Methods for crosslinking polymers

Abstract

The present invention relates to a method for crosslinking a polymer, the method comprising at least the following steps: a) providing a polymer; b) providing a crosslinking agent; c) performing one or more crosslinking steps in the presence of the polymer and the crosslinking agent; d) obtaining a crosslinked polymer; characterized in that the crosslinking step or each of the crosslinking steps is performed at a constant temperature or at a linearly varying or gradually varying temperature, said constant or varying temperature being less than or equal to 15°C, and characterized in that the duration of the crosslinking step c) is from 3 hours to 72 hours.

Description

[0001] The present invention relates to the field of polymer-based formulations used as biomaterials and more particularly in the medical and aesthetic fields. In all these applications, the formulations must have optimized rheological properties and must guarantee good injectability and good in vivo performance.

[0002] There is a need for a product that offers optimal characteristics and significant advantages in aesthetic applications, with good damping capacity conferred by an optimized Tan Delta (Tn Delta) and good persistence in the injection zone conferred by a very wide plastic range.

[0003] Surprisingly, it has been proven that such characteristics can be obtained using a crosslinking method under specific temperature and time conditions.

[0004] As shown in Figure 1 These characteristics can be obtained by optimizing the damping factor or tangent of the phase angle (Tan Delta (Tn Delta)) while maintaining sufficient rigidity (elastic modulus G') and increasing the plastic range, as shown in

[0005] The plastic range is characterized by the variation of the elastic modulus G' and the viscous modulus G" as a function of the deformation applied to the product.

[0006] When the deformation applied to the gel is within the plastic range, it deforms the product without breaking it. When the yield point (G' / G" intersection) is exceeded, the product yields and the damping and filling properties of the product are no longer optimal.

[0007] Figure 1 A succession of ranges observed during an oscillatory strain sweep is described.

[0008] It is generally observed that the more rigid (high G') the product, the smaller its plastic range. In essence, a rigid product will generally be more brittle and less able to deform.

[0009] Surprisingly, the method of the invention makes it possible to obtain a crosslinked product that is more rigid but also able to withstand very high levels of deformation, with the quality of a very wide plastic range.

[0010] The product obtained thus has optimal characteristics in aesthetic applications, in which its good damping capacity conferred by Tan Delta (Tn Delta) and its good persistence in the injection zone conferred by a highly optimized plastic range make it possible to offer significant advantages.

[0011] The present invention relates to a method for preparing a crosslinked polymer-based formulation, such as a crosslinked hyaluronic acid-based formulation, and more particularly to a crosslinking method that makes it possible to obtain specific characteristics, in particular an optimized Tan Delta (Tn Delta) and a wide plastic range t.

[0012] In the context of the present application, "cross-linking" is understood to mean the creation of covalent bonds between monomers of the polymer.

[0013] When cross-linking is achieved by means of a cross-linking agent, the cross-linking rate (X) can be calculated theoretically using the following formula:

[0014]

[0015] Thus, for example, if the medium comprises 100 disaccharide units, and the medium further comprises 10 molecules of cross-linking agent, the cross-linking rate (X) will be as follows: X = 10 / 100 = 0.1. This cross-linking rate is thus not influenced by the degree of polymerization, the molecular weight of the chosen polymer, or the proportion of cross-linking agent that actually reacts with at least one function of the polymer. It only takes into account the theoretical determination of the amount of cross-linking agent placed in contact and of the repeating units.

[0016] Cross-linking can also be evaluated a posteriori (after cross-linking) by means of the degree of modification (Mod). Unlike the cross-linking rate X, Mod takes into account the proportion of cross-linking agent that actually reacts with at least one function of the polymer.

[0017] The degree of modification can be expressed as follows:

[0018]

[0019] When the polymer is hyaluronic acid, the repeating unit (or monomer) is a disaccharide unit.

[0020] The determination of the values of the numerator and the denominator depends on the chosen polymer and the chosen cross-linking agent, and is well known to the person skilled in the art. For example, in the particular case of a hyaluronic acid-based formulation cross-linked with BDDE, the method described in the publication L. Nord, A. Emilson, C. Sturesson, A. H. Kenne, Degree of Modification of Hyaluronic Acid Dermal Fillers, EADV 18th Congress, Berlin, 2009, can be used.

[0021] In the particular case of a hyaluronic acid-based formulation cross-linked with BDDE, the degree of modification can be expressed as follows:

[0022]

[0023] For example, a Mod of 1% for a hyaluronic acid-based formulation cross-linked with BDDE indicates that it has one (single or double bond) BDDE molecule per 100 disaccharide units.

[0024] Traditionally, the cross-linking step is carried out at a temperature much higher than ambient temperature for a relatively short time.

[0025] Thus, for example in example 1 of the application WO2009071697 in the name of the Applicant, the cross-linking conditions are as follows: 50°C for 2 hours 20 minutes (2:20). These cross-linking conditions are quite conventional and are applied almost systematically.

[0026] It has only been proposed recently to use cross-linking temperatures lower than those conventionally used.

[0027] The application CN108774330 in the name of the company Huaxi Furuida Biopharmaceutical Co., Ltd. proposes to implement the cross-linking at varying temperatures in the case of the preparation of a formulation intended to be applied to the skin. More particularly, for example in example 2, a cross-linking is proposed in which the temperature is in turn 1 °C to 4 °C, then 50 °C, and the operation is repeated a number of times. The conclusion of table 1 is that the temperature is ideal in the high temperature phase, 50 °C to 80 °C. It should be noted that the formulation disclosed is a biphasic formulation for external use (not injected, only applied to the skin), and there is no mention of the rheology of the formulation and of the quantification of the cross-linking carried out. Finally, in this application, it is not determined that the cross-linking occurs at 1 °C to 4 °C.

[0028] The application CN107936272 in the name of the company Huaxi Furuida Biopharmaceutical Co., Ltd. proposes a cross-linking method which also provides for alternation of low temperature (0 °C to 10 °C) and high temperature (30 °C to 60 °C). It should be noted that there is no mention of the rheology of the formulation.

[0029] The application CN108774330 in the name of the company Huaxi Furuida Biopharmaceutical Co., Ltd. proposes a cross-linking method which also provides for alternation of low temperature (1 °C to 4 °C) and high temperature (50 °C to 80 °C).

[0030] The application W017016917 in the name of the company GALDERMA SA proposes a cross-linking with a high concentration of hydroxyl ions (1.5% to 8%), a high concentration of hyaluronic acid (more than 10%) and very specific temperature and time conditions. For example, the method of example 3 corresponds to the following conditions: 29 °C, 16 hours.

[0031] The application CN103146003 in the name of the company Shanghai Qisheng Biological Preparations Co., Ltd. discloses a cross-linking method which also comprises alternation of low temperature and high temperature, for example in embodiment 1, the cross-linking starts at 4 °C and ends at 40 °C. It should be noted that there is no mention of the rheology of the formulation or of the effect of the low temperature on the cross-linking reaction.

[0032] Application US2010 / 0261893, filed under the name of Tor-Chern Chen, discloses examples of crosslinking at temperatures ranging from 10°C to 30°C, specifically aimed at reducing the percentage of crosslinking agent containing free ends after the reaction. When the method operates at temperatures below 20°C, the reaction time is typically greater than 10 days and can be as long as 28 days. Furthermore, no analysis of rheological properties was performed; the goal was solely to reduce the crosslinking agent content in the finished product.

[0033] Application KR1018666678, filed in the name of Seoul National University, discloses an exemplary embodiment of a method for crosslinking at temperatures below 20°C and with a reaction time greater than 14 days.

[0034] In summary, in the above applications, the reaction time for complete or partial crosslinking at temperatures above 30°C is less than one day, or the reaction time is very long when the crosslinking temperature is below 20°C. Furthermore, when describing rheological properties, the rheological properties do not correspond to those sought and obtained under the conditions according to the present invention.

[0035] As stated above, the applicant has demonstrated that polymer-based formulations with particularly advantageous rheological properties and good damping capacity imparted by optimized TanΔ (Tnδ) and good durability in the injection region imparted by a very wide plasticity range can be obtained by crosslinking only at low temperatures (below or equal to 15°C) for a time suitable for industrial production, such as 3 to 72 hours.

[0036] For example, in the case of hyaluronic acid-based formulations, it has been demonstrated that using a low crosslinking temperature enables the following:

[0037] - The acquisition of polymers with a modification degree Mod (%) less than that obtained at higher temperatures, wherein:

[0038] The elastic modulus G' is optimized and is always less than 1000 Pa;

[0039] The viscosity modulus G” was optimized (the value of G” was significantly higher than that of the formulation based on the prior art);

[0040] o and;

[0041] - Reduced degradation of the polymer during crosslinking;

[0042] - Optimization of the value of TanΔ(Tnδ) (the ratio of viscous modulus G” to elastic modulus G’) to obtain formulations that can better withstand stresses associated with product deformation with an improved target value in the range of 0.25 to 1 (0.25≤TanΔ(Tnδ)≤1);

[0043] - Excellent injectability;

[0044] - Reduced energy consumption (relative to high temperatures, or even relative to changing temperatures);

[0045] - A reliable, repeatable method that requires almost no human intervention;

[0046] - A simplified method;

[0047] - Highly effective and achieves minimal modification of the polymer for crosslinking to ensure better biocompatibility (achieving good rheological properties with fairly low Mod).

[0048] Even more surprisingly, it has been demonstrated that when preparations based on formulations of several polymers (which may be, for example, hyaluronic acid) crosslinked at low temperatures prior to interpenetration by mixing are prepared according to the method of the invention, the formulations obtained by the method of the invention have further improved properties.

[0049] For example, as will be demonstrated in the examples, the crosslinking method according to the invention allows for obtaining a particularly advantageous TanΔ value (target value > 0.25). It has been observed that for values ​​of TanΔ > 0.25, the resulting material exhibits reduced brittleness and increased deformability; this appears ideal for medical filling applications where damping of deformation is important. In aesthetically specific cases, this property is a significant advantage due to its natural correction after injection. Therefore, in this method, we seek to optimize the damping factor while maintaining a satisfactory and prior art-comparable stiffness G'.

[0050] Finally, it was demonstrated that the crosslinking efficiency was very good due to the very good rheological properties obtained with a relatively low Mod (%); this makes it possible to ensure improved biocompatibility.

[0051] This invention relates to a method for crosslinking polymers, the method comprising at least the following steps:

[0052] a) Provide polymers;

[0053] b) Provide crosslinking agents;

[0054] c) Perform one or more crosslinking steps in the presence of the polymer and the crosslinking agent;

[0055] d) Obtain cross-linked polymers;

[0056] The crosslinking step or each of the crosslinking steps is carried out at a constant temperature or at a temperature that varies linearly or gradually, wherein the constant or varying temperature is less than or equal to 15°C (temperature ≤ 15°C).

[0057] The method for crosslinking a polymer according to the present invention is further characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 1000 Pa.

[0058] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 800 Pa.

[0059] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 600 Pa.

[0060] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 500 Pa.

[0061] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 300 Pa.

[0062] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 200 Pa.

[0063] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 100 Pa.

[0064] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the crosslinked polymer obtained in step d) has a G' ≤ 50 Pa.

[0065] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the value of G' of the crosslinked polymer obtained in step d) is in the range of 50 Pa to 610 Pa (50 ≤ G' ≤ 610).

[0066] The method for crosslinking a polymer according to the present invention is further characterized in that the value of TanΔ(Tnδ) of the crosslinked polymer obtained in step d) is 0.25 to 1 (0.25 ≤ TanΔ(Tnδ)) ≤ 1).

[0067] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the value of TanΔ(Tnδ) of the crosslinked polymer obtained in step d) is 0.50 to 1 (0.50 ≤ TanΔ(Tnδ)) ≤ 1).

[0068] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the value of TanΔ(Tnδ) of the crosslinked polymer obtained in step d) is 0.75 to 1 (0.75 ≤ TanΔ(Tnδ)) ≤ 1).

[0069] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the tan value of the crosslinked polymer obtained in step d) is in the range of 0.3 to 0.6 (0.3 ≤ TanΔ(Tnδ) ≤ 0.6).

[0070] The present invention also relates to polymers obtained by the method according to the invention.

[0071] In one embodiment, the polymer according to the invention has a G' ≤ 1000 Pa and a TanΔ(Tnδ) value of 0.25 to 1 (0.25 ≤ TanΔ(Tnδ)) ≤ 1).

[0072] In one embodiment, the polymer according to the invention has a G' ≤ 800 Pa.

[0073] In one embodiment, the polymer according to the invention has a G' ≤ 600 Pa.

[0074] In one embodiment, the polymer according to the invention has a G' ≤ 500 Pa.

[0075] In one embodiment, the polymer according to the invention has a G' ≤ 300 Pa.

[0076] In one embodiment, the polymer according to the invention has a G' ≤ 200 Pa.

[0077] In one embodiment, the polymer according to the invention has a G' ≤ 100 Pa.

[0078] In one embodiment, the polymer according to the invention has a G' ≤ 50 Pa.

[0079] In one embodiment, the value of G' of the polymer according to the invention is in the range of 50 Pa to 610 Pa (50 ≤ G' ≤ 610).

[0080] In one embodiment, the value of TanΔ(Tnδ) of the polymer according to the invention is 0.50 to 1 (0.50 ≤ tanΔ(Tnδ)) ≤ 1).

[0081] In one embodiment, the value of TanΔ(Tnδ) of the polymer according to the invention is 0.75 to 1 (0.75 ≤ TanΔ(Tnδ)) ≤ 1).

[0082] In one embodiment, the value of TanΔ(Tnδ) of the polymer according to the invention is 0.3 to 0.6 (0.3 ≤ TanΔ(Tnδ) ≤ 0.6).

[0083] In one embodiment, the polymer according to the invention is characterized in that it is selected from the group consisting of polysaccharides.

[0084] In one embodiment, the polymer according to the invention is characterized in that it comprises a mixture of polymers.

[0085] In one embodiment, the polymer according to the invention is hyaluronic acid or a mixture of hyaluronic acid salts.

[0086] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the crosslinking step or each of the crosslinking steps is carried out at a constant temperature.

[0087] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that at least one crosslinking step is carried out at a varying temperature.

[0088] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that at least one crosslinking step is carried out at a linearly varying temperature.

[0089] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that at least one crosslinking step is carried out at a temperature that varies in a stepwise manner.

[0090] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the constant or variable temperature is less than or equal to 15°C (temperature ≤ 15°C).

[0091] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that at least 90% of the crosslinking is carried out at a constant or varying temperature (temperature ≤ 15°C) below or equal to 15°C.

[0092] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that at least 80% of the crosslinking is carried out at a constant or varying temperature (temperature ≤ 15°C) below or equal to 15°C.

[0093] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that at least 70% of the crosslinking is carried out at a constant or varying temperature (temperature ≤ 15°C) below or equal to 15°C.

[0094] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the crosslinking step at a constant or varying temperature (temperature ≤ 15°C) below or equal to 15°C accounts for at least 90% of the contact time with the reagent.

[0095] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the crosslinking step at a constant or varying temperature (temperature ≤ 15°C) below or equal to 15°C accounts for at least 80% of the contact time with the reagent.

[0096] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the crosslinking step at a constant or varying temperature (temperature ≤ 15°C) below or equal to 15°C accounts for at least 70% of the contact time with the reagent.

[0097] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the constant or variable temperature is less than or equal to 12°C (temperature ≤ 12°C).

[0098] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the constant or variable temperature is less than or equal to 10°C (temperature ≤ 10°C).

[0099] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the constant or variable temperature is less than or equal to 9°C (temperature ≤ 9°C).

[0100] The solidification temperature of the reaction medium should be understood as the temperature at which the medium solidifies. For aqueous media, this temperature will be 0°C or slightly lower as a function of the salt concentration in the medium.

[0101] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the constant or varying temperature is between the solidification temperature and 15°C.

[0102] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the constant or variable temperature is between the solidification temperature and 10°C.

[0103] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the constant or variable temperature is between the solidification temperature and 9°C.

[0104] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized by performing a step for dissolving the polymer prior to step c).

[0105] The polymer is dissolved by adding water or a saline solution (e.g., a phosphate buffer solution, such as PBS) or by adding sodium hydroxide or an acid solution to obtain a pH suitable for carrying out the crosslinking reaction.

[0106] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that, at the latest in step c), a step for adjusting the pH to the crosslinking pH is performed.

[0107] pH adjustment is achieved by adding a preferred inorganic acid solution (e.g., hydrochloric acid) or a preferred inorganic base (e.g., sodium hydroxide or potassium hydroxide) in an amount such that the target crosslinking pH can be obtained.

[0108] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, at the latest in step c), a step of adjusting the pH to a crosslinking pH suitable for the crosslinking agent is performed.

[0109] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, at the latest in step c), a step for adjusting the pH to a crosslinking pH greater than 10 is performed.

[0110] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, at the latest in step c), a step for adjusting the pH to a crosslinking pH of less than 3 is performed.

[0111] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, at the latest in step c), a step for adjusting the pH to a crosslinking pH greater than 10 is performed, wherein the crosslinking agent is BDDE.

[0112] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, at the latest in step c), a step for adjusting the pH to a crosslinking pH of less than 3 is performed, wherein the crosslinking agent is BDDE.

[0113] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, at the latest in step c), a step for adjusting the pH to a crosslinking pH greater than 10 is performed.

[0114] Crosslinking begins when the following three conditions are combined: the presence of a polymer, the presence of a crosslinking agent, and a reaction medium at an appropriate pH.

[0115] In one embodiment, the method for crosslinking a polymer according to the invention is characterized by initiating crosslinking by adding the crosslinking agent.

[0116] In one embodiment, the method for crosslinking a polymer according to the invention is characterized by initiating crosslinking by adding the polymer.

[0117] In one embodiment, the method for crosslinking a polymer according to the invention is characterized by initiating crosslinking by applying a crosslinking pH.

[0118] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that, after step c), a step for adjusting the pH to 6 to 8 is performed.

[0119] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that, after step c), a step for adjusting the pH to 6 to 8 is performed.

[0120] As a function of the pH of the reaction medium at the end of the crosslinking reaction, the pH is adjusted by adding a preferred inorganic acid solution (e.g., hydrochloric acid) or a preferred inorganic base (e.g., sodium hydroxide or potassium hydroxide) in an amount such that a pH of 6 to 8 can be obtained.

[0121] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, after step c), the step of adjusting the pH to a pH of 6 to 8 is carried out by adding at least one acid, namely hydrochloric acid (HCl).

[0122] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized by a purification step prior to step d).

[0123] In one embodiment, the method for crosslinking a polymer according to the invention is characterized by performing a purification step by dialysis prior to step d).

[0124] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that, prior to step d), the step of purification by dialysis is performed using a dialysis solution or solvent selected from phosphate buffers such as PBS and water.

[0125] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized by performing a step for removing the crosslinking agent prior to step d).

[0126] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized by performing a step of cooling to the crosslinking temperature prior to step c).

[0127] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the polymer in step a) is a mixture of polymers.

[0128] In the context of this application, during step a), all the referenced polymers may be placed in contact as a mixture of polymers having the same properties (e.g., a mixture of hyaluronic acids having a wide variety of molecular weights) or a mixture of polymers having different properties (e.g., a mixture of hyaluronic acid and chitosan). During the crosslinking step, co-crosslinking may occur between a wide variety of polymers.

[0129] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the polymer in step a) is a mixture of hyaluronic acid or hyaluronic acid salt.

[0130] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the polymer in step a) is a mixture of two hyaluronic acids or hyaluronic acid salts.

[0131] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the polymer in step a) is a mixture of three hyaluronic acids or hyaluronic acid salts.

[0132] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the polymer in step a) is a mixture of four hyaluronic acids or hyaluronic acid salts.

[0133] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the contact between the polymer and at least one crosslinking agent occurs in a solvent.

[0134] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is selected from ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether (BDDE), polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, diepoxides or polyepoxides (e.g., 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane), dialkyl sulfones, divinyl sulfones, formaldehyde, epichlorohydrin or glutaraldehyde, carbodiimide (e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)), and trimetaphosphates (e.g., sodium trimetaphosphate, calcium trimetaphosphate or barium trimetaphosphate).

[0135] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is selected from ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether (BDDE), polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, diepoxides or polyepoxides (e.g., 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane), and trimetaphosphates (e.g., sodium trimetaphosphate, calcium trimetaphosphate or barium trimetaphosphate).

[0136] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is selected from ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether (BDDE), polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, diepoxides or polyepoxides (e.g., 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane).

[0137] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is selected from trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate or barium trimetaphosphate.

[0138] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is selected from epoxides, such as 1,4-butanediol diglycidyl ether (BDDE), epoxy halopropane, and divinyl sulfone (DVS).

[0139] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is divinyl sulfone (DVS).

[0140] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the at least one crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0141] In the context of this application, BDDE is particularly preferred.

[0142] In one embodiment, the method for crosslinking a polymer according to the invention is characterized in that the at least one crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE), and step c) is carried out at a pH greater than 10.

[0143] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is from 3 hours to 72 hours.

[0144] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is from 3 hours to 60 hours.

[0145] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is from 3 hours to 50 hours.

[0146] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is from 5 hours to 50 hours.

[0147] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is 10 hours to 50 hours.

[0148] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is 15 hours to 48 hours.

[0149] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is 20 to 30 hours.

[0150] In one embodiment, the method for crosslinking a polymer according to the present invention is characterized in that the duration of the crosslinking step c) is 21 to 28 hours.

[0151] When multiple consecutive crosslinking operations are performed, the duration mentioned refers to the total duration (the sum of the durations of consecutive crosslinking operations).

[0152] In one embodiment, during step c), the crosslinking step is carried out in the presence of the polymer and the crosslinking agent in a reaction medium in which the polymer is hydrated and / or swollen.

[0153] In one embodiment, during step c), the crosslinking step is carried out in the presence of the polymer and the crosslinking agent in a reaction medium in which the polymer is hydrated and / or swollen by adding water or an aqueous salt solution (e.g., a phosphate buffer solution, such as PBS).

[0154] In one embodiment, during step c) of the crosslinking step in the presence of the polymer and the crosslinking agent, the polymer concentration is from 0.05% by mass to 30% by mass relative to the total mass of the crosslinking reaction medium.

[0155] In one embodiment, during step c) of the crosslinking step in the presence of the polymer and the crosslinking agent, the polymer concentration is from 1% to 30% by mass relative to the total mass of the reaction medium.

[0156] In one embodiment, during step c) of the crosslinking step in the presence of the polymer and the crosslinking agent, the polymer concentration is from 5% to 25% by mass relative to the total mass of the reaction medium.

[0157] In one embodiment, during step c) of the crosslinking step in the presence of the polymer and the crosslinking agent, the polymer concentration is 10% to 15% by mass relative to the total mass of the reaction medium.

[0158] In one embodiment, during step c) the crosslinking step is carried out in the presence of hyaluronic acid alone or in mixtures, or any biologically acceptable salt thereof, and the crosslinking agent, the concentration of hyaluronic acid is from 0.05% by mass to 30% by mass relative to the total mass of the reaction medium.

[0159] In one embodiment, during step c), the hyaluronic acid concentration is from 1% to 30% by mass relative to the total mass of the reaction medium, in the presence of hyaluronic acid alone or in mixtures with any biologically acceptable salt thereof and the crosslinking agent during the crosslinking step in the crosslinking reaction medium.

[0160] In one embodiment, during step c), the crosslinking step is carried out in the presence of hyaluronic acid alone or in mixtures thereof, or any biologically acceptable salt thereof, and the crosslinking agent, with the polymer concentration being 5% to 25% by mass relative to the total mass of the reaction medium.

[0161] In one embodiment, during step c), the hyaluronic acid concentration is 10% to 15% by mass relative to the total mass of the reaction medium, during the crosslinking step performed in the presence of hyaluronic acid alone or in mixtures of hyaluronic acid or any biologically acceptable salt thereof and the crosslinking agent.

[0162] In one embodiment, during step c), the crosslinking reaction medium comprises sodium hydroxide (NaOH) in the presence of the polymer and the crosslinking agent.

[0163] In one embodiment, during step c) of the crosslinking step in the presence of the polymer and the crosslinking agent, the sodium hydroxide concentration is 0.5% to 1.5% by mass relative to the total mass of the reaction medium.

[0164] In one embodiment, during step c) of the crosslinking step in the presence of the polymer and the crosslinking agent, the sodium hydroxide concentration is 0.8% to 1% by mass relative to the total mass of the reaction medium.

[0165] The present invention also relates to a method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention.

[0166] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step for hydration and / or swelling.

[0167] In one embodiment, the steps for hydration and / or swelling in the liquid are performed by adding water or an aqueous salt solution (e.g., a phosphate buffer solution, such as PBS).

[0168] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of hydrating and / or swelling in an aqueous solution to obtain a polysaccharide concentration of 2 mg / g to 50 mg / g relative to the total mass of the formulation.

[0169] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of hydrating and / or swelling in an aqueous solution to obtain a polysaccharide concentration of 4 mg / g to 40 mg / g relative to the total mass of the formulation.

[0170] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of hydrating and / or swelling in an aqueous solution to obtain a polysaccharide concentration of 5 mg / g to 30 mg / g relative to the total mass of the formulation.

[0171] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of hydrating and / or swelling in an aqueous solution to obtain a polysaccharide concentration of 10 mg / g to 30 mg / g relative to the total mass of the formulation.

[0172] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of hydrating and / or swelling in an aqueous solution to obtain a polysaccharide concentration of about 20 mg / g relative to the total mass of the formulation.

[0173] In one embodiment, a method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention includes a step of uniformly mixing Y identical or different crosslinked polymers crosslinked prior to interpenetration by mixing, wherein Y is 2 to 5, characterized in that at least one of the Y polymers is crosslinked according to the crosslinking method of the invention.

[0174] In one embodiment, Y = 2, and one polymer is cross-linked according to the method for preparing a cross-linked polymer according to the present invention.

[0175] In one embodiment, Y = 2, and the two polymers are cross-linked according to the method for preparing cross-linked polymers according to the present invention.

[0176] In one embodiment, Y = 3, and one polymer is cross-linked according to the method for preparing a cross-linked polymer according to the present invention.

[0177] In one embodiment, Y = 3, and the two polymers are cross-linked according to the method for preparing cross-linked polymers according to the present invention.

[0178] In one embodiment, Y = 3, and the three polymers are cross-linked according to the method for preparing cross-linked polymers according to the present invention.

[0179] In one embodiment, Y = 2, and the two polymers are hyaluronic acid or hyaluronic acid salts with different molecular weights.

[0180] In one embodiment, the Y polymers are mixed before each of the Y polymers swells.

[0181] In one embodiment, the Y polymers are mixed after each of the Y polymers has swelled.

[0182] In one embodiment, the Y polymers are mixed before swelling.

[0183] In one embodiment, the Y polymers are mixed after swelling.

[0184] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one final sterilization step.

[0185] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer according to the invention further includes a final sterilization step.

[0186] In one embodiment, the final sterilization step is performed by heat, by moist heat, by gamma radiation, or by an accelerated electron beam.

[0187] In one embodiment, the final sterilization step is performed by autoclaving.

[0188] In one implementation, autoclaving is performed for F0 ≥ 4 minutes.

[0189] In one implementation, autoclaving is performed for F0 ≥ 10 minutes.

[0190] In one implementation, autoclaving is performed for F0 ≥ 15 minutes.

[0191] In one implementation, autoclaving is performed for F0 ≥ 20 minutes.

[0192] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one active agent.

[0193] In one embodiment, at least one active agent is added in powder form.

[0194] In one embodiment, at least one active agent is added in the form of a solution or suspension.

[0195] In one embodiment, at least one active agent is added in solution or suspension form to a solvent or solution selected from water and aqueous saline solutions (e.g., phosphate buffer solutions, such as PBS).

[0196] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one active ingredient selected from local anesthetics, vitamin C derivatives, anti-inflammatory agents, polyols, and mixtures thereof.

[0197] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic.

[0198] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic to obtain a local anesthetic concentration of 0.1% to 5% relative to the total mass of the formulation.

[0199] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic to obtain a local anesthetic concentration of 0.1% to 4% relative to the total mass of the formulation.

[0200] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic to obtain a local anesthetic concentration of 0.1% to 2% relative to the total mass of the formulation.

[0201] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic to obtain a local anesthetic concentration of 0.1% to 1% relative to the total mass of the formulation.

[0202] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic to obtain a local anesthetic concentration of 0.1% to 0.5% relative to the total mass of the formulation.

[0203] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic to obtain a local anesthetic concentration of about 0.3% relative to the total mass of the formulation.

[0204] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic selected from lidocaine, mepivacaine, and mixtures thereof.

[0205] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic, namely lidocaine.

[0206] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely lidocaine, to obtain a lidocaine concentration of 0.1% to 5% relative to the total mass of the formulation.

[0207] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely lidocaine, to obtain a lidocaine concentration of 0.1% to 4% relative to the total mass of the formulation.

[0208] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely lidocaine, to obtain a lidocaine concentration of 0.1% to 2% relative to the total mass of the formulation.

[0209] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely lidocaine, to obtain a lidocaine concentration of 0.1% to 1% relative to the total mass of the formulation.

[0210] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely lidocaine, to obtain a lidocaine concentration of 0.1% to 0.5% relative to the total mass of the formulation.

[0211] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic, namely lidocaine, to obtain a local anesthetic concentration of about 0.3% relative to the total mass of the formulation.

[0212] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine.

[0213] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine, to obtain a mepivacaine concentration of 0.1% to 5% relative to the total mass of the formulation.

[0214] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine, to obtain a mepivacaine concentration of 0.1% to 4% relative to the total mass of the formulation.

[0215] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine, to obtain a mepivacaine concentration of 0.1% to 2% relative to the total mass of the formulation.

[0216] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine, to obtain a mepivacaine concentration of 0.1% to 1% relative to the total mass of the formulation.

[0217] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine, to obtain a mepivacaine concentration of 0.1% to 0.5% relative to the total mass of the formulation.

[0218] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one local anesthetic, namely mepivacaine, to obtain a mepivacaine concentration of about 0.3% relative to the total mass of the formulation.

[0219] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one vitamin C derivative.

[0220] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one vitamin C derivative selected from ascorbic acid phosphate (e.g., magnesium ascorbic acid phosphate, sodium ascorbic acid phosphate), ascorbic acid glycoside (e.g., ascorbic acid-2-glucosidase) and mixtures thereof.

[0221] In one embodiment, the at least one vitamin C derivative is magnesium ascorbate phosphate.

[0222] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one anti-inflammatory agent.

[0223] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one anti-inflammatory agent selected from steroidal anti-inflammatory agents and nonsteroidal anti-inflammatory agents.

[0224] In one embodiment, the at least one anti-inflammatory agent is selected from steroidal anti-inflammatory agents (e.g., dexamethasone, prednisolone, corticosteroids, budesonide, sulfasalazine, mesalamine, cetirizine, diphenhydramine, antipyrine, methyl salicylate, loratadine, thymol, isothymol, bisabolol, allantoin, eucalyptol, ampicillin (antipyrine), profenazole) and nonsteroidal anti-inflammatory agents (e.g., ibuprofen, naproxen, fenofofen, ketoprofen, flurbiprofen, oxaprazine). Indomethacin, sulindac, etodoxacin, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, doxicam, lornoxicam, isoxicam, mefenamic acid, flufenamic acid, tofenamic acid, celecoxib, lofencoxib, valdecoxib, parecoxib, rumicoxib, ilecoxib, firocoxib, or sucrose octasulfate and / or its salts).

[0225] In one embodiment, the at least one anti-inflammatory agent is selected from sucrose octasulfate and its salts.

[0226] In one embodiment, the at least one anti-inflammatory agent is selected from sucrose octasulfate and its sodium and potassium salts.

[0227] In one embodiment, the water-soluble salt of the sucrose octasulfate is selected from alkali metal salts, alkaline earth metal salts, silver salts, ammonium salts, and amino acid salts.

[0228] In one embodiment, the water-soluble salt of sucrose octasulfate is selected from alkali metal salts or alkaline earth metal salts.

[0229] In one embodiment, the water-soluble salt of sucrose octate is sodium sucrose octate or potassium sucrose octate.

[0230] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol.

[0231] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol selected from mannitol, sorbitol, glycerol, maltitol, lactitol and erythritol.

[0232] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol selected from mannitol, sorbitol and glycerol.

[0233] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol to obtain a polyol concentration of 0.1 mg / ml to 50 mg / ml relative to the total mass of the formulation.

[0234] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol to obtain a polyol concentration of 5 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0235] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol to obtain a polyol concentration of 10 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0236] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol to obtain a polyol concentration of 20 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0237] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol to obtain a polyol concentration of 30 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0238] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol, namely mannitol.

[0239] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding mannitol to obtain a mannitol concentration of 5 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0240] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding mannitol to obtain a mannitol concentration of 10 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0241] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding mannitol to obtain a mannitol concentration of 20 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0242] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding mannitol to obtain a mannitol concentration of 30 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0243] In one embodiment, the method for preparing an formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding at least one polyol, namely sorbitol.

[0244] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding sorbitol to obtain a sorbitol concentration of 5 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0245] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding sorbitol to obtain a sorbitol concentration of 10 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0246] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the invention further includes at least one step of adding sorbitol to obtain a sorbitol concentration of 20 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0247] In one embodiment, the method for preparing a formulation comprising at least one crosslinked polymer obtained according to the method of the present invention further includes at least one step of adding sorbitol to obtain a sorbitol concentration of 30 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0248] The present invention also relates to formulations comprising at least one crosslinked polymer obtained according to the method of the present invention.

[0249] In one embodiment, the formulation is characterized in that the polymer concentration is from 2 mg / g to 50 mg / g relative to the total mass of the formulation.

[0250] In one embodiment, the formulation is characterized in that the polymer concentration is from 4 mg / g to 40 mg / g relative to the total mass of the formulation.

[0251] In one embodiment, the formulation is characterized in that the polymer concentration is from 5 mg / g to 30 mg / g relative to the total mass of the formulation.

[0252] In one embodiment, the formulation is characterized in that the polymer concentration is from 10 mg / g to 30 mg / g relative to the total mass of the formulation.

[0253] In one embodiment, the formulation is characterized in that the polymer concentration is about 20 mg / g relative to the total mass of the formulation.

[0254] In one embodiment, the formulation is characterized by being injectable.

[0255] In one embodiment, the formulation is characterized by being sterile.

[0256] In one embodiment, the formulation is characterized by being monophasic.

[0257] In one embodiment, the formulation is characterized by being injectable and sterile.

[0258] In one embodiment, the formulation is characterized as being injectable, sterile, and monophasic.

[0259] In one embodiment, the formulation comprising at least one polymer crosslinked according to the method of the invention comprises a homogeneous mixture of Y identical or different crosslinked polymers crosslinked prior to interpenetration by mixing, wherein Y is 2 to 5, characterized in that at least one of the Y polymers is crosslinked according to the method of the invention for preparing crosslinked polymers.

[0260] In one embodiment, Y = 2, and one polymer is cross-linked according to the method for preparing a cross-linked polymer according to the present invention.

[0261] In one embodiment, Y = 2, and the two polymers are cross-linked according to the method for preparing cross-linked polymers according to the present invention.

[0262] In one embodiment, Y = 3, and one polymer is cross-linked according to the method for preparing a cross-linked polymer according to the present invention.

[0263] In one embodiment, Y = 3, and the two polymers are cross-linked according to the method for preparing cross-linked polymers according to the present invention.

[0264] In one embodiment, Y = 3, and the three polymers are cross-linked according to the method for preparing cross-linked polymers according to the present invention.

[0265] In one embodiment, Y = 2, and the two polymers are hyaluronic acid or hyaluronic acid salts with different molecular weights.

[0266] In one embodiment, the formulation further comprises at least one active agent selected from the group consisting of local anesthetics, vitamin C derivatives, anti-inflammatory agents, polyols, and mixtures thereof.

[0267] In one embodiment, the formulation further comprises at least one local anesthetic.

[0268] In one embodiment, the mass percentage of the at least one local anesthetic is 0.1% to 5% relative to the total mass of the preparation.

[0269] In one embodiment, the mass percentage of the at least one local anesthetic is 0.1% to 4% relative to the total mass of the preparation.

[0270] In one embodiment, the mass percentage of the at least one local anesthetic is 0.1% to 2% relative to the total mass of the preparation.

[0271] In one embodiment, the mass percentage of the at least one local anesthetic is 0.1% to 1% relative to the total mass of the preparation.

[0272] In one embodiment, the mass percentage of the at least one local anesthetic is 0.1% to 0.5% relative to the total mass of the preparation.

[0273] In one embodiment, the mass percentage of the at least one local anesthetic is about 0.3% relative to the total mass of the preparation.

[0274] In one embodiment, the formulation further comprises at least one active agent.

[0275] In one embodiment, the formulation further comprises at least one local anesthetic selected from lidocaine, mepivacaine, and mixtures thereof.

[0276] In one embodiment, the local anesthetic is lidocaine.

[0277] In one embodiment, the local anesthetic is lidocaine at a mass percentage of 0.1% to 5% relative to the total mass of the preparation.

[0278] In one embodiment, the local anesthetic is lidocaine, and the mass percentage of lidocaine relative to the total mass of the preparation is 0.1% to 4%.

[0279] In one embodiment, the local anesthetic is lidocaine, and the mass percentage of lidocaine relative to the total mass of the preparation is 0.1% to 2%.

[0280] In one embodiment, the local anesthetic is lidocaine, and the mass percentage of lidocaine relative to the total mass of the preparation is 0.1% to 1%.

[0281] In one embodiment, the local anesthetic is lidocaine, and the mass percentage of lidocaine relative to the total mass of the preparation is 0.1% to 0.5%.

[0282] In one embodiment, the local anesthetic is mepivacaine.

[0283] In one embodiment, the local anesthetic is mepivacaine, and the mass percentage of mepivacaine relative to the total mass of the preparation is 0.1% to 5%.

[0284] In one embodiment, the local anesthetic is mepivacaine, and the mass percentage of mepivacaine relative to the total mass of the preparation is 0.1% to 4%.

[0285] In one embodiment, the local anesthetic is mepivacaine, and the mass percentage of mepivacaine relative to the total mass of the preparation is 0.1% to 2%.

[0286] In one embodiment, the local anesthetic is mepivacaine, and the mass percentage of mepivacaine relative to the total mass of the preparation is 0.1% to 1%.

[0287] In one embodiment, the local anesthetic is mepivacaine, and the mass percentage of mepivacaine relative to the total mass of the preparation is 0.1% to 0.5%.

[0288] In one embodiment, the formulation further comprises at least one vitamin C derivative.

[0289] In one embodiment, the at least one vitamin C derivative is selected from ascorbate phosphate (e.g., magnesium ascorbate phosphate, sodium ascorbate phosphate), ascorbate glycoside (e.g., ascorbate-2-glucosidate) and mixtures thereof.

[0290] In one embodiment, the at least one vitamin C derivative is magnesium ascorbate phosphate.

[0291] In one embodiment, the formulation further comprises at least one anti-inflammatory agent.

[0292] In one embodiment, the at least one anti-inflammatory agent is selected from steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents.

[0293] In one embodiment, the at least one anti-inflammatory drug is selected from steroidal anti-inflammatory agents (e.g., dexamethasone, prednisolone, corticosteroids, budesonide, sulfasalazine, mesalamine, cetirizine, diphenhydramine, antipyrine, methyl salicylate, loratadine, thymol, isothymol, bisabolol, allantoin, eucalyptol, ampicillin (antipyrine), profenazole) and nonsteroidal anti-inflammatory agents (e.g., ibuprofen, naproxen, etc.). Noprofen, ketoprofen, flurbiprofen, oxaprazine, indomethacin, sulindac, etodoxacin, ketoroxyprofen, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, doxicam, lornoxicam, isoxicam, mefenamic acid, flufenamic acid, tofenamic acid, celecoxib, lofencoxib, vardicoxib, parecoxib, rumicoxib, etorcoxib, filocoxib, or sucrose octasulfate and / or its salts).

[0294] In one embodiment, the at least one anti-inflammatory agent is selected from sucrose octasulfate and its salts.

[0295] In one embodiment, the at least one anti-inflammatory agent is selected from sucrose octasulfate and its sodium and potassium salts.

[0296] In one embodiment, the water-soluble salt of the sucrose octasulfate is selected from alkali metal salts, alkaline earth metal salts, silver salts, ammonium salts, and amino acid salts.

[0297] In one embodiment, the water-soluble salt of sucrose octasulfate is selected from alkali metal salts or alkaline earth metal salts.

[0298] In one embodiment, the water-soluble salt of sucrose octate is sodium sucrose octate or potassium sucrose octate.

[0299] In one embodiment, the formulation further comprises at least one polyol.

[0300] In one embodiment, the formulation further comprises at least one polyol selected from mannitol, sorbitol, glycerol, maltitol, lactitol, and erythritol.

[0301] In one embodiment, the formulation further comprises at least one polyol selected from mannitol, sorbitol, and glycerol.

[0302] In one embodiment, the formulation further comprises at least mannitol.

[0303] In one embodiment, the mass percentage of the polyol relative to the total mass of the formulation is from 0.1 mg / ml to 50 mg / ml.

[0304] In one embodiment, the mass percentage of the polyol relative to the total mass of the preparation is from 5 mg / ml to 40 mg / ml.

[0305] In one embodiment, the mass percentage of the polyol relative to the total mass of the formulation is from 10 mg / ml to 40 mg / ml.

[0306] In one embodiment, the mass percentage of the polyol relative to the total mass of the formulation is 20 mg / ml to 40 mg / ml.

[0307] In one embodiment, the mass percentage of the polyol relative to the total mass of the formulation is 30 mg / ml to 40 mg / ml.

[0308] In one embodiment, the formulation further comprises at least mannitol, wherein the mass percentage of mannitol is from 0.1 mg / ml to 50 mg / ml relative to the total mass of the formulation.

[0309] In one embodiment, the formulation further comprises at least mannitol, wherein the mass percentage of mannitol is 5 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0310] In one embodiment, the formulation further comprises at least mannitol, wherein the mass percentage of mannitol is from 10 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0311] In one embodiment, the formulation further comprises at least mannitol, wherein the mass percentage of mannitol is 20 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0312] In one embodiment, the formulation further comprises at least mannitol, wherein the mass percentage of mannitol is 30 mg / ml to 40 mg / ml relative to the total mass of the formulation.

[0313] The formulations obtained by the subject method of this invention have many applications.

[0314] Medical applications include, for example, injections to replace defective biological fluids (e.g., in joints to replace synovial fluid), postoperative injections to prevent peritoneal adhesions, periurethral injections to treat incontinence, and injections after presbyopia surgery. Aesthetic applications include, for example, injections to fill wrinkles, fine lines, and skin defects, or injections to increase volume (e.g., the volume of lips, cheeks, etc.).

[0315] More specific anticipated applications are those typically associated with injectable viscoelastics and polysaccharides used or potentially used in the following pathologies or treatments:

[0316] - Aesthetic injections for the face: used to fill wrinkles or skin defects or to augment volume (cheeks, chin, lips);

[0317] -Body augmentation injections: breast and buttock enlargement, G-spot enlargement, vaginoplasty, labia majora reconstruction, penis enlargement;

[0318] - Used in joint and dental surgeries, for example, to fill periodontal pockets.

[0319] - In the treatment of arthritis, it is injected into the joint to replace or increase the defective synovial fluid;

[0320] - Periurethral injection for the treatment of urinary incontinence due to sphincter defects;

[0321] - Postoperative injection, especially for preventing peritoneal adhesions;

[0322] - Injection after presbyopic surgery via scleral laser incision;

[0323] - Injected into the vitreous cavity;

[0324] - Injections during cataract surgery;

[0325] - An injection used to treat vaginal dryness;

[0326] - Injected into the genital structures.

[0327] More specifically, in aesthetic surgery, formulations obtained through the subject method of this invention can be used as a function of their viscoelasticity and durability properties:

[0328] - Used to fill fine lines, medium or deep wrinkles, and injected with a small diameter needle (e.g., 27 gauge);

[0329] - As a vomulizer, it is injected by a needle with a larger diameter (e.g., 22 to 26) and a longer length (e.g., 30 mm to 40 mm); in this case, its cohesive properties ensure that it remains at the injection site.

[0330] These exemplary uses are by no means limiting; more generally, formulations obtained according to the subject matter method of the invention are provided for:

[0331] -Fill volume;

[0332] - Create space within certain organizations to facilitate their optimal function;

[0333] - Replace defective physiological fluids.

[0334] The following embodiments are applicable to methods for crosslinking polymers according to the present invention, methods for preparing formulations comprising polymers obtained by the methods according to the present invention, formulations according to the present invention, and various uses applicable to said formulations.

[0335] In one embodiment, the polymer is selected from the group consisting of polysaccharides.

[0336] In one embodiment, the polymer is selected from glycosaminoglycans (GAG).

[0337] In one embodiment, the polymer is selected from glycosaminoglycans (GAGs), such as chondroitin, keratin, heparin, heparin precursor, or hyaluronic acid, and mixtures thereof.

[0338] In one embodiment, the polymer is selected from hyaluronic acid, keratin, heparin, cellulose, cellulose derivatives, alginic acid, xanthan gum, carrageenan, chitosan, chondroitin, heparin precursors, and biologically acceptable salts thereof, either alone or in mixtures.

[0339] In one embodiment, the polymer is hyaluronic acid alone or in mixtures, or any biologically acceptable salt thereof.

[0340] In the context of this application, hyaluronic acid, either alone or in mixtures, or any of its biologically acceptable salts, is preferred.

[0341] In one embodiment, the polymer is selected from hyaluronic acid, sodium hyaluronate, and mixtures thereof.

[0342] In one embodiment, the polymer is hyaluronic acid.

[0343] In one embodiment, the polymer is selected from sodium hyaluronate and potassium hyaluronate.

[0344] In one embodiment, the polymer is sodium hyaluronate.

[0345] In the context of this application, sodium hyaluronate is a particularly preferred polymer.

[0346] In one embodiment, the polymer is hyaluronic acid or a salt thereof that has been chemically modified by substitution.

[0347] In one embodiment, the polymer is hyaluronic acid or a salt thereof that has been substituted with groups that impart lipophilic or hydrating properties, such as substituted hyaluronic acid as described in the applicant’s patent application FR 2 983483.

[0348] In the context of this application, Mw or “molecular weight” means the average molecular weight of the polymer measured in Daltons.

[0349] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 0.01 MDa to 5 MDa.

[0350] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 0.01 MDa to 3.5 MDa.

[0351] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 0.5 MDa to 3.5 MDa.

[0352] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 2.75 MDa to 3.25 MDa.

[0353] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 0.75 MDa to 1.25 MDa.

[0354] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 2 MDa to 5 MDa.

[0355] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 2 MDa to 4 MDa.

[0356] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 0.5 MDa to 2 MDa.

[0357] In one embodiment, the hyaluronic acid or a salt thereof has a molecular weight of 0.5 MDa to 1.5 MDa.

[0358] In one embodiment, the at least one crosslinking agent is selected from ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether (BDDE), polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, diepoxides or polyepoxides (e.g., 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane), dialkyl sulfones, divinyl sulfones, formaldehyde, epichlorohydrin or glutaraldehyde, carbodiimide (e.g., 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)), and trimetaphosphates (e.g., sodium trimetaphosphate, calcium trimetaphosphate or barium trimetaphosphate).

[0359] In one embodiment, the at least one crosslinking agent is selected from ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether (BDDE), polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, diepoxides or polyepoxides (e.g., 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane), and trimetaphosphates (e.g., sodium trimetaphosphate, calcium trimetaphosphate or barium trimetaphosphate).

[0360] In one embodiment, the at least one crosslinking agent is selected from ethylene glycol diglycidyl ether, butylene glycol diglycidyl ether (BDDE), polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, diepoxides or polyepoxides (e.g., 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane).

[0361] In one embodiment, the at least one crosslinking agent is selected from trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate, or barium trimetaphosphate.

[0362] In one embodiment, the at least one crosslinking agent is selected from epoxides, such as 1,4-butanediol diglycidyl ether (BDDE), epoxy halopropane, and divinyl sulfone (DVS).

[0363] In one embodiment, the at least one crosslinking agent is divinyl sulfone (DVS).

[0364] In one embodiment, the at least one crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0365] In the context of this application, BDDE is particularly preferred.

[0366] In one implementation, the crosslinking rate X is from 0.001 to 0.5.

[0367] In one implementation, the crosslinking rate X is from 0.01 to 0.4.

[0368] In one implementation, the crosslinking rate X is from 0.03 to 0.23.

[0369] In one implementation, the crosslinking rate X is from 0.03 to 0.20.

[0370] In one implementation, the crosslinking rate X is from 0.03 to 0.15.

[0371] In one implementation, the crosslinking rate X is from 0.03 to 0.10.

[0372] In one implementation, the crosslinking rate X is 0.10 to 0.15.

[0373] In one implementation, the crosslinking rate X is from 0.08 to 0.15.

[0374] In one embodiment, the degree of modification of the crosslinked polymer is less than 5%.

[0375] In one embodiment, the degree of modification of the crosslinked polymer is less than 4%.

[0376] In one embodiment, the degree of modification of the crosslinked polymer is less than 3.5%.

[0377] In one embodiment, the degree of modification of the crosslinked polymer is less than 3%.

[0378] In one embodiment, the degree of modification of the crosslinked polymer is less than 2.5%. Example

[0379] In the context of this embodiment, a number of parameters were measured.

[0380] Determination of rheological parameters G', G” and TanΔ(Tnδ):

[0381] TAInstruments DHR-2 device. Conical geometry with a 2° angle and a 40mm diameter. Frequency scanning method (logarithmic scan), 0.8% deformation (strain) (deformation within the linear range), frequency range from 0.08Hz to 5Hz, with values ​​read at 1Hz.

[0382] Determination of MoD:

[0383] Proton NMR spectra were obtained using a 400 MHz spectrometer. The MoD value was calculated by integrating the N-acetyl signal group of hyaluronic acid and the BDDE signal (two -CH2- groups). The ratio of the integrals of these two signals (crosslinker / NAc HA) corresponds to the MoD.

[0384] The above method (hyaluronic acid cross-linked via BDDE) and the above formula were used to determine the value of Mod (%).

[0385] Injectability measurement:

[0386] A traction stage and a force gauge (N) were used. The traction stage applied a displacement velocity to the plunger rod of the syringe, and the gel was ejected from the needle (27G1 / 2); the force, measured in Newtons, was recorded by the force gauge during the gel ejection at a rate of 13 mm / min.

[0387] Assessment of resistance to enzymatic degradation (hyaluronidase):

[0388] Prepare the hyaluronidase solution (Sigma Aldrich H3506) (see Table 7 for U / g values ​​in phosphate buffer). Mix this solution (20 μL) with 1 g of the gel to be tested and incubate everything at 37°C for 5 to 10 minutes.

[0389] Rheological analysis was then performed on the enzyme-mixed gel using a TA Instruments DHR-2 instrument. The geometry was 2° angled and 40 mm in diameter. Frequency oscillation (logarithmic scan) was used, with 0.8% deformation (strain) at 37°C and a fixed frequency of 1 Hz.

[0390] This analysis includes monitoring the loss of G' (Pa) as a function of time. The time when the initial G' of the formulation decreases by half corresponds to the half-life of the product being analyzed.

[0391] Measurement of plastic range and flow deformation:

[0392] TAInstruments DHR-2 device. Geometry with an angle of 2° and a diameter of 40mm. Strain scanning method: logarithmic scan, deformation (strain) from 0.1% to 1000%, frequency 1Hz.

[0393] Observe the plastic range of deformation (in percentage) from the point where G'(Pa) decreases by 10% relative to the initial G' to the intersection of G' and G”.

[0394] Example 1: Rheological properties of the formulation obtained according to the method of the present invention.

[0395] Example 1 illustrates the effect of implementing the method according to the invention on the properties (G', G”, and TanΔ(Tnδ), Mod) of the obtained formulation. In this example, the properties (G', G”, TanΔ(Tnδ), and Mod) of the formulation obtained according to the method according to the invention are compared with those of a formulation obtained by conventional crosslinking (described in application WO2009071697).

[0396] Methods for preparing formulations

[0397] The two formulations being compared were each prepared according to the following methods:

[0398] Weigh 1 g of injectable sodium hyaluronate fiber (molecular weight: 3 MDa) into a container. Add 7.4 g of 1% sodium hydroxide aqueous solution to water and homogenize everything with a spatula at ambient temperature and 900 mmHg for about 1 hour.

[0399] Add an appropriate amount of BDDE to the non-crosslinked sodium hyaluronate gel obtained in the preceding steps to obtain a crosslinking rate X of about 0.14, and homogenize everything with a spatula at ambient temperature and 900 mm Hg for about 30 minutes.

[0400] The crosslinking conditions are as follows:

[0401] - For the (comparative) method of the prior art, at 50°C for 3 hours and 10 minutes; and

[0402] - For the method of the present invention, the temperature is maintained at about 9°C for 23 to 26 hours.

[0403] For each method, the final cross-linked gel was then neutralized by adding HCl 1N and placed in a phosphate buffer bath to stabilize the pH and allow it to hydrate or swell to a hyaluronic acid concentration of 30 mg / g. The gel was then dialyzed in a phosphate buffer bath until a hyaluronic acid concentration of 20.9 mg / g was obtained. At the end of this step, the pH of the gel corresponded to the pH of the buffer, or approximately 7.2. The final gel was then homogenized, and parameters (G', G”, Mod) were measured.

[0404] In summary, the only difference between these two methods is the crosslinking temperature and crosslinking time conditions.

[0405] Characteristics of the obtained formulation (before sterilization)

[0406] The results of determining the rheological parameters and Mod are given in Table 1 below:

[0407]

[0408] Table 1: Characteristics of the formulation prior to sterilization in Example 1

[0409] It is noted that the formulation obtained by the method according to the invention has a much higher G' (401 Pa) than the composition obtained by the method according to the prior art.

[0410] Furthermore, the G” value in the case of the method according to the present invention is more than three times that of the conventionally used method.

[0411] Finally, quite unexpectedly, these improved rheological properties were obtained even when the formulation prepared by the method according to the invention had a low Mod percentage.

[0412] Characteristics of the obtained formulation (after sterilization)

[0413] Both formulations were sterilized by autoclaving (F0 = 44 minutes), and the same parameters (G', G") were measured a second time.

[0414] The results of determining the rheological parameters and Mod are given in Table 2 below:

[0415]

[0416] Table 2: Characteristics of the Sterilized Formulation in Example 1

[0417] It is noted that the G' (401 Pa / 153 Pa) of the formulation prepared according to the method of the present invention is more affected by sterilization than that of the formulation prepared by conventional methods (253 Pa / 159 Pa), and the G' values ​​of the two formulations are similar after sterilization.

[0418] It should be noted that, after sterilization, the G” of the formulation prepared according to the method of the present invention is still much higher than that of the formulation prepared by conventional methods.

[0419] From the above, it can be concluded that the TanΔ (Tanδ) of the formulation prepared according to the method of the present invention doubles during sterilization (0.27 / 0.52), while the TanΔ (Tnδ) value of the formulation prepared by conventional methods changes relatively little (0.13 / 0.17).

[0420] In summary, the formulation prepared according to the present invention already has a high TanΔ value before sterilization, which further increases during the sterilization step.

[0421] Therefore, the method according to the invention enables the production of formulations with very good rheological properties while maintaining a relatively low Mod(%) (good crosslinking efficiency). In our embodiments, the gel has considerable rigidity G' and exhibits deformation (optimized damping), and the gel is characterized by being less prone to breakage.

[0422] Injectability of the formulation prepared according to the method of the present invention

[0423] The injectability of the formulation prepared according to the method of the present invention is measured.

[0424] After sterilization, for both gels obtained by conventional methods and gels obtained by the method according to the invention, an injection force of less than 35 N was observed at 13 mm / min. This makes it suitable for the applications envisioned in this application.

[0425] Therefore, the formulations according to the present invention can be considered injectable formulations.

[0426] Example 2: Rheological properties of the formulation obtained by the method according to the present invention.

[0427] The method used in Example 2 is the same as that in Example 1, except that both formulations are based on hyaluronic acid with an average molecular weight of 0.9 MDa by weight and have a crosslinking rate X of approximately 0.09.

[0428] The results of determining the rheological parameters and Mod are given in Table 3 below:

[0429]

[0430] Table 3: Characteristics of the formulation prior to sterilization in Example 2

[0431] It should be noted that the formulation obtained by the method according to the invention has a lower G' than that obtained by the method according to the prior art.

[0432] The G” value of the formulation obtained by the subject method of this invention is twice that of the formulation obtained by conventionally used methods.

[0433] From the above, it can be concluded that the value of TanΔ1Hz is optimized in the formulation obtained by the method according to the present invention.

[0434] Similarly, the formulations obtained by the subject method of the present invention have improved rheological properties while also having a relatively low Mod (%).

[0435] Example 3: Rheological properties of the formulation obtained by the method according to the present invention.

[0436] The two formulations from Examples 1 and 2, prepared according to conventional methods (before sterilization), were mixed in a 50 / 50 ratio. This produced a formulation comprising two pre-crosslinked and mixed / interpenetrating formulations.

[0437] The two formulations of Examples 1 and 2 prepared according to the method according to the invention (before sterilization) were also mixed in a 50 / 50 ratio. This produced a formulation comprising two pre-crosslinked and mixed / interpenetrating formulations.

[0438] The results of determining the rheological parameters and Mod are given in Table 4 below:

[0439]

[0440] Table 4: Example 3 - Interpenetrating Formulation

[0441] Note that the G' value (358 Pa) of the interpenetrating formulation according to the present invention is closer to the high value (Example 1; 401 Pa) than the low value (Example 2; 198 Pa) of the formulation constituting the interpenetrating formulation.

[0442] It is also noted that the G” value (119 Pa) of the formulation according to the invention is higher than that of each of the formulations constituting the formulation (Example 1, 107 Pa; Example 2, 89 Pa).

[0443] In summary, the rheological properties of the interpenetrating formulations prepared according to the method of the present invention are particularly unexpected and unforeseen.

[0444] The preparation obtained according to the method of the invention is then sterilized (F0 = 14.5 minutes) and its injectability is tested.

[0445] It was demonstrated that at a speed of 13 mm / min and an ambient temperature of (27) 1 / 2 Under the specified conditions (see figure), the injectability is less than 35N. Therefore, the formulation obtained according to the method of the present invention is completely injectable.

[0446] Example 4: Application of the present invention in high-quality hyaluronic acid

[0447] Weigh 1 g of injectable sodium hyaluronate fiber (molecular weight: 3 MDa) into a container. Add 7.4 g of 1% sodium hydroxide aqueous solution to water and homogenize everything with a spatula at ambient temperature and 900 mmHg for about 1 hour.

[0448] Add an appropriate amount of BDDE to the non-crosslinked sodium hyaluronate gel obtained in the preceding steps to obtain a crosslinking rate X of about 0.14, and homogenize all of the mixture with a spatula at ambient temperature and 900 mm Hg for about 30 minutes.

[0449] The crosslinking conditions for the four tests are as follows:

[0450] - For the existing (comparative) method, at 50°C for 3 hours and 10 minutes,

[0451] -For the method according to the invention, 24 hours at 9°C,

[0452] - After 24 hours at 2℃,

[0453] - 48 hours at 9°C.

[0454] For each method, the final cross-linked gel was then neutralized by adding HCl 1N and placed in a phosphate buffer bath to stabilize the pH and allow it to hydrate or swell to a hyaluronic acid concentration of approximately 40 mg / g. The gel was then dialyzed in a phosphate buffer bath until a hyaluronic acid concentration of approximately 26 mg / g was obtained. At the end of this step, the pH of the gel corresponded to the pH of the buffer, or approximately 7.2. The final gel was then homogenized, sterilized in an autoclave, and the following measurements were performed:

[0455] -G', G”; for all reaction times and temperatures.

[0456] -MoD; for reaction times of 24 hours and 48 hours, and temperatures of 9°C.

[0457] - Half-life (tolerance to hyaluronidase); for a reaction time of 48 hours and a temperature of 9°C.

[0458]

[0459] Table 5: Rheological properties of the formulation after sterilization in Example 4

[0460] It was noted that the G' value of the formulation at a 24-hour reaction time was relatively close to that of the reference formulation, while TanΔ(Tnδ) was optimized.

[0461] We observed that the greater the increase in reaction time and temperature (according to the present invention), the closer TanΔ(Tnδ) is to TanΔ(Tnδ) of conventionally used methods.

[0462]

[0463] Table 6: MoD of the formulation in Example 4 -

[0464] Surprisingly, the 48-hour -9°C method allowed for the simultaneous attainment of optimized G' and TanΔ(Tnδ) and a reduction in MoD(%). This formulation thus imparts optimized properties for filler applications (a formulation that is both more rigid and has better damping capabilities) and improved biocompatibility.

[0465]

[0466] Table 7: Enzyme resistance (durability) of the formulation in Example 4

[0467] The results in the table above are unexpected. The MoD of the formulation at 48 hours -9°C is lower than that of the reference (the conventional method). Hyaluronic acid has fewer cross-linking bridges and should have lower durability.

[0468] Surprisingly, the measurements showed the opposite; the half-lives were actually similar, but the enzyme concentration was higher for this invention.

[0469] In summary, the formulation crosslinked at 9°C for 48 hours exhibits significant and unexpected advantages, such as good deformability, maintained rigidity, and optimized biocompatibility and resistance to enzyme degradation.

[0470] Example 5: The present invention has Average quality Use of hyaluronic acid

[0471] The method used in this embodiment is the same as that in Example 4, except that both formulations are based on hyaluronic acid with an average molecular weight of 0.9 MDa by weight and have a crosslinking rate X of approximately 0.09.

[0472] The crosslinking conditions for the four tests are as follows:

[0473] - For the existing (comparative) method, at 50°C for 3 hours and 10 minutes,

[0474] -For the method according to the invention, 24 hours at 9°C,

[0475] - After 24 hours at 2℃,

[0476] - 48 hours at 9°C.

[0477] For each method, the final cross-linked gel was then neutralized by adding HCl 1N and placed in a phosphate buffer bath to stabilize the pH and allow it to hydrate or swell to a hyaluronic acid concentration of approximately 40 mg / g. The gel was then dialyzed in a phosphate buffer bath until a hyaluronic acid concentration of approximately 26 mg / g was obtained. At the end of this step, the pH of the gel corresponded to the pH of the buffer, or approximately 7.2. The final gel was then homogenized and the G' / G” ratio was analyzed for all reaction times. Measurements of MoD at reaction times of 24 h and 48 h and a temperature of 9 °C are also shown.

[0478] The formulation was then sterilized in an autoclave, and the G' / G” of all reaction times was measured again.

[0479]

[0480] Table 8: Rheological properties of the formulation before sterilization in Example 5

[0481] Prior to sterilization, we also observe the optimization of G” and thus TanΔ(Tnδ) as a result of the present invention.

[0482] Figure 2 The strain scan (strain scan curve) shown in the figure also unexpectedly demonstrated a highly optimized plasticity range for the formulation at 48 hours -9°C. It is noted on this curve that, at a corresponding G', the product obtained by the method according to the invention exhibits the widest plasticity range.

[0483]

[0484] Table 9: MoD of the formulation in Example 5 -

[0485] The obtained MoD value is relatively low. Surprisingly, the reference G' and the G' of the 48-hour -9°C method were observed to be the same, while the MoD (%) of the present invention was lower. The obtained product exhibits the same rigidity properties and minimal hyaluronic acid transformation.

[0486]

[0487] Table 10: Rheological properties of the formulation after sterilization in Example 5

[0488] Furthermore, the 48-hour -9°C formulation is highly advantageous and allows for a relatively constant G' to significantly improve the product's deformability.

Claims

1. A process for the preparation of a preparation comprising at least one crosslinked polymer obtained according to a method of crosslinking, comprising at least the following steps: a) providing a polymer, said polymer being hyaluronic acid or any of its biologically acceptable salts, alone or in mixture; b) providing a crosslinking agent; c) carrying out one or more crosslinking steps in the presence of said polymer and of said crosslinking agent; d) obtaining a crosslinked polymer; characterized in that said crosslinking step or each of said crosslinking steps is carried out at a constant temperature or at a temperature varying linearly or in a stepwise manner, said constant or varying temperature being between the freezing temperature and 9°C, and in that the duration of said crosslinking step c) is comprised between 3 hours and 72 hours, and in that said process further comprises at least one step for hydration and / or swelling.

2. The process for the preparation of a preparation according to claim 1, characterized in that said crosslinking step or each of said crosslinking steps is carried out at a constant temperature.

3. The process for the preparation of a preparation according to claim 1 or 2, characterized in that the crosslinked polymer obtained in step d) has a Tan Delta (TnD) > 0.25 as determined by frequency sweep method with TA Instruments DHR-2 equipment, cone geometry with angle 2° and diameter 40 mm, logarithmic sweep, deformation in the linear range of 0.8%, and reading at a frequency of 1 Hz in a frequency range of 0.08 Hz to 5 Hz.

4. The process for the preparation of a preparation according to claim 1 or 2, characterized in that the crosslinked polymer obtained in step d) has a G' < 1000 Pa as determined by frequency sweep method with TA Instruments DHR-2 equipment, cone geometry with angle 2° and diameter 40 mm, logarithmic sweep, deformation in the linear range of 0.8%, and reading at a frequency of 1 Hz in a frequency range of 0.08 Hz to 5 Hz.

5. The process for the preparation of a preparation according to claim 1 or 2, characterized in that the polymer provided in step a) is a mixture of hyaluronic acids or a mixture of hyaluronic acid salts.

6. The process for the preparation of a preparation according to claim 1 or 2, characterized in that the preparation comprises a homogeneous mixture of Y identical or different crosslinked polymers crosslinked before being interpenetrated by mixing, Y being comprised between 2 and 5.

7. A preparation obtained according to the process of any one of claims 1 to 6, having a Tan Delta (TnD) > 0.25 as determined by frequency sweep method with TA Instruments DHR-2 equipment, cone geometry with angle 2° and diameter 40 mm, logarithmic sweep, deformation in the linear range of 0.8%, and reading at a frequency of 1 Hz in a frequency range of 0.08 Hz to 5 Hz. ​ ​ ​ ​ ​ ​ 8. A preparation, obtained according to the method of any one of claims 1 to 6, characterized in that it further comprises at least one active agent selected from the group consisting of local anesthetics, vitamin C derivatives, anti-inflammatory agents, polyols, and mixtures thereof.

9. The preparation according to claim 7 or 8, characterized in that the polymer comprises a mixture of hyaluronic acid or a mixture of hyaluronic acid salts.

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