PROCESS FOR PREPARING HYDROGELS

Abstract

The present invention relates to a process for preparing a crosslinked gel of at least one polysaccharide or a salt thereof, comprising at least the steps consisting in: a) providing a solution formed from an aqueous medium comprising at least said polysaccharide(s) or a salt thereof in a non-crosslinked form, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt; b) crosslinking the solution from step a) and, where appropriate; c) recovering said crosslinked gel formed.

Claims

1. A process for preparing a crosslinked gel of at least one polysaccharide or a salt thereof, comprising: a) providing a solution formed from an aqueous medium comprising at least one polysaccharide or a salt thereof in a non-crosslinked form, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt; b) crosslinking the solution from step a) to form a crosslinked gel, and c) recovering said crosslinked gel.

2. The process according to claim 1, further comprising homogenizing the solution from step a), prior to and/or simultaneously with the crosslinking step b).

3. The process according claim 1, wherein the crosslinking step b) is performed at a temperature from 15 to 35° C.

4. The process according to claim 1, wherein the crosslinking step b) is performed over a period of between 5 hours and 336 hours.

5. The process according to claim 1, wherein the phosphate salt is chosen from the sodium salts, potassium salts, lithium salts, caesium salts, silver salts, and mixtures thereof.

6. The process according to claim 1, wherein the phosphate salt is chosen from sodium phosphate, sodium triphosphate and sodium trimetaphosphate, and mixtures thereof.

7. The process according to claim 1, wherein the difunctional or multifunctional epoxide crosslinking agent is butanediol diglycidyl ether.

8. The process according to claim 1, wherein the polysaccharide is hyaluronic acid or a salt thereof.

9. The process according to claim 8, wherein the hyaluronic acid salt is chosen from sodium salt, potassium salt, zinc salt, a silver salt, and mixtures thereof.

10. The process according to claim 1, wherein the solution from step a) comprises a number of moles of phosphate salt(s)/total number of moles of polysaccharide units molar ratio of between 0.005 and 1.

11. The process according to claim 1, wherein the solution from step a) comprises a number of moles of difunctional or multifunctional epoxide crosslinking agent(s)/total number of moles of polysaccharide units molar ratio of between 0.005 and 1.

12. The process according to claim 1, further comprising adding at least one non-crosslinked polysaccharide; after step b) but prior to, simultaneously with or subsequent to the recovery of step c).

13. The process according to claim 1, wherein the crosslinked polysaccharide(s) included in the crosslinked gel have a degree of modification of between 0.1% and 10%.

14. The process according to claim 1, wherein the crosslinked gel has an elastic modulus (G′) of between 20 and 1000 Pa associated with a phase angle (δ) of less than 45°.

15. An injectable and sterile dermatological composition comprising, in a physiologically acceptable medium, at least one crosslinked gel having at least one polysaccharide or a salt thereof, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt.

16. A cosmetic or dermatological composition comprising at least one crosslinked gel having at least one polysaccharide or a salt thereof, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt.

17. A kit comprising: packaging containing at least one dose of a crosslinked gel, the crosslinked gel having at least one polysaccharide or a salt thereof. at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane, 1,2-bis(2,3-epoxypropyI)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt; and a device for injection into or through the skin or a skin microperforation device for administration of the one dose of the crosslinked gel.

18. A method for filling skin volume defects by administering a crosslinked gel having at least one polysaccharide or a salt thereof, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxvoctane. 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt.

19. A crosslinked gel for treating gingival deficiencies comprising at least one polysaccharide or a salt thereof, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxvoctane. 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt.

20. A crosslinked gel for articular viscosupplementation comprising at least one polysaccharide or a salt thereof at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethylene, and mixtures thereof and at least one phosphate salt.

21. A crosslinked gel for treating ophthalmic disorders comprising at least one polysaccharide or a salt thereof, at least one difunctional or multifunctional epoxide crosslinking agent chosen from butanediol diglycidyl ether, diepoxyoctane. 1,2-bis(2,3-epoxypropyI)-2,3-ethylene, and mixtures thereof, and at least one phosphate salt.

Description

EXAMPLES

Example 1

Effect of a Phosphate Salt (STMP)

[0194] Three crosslinked hyaluronic acid gels 1b, 1c and 1d, crosslinked in the presence of different concentrations of sodium trimetaphosphate (SMTP) as compound A, are prepared according to the abovementioned procedure.

[0195] Gel 1a is free of STMP and is thus the control. It is also prepared according to the abovementioned procedure.

[0196] Table 1 below details the nature and amounts of the compounds used.

TABLE-US-00001 TABLE 1 Parameters R.sub.A R.sub.BDDE Crosslinking (in hours and in ° C.) [HA] (mg/g) [00001]$\%.Math.\frac{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}}}{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}.Math.+.Math.\mathrm{crosslinked}}}$ 1a (comparative) 0 0.02 72 hours at 21 ± 2° C. 23 10% 1b (invention) 0.03 (BDDE + STMP) 1c (invention) 0.06 1d (invention) 0.09

[0197] The viscoelastic properties of gels 1a, 1b, 1c and 1d, measured according to the abovementioned protocols, are presented in Table 2 below.

TABLE-US-00002 TABLE 2 Compression Injection G′ (in Pa) δ (in °) strength (in N.s.) force (in N) 1a (comparative) 14 50.9 3.1 10.3 1b (invention) 36 37.9 6.4 12.5 1c (invention) 49 33.9 10.3 14.0 1d (invention) 54 31.8 14.8 14.8

[0198] Gel 1a (control) is not suited to wrinkle-filling properties, since its phase angle δ is greater than 45° . In other words, the amount of crosslinking agent used is too low to obtain a crosslinked gel that is satisfactory as regards filling wrinkles.

[0199] On the other hand, gels 1b, 1c and 1d according to the present invention, although manufactured with the same amount of BDDE, have an angle δ of less than 45° and are thus satisfactory as regards filling wrinkles.

[0200] This effect is associated with the presence of STMP in the crosslinking system. What is more, it is observed that the more the amount of STMP increases, the more the elasticity part of the gel increases (i.e. decrease in δ), and the more the elastic modulus increases.

[0201] The increase in the amount of STMP between gels 1b, 1c and 1d according to the invention is also accompanied by an increase in the compression strength, thus revealing gels that are increasingly consistent and resistant to deformation.

[0202] The degradation-resistance properties of gels 1a, 1b, 1c and 1d, measured according to the abovementioned protocols, are presented in Table 3 below.

TABLE-US-00003 TABLE 3 Loss of G′ after Loss of G′ after sterilization (%) oxidative stress (%) 1a (comparative) 43 89 1b (invention) 14 65 1c (invention) 9 65 1d (invention) 19 48

[0203] Degree of Modification

[0204] Gels 1a and 1d are then evaluated as regards the degree of modification of hyaluronic acid. To this end, gels 1a and 1d are washed/precipitated using isopropanol. The solids obtained are dried and then dissolved in D.sub.2O, and treated in the presence of hyaluronidase (type VI-S, Sigma, 3kU) in 1 ml of D.sub.2O for degradation of the gel, so as to obtain a liquid matrix for analysis. Each homogeneous mixture obtained is then analysed by .sup.1H NMR.

[0205] Protocol for Measuring the Degree of Modification

[0206] Characterization of the degree of modification is performed by NMR spectroscopy. The degree of modification is obtained by applying the method developed by L. Nord et al. on samples of HA crosslinked with BDDE. The degree of modification is obtained by integrating the 1H NMR signal of the N-acetyl group (δ≈2 ppm) present in the HAs and a signal present in the crosslinking agent (two-CH2-groups, δ≈1.6 ppm). The ratio of the

[00002]$\mathrm{MoD}=\frac{\left[\frac{\mathrm{Integral}.Math..Math.{\delta }_H.Math..Math.1.6}{4}\right]}{\left[\frac{\mathrm{Integral}.Math..Math.{\delta }_H.Math..Math.2.0}{3}\right]}$

integrals of these two signals (crosslinking agent/NAc HA) corresponds to the degree of modification, after correction for the number of protons associated with each signal. The NMR analysis is performed on a Briiker Avance 1 spectrometer operating at 400 MHz (.sup.1H).

[0207] The degrees of modification measured according to the above protocol for gels 1a and 1d are presented in the table below.

TABLE-US-00004 GEL Degree of modification 1a (comparative) 1.4% 1d (invention) 1.4%

[0208] The same type of NMR analysis, but performed by phosphorus NMR (.sup.31P NMR) was performed. In this respect, the primary reference in .sup.31P NMR is aqueous 58% phosphoric acid (δ=0 ppm). This NMR analysis was performed on a Brüker Avance 1 spectrometer operating at 400 MHz (.sup.1H) and 161.97 MHz (31P).

[0209] This analysis did not make it possible to reveal any attachment between the hyaluronic acid and a phosphorylated species derived from STMP.

[0210] The use of STMP in the presence of BDDE for performing the crosslinking reaction thus makes it possible to obtain crosslinked gels with satisfactory filling properties (mechanical properties and degradation resistance), such properties not being achievable, however, at the (nBDDE/nHA) molar ratio under consideration.

[0211] What is more, this effect of STMP occurs without increasing the degree of modification of the hyaluronic acid (HA).

Example 2

Confirmation of the Effect of a Phosphate Salt (STMP) on more Crosslinked Gels (i.e. r.SUB.BDDE .Greater than that of Example 1)

[0212] Three crosslinked hyaluronic acid gels 3b, 3c and 3d comprising the use of different concentrations of STMP as compound A are prepared according to the abovementioned procedure.

[0213] Gel 3a is free of STMP and is thus the control. It is also prepared according to the abovementioned procedure.

[0214] Table 4 below details the nature and amounts of the compounds used.

TABLE-US-00005 TABLE 4 Parameters R.sub.A R.sub.BDDE Crosslinking [HA] (in mg/g) [00003]$\%.Math.\frac{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}}}{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}.Math.+.Math.\mathrm{crosslinked}}}$ 3a (comparative) 0 0.04 72 hours at 21 ± 2° C. 18 10% 3b (invention) 0.02 (BDDE + STMP) 3c (invention) 0.04 3d (invention) 0.06

[0215] The viscoelastic properties of gels 3a, 3b, 3c and 3d, measured according to the abovementioned protocols, are presented in Table 5 below.

TABLE-US-00006 TABLE 5 Compression Injection G′ (in Pa) δ (in °) strength (in N.s.) force (in N) 3a (comparative) 96 16.2 16.4 12.4 3b (invention) 107 15.3 17.4 11.3 3c (invention) 117 14.0 18.0 10.8 3d (invention) 197 10.8 20.2 9.5

[0216] Despite an HA concentration lower than that used in Example 1, gel 3a has satisfactory properties for the function of filling wrinkles, with a phase angle δ of less than 45° and far superior mechanical properties. This increase in the mechanical properties is associated with the amount of BDDE used (R.sub.BDDE=0.04) which is higher than that of Example 1. The amount of BDDE crosslinking agent used in this gel 3a is thus satisfactory for obtaining a gel that is efficient as regards filling wrinkles.

[0217] Gels 3b, 3c and 3d according to the present invention have mechanical properties superior to those of the control gel 3a. This effect is associated with the presence of STMP in combination with the BDDE. The more the amount of STMP increases, the greater the increase in the mechanical properties. Thus, gel 3d has an elastic modulus G′ more than 2 times higher than that of the control gel 3a.

[0218] The compression strength also increases with the amount of STMP used.

[0219] Finally, it is interesting to note that the injection force has a tendency to decrease with gels 3b, 3c and 3d according to the present invention; in all cases, the injection force is less than that obtained in Example 1, by virtue of the use of a lower concentration of HA. The improvement in the mechanical properties of a gel crosslinked according to a process according to the invention therefore does not take place at the expense of the injection force for a customary degree of crosslinking

[0220] The degradation-resistance properties of gels 3a, 3b, 3c and 3d, measured according to the abovementioned protocols, are presented in Table 6 below.

TABLE-US-00007 TABLE 6 Loss of G′ after Loss of G′ after sterilization (%) oxidative stress (%) 3a (comparative) 40 73 3b (invention) 15 47 3c (invention) 18 45 3d (invention) 15 30

[0221] Gels 3b, 3c and 3d crosslinked in the presence of STMP show a much higher resistance to degradation than that of gel 3a crosslinked without STMP. Gel 3d is the most resistant, with a minimal relative loss of elastic modulus G′.

Example 3

Effect of Different Phosphate Salts

[0222] Unlike sodium trimetaphosphate (STMP) which is cyclic, sodium triphosphate (STPP) is a linear phosphate salt. STMP and STPP are both triphosphates, and the amount tested is thus R.sub.A=0.06, for comparison with gel 3d of Example 2, identical but with STMP.

[0223] Unlike STMP and STPP which are triphosphates, sodium phosphate (SP) is a monophosphate. The amount tested is always R.sub.A=0.06 for gel 4c.

[0224] Thus, three crosslinked hyaluronic acid gels 3d, 4b and 4c comprising the use of sodium trimetaphosphate (STMP), sodium triphosphate (STPP) or sodium phosphate (SP) as compound A are prepared according to the abovementioned procedure.

[0225] Gel 3a, which is free of phosphate salt, is thus the control. It is also prepared according to the abovementioned procedure. Gels 3a and 3d are those of Example 2.

[0226] Table 7 below details the nature and amounts of the compounds used.

TABLE-US-00008 TABLE 7 Parameters A R.sub.A R.sub.BDDE Crosslinking [HA] (mg/g) [00004]$\%.Math.\frac{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}}}{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}.Math.+.Math.\mathrm{crosslinked}}}$ 3a (comparative) / 0 0.04 72 hours at 21 ± 2° C. 18 10% 3d (invention) STMP 0.06 (BDDE + STMP) 4b (invention) STPP 0.06 3d (invention) SP 0.06

[0227] The viscoelastic properties of gels 3a, 3d, 4b and 4c, measured according to the abovementioned protocols, are presented in Table 8 below.

TABLE-US-00009 TABLE 8 Compression strength Injection force G′ (Pa) δ (°) (in N.s.) (in N) 3a (comparative) 96 16.2 16.4 12.4 3d (invention) 197 10.8 20.2 9.5 4b (invention) 123 12.0 16.8 10.6 4c (invention) 106 16.4 16.6 9.3

[0228] The mechanical properties of gels 4b and 4c remain better than those of the control gel 3a free of phosphate salt. STPP and SP thus also have an advantageous effect as regards the viscoelastic properties.

[0229] The degradation-resistance properties of gels 3a, 3d, 4b and 4c, measured according to the abovementioned protocols, are presented in Table 9 below.

TABLE-US-00010 TABLE 9 Loss of G′ after Loss of G′ after sterilization (%) oxidative stress (%) 3a (comparative) 40 73 3d (invention) 15 30 4b (invention) 16 29 4c (invention) 31 62

[0230] As for gel 3d crosslinked in the presence of STMP, the degradation resistance is improved for gels 4d crosslinked in the presence of STPP, and for gel 4c crosslinked in the presence of SP.

[0231] The use of STPP or SP in the presence of BDDE in the crosslinking medium also makes it possible to obtain an advantageous effect on the mechanical properties. The degradation resistance is also improved with the use of these phosphate salts STPP and SP.

Example 4

Demonstration of a Synergistic Effect of a Phosphate Salt (STMP) used during Crosslinking with BDDE

[0232] Four crosslinked hyaluronic acid gels 5a, 5b, 5c and 5d, comprising the use of BDDE and STMP as compound A, are prepared according to the abovementioned procedure.

[0233] Gel 5a is in accordance with the present invention since it is obtained after a step of crosslinking in the simultaneous presence of BDDE and STMP.

[0234] Gels 5b, 5c and 5d are not in accordance with the present invention since they are obtained after a dissociated crosslinking step. Specifically, the BDDE and STMP were integrated sequentially and with a significant time interval with regard to the crosslinking

[0235] Table 10 below details the nature and amounts of compounds used.

TABLE-US-00011 TABLE 10 Parameters R.sub.A R.sub.BDDE Crosslinking at 21 ± 2° C. [HA], mg/g [00005]$\%.Math.\frac{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}}}{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}.Math.+.Math.\mathrm{crosslinked}}}$ 5a (invention) 0.06 0.04 72 hours 18 0 STMP + BDDE 5b (comparative) 72 hours STMP then 72 hours BDDE 5c (comparative) 24 hours STMP then 72 hours BDDE 5d (comparative) 72 hours BDDE then STMP added post-crosslinking

[0236] The viscoelastic properties of gels 5a, 5b, 5c and 5d, measured according to the abovementioned protocols, are presented in Table 11 below.

TABLE-US-00012 TABLE 11 Compression G′ (Pa) δ (°) strength (N.s.) Injection force (N) 5a (invention) 136 9.6 22.6 38.0 5b (comparative) 17 18.5 3.2 11.5 5c (comparative) 87 13.5 17.7 31.8 5d (comparative) 76 13.7 17.8 34.9

[0237] Gel 5a in accordance with the invention has optimum rheological properties, by virtue of crosslinking performed with BDDE in the presence of STMP.

[0238] If the same amount of STMP is incorporated after crosslinking with BDDE, the effect on the mechanical properties is no longer observed: thus, gel 5d has significantly lower mechanical properties.

[0239] Additionally, there is no improvement in the mechanical properties of the gel if STMP is incorporated into the HA 24 hours before the addition of BDDE (gel 5c), and there is even degradation of the mechanical properties of the gel if STMP is added 72 hours before the addition of BDDE (gel 5b).

[0240] In other words, these results demonstrate that the effect of the reaction {STMP +BDDE} is not equivalent to {STMP} +{BDDE}; there is thus a synergistic effect when the crosslinking reaction of HA is performed via a non-dissociated combination between the BDDE and STMP.

Example 5

Effect of STMP with Faster Crosslinking Conditions

[0241] Three crosslinked hyaluronic acid gels 6a, 6b and 6c comprising the use of BDDE alone (i.e. 6a) or with STMP as compound A (i.e. 6b and 6c) are prepared according to the abovementioned procedure.

[0242] Table 12 below details the nature and amounts of the compounds used.

TABLE-US-00013 TABLE 12 Parameters R.sub.A R.sub.BDDE Crosslinking [HA], mg/g [00006]$\%.Math.\frac{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}}}{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}.Math.+.Math.\mathrm{crosslinked}}}$ 6a (comparative) 0 0.15 3 hours at 52 ± 2° C. 20 10% 6b (invention) 0.06 (BDDE + STMP) 6c (invention) 0.15

[0243] This mode of heat-mediated crosslinking (52° C.) is interesting in that it is faster than at room temperature.

[0244] The viscoelastic properties of gels 6a, 6b and 6c, measured according to the abovementioned protocols, are presented in Table 13 below.

TABLE-US-00014 TABLE 13 Compression G′ (Pa) δ (°) strength (N.s.) Injection force (N) 6a (comparative) 217 10.5 18.1 10.1 6b (invention) 299 10.2 19.0 11.1 6c (invention) 426 9.5 18.9 10.2

[0245] In this case also, an increase in the elastic modulus G′ is observed when the crosslinking is performed in the presence of the phosphate salt (STMP).

Example 6

Comparison STMP vs NaCl

[0246] Four hyaluronic acid crosslinked gels 7a, 7b, 7c and 7d comprising the use of BDDE alone (i.e. 7a), with STMP as compound A (i.e. 7b), or with NaCl as compound A (i.e. 7c and 7d), were prepared according to the above protocol.

[0247] Gel 7a is devoid of any STMP and NaCl and is therefore the reference.

TABLE-US-00015 TABLE 14 Parameters A R.sub.A R.sub.BDDE Crosslinking [HA], mg/g [00007]$\%.Math.\frac{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}}}{{\mathrm{HA}}_{\mathrm{non}.Math.\text{-}.Math.\mathrm{crosslinked}.Math.+.Math.\mathrm{crosslinked}}}$ 7a (reference) / 0 0.02 72 H at 21 ± 2° C. 23 10% 7b (invention) STMP 0.06 (BDDE + A) 7c (comparative) NaCl 0.18 7d (comparative) NaCl 1.5

[0248] Since SMTP is a trivalent salt and NaCl a monovalent salt, gel 7c was made with an amount of NaCl corresponding to 3 times that of the gel 7b. Gel 7d is manufactured with a greater amount of NaCl (R.sub.A=1.5), corresponding to a massic concentration of NaCl in the crosslinking medium of 3%, as described in FR 2 997 085.

[0249] The viscoelastic properties of gels 7a, 7b, 7c et 7d, measured according to the above described protocols, are given in Table 15.

TABLE-US-00016 TABLE 15 Compression Injection G′ (in Pa) δ (in °) strength (N.s.) force (N) 7a (reference) 14 50.9 3.1 10.3 7b (invention) 49 33.9 10.3 14.0 7c (comparative) 23 45.7 3.2 9.4 7d (comparative) 6.3 63.0 1.1 8.3

[0250] Contrary to gel 7b according to the invention, reference gel 7a and comparative gels 7c and 7d are not suitable for filling wrinkles because their phase angle δ is greater than 45°.