GP concentration (at equal polymer concentration) due to the fact that the starting polymer is a chitosan substituted water-soluble, the concentration of GP being adjustable to obtain the gel character, osmolarity and pH desired.
[0060] Example 7 - Capacity of chitosan succinamide solutions of final concentration 0.88% to be gelled over a wide pH range In this example, the pH of the solutions was adjusted by adding concentrated acetic acid (96% w / v) to to minimize the dilution of the hydrogel. All solutions are soluble and easily injectable.
[0061] For a given molecular weight and degree of substitution, it is possible to obtain the gelling character over a wide pH range, for example from 7.2 to 8.0, for example from a molecular weight CSS " medium "and a mass ratio of 0.133. Example 8 - Ability to Decrease the Substituted Chitosan Concentration (CSS) While Retaining the Gelability of the Solution Table 9. Low-Level CSS Solutions Mv Range CSS No. Range CSS Range Range (% m / m) This osmolarity concentration at GP 37 ° C (% m / m) 7.0 3.5 - 4.1 310 - 376 No 0.75% high CSS7 7.5 3.4 - 4.2 300 - 375 Yes 7.0 3.4 - 4.4 300 - 373 No 0.88% 7.5 3.4 - 4.4 300 - 380 Yes 1.34% 7.0 3 - 3.7 300 - 380 Yes 7.0 3.4 - 3.8 300 - 375 No 0.88% medium CSS17 7.5 3.4 - 4.5 300 - 400 No 7.0 2.4 - 2.7 300 - 360 No 1 , 35% 7.5 2.4 - 2.7 300 - 340 Yes Table 8. Solution based on CSS at a concentration of 0.88% (m / m) and GP at a concentration of 4.16% Range No pH CSS Mv Gel at 37 ° C 6.8 No medium CSS10 7.0 No 7.2 Yes 7.5 Yes 7.6 Yes 8.0 Yes It is sometimes advantageous to form the final low concentration solutions in substituted chitosan in certain applications, for example below 1%, for example 0.85%, t while retaining the ability to produce a gel at physiological temperature, for example 7.5. Example 9 - Injectability of Commercial Products and Solutions of Substituted Chitosan and GP at Room Temperature This example aims to verify that the product can be easily injected through a syringe equipped with a small diameter needle, as used for injections. for example in intra-dermal or intra-articular. In particular, it is important to be able to inject easily in small joints like those of the hand.
[0062] The force required to inject the solutions at room temperature during the injection of the solution via a 22-gauge needle is evaluated according to the protocol described in the text. The force required for the injection is measured after a movement of the plunger of the syringe by about 15 mm, which makes it easy to compare the different products with each other. Three commercially available hyaluronic acid solutions for intra-articular injection were tested, and compared with solutions of chitosan succinamide of "medium" molecular weight and variable concentration. Table 10. Force Required for Injection After Piston Movement Approximately 15mm for Needle 22 Gauge Product Strength (N) Pure Water 0.5 Commercial Solution "A" (Hyaluronic Acid) 2N Commercial Solution "C" (Acid hyaluronic) 6 - 8 N Solution based on 3N CSS18 at a concentration of 1.0% at pH 7.5 Solution based on 3N CSS19 at a concentration of 1.5% at pH 7.5 We see that the solutions of Chitosan succinamide are as easily injectable through a 22 gauge needle as a commercial solution of hyaluronic acid (A), and more easily injectable than a commercial solution of hyaluronic acid (C). Example 10 - Rheological properties of hyaluronic acid-based commercial products and thermogelifiable solutions based on chitosan succinamide The G 'and G "modules of commercial products based on hyaluronic acid are measured at a temperature ranging from 4 ° C. at 37 ° C., as a function of time, using an ARES rheometer, according to the method described above, Then the value of the modules G 'and G "is determined at 60 minutes (Table 11). Table 11. G 'and G "Modules at 60 minutes Product Sol or Gel G' G" Soil or gel at 4 ° C at 37 ° C at 37 ° C at 37 ° C (Pa) (Pa) (G 'versus G ") Commercial solution (hyaluronic acid)" A "Sol 0.1 1 Sol (G '<G") Commercial solution (hyaluronic acid) "g" Gel 40 38 Gel (G'> G ") Commercial solution (hyaluronic acid)" D Gel 640 120 Gel (G '> G ") Solution based on CSS16 at a concentration of 1.2% at pH 7.5 (solution of Example 12) Sol 16 1 Gel (G'> G") For the compositions of the invention, for example for the composition based on chitosan succinamide and GP at pH = 7.5, the module G 'crosses the module G "after a few seconds, and G' is greater than G" beyond the time at which the crossing occurred, characterizing a rapid sol-gel transition when the product passes from 4 ° C. to 37 ° C. The commercial compositions based on hyaluronic acid that are tested are not thermogelifiable.
[0063] Example 11 Effect of intra-articular injection of a chitosan succinamide solution in the rabbit after transection of the anterior ligament The solution of this example is a sterilized solution comprising 1.2% (m / m) of degree chitosan succinamide 15% substitution and "medium" molecular weight (No. CSS16 in Table 2), 3.5% (w / w) GP, 0.4% (w / w) D-mannitol and 11 mmol / l of sodium acetate trihydrate. The solution is sterilized with steam by autoclave, the concentration is adjusted by dilution by a factor of 1.33, then the solution is packaged in a syringe. After sterilization and dilution, the pH is 7.5, the osmolarity is 380mosm / kg, and the apparent dynamic viscosity is 126mPa.s at 9 ° C. Anterior cruciate ligament transection (ACLT) in rabbits consistently and well documented symptomatic changes in osteoarthritis (OA), including cartilage fissures, lesions, and inflammation. significant synovial membrane without loss of cartilage over the entire height of the joint. This model is used in particular to study the effects of new therapies such as viscosupplement injected intraarticularly on the cartilage structure, the initial lesions may be of very variable origin and nature (in Laverty et al., Osteoarthr Cartil, 2010; Phys Ther Sport, 2013, Mainil-Varlet et al., Cartilage, 2013, Oprenyeszk et al., Osteoarthr Cartil, 2013). In this model, erosion of the cartilage appears as early as 4 weeks after the surgery, which consists of transection of the anterior ligament of the right knee. It is recommended that the study be conducted for up to 8 weeks after surgery, resulting in cartilage erosion in at least 40% of the femoral condyles.
[0064] The intra-articular injection of the product to be tested is done before the appearance of the first signs of osteoarthritis, ie 1 week after the surgery on the knee having undergone the transection. The right knee of the 10 animals of the test group received an injection of 6001.11 of the thermogelifiable solution to be tested. The right knee of the 9 animals in the control group received an injection of 6001.11 0.9% saline. Products are injected into the knee joint of rabbits via a syringe with a 22 gauge needle. The ability of the product to slow down or halt the progression of surgery-induced osteoarthritis is characterized by examining several macroscopic, histological and radiological indicators, characterized according to the OARSI-recommended scores (in Laverty et al., Osteoarthr Cartil 2010). Joint radiology results are evaluated according to the Kellgren & Lawrence scale, based on the presence of osteophytes and inter-articular space. The study is double blind, that is to say that neither the surgeon who carries out the operation and the injection of the products to be tested, nor the persons who make the macroscopic observations during the life of the At the time of their euthanasia, neither the persons who analyze the macroscopic or histological results and establish the statistical calculations know the correspondence between the numbers of the animals and the groups by product tested. This correspondence is not communicated until the calculations are completed and written by an independent person. The results are reported in Tables 11 to 13. In comparison with the injection of a saline solution, the injection of the thermogelifiable solution HG makes it possible to statistically reduce all the indicators of osteoarthritis at 8 weeks, as demonstrated by a decrease in the inter-articular space and the presence of osteophytes visible on the radiological examination (Table 12, according to the Kellgren & Lawrence scale), the decrease in the severity and the size of the cartilage lesions that appear on the lateral side of the joints (Table 13), and the decrease of inflammatory infiltrates of the synovial membrane (Table 14). No undesirable side effects of the thermogelifiable solution were detected compared to the control group, reflecting the good tolerance of the product under the conditions of the study. Table 12. Kellgren & Lawrence Scale Scale (0-4) Evaluating Inter-articular Space and Presence of Osteophytes (p-value calculated by the non-parametric method of the Mann-Whitney U-test) ) Radiological score Saline solution K & L solution (0-4) thermogelatable Number of animals 9 10 N observation 9 10 Mean score 2.0 0.0 Median score 1.8 0.4 Min score 0.0 0.0 Max score 3.0 3.0 Mean difference 0.7 0.6 Difference between the 2 groups and p-value (probability) - Statistically significant P-value = 0.0079 Table 13. Overall macroscopic score evaluating the size and severity of lesions in the lateral cartilage compartments of the tibial plateau and femoral condyles (p-value calculated by the non-parametric method of the Mann-Whitney U-test) Overall macroscopic score Saline solution Thermoceliable solution (size and severity of lesions) Number of animals 9 10 N observation 18 20 Average score 32.0 17.5 Median score 32 , 8 21.4 Min score 3.0 3.0 Max score 68.0 44.0 Mean-score 13.4 10.2 Difference between the 2 groups and p-value (probability) Statistically significant Value-p <0, 0041 Table 14- Synovial synovial membrane inflammatory infiltrate score (p-value calculated by non-parametric method of Mann-Whitney U test) Infiltrate score Saline solution Inflammatory solution (0-5) thermogelatable Number of animals 9 10 N observation 27 28 Mean score 2.0 1.4 Median score 2.6 1.8 Min score 1 0.5 Max score 5 5.0 Gap 1.0 1.0 Difference between the 2 groups and p-value (probability) Statistically significant Value p = 0.0011

权利要求:
Claims (16)
[0001]
1. Thermogelifiable composition comprising a chitosan having N-acetyl-glucosamine units, glucosamine units, and substituted glucosamine units other than N-acetyl-glucosamine units, said substituted chitosan preferably having a degree of substitution of glucosamine units ranging from from 10 to 50%, expressed as the number of moles of the substituent relative to the number of moles of total units.
[0002]
2.-thermogelifiable composition according to claim 1, characterized in that the composition has a thermoreversible sol-gel transition.
[0003]
3.- thermogelifiable composition according to claim 1 or 2, characterized in that the concentration of chitosan is less than 4%, for example less than 3%, or for example less than 2% by weight relative to the total mass of the composition (m / m).
[0004]
4.- thermogelifiable composition according to any one of claims 1 to 3, characterized in that said composition comprises a gelling agent, preferably a gelling agent inducing a sol-gel transition of the composition, for example a glycerol salt phosphate, for example in the sodium or calcium form, for example in its pentahydrate form, said gelling agent being preferably present in the composition at a concentration of between 1 and 20%, preferably 3 and 9% by weight relative to the total mass of the final composition (m / m).
[0005]
5.- thermogelifiable composition according to any one of claims 1 to 4, characterized in that it has a pH greater than or equal to 7, for example greater than or equal to 7.1, and for example a pH of 7, 2 to 8.5.
[0006]
6.- thermogelifiable composition according to any one of claims 1 to 5, characterized in that said composition has an osmolarity of 100 to 700mosm / kg, preferably 200 to 500mosm / kg.
[0007]
7.- thermogelifiable composition according to any one of claims 1 to 6, characterized in that said composition has in the fluid state a viscosity of between 20 and 800mPa.s, for example from 40 to 500mPa.s.
[0008]
8.- thermogelifiable composition according to any one of claims 1 to 7, characterized in that said composition comprises a buffer, for example an acetate buffer.
[0009]
9.- thermogelifiable composition according to any one of claims 1 to 6, characterized in that said composition comprises a reducing sugar, for example D-mannitol.
[0010]
10.- injectable composition characterized in that it comprises or consists of a thermogelifiable composition according to any one of claims 1 to 9.
[0011]
11.- injectable composition according to claim 10, characterized in that it is used as an injectable pharmaceutical composition or injectable or implantable medical device.
[0012]
12. The composition according to claim 11, for use for a therapeutic treatment, for example comprising subcutaneous, intradermal, intraocular or intra-articular injection of said composition, for example for repair or filling. at least one body tissue requiring repair or filling.
[0013]
13.- composition according to claim 12, characterized in that the body tissue is selected from the tissues belonging to the vocal cords, muscles, ligaments, tendons, cartilage, sexual organs, bones, joints, eyes, dermis, or any any of their combinations, and more particularly to joint joints.
[0014]
14. The composition of claim 12 or 13 for the treatment of osteoarthritis, or the repair of a cartilage defect, for example by injection into the synovial fluid or after mixing with the blood and implantation into the cartilage.
[0015]
15. A medical device, for example a medical implant, characterized in that it comprises or consists of a composition as defined in any one of claims 1 to 14.
[0016]
A process for preparing a thermally derivable composition comprising a substituted chitosan having N-acetyl glucosamine units, glucosamine units, and substituted glucosamine units other than N-acetyl glucosamine units; said process comprising: dissolving in an aqueous solution, preferably water, optionally buffered, preferably at a pH of between 6.2 and 8.5, and preferably between 6.5 and 7.5 substituted chitosan preferably having a degree of substitution of glucosamine units ranging from 10 to 50%, expressed as the number of moles of the substituent relative to the number of moles of total units; the eventual adjustment of the pH to a physiological pH; - mixing with a solution of sugar salt or polyol, such as a glycerol phosphate salt solution; the eventual adjustment of the pH to a physiological pH, for example by addition of a buffering agent; the possible adjustment of the osmolarity of the composition; the possible adjustment of the viscosity of the composition.

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CN1830420B|2005-03-08|2012-07-25|中国科学院过程工程研究所|Injection type pH sepsitive chitin quarternary ammonium salt aquagel and its preparation method|

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CN101972470B|2010-09-13|2012-10-17|舒泰神（北京）生物制药股份有限公司|In-situ gel composition for eyes|

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CN103834045B|2013-11-07|2016-09-28|浙江科技学院|A kind of bidirectional reversible thermosensitive hydrogel and preparation method thereof|FR3024362A1|2014-08-01|2016-02-05|Synolyne Pharma Sa|STERILIZED THERMOGELIFIABLE COMPOSITION|

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CN109810263A|2017-11-21|2019-05-28|中国科学院大连化学物理研究所|A kind of preparation method and application of chitosan thermo-sensitive gel|

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FR3074043B1|2017-11-28|2020-11-13|Kiomed Pharma|ANIONIC CHARGED CHITOSAN|

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FR3074044B1|2017-11-28|2019-11-01|Kiomed Pharma|CARBOXYALKYL CHITOSANE|

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FR3096260A1|2019-05-24|2020-11-27|Kiomed Pharma|CHITOSAN AND ITS APPLICATIONS|

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US20210236398A1|2020-01-31|2021-08-05|L'oreal|Compositions and methods for hair|

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FR3107832A1|2020-03-05|2021-09-10|L'oreal|Compositions and methods for the hair|

法律状态:
2015-08-21| PLFP| Fee payment|Year of fee payment: 2 |

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2016-02-05| PLSC| Publication of the preliminary search report|Effective date: 20160205 |

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2016-08-24| PLFP| Fee payment|Year of fee payment: 3 |

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2016-10-07| CD| Change of name or company name|Owner name: SYNOLYNE PHARMA, BE Effective date: 20160905 |

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2016-10-07| RM| Correction of a material error|Effective date: 20160905 |

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2017-07-20| PLFP| Fee payment|Year of fee payment: 4 |

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2018-07-25| PLFP| Fee payment|Year of fee payment: 5 |

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2020-05-08| ST| Notification of lapse|Effective date: 20200406 |

优先权:

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申请号 | 申请日 | 专利标题

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FR1457547|2014-08-01|

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FR1457547A|FR3024363B1|2014-08-01|2014-08-01|THERMOGELIFIABLE COMPOSITION|FR1457547A| FR3024363B1|2014-08-01|2014-08-01|THERMOGELIFIABLE COMPOSITION|

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EP15742325.2A| EP3016662A1|2014-08-01|2015-07-31|Thermogelling composition|

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PCT/EP2015/067754| WO2016016463A1|2014-08-01|2015-07-31|Thermogelling composition|

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CA2956789A| CA2956789A1|2014-08-01|2015-07-31|Thermogelling composition|

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CN201580048950.4A| CN106714780A|2014-08-01|2015-07-31|Thermogelling composition|

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US15/420,462| US9901543B2|2014-08-01|2017-01-31|Thermogelling composition|