BIODEGRADABLE SINGLE-PHASE COHESIVE HYDROGELS

Abstract

A biodegradable single-phase cohesive hydrogel includes a homogenous mixture of 2 to 5 identical or different polymers interpenetrating each other in a single phase, wherein the polymers are independently crosslinked prior to interpenetration by mixing, and the crosslinked polymers are insoluble in water and miscible with each other.

Claims

1-24. (canceled)

25. A biodegradable single-phase cohesive hydrogel, comprising: a homogenous mixture of 2 to 5 identical or different polymers interpenetrating each other in a single phase, wherein: the polymers are independently crosslinked prior to interpenetration by mixing; and the crosslinked polymers are insoluble in water and miscible with each other.

26. The hydrogel according to claim 25, wherein the polymers have different degrees of crosslinking.

27. The hydrogel according to claim 25, wherein the polymers have identical degrees of crosslinking.

28. The hydrogel according to claim 25, wherein each polymer is a polysaccharide.

29. The hydrogel according to claim 28, wherein each polysaccharide is selected from the group consisting of hyaluronic acid, keratan, heparin, cellulose, cellulose derivatives, alginic acid, xanthan, carrageenan, chitosan, chondroitin, and biologically acceptable salts thereof.

30. The hydrogel according to claim 28, wherein each polysaccharide is hyaluronic acid or a biologically acceptable salt thereof.

31. The hydrogel according to claim 28, wherein at least one polysaccharide is selected from the group consisting of cellulose derivatives and biologically acceptable salts thereof.

32. The hydrogel according to claim 28, wherein at least one polysaccharide is selected from the group consisting of chondroitin and biologically acceptable salts thereof.

33. The hydrogel according to claim 28, wherein at least one polysaccharide is selected from the group consisting of chitosan and biologically acceptable salts thereof.

34. The hydrogel according to claim 25, wherein the homogenous mixture is of 2 polymers.

35. The hydrogel according to claim 25, further comprising one or more active principles selected from the group consisting of antioxidants, antiseptics, anti-inflammatories, local anesthetics, and mixtures thereof.

36. The hydrogel according to claim 25, further comprising at least one antioxidant selected from the group consisting of mannitol and sorbitol.

37. The hydrogel according to claim 25, further comprising a local anesthetic.

38. A kit comprising the hydrogel according to claim 25 packaged in a sterile syringe.

39. A process of preparing a biodegradable single-phase cohesive hydrogel, comprising: independently crosslinking 2 to 5 polymers to obtain 2 to 5 identical or different crosslinked polymers; and interpenetrating the crosslinked polymers by mixing to obtain a homogenous mixture of the crosslinked polymers in a single phase, wherein the crosslinked polymers are insoluble in water and miscible with each other.

40. The process according to claim 39, wherein crosslinking is performed using a bi- or polyfunctional crosslinking agent selected from the group consisting of bi- or polyfunctional epoxy compounds and divinyl sulfone.

41. The process according to claim 39, wherein each polymer has a degree of crosslinking within a range from 0.02 to 0.4.

42. A method of cosmetic injection comprising injecting the hydrogel according to claim 25 to fill a wrinkle, skin defect, or volume defect of a patient.

Description

EXAMPLES

[0101] Degree of Crosslinking:

[0102] The degrees of crosslinking x in the examples which follow are defined by:

[0103] x=number of moles of crosslinking agent introduced into the reaction medium/total number of disaccharide units introduced into the reaction medium.

Example 1

[0104] Crosslinking Gel 1

[0105] Stage a): Hydration of Sodium Hyaluronate Fibers in the Form of a Noncrosslinked Gel

[0106] Sodium hyaluronate fibers of injectable grade (1 g: molecular weight: approximately 2.7 MDa) are weighed out in a container. A 1% aqueous solution of sodium hydroxide in water (7.4 g) is added and the combined mixture is homogenized for approximately 1 hour using a spatula at ambient temperature and 900 mm Hg.

[0107] Stage b): Crosslinking

[0108] BDDE (65 mg) is added to the noncrosslinked sodium hyaluronate (NaHA) gel obtained in the preceding stage, the combined mixture being homogenized with a spatula for approximately 30 minutes at ambient temperature. The combined mixture is subsequently placed on a water bath at 50° C. for 2 h 20 in order to obtain a degree of crosslinking x1 of approximately 0.14.

[0109] Stage c): Neutralization, Purification

[0110] The crosslinked final gel is subsequently neutralized by addition of 1N HCl and placed in a phosphate buffer bath in order to stabilize the pH and to make possible its hydration or swelling as far as 30 mg/g of HA. An NaHa hydrogel crosslinked by the route conventionally used is thus obtained: G1 with an HA concentration of approximately 30 mg/g.

[0111] A portion of the gel is stored at this concentration and the other portion is diluted by addition of phosphate buffer in order to obtain, at the end, 20 mg/g of HA. This gel is subsequently homogenized before being filled into syringes which are sterilized by autoclaving: sterile syringes comprising gel G1 at 20 mg/g.

[0112] Crosslinking Gel 2

[0113] Stage a): Hydration of Sodium Hyaluronate Fibers in the Form of a Noncrosslinked Gel

[0114] Sodium hyaluronate fibers of injectable grade (1 g; molecular weight: approximately 1.5 MDa) are weighed out and dried beforehand in a container. A 1% aqueous solution of sodium hydroxide in water (6.3 g) is added and the combined mixture is homogenized for approximately 1 hour using a spatula at ambient temperature and 900 mmHg.

[0115] Stage b): Crosslinking

[0116] BDDE (43 mg) is added to the noncrosslinked sodium hyaluronate (NaHA) gel obtained in the preceding stage, the combined mixture being homogenized with a spatula at ambient temperature and atmospheric pressure for approximately 30 minutes. The combined mixture is subsequently placed on a water bath at 50° C. for 2 h 20 in order to obtain a degree of crosslinking x2 of approximately 0.09.

[0117] Stage c): Neutralization, Purification

[0118] The crosslinked final gel is subsequently neutralized by addition of 1N HCl and placed in a phosphate buffer bath in order to stabilize the pH and to make possible its hydration or swelling as far as 30 mg/g of I-IA. An NaHa hydrogel crosslinked by the route conventionally used is thus obtained: G2 with an HA concentration of approximately 30 mg/g.

Example 2

[0119] Mixing/interpenetration of Gel 1 and Gel 2 in the proportions 10% G1-90% G2

[0120] Mixing/interpenetration of the gels G1 and G2 at 10%/90%

[0121] 18 g of gel G2 at 30 mg/g are weighed out and 2 g of gel G1 obtained at the end of the preceding stage c) (G1 at 30 mg/g) are added thereto. 10 g of phosphate buffer are added and the 2 gels are placed under slow mechanical stirring for 1 h under hyperbaric pressure.

[0122] The mixture thus obtained is a homogeneous gel comprising 20 mg/g of HA and composed of 2 inter-penetrating networks; this gel is then packaged into syringes and autoclaved.

Example 3

[0123] Mixing/interpenetration of Gel 1 and Gel 2 in the proportions of 50%-50%

[0124] The gels obtained at the end of stage c) of each example above: gel 1 crosslinked to x1 approximately 0.14 and G2 crosslinked to x2 approximately 0.09, both with a concentration of approximately 30 mg/g of HA, are weighed out: 10 g of G1+10 g of G2.

[0125] 10 g of phosphate buffer are also added and the 2 gels are placed under slow mechanical stirring for 1 h under hyperbaric pressure.

[0126] The mixture thus obtained is a homogeneous gel comprising 20 mg/g of HA and composed of 2 inter-penetrating networks; this gel is then packaged into syringes and autoclaved.

Example 4

[0127] Characterization of the gels of examples 1 and 2: [0128] gel G1 crosslinked to x1, [0129] mixture of 10% G1 and 90% G2 crosslinked to x2, [0130] mixture of 50% G1 and 50% G2,
these 3 final products being all 3 at a final concentration of 20 mg/g of HA.

[0131] Characterization of the extrusion force or “injectability”:

[0132] This test is carried out on the gels packaged into syringes and sterilized, with 27G½ needles, on a tensile compression testing machine with a rate of compression of 13 mm/min. The results of the extrusion forces of each of examples 1, 2 and 3 are given in the table below:

TABLE-US-00001 Gel tested Injectability (N) Gel 1 37 10% Gel 1 + 90% Gel 2 21 50% Gel 1 + 50% Gel 2 31

[0133] A lower injectability of the interpenetrating networks of crosslinked gels in the comparison with the gel G1 alone is clearly observed.

[0134] Decomposition Test

[0135] These various gels were also characterized by an in vitro temperature decomposition test. This test makes it possible to simulate the subsequent in vivo persistence of the gels injected intradermally. It was developed on the basis of the specifications of the test of persistence described in patent FR 2 861 734. The gels were all placed in an oven at 93° C. for 14 h, 24 h and 48 h, with characterization of the elasticity after each time. The curves of the trend in the decomposition results for these various gels subsequently make it possible to evaluate the half-life of these various gels (period of time necessary to have G′=G′0/2, in hours, with G′0=elasticity at t0 of the gel characterized). The half-lives obtained are also given in the table below.

TABLE-US-00002 Gel tested 1/2 life (hours) Gel 1 19 10% Gel 1 + 90% Gel 2 22.5 50% Gel 1 + 50% Gel 2 20.5

[0136] A greater decomposition is observed for Gel 1 alone, in comparison with the two interpenetrating networks of gels crosslinked beforehand.

[0137] Thus, for lower injectability and thus better control of the surgical action, the half-lives of the interpenetrating networks of gels obtained according to the invention are longer, guaranteeing a greater time of in vivo persistence.

Example 5

[0138] In order to confirm the cohesiveness and the single-phase nature of the hydrogels according to the invention, manual centrifuging tests of 3 times 5 minutes were carried out on the 10/90 and 50/50 mixtures comprising 20 mg/g of NaHA obtained in the preceding examples.

[0139] By comparison, a product of “two-phase” type, such as described in the prior art, was prepared according to the procedure of patent EP 0 466 300 with 50% of crosslinked NaHA particles dispersed in 50% of noncrosslinked NaHA viscous product, the two phases having been hydrated beforehand in phosphate buffer, comprising 20 mg/g of NaHA.

[0140] The products according to the invention obtained in the preceding examples do not show any separation on settling; the product, if ejected after the centrifuging operations, still has a homogeneous appearance.

[0141] On the other hand, the product of “two-phase” type shows, after centrifuging, separated particles at the bottom of the syringe. If the product is ejected from the syringe, the viscous product exits first, followed by the particles, which have no cohesiveness with one another, agglomerated at the bottom of the syringe, and which render the injectability particularly difficult.

Example 6

[0142] Mixing/interpenetrating of the gels G1 and G2 of example 1, in order to finally obtain gels and mixtures of gels at a concentration of 25.5 mg/g according to the process described in example 2 with adjustment of the NaHA concentrations by addition of phosphate buffer, in the following proportions:

[0143] IPN-Like Gel 1: 70% Gel 1 cross. x1+30% Gel 2 cross. x2

[0144] IPN-Like Gel 2: 50% Gel 1 cross. x1+50% Gel 2 cross. x2

[0145] IPN-Like Gel 3: 30% Gel 1 cross. x1+70% Gel 2 cross. x2

[0146] These gels are then packaged into syringes and sterilized by autoclaving.

[0147] Characteriziation of the extrusion force and of the elasticity of these IPN-like gels and of Gel 1 cross-linked to x1 and brought to an NaHA concentration of 25.5 mg/g:

[0148] The extrusion force is characterized on a Mecmesin tensile/compression testing machine under a rate of compression of 50 mm/min with 23G1 ¼ needles; the results are given in the table below.

[0149] The elasticity is characterized on a TA Instruments AR2000 Ex rheometer in oscillation at 25° C., the value of the elasticity being recorded at a frequency of 1 Hz; the results are given in the table below.

TABLE-US-00003 Inter- Inter- Inter- penetrating penetrating penetrating Gel 1 at Gel 1 Gel 2 Gel 3 25.5 mg/g 25.5 mg/g 25.5 mg/g 25.5 mg/g Extrusion force (N)  63  61  61  57 23G1¼ need. Rate 50 mm/min Elasticity: G′ (Pa) at 200 225 244 265 1 Hz

[0150] It is observed, with regard to the 3 interpenetrating gels, that the extrusion forces are fairly close but all less than that of Gel 1, for increasing elasticities. Thus, the use of this technique of interpenetrating crosslinked gels makes it possible to obtain finished products of variable rheology: increasing elasticity (thus a better volumizing effect and a greater expected persistence) for lower levels of injectability.

Example 7

[0151] Synthesis of Gel 3: A gel is synthesized according to the protocol/operating conditions of example 1, Gel 1:

[0152] Stage a): Hydration of Sodium Hyaluronate Fibers in the Form of a Noncrosslinked Gel

[0153] This stage is identical to stage a) of the synthesis of Gel 1 of example 1.

[0154] Stage b): Crosslinking the Gel

[0155] This stage is identical to stage b) of the synthesis of Gel 1 of example 1, with 81 mg of PDDE. A Gel 3 with a degree of crosslinking x3 of approximately 0.17 is obtained.

[0156] Stage c): Neutralization, Purification

[0157] This stage is identical to stage c) of the synthesis of Gel 1 of example 1, in order to obtain a gel G3 with an HA concentration of approximately 30 mg/g.

[0158] A portion of the gel is stored at this concentration and the other portion is diluted by addition of phosphate buffer in order to finally obtain 24 mg/g of HA; this gel is subsequently homogenized before being filled into syringes which are sterilized by autoclaving: sterile syringes comprising gel G3 at 24 mg/g.

[0159] Interpenetration Gel 1/Gel 3 in the proportions 80/20:

[0160] 16 g of gel G1 at 30 mg/g are weighed out and 4 g of gel G3 at 30 mg/g, obtained at the end of the preceding stage c), are added thereto. 5 g of phosphate buffer are added and the 2 gels are placed under slow mechanical stirring for 1 h.

[0161] The mixture thus obtained is a homogeneous gel comprising 24 mg/g of HA and composed of 2 inter-penetrating networks; this gel is then packaged into syringes and autoclaved.

[0162] Characterization of the gels and interpenetrating gels described above: [0163] Gel 3 with a degree of crosslinking x3, at 24 mg/g, [0164] Gel 1 with a degree of crosslinking x1 and brought beforehand to 24 mg/g, packaged into syringes and sterilized, [0165] and the mixture of interpenetrating gels 80% Gel 1+20% Gel 3, at 24 mg/g.

[0166] These gels are characterized by extrusion force. The tests are carried out with 27G½ needles on a Mecmesim tensile/compression testing machine with a rate of compression of 13 ram/min. The results for the extrusion forces of each of these gels are given in the table below.

[0167] These gels are also characterized by the in vitro temperature decomposition test described in example 4. The ½ lives obtained are also given in the table below.

TABLE-US-00004 Extrusion force (N) 27G1/2 needle- 1/2 life 13 mm/min (hours) Gel 1-x1-24 mg/g 27 17 Gel 3-x3-24 mg/g 30 21 80% Gel 1/20% Gel 3-24 mg/g 23 20.5

[0168] Thus, an equivalent persistence is observed for the interpenetrating gel and for Gel 3 crosslinked to the highest degree x3, for a lower level of injectability of this interpenetrating gel.

Example 8

[0169] Synthesis of 3 single-phase crosslinked gels according to examples 1 and 2: [0170] Gel 4:

[0171] Stage a): identical to stage a) for the synthesis of Gel 1 of example 1 with 1 g of HA with a molecular weight of approximately 2.7 MDa and 6.8 g of a 1% aqueous solution of sodium hydroxide in water. The homogenization conditions are the same as in example 1.

[0172] Stage b): Crosslinking: identical to stage b) of the synthesis of Gel 1 of example 1 with 62 mg of BDDE. The combined product is brought to 50° C. on a water bath for 3 hours, in order to obtain a degree of crosslinking x4 of approximately 0.13.

[0173] Stage c): Neutralization, purification: identical to stage c) of the synthesis of Gel 1 of example 1, in order to obtain a Gel 4 at 30 mg/g. A portion of the gel is stored at this concentration and the other portion is diluted by addition of phosphate buffer in order to finally obtain 24 mg/g of HA; this gel is subsequently homogenized before being filled into syringes which are sterilized by autoclaving: sterile syringes comprising gel G4 at 24 mg/g. [0174] Gel 5:

[0175] Stage a): identical to stage a) of the synthesis of Gel 4.

[0176] Stage b): Crosslinking: identical to stage h) of the synthesis of Gel 4 with 80 mg of BDDE. The combined product is brought to 50° C. on a water bath for 3 hours, in order to obtain a degree of crosslinking x5 of approximately 0.17.

[0177] Stage c): Neutralization, purification: identical to stage c) of the synthesis of Gel 4 in order to obtain a Gel 5 at 30 mg/g. A portion of the gel is stored at this concentration and the other portion is diluted by addition of phosphate buffer in order to finally obtain 24 mg/g of HA; this gel is subsequently homogenized before being filled into syringes which are sterilized by autoclaving: sterile syringes comprising gel G5 at 24 mg/g. [0178] Gel 6:

[0179] Stage a): identical to stage a) of the synthesis of Gel 2 of example 1 with 1 g of sodium hyaluronate with a molecular weight of approximately 1.3 MDa and 5.7 g of a 1% aqueous solution of sodium hydroxide in water.

[0180] Stage b): Crosslinking

[0181] Identical to stage c) of example 1 with 41 mg of BDDE. The combined product is brought to 50° C. on a water bath for 3 hours, in order to obtain a degree of crosslinking x6 of approximately 0.09.

[0182] Stage c): Neutralization, purification

[0183] Identical to stage c) of the synthesis of the preceding Gel 5 in order to obtain a Gel 6 at 30 mg/g. A portion of the gel is stored at this concentration and the other portion is diluted by addition of phosphate buffer in order to finally obtain 24 mg/g of HA; this gel is subsequently homogenized before being filled into syringes which are sterilized by autoclaving: sterile syringes comprising gel G6 at 24 mg/g.

[0184] Interpenetration of Gels 4, 5 and 6 (respective proportions: 25%, 5% and 70%)

[0185] 5 g of gel G4 at 30 mg/g are weighed out, 1 g of gel G5 at 30 mg/g is weighed out and then 14 g of gel G6 at 30 mg/g are weighed out. 5 g of phosphate buffer are added and the 3 gels are placed under slow mechanical stirring for 1 h. A final single-phase gel G7 comprising 24 mg/g of sodium hyaluronate and composed of 3 interpenetrating single-phase crosslinked gels is thus obtained.

[0186] Characterization of the Elasticity and of the Extrusion Force of the 3 Conventional Gels and of the Interpenetrating Mixture:

according to the methods described in the preceding examples.

TABLE-US-00005 Gel G7 (interpenetrating 4, Gel 4 Gel 5 Gel 6 5 and 6) 24 mg/g 24/mg 24 mg/g 24 mg/g Extrusion force (N)  31  38  18  16 23G1¼ need. Rate 13 mm/min Elasticity: G′ (Pa) 245 415 186 224 at 1 Hz

[0187] Gel G7, composed of the interpenetration of the 3 crosslinked gels (G4, G5 and G6), has the lowest extrusion force, for an elasticity value greater by approximately 20% than that of the gel G6 with a close but slightly greater level of injectability.

[0188] Its elasticity is lower by only 10% with respect to that of Gel 4, the level of injectability of which is greater by more than 40%.

[0189] The advantage of these interpenetrating gels is clearly perceived.

Example 9

[0190] Interpenetration of crosslinked HA and crosslinked CMC (carboxymethyl cellulose) gels [0191] Crosslinked CMG gel: gel G8

[0192] Stage a): Hydration of NaCMC in the form of a noncross-linked gel

[0193] 1 g of sodium carboxymethyl cellulose with an intrinsic viscosity (supplied by Sigma) is weighed out in a container. A 1% aqueous solution of sodium hydroxide in water (7.3 g) is added and the combined mixture is homogenized for approximately 90 minutes using a spatula at ambient temperature and 900 mmHg.

[0194] Stage b): Crosslinking

[0195] BDDE (37 mg) is added to the noncrosslinked CMC gel obtained in the preceding stage, the combined mixture being homogenized with a spatula for approximately 30 minutes at ambient temperature. The combined mixture is subsequently placed on a water bath at 50° C. for 3 h in order to obtain a degree of crosslinking x8 of approximately 0.19.

[0196] Stage c): Neutralization, purification

[0197] The crosslinked final gel is subsequently neutralized by addition of 1N HCl and placed in a phosphate buffer bath in order to stabilize the pH and to make possible its hydration or swelling as far as 45 mg/g of CMC. An NaCMC hydrogel crosslinked by the route conventionally used is thus obtained: G8 with a CMC concentration of approximately 45 mg/g. [0198] Interpenetration of HA gel G1 and CMC gel G8

[0199] The HA gel G1 crosslinked to a level of 0.14, at a concentration of 30 mg/g, is added in various proportions to the crosslinked NaCMC gel G8, the phosphate buffer is added in order to adjust the final concentrations to 26 mg/9 of HA and 37 mg/g of CMC and the 2 gels are placed under slow mechanical stirring with the phosphate buffer for 1 hour under hyperbaric pressure. 3 interpenetrating gels as described below are thus obtained: [0200] Gel 9: 30% G1+70% G8 [0201] Gel 10: 50% G1+50% G8 [0202] Gel 11: 70% G1+30% G8

[0203] These 3 interpenetrating gels are subsequently packaged into syringes and characterized by rheology (elastic modulus G′) and by injectability under a rate of 13 mm/min with 27G½ needles. The gels G1 and G8 are also adjusted to the concentrations of 26 mg/g for G1 and 37 mg/g for G8 in order to compare them with the 3 interpenetrating gels.

[0204] The results of the characterizations are combined in the table below.

TABLE-US-00006 G9 G10 G11 G1 G8 Inter- Inter- Inter- (cross- (cross- penetrating penetrating penetrating linked linked gel gel gel HA, CMC, 30% Gl + 50% Gl + 70% Gl + 26 mg/g) 37 mg/g) 70% G8 50% G8 30% G8 Elastic 235 265 240 243 264 modulus G′ at 1 Hz (Pa) Inject-  33  18  18  12  16 ability 27G1/2 need. (N)

[0205] A virtually constant elastic modulus is observed for the 5 interpenetrating or non-interpenetrating gels but with lower levels of injectability for the inter-penetrating gels than for each independent crosslinked gel, with a high synergistic effect with regard to the 50/50 mixture (Gel 10).