PROCESS FOR CROSSLINKING A POLYMER

Abstract

A process for crosslinking a polymer, includes at least the following steps: a) providing a polymer; b) providing a crosslinking agent; c) carrying out one or more crosslinking steps in the presence of the polymer and the crosslinking agent; d) obtaining a crosslinked polymer; wherein the crosslinking step or each of the crosslinking steps is carried out at constant temperature or at a temperature that varies linearly or in a stepwise manner, the constant or variable temperature being less than or equal to 15° C., and in that the crosslinking step c) has a duration of between 3 and 72 hours.

Claims

1. A process for crosslinking a polymer, comprising at least the following steps: a) providing a polymer; b) providing a crosslinking agent; c) carrying out one or more crosslinking step(s) in the presence of said polymer and said crosslinking agent; d) obtaining a crosslinked polymer; wherein the crosslinking step or each of the crosslinking steps is carried out at a constant temperature or at a temperature that varies linearly or in a stepwise manner, said constant or variable temperature being less than or equal to 15° C., and in that said crosslinking step c) has a duration of between 3 and 72 hours.

2. The process for crosslinking a polymer according to claim 1, wherein the crosslinking step or each of the crosslinking steps is carried out at a constant temperature.

3. The process for crosslinking a polymer according to claim 1, wherein the crosslinked polymer obtained in step d) has Tan Δ (Tn δ)≥0.25.

4. The process for crosslinking a polymer according to claim 1, wherein the crosslinked polymer obtained in step d) has a G′≤1000.

5. The process for crosslinking a polymer according to claim 1, wherein said polymer is chosen from the group of polysaccharides.

6. A process for preparing a formulation comprising at least one crosslinked polymer obtained according to the process of claim 1.

7. The process according to claim 5, said formulation comprising at least one polymer crosslinked according to the process comprises a homogeneous mixture of Y identical or different crosslinked polymers, crosslinked prior to their interpenetration by mixing, Y being between 2 and 5, wherein at least one of the Y polymers is crosslinked according to the process for preparing a crosslinked polymer.

8. The process according to claim 5, wherein said polymer is chosen from the group of polysaccharides.

9. A formulation comprising at least one crosslinked polymer obtained according to the process of claim 1.

10. The formulation according to claim 9, wherein it also comprises at least one active agent chosen from the group composed of local anesthetics, vitamin C derivatives, anti-inflammatories, polyols, and their mixtures.

11. A polymer wherein it has a G′≤1000 Pa and a tan Δ (Tn δ) whose value is between 0.25 and 1 (0.25≤Tan Δ (Tn δ))≤1).

12. The polymer according to claim 11, wherein it is chosen from the group of polysaccharides.

13. The polymer according to claim 11 wherein it is comprised of a mixture of hyaluronic acids, or of hyaluronic acid salts.

Description

EXAMPLES

[0367] In the context of the examples, a certain number of parameters were measured.

Determination of the Rheological Parameters G′, G″ and Tan Δ (Tn δ):

[0368] TA Instruments DHR-2 equipment. Cone type geometry with an angle of 2° and a diameter of 40 mm. Frequency sweep method (logarithmic sweep), deformation (strain) of 0.8% (deformation in the linear range), frequency range of 0.08 to 5 Hz, values read at the frequency of 1 Hz.

Determination of the MoD:

[0369] The proton NMR spectra are obtained in a 400 MHz spectrometer. The MoD value is calculated from the integrals of the N-acetyl signal group of the hyaluronic acid and a BDDE signal (two —CH.sub.2— groups). The ratio of the integrals of these two signals (crosslinking agent/NAc HA) corresponds to the MoD.

[0370] The value of the Mod (%) was determined using the process mentioned above (hyaluronic acid crosslinked by BDDE), and using the formula also mentioned above.

Injectability Measurement:

[0371] A traction bench and a force gauge (N) are used. The traction bench applies a displacement speed to the plunger rod of a syringe and the gel is expelled from the needle (27 G1/2); the force in Newtons is recorded by the gauge during the ejection of the gel at a speed 13 mm/min.

Evaluation of the Enzyme Degradation Resistance (Hyaluronidases):

[0372] A hyaluronidase solution (Sigma Aldrich H3506) (see Table 7 for the U/g value in a phosphate buffer) is prepared. This solution (20 μL) is mixed with 1 g of the gel to test, and everything is maintained at 37° C. for 5 to 10 minutes.

[0373] The gel mixed with the enzymes is then rheologically analyzed, TA Instruments DHR-2 equipment. Geometry with an angle of 2° and a diameter of 40 mm. Frequency oscillation method (logarithmic sweep), deformation (strain) of 0.8%, temperature of 37° C., fixed frequency of 1 Hz applied.

[0374] The analysis consists of monitoring the loss of G′ (Pa) as a function of time. The time at which the initial G′ of the formulation is reduced by half corresponds to the half-life of the analyzed product.

Measurement of the Plastic Range, Rheological Deformation Measurements:

[0375] TA Instruments DHR-2 equipment. Geometry with an angle of 2° and a diameter of 40 mm. Strain sweep method: logarithmic sweep, deformation (strain) of 0.1 to 1000%, frequency of 1 Hz.

[0376] Observation of the plastic range, running from the deformation (in %) at which the G′ (Pa) drops by 10% relative to the initial G′ to the deformation (in %) at the crossover of the G′ and the G″.

Example 1: Rheological Properties of a Formulation Obtained According to the Process of the Invention

[0377] Example 1 illustrates the influence of the implementation of the process according to the invention on the properties (G′, G″ and Tan Δ (Tn δ), Mod) of the formulation obtained. In this example, the properties (G′, G″, Tan Δ (Tn δ) and Mod) of a formulation obtained according to the process of the invention were compared to those of a formulation obtained by means of a conventionally used crosslinking (of the described in the application WO2009071697).

Processes for Preparing the Formulations

[0378] The two compared formulations are each prepared according to the following process:

[0379] Sodium hyaluronate fibers of injectable quality (1 g; molecular mass: 3 MDa) are weighed in a vessel. A 1% aqueous solution of sodium hydroxide in water (7.4 g) is added, and everything is homogenized with a spatula for about 1 hour at ambient temperature and at 900 mm Hg.

[0380] An appropriate quantity of BDDE for obtaining a crosslinking rate X of around 0.14 is added to the non-crosslinked sodium hyaluronate gel obtained in the preceding step, and everything is homogenized with a spatula for about 30 minutes at ambient temperature and at 900 mm Hg.

[0381] The crosslinking conditions are as follows: [0382] 3 h 10 m at 50° C. for the (comparative) process of the prior art; and [0383] 23 to 26 h at around 9° C. for the process of the invention.

[0384] For each of the processes, the final crosslinked gel is then neutralized by adding HCl 1N and placed in a phosphate buffer bath in order to stabilize the pH and enable it to be hydrated or swollen to a hyaluronic acid concentration of 30 mg/g. The gel is then dialyzed in a phosphate buffer bath until a hyaluronic acid concentration of 20.9 mg/g is obtained. The pH of the gels at the end of this step corresponds to the pH of the buffer, or around 7.2. The final gels are then homogenized, and a measurement of the parameters (G′, G″, Mod) is performed.

[0385] In summary, the two compared processes differ only by the crosslinking temperature and crosslinking time conditions.

[0386] Properties of the Formulations Obtained (Before Sterilization)

[0387] The results of the determination of the rheological parameters and the Mod are given in Table 1 below:

TABLE-US-00001 TABLE 1 Example 1 - Properties of the formulations before sterilization Conventionally Process according used process to the invention Crosslinking conditions 3 h 10 m - 50° C. 23 to 26 h - around 9° C. Hyaluronic acid 3 MDa 3 MDa G′ (Pa) 1 Hz of the 253 401 formulation obtained G″ (Pa) 1 Hz of the 33 107 formulation obtained Tan Δ (tan δ) 1 Hz of the 0.13 0.27 formulation obtained Mod as a % of the 5.9 3.4 formulation obtained

[0388] It is noted that the formulations obtained by means of the process according to the invention have a G′ (401 Pa) much higher than that of the compositions obtained according to the process of the prior art (253 Pa).

[0389] Also, the value of G″ is more than three times higher with the process according to the present invention compared to the conventionally used process.

[0390] Lastly, quite surprisingly, these improved rheological properties are obtained even though the Mod as a % of the formulation prepared by means of the process according to the invention is lower.

Properties of the Formulations Obtained (after Sterilization)

[0391] The two formulations are sterilized by autoclaving (F0=44 min), and a second measurement of the same parameters (G′, G″) is performed.

[0392] The results of the determination of the rheological parameters and the Mod are given in Table 2 below:

TABLE-US-00002 TABLE 2 example 1 - Properties of the formulations after sterilization Conventionally Process according used process to the invention Crosslinking conditions 3 h 10 m - 50° C. 23 à 26 h - around 9° C. Hyaluronic acid 3 MDa 3 MDa Sterilization conditions F0 = 44 F0 = 44 (autoclaving) F0 in min G′ (Pa) 1 Hz of the 159 153 formulation obtained (after sterilization) G″ (Pa) 1 Hz of the 27 80 formulation obtained (after sterilization) Tan Δ (tan δ) 1 Hz of the 0.17 0.52 formulation obtained (after sterilization) pH (after sterilization) 7.2 7.3

[0393] It is noted that the G′ of the formulation prepared with the process according to the invention (401 Pa/153 Pa) is more affected by the sterilization than the G′ of the formulation prepared with the conventionally used process (253 Pa/159 Pa), the G′ values of the two formulations being similar after sterilization.

[0394] It is noted that the G″ of the formulation prepared with the process according to the invention remains much higher than the G″ of the formulation prepared with the conventionally used process after sterilization.

[0395] It follows from the above that the Tan Δ (tan δ) of the formulation prepared with the process according to the invention is doubled during sterilization (0.27/0.52), while the Tan Δ (tan δ) value of the formulation prepared with the conventionally used process is modified relatively little (0.13/0.17).

[0396] In summary, the Tan Δ value, already higher for the formulation prepared according to the invention before sterilization, is further increased during the sterilization step.

[0397] The process according to the invention therefore makes it possible to obtain formulations having very good rheological properties while retaining a relatively low Mod (%) (good crosslinking efficiency). In our example, the gel has an equivalent rigidity G′ and reveals a deformation (optimized damping), the gel is characterized as less brittle.

Injectability of the Formulation Prepared According to the Process of the Invention

[0398] The Injectability of the formulation prepared according to the process of the invention was measured.

[0399] After sterilization, an injection force of less than 35 N at 13 mm/min is observed for the gel obtained by the conventionally used process and by the process according to the invention. This makes it suitable for the applications envisioned in this application.

[0400] The formulation according to the invention is therefore a formulation that can be qualified as injectable.

Example 2: Rheological Properties of a Formulation Obtained According to the Subject Process of the Invention

[0401] The processes used in Example 2 are identical to those of Example 1, except for the fact that the two formulations are based on hyaluronic acid with an average molecular mass by weight of 0.9 MDa and have a crosslinking rate X approximately equal to 0.09.

[0402] The results of the determination of the rheological parameters and the Mod are given in Table 3 below:

TABLE-US-00003 TABLE 3 Example 2 - Properties of the formulations before sterilization Conventionally Process according used process to the invention Crosslinking conditions 3 h 10 m - 50° C. 23 a 26 h - around 9° C. Hyaluronic acid 0.9 MDa 0.9 MDa G′ (Pa) 1 Hz of the 432 198 formulation obtained G″ (Pa) 1 Hz of the 46 89 formulation obtained Tan Δ (tan δ) 1 Hz of the 0.11 0.45 formulation obtained Mod as a % of the 4.5 2.4 formulation obtained

[0403] It is noted that the formulation obtained by means of the process according to the invention has a G′ lower than that of the formulation obtained according to the process of the prior art.

[0404] The G″ value of the formulation obtained by means of the subject process of the invention is two times greater than that of the formulation obtained according to the conventionally used process.

[0405] It follows from the above that the value of Tan Δ 1 Hz is optimized in the formulation obtained according to the process of the invention.

[0406] Here again, the formulation obtained by the subject process of the invention has improved rheological properties while also having a relatively low Mod (%).

Example 3: Rheological Properties of a Formulation Obtained According to the Subject Process of the Invention

[0407] The two formulations of Examples 1 and 2 prepared according to the conventionally used processes (before sterilization) are mixed in 50/50 proportions. This results in a formulation comprising two pre-crosslinked and mixed/interpenetrated formulations.

[0408] The two formulations of Examples 1 and 2 prepared according to the processes according to the invention (before sterilization) are also mixed in 50/50 proportions. This results in a formulation comprising two pre-crosslinked and mixed/interpenetrated formulations.

[0409] The results of the determination of the rheological parameters and the Mod are given in Table 4 below:

TABLE-US-00004 TABLE 4 Example 3 - Interpenetrated formulations Conventionally Interpenetrated used interpenetrated formulation according formulation to the invention G′ (Pa) 1 Hz of the 374 358 formulation obtained G″ (Pa) 1 Hz of the 42 119 formulation obtained Tan Δ (tan δ) 1 Hz of the 0.11 0.33 formulation obtained Mod as a % of the 5.2 2.7 formulation obtained

[0410] It is noted that the G′ value (358 Pa) of the interpenetrated formulation according to the invention is much closer to the high value (Example 1; 401 Pa) than to the low value (Example 2; 198 Pa) of the formulations that constitute it.

[0411] It is also noted that the G″ value (119 Pa) of the formulation according to the invention is higher than each of the values of the formulations that constitute it (Example 1, 107 Pa; Example 2, 89 Pa).

[0412] In summary, the rheological properties of the interpenetrated formulations prepared according to the process of the invention have particularly surprising and unexpected rheological characteristics.

[0413] The formulation obtained according to the process according to the invention was then sterilized (F0=14.5 minutes) and tested for injectability.

[0414] It was demonstrated that at a speed of 13 mm/min and at ambient temperature (Gauge 27.sup.1/2), the injectability is less than 35 N. The formulation obtained according to the process according to the invention is therefore perfectly injectable.

Example 4: Use of the Invention on Hyaluronic Acids with High Masses

[0415] Sodium hyaluronate fibers of injectable quality (1 g; molecular mass: 3 MDa) are weighed in a vessel. A 1% aqueous solution of sodium hydroxide in water (7.4 g) is added, and everything is homogenized with a spatula for about 1 hour at ambient temperature and at 900 mm Hg.

[0416] An appropriate quantity of BDDE for obtaining a crosslinking rate X of around 0.14 is added to the non-crosslinked sodium hyaluronate gel obtained in the preceding step, everything being homogenized with a spatula for about 30 minutes at ambient temperature and at 900 mm Hg.

[0417] The crosslinking conditions of the four tests are as follows: [0418] 3 h 10 m at 50° C. for the (comparative) process of the prior art, [0419] 24 h at 9° C., [0420] 24 h at 2° C., [0421] 48 h at 9° C. for the process according to the invention.

[0422] For each of the processes, the final crosslinked gel is then neutralized by adding HCl 1N, and placed in a phosphate buffer bath in order to stabilize the pH and enable it to be hydrated or swollen to a hyaluronic acid concentration of about 40 mg/g. The gel is then dialyzed in a phosphate buffer bath until a hyaluronic acid concentration of about 26 mg/g is obtained. The pH of the gels at the end of this step corresponds to the pH of the buffer, or about 7.2. The final gels are subsequently homogenized, then sterilized in the autoclave, and the following measurements are performed: [0423] G′, G″; for all of the reaction times and temperatures. [0424] MoD; for the reaction times of 24 and 48 h and the temperature of 9° C. [0425] Half-life (resistance to hyaluronidase enzymes); for the reaction time of 48 h and the temperature of 9° C.

TABLE-US-00005 TABLE 5 Example 4 - Rheological properties of the formulations after sterilization Conventionally used process Process according to the invention Crosslinking 3 h 10 m - 24 h - 24 h - 48 h - conditions 50° C. 9° C. 2° C. 9° C. Hyaluronic acid 3 MDa 3 MDa 3 MDa 3 MDa G′ (Pa) 1 Hz of 208 257 162 309 the formulation obtained G″ (Pa) 1 Hz of 46 132 122 110 the formulation obtained Tan Δ (Tn δ) 1 0.22 0.51 0.75 0.36 Hz of the formulation obtained

[0426] It is noted that the G′ values of the formulas at the reaction time of 24 h are relatively close to the reference formula while the Tan Δ (Tn δ) is optimized.

[0427] We observe that the more the reaction time and temperature increase (according to the invention), the closer the Tan Δ (Tn δ) approaches that of the conventionally used process.

TABLE-US-00006 TABLE 6 Example 4 - MoD of the formulations Conventionally used process Process according to the invention Crosslinking 3 h 10 m - 24 h - 48 h - conditions 50° C. 9° C. 9° C. Hyaluronic acid 3 MDa 3 MDa 3 MDa MoD as a % of 5.8 3.1 4.7 the formulation obtained

[0428] Surprisingly, the 48 h-9° C. process makes it possible to simultaneously obtain an optimized G′ and Tan Δ (Tn δ) and reduce the MoD (%). The formula therefore imparts properties that are optimized for a filling application (a formula both more rigid and having a better damping capacity), with improved biocompatibility.

TABLE-US-00007 TABLE 7 Example 4 - Enzymatic resistances (persistence) of the formulations Conventionally Process according used process to the invention Crosslinking conditions 3 h 10 m - 50° C. 48 h - 9° C. Hyaluronic acid 3 MDa 3 MDa Half-life (min) 18.2 19.4 Concentration of the 1750 2500 hyaluronidase solution placed in contact with the gel, in U/g

[0429] The results of the above table are unexpected in that, the MOD of the 48 h-9° C. formulation being lower than the reference (conventionally used process), the hyaluronic acid has fewer crosslinking bridges and should have a lower persistence.

[0430] Surprisingly, the measurements show the opposite; the half-lives are in fact similar, but with a higher enzyme concentration for the invention.

[0431] In summary, the formula crosslinked for 48 h at 9° C. has significant and surprising advantages such as good deformation capacity, retained rigidity, as well as optimized biocompatibility and resistance to enzymatic degradation.

Example 5: Use of the Invention on Hyaluronic Acids with Average Masses

[0432] The processes used in this example are identical to those of Example 4, except for the fact that the two formulations are based on hyaluronic acid with an average molecular mass by weight of 0.9 MDa and have a crosslinking rate X approximately equal to 0.09.

[0433] The crosslinking conditions for the four tests are as follows: [0434] 3 h 10 m at 50° C. for the (comparative) process of the prior art, [0435] 24 h at 9° C., [0436] 24 h at 2° C., [0437] 48 h at 9° C. for the process according to the invention.

[0438] For each of the processes, the final crosslinked gel is then neutralized by adding HCl 1N and placed in a phosphate buffer bath in order to stabilize the pH and enable it to be hydrated or swollen to a hyaluronic acid concentration of about 40 mg/g. The gel is then dialyzed in a phosphate buffer bath until a hyaluronic acid concentration of about 26 mg/g is obtained. The pH of the gels at the end of this step corresponds to the pH of the buffer, or about 7.2. The final gels are then homogenized and analyzed for G′/G″ for all the reaction times. The measurements of the MoD are also shown for the reaction times of 24 and 48 h and the temperature of 9° C.

[0439] The formulations are then sterilized in the autoclave and the measurements of G′/G″ for all the reaction times are performed again.

TABLE-US-00008 TABLE 8 Example 5 - Rheological properties of the formulations before sterilization Conventionally used process Process according to the invention Crosslinking 3 h 10 m - 24 h - 24 h - conditions 50° C. 9° C. 2° C. Hyaluronic acid 0.9 MDa 0.9 MDa 0.9 MDa G′ (Pa) 1 Hz of 557 397 285 the formulation obtained G″ (Pa) 1 Hz of 55 140 152 the formulation obtained Tan Δ (Tn δ) 1 0.10 0.35 0.53 Hz of the formulation obtained

[0440] Before sterilization, we observe here again an optimization of the G″ and thus of the Tan Δ (Tn δ) as a result of the invention.

[0441] The strain sweep represented in FIG. 2 (Strain sweep curves) also surprisingly demonstrates a highly optimized plastic range for the 48 h-9° C. formula. It is noted on this curve that at equivalent G′, the product obtained by the process according to the invention has the widest plastic range.

TABLE-US-00009 TABLE 9 Example 5 - MoD of the formulations Conventionally used process Process according to the invention Crosslinking 3 h 10 m - 24 h - 48 h - conditions 50° C. 9° C. 9° C. Hyaluronic acid 0.9 MDa 0.9 MDa 0.9 MDa MoD as a % of the 4.5 2.4 3.1 formulation obtained

[0442] The MoD values obtained are relatively low. Surprisingly, it is observed that the G′ of the reference and of the 48 h-9° C. process are identical, while the MoD (%) of the invention is lower. The product obtained has the same rigidity performance with a minimal transformation of the hyaluronic acid.

TABLE-US-00010 TABLE 10 Example 5 - Rheological properties of the formulations after sterilization Conventionally used process Process according to the invention Crosslinking 3 h 10 m - 24 h - 48 h - conditions 50° C. 9° C. 9° C. Hyaluronic acid 0.9 MDa 0.9 MDa 0.9 MDa G′ (Pa) 1 Hz of 394 142 288 the formulation obtained G″ (Pa) 1 Hz of 46 88 85 the formulation obtained Tan Δ (Tn δ) 1 0.12 0.62 0.30 Hz of the formulation obtained

[0443] Here again, the 48 h-9° C. formula is very advantageous, and makes it possible with a relatively unchanged G′ to significantly improve the deformability of the product.