CONTINUOUS RELEASE COMPOSITIONS MADE FROM HYALURONIC ACID, AND THERAPEUTIC APPLICATIONS OF SAME

Abstract

The present invention concerns polymer particles made from poly(lactic-co-glycolic acid) (PLGA) polymer, poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer, or the mixture of same, combined with hyaluronic acid molecules or hyaluronic acid salts, and the method for preparing same. The present invention also concerns injectable pharmaceutical or cosmetic compositions comprising such polymer particles, the method for preparing such compositions, and the use thereof for medical purposes, in particular for the prevention and/or treatment of musculoskeletal diseases, diseases and traumatic conditions of the skin, oral disorders, vaginal mucosa dryness and urinary infections or cystitis, dryness of the eye membrane and eye infections, obesity, and the use of same to combat ageing of the skin and/or for repairing the dermal tissue (mesotherapy).

Claims

1-28. (canceled)

29. A process for the preparation of polymer particles consisting essentially of at least a poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer or a mixture of a poly(lactic-co-glycolic acid) (PLGA) polymer and a poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer, combined with hyaluronic acid molecules or hyaluronic acid salts without formation of covalent bonds between the hyaluronic acid molecules or hyaluronic acid salts and the polymer, wherein the poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer has a molecular weight ranging from 50 000 to 70 000 g.mol.sup.−1, wherein said polymer particles continuously release the hyaluronic acid molecules or hyaluronic acid salts, and have a size ranging from 10 to 130 μm, wherein the continuous release of the hyaluronic acid or hyaluronic acid salts continues to occur for between 8 to 12 weeks when the polymer particles are in synovial fluid, and wherein the content by weight of hyaluronic acid or of hyaluronic acid salts combined with the polymer particles is in a range from 1 to 50 μg.mg.sup.−1, comprising: (i) with stirring, emulsifying an aqueous solution of hyaluronic acid or of hyaluronic acid salts in an organic solution comprising at least a poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer or a mixture of a poly(lactic-co-glycolic acid) (PLGA) polymer and of a poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer, (ii) with stirring, mixing the emulsion obtained from stage (i) with an aqueous solution of polyvinyl alcohol (PVA), (iii) with stirring, adding an aqueous solvent to the emulsion obtained from stage (ii), said aqueous solvent preferably being purified water, (iv) filtering the emulsion obtained from stage (iii), and (v) freeze-drying the polymer particles obtained from stage (iv), and (vi) optionally sterilizing the freeze-dried polymer particles obtained from stage (v).

30. The process as claimed in claim 29, in which the aqueous solution of hyaluronic acid or of hyaluronic acid salts of stage (i) is an aqueous solution of polyvinyl alcohol (PVA) having a concentration by weight of polyvinyl alcohol (PVA) of 1 to 10%.

31. The process as claimed in claim 30, in which the aqueous solution of hyaluronic acid or of hyaluronic acid salts of stage (i) is an aqueous solution of polyvinyl alcohol (PVA) having a concentration by weight of polyvinyl alcohol (PVA) of 2 to 5%.

32. The process as claimed in claim 31, in which the aqueous solution of hyaluronic acid or of hyaluronic acid salts of stage (i) is an aqueous solution of polyvinyl alcohol (PVA) having a concentration by weight of polyvinyl alcohol (PVA) of 4%.

33. The process as claimed in claim 29, in which the aqueous solution of polyvinyl alcohol (PVA) employed during stage (ii) has a concentration by weight of polyvinyl alcohol (PVA) of 1 to 10%.

34. The process as claimed in claim 29, in which the aqueous solution of polyvinyl alcohol (PVA) employed during stage (ii) has a concentration by weight of polyvinyl alcohol (PVA) of 2 to 5%.

35. The process as claimed in claim 29, in which the aqueous solution of polyvinyl alcohol (PVA) employed during stage (ii) has a concentration by weight of polyvinyl alcohol (PVA) of 4%.

36. The process as claimed in claim 29, in which the solvent present in the emulsion obtained from stage (iii) is evaporated, before being filtered according to stage (iv).

37. The process as claimed in claim 29, in which the polymer particles obtained from stage (iv) are washed with purified water, before being freeze-dried according to stage (v).

38. The process as claimed in claim 29, in which an uptake solution comprising a pharmaceutically or cosmetically acceptable excipient and/or another active principle is added to the polymer particles obtained from stage (iv), before being freeze-dried according to stage (v).

39. The process as claimed in claim 38, in which the uptake solution is purified water.

40. The process as claimed in claim 38, in which the excipient is chosen from sorbitol, mannitol or trehalose.

41. The process as claimed in claim 38, in which the other active principle is an anesthetic agent.

42. The process as claimed in claim 29, in which the sterilization stage (vi) is carried out by sterilization with gamma rays.

43. The process as claimed in claim 29, in which the hyaluronic acid molecules or hyaluronic acid salts are bonded via low-energy bonds to the poly(lactic-co-glycolic acid)-polyethylene polymer and/or to the poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) copolymer.

44. The process as claimed in claim 29, in which the polymer particules consiste solely of poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer combined with hyaluronic acid molecules or hyaluronic acid salts.

45. The process as claimed in claim 29, in which the polymer particules are composed of the mixture of poly(lactic-co-glycolic acid) (PLGA) polymer and poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer.

46. The process as claimed in claim 45, in which the ratio by weight of the poly(lactic-co-glycolic acid) (PLGA) polymer to the poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer is in a range from 40/60 to 60/40.

47. The process as claimed in claim 45, in which the ratio by weight of the poly(lactic-co-glycolic acid) (PLGA) polymer to the poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer is 50/50.

48. The process as claimed in claim 29, in which the hyaluronic acid molecules have a molecular weight in a range from 0.8×10.sup.6 to 1.2×10.sup.6 g.mol.sup.−1.

49. The process as claimed in claim 29, in which the polymer particles have a size ranging from 20 to 85 μm.

50. A process for the preparation of a composition comprising polymer particles in suspension in an uptake solution, said polymer particles consisting essentially of at least a poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer or a mixture of a poly(lactic-co-glycolic acid) (PLGA) polymer and a poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer, combined with hyaluronic acid molecules or hyaluronic acid salts without formation of covalent bonds between the hyaluronic acid molecules or hyaluronic acid salts and the polymer, wherein the poly(lactic-co-glycolic acid)-polyethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) copolymer has a molecular weight ranging from 50 000 to 70 000 g.mol.sup.−1, wherein said polymer particles continuously release the hyaluronic acid molecules or hyaluronic acid salts, and have a size ranging from 10 to 130 μm, wherein the continuous release of the hyaluronic acid or hyaluronic acid salts continues to occur for between 8 to 12 weeks when the polymer particles are in synovial fluid, and wherein the content by weight of hyaluronic acid or of hyaluronic acid salts combined with the polymer particles is in a range from 1 to 50 μg.mg.sup.−1, said process comprising stages (i) to (vi) as defined in claim 1, and a stage (vii) of suspending the freeze-dried polymer particles obtained from stage (v) or (vi) in an uptake solution.

51. The process as claimed in claim 50, wherein said uptake solution comprises at least one selected from the group of a pharmaceutically or cosmetically acceptable excipient and a second active principle.

52. The process as claimed in claim 50, in which the uptake solution is a solution of purified water, an aqueous solution of crosslinked or uncrosslinked hyaluronic acid or a salt thereof, or an aqueous solution of another salt.

53. The process as claimed in claim 50, in which the uptake solution is an aqueous solution of hyaluronic acid or a salt thereof, the concentration by weight of hyaluronic acid or of hyaluronic acid salts of which varies from 0.5 to 10%.

54. The process as claimed in claim 53, in which the concentration by weight of hyaluronic acid or of hyaluronic acid salts of which varies from 0.5 to 4%.

55. The process as claimed in claim 51, in which the pharmaceutically or cosmetically acceptable excipient is present and is chosen from sorbitol, mannitol or trehalose.

56. The process as claimed in claim 50, in which the second active principle is present and is an anesthetic agent.

Description

[0063] In addition to the preceding provisions, the invention also comprises other provisions which will emerge from the remainder of the description which follows, which relates to examples of preparations of polymer particles and of compositions according to the invention and to the in vivo evaluation of the tolerance of these compositions during injections into the intraarticular space of rabbits, and also to the appended drawings, in which:

[0064] FIGS. 1a and 1b are respectively images of polymer particles in which are bonded hyaluronic acid molecules according to the invention prepared according to example 1 and example 2 (scanning electron microscopy images),

[0065] FIGS. 2a and 2b respectively represent PLGA polymer particles according to the invention combined with hyaluronic acid molecules (FIG. 2a) and PLGA-PEG-PLGA polymer particles according to the invention combined with hyaluronic acid molecules (FIG. 2b),

[0066] FIG. 3 represents the release profile of the polymer particles combined with hyaluronic acid molecules prepared according to example 1, and

[0067] FIG. 4 represents the release profile of the polymer particles combined with hyaluronic acid molecules prepared according to example 2.

EXAMPLES

[0068] Starting materials used:

TABLE-US-00001 TABLE 1 Commercial Reactants references Suppliers Hyaluronic AH HMW Altergon acid Polyvinyl Gohsenol Nippon Gohsei alcohol (PVA) EG-05PW PLGA-PEG- LSB 5050 DLG Evonik (Birmingham Labs) PLGA triblock PEG 6000 PLGA Resomer RG 505 Evonik Ultrapure — MilliPore water Acetone 24201 Sigma-Aldrich Dichloromethane 24233 Sigma-Aldrich Sorbitol S1876 Sigma-Aldrich

Example 1

Preparation of the Polymer Particles According to the Invention

[0069] 1.sup.st Stage: “Primary” Emulsification

[0070] An aqueous solution of hyaluronic acid is prepared by dissolving 50 mg of hyaluronic acid in 5 ml of a 4% by weight polyvinyl alcohol (PVA) solution. An organic solution of polymer is also prepared by dissolving 900 mg of a PLGA-PEG-PLGA triblock polymer in 12 ml of a dichloromethane/acetone (3/1 v/v) mixture. These two solutions are emulsified at room temperature for two minutes with a stirrer of UltraTurrax® IKA T25 Basic type at a speed of 16 000 revolutions/minute and simultaneously with magnetic stirring using a magnetic bar at a speed of 500 revolutions/minute. A second stirring cycle is carried out in identical fashion in an ice bath.

[0071] 2.sup.nd Stage: “Secondary” Emulsification

[0072] The stable emulsion obtained from the 1.sup.st stage is introduced into a glass syringe and then injected into a formulation reactor containing 450 ml of 4% by weight polyvinyl alcohol (PVA), with magnetic stirring at a rate of 750 revolutions/minute.

[0073] 3.sup.rd Stage: Addition of an Aqueous Solvent

[0074] Stirring is maintained for two minutes and then 500 ml of ultrapure water (resistivity greater than 18 MΩ.cm.sup.−1) are added to the reactor. Stirring is subsequently maintained for an additional ten minutes, at a rate of 750 revolutions/minute. The solvent present in the preformed droplets is then diffused into the aqueous phase containing the polyvinyl alcohol (PVA) and polymer particles combined with hyaluronic acid molecules are then formed.

[0075] 4.sup.th Stage: Purification and Extraction of the Solvent

[0076] The solvent present in the emulsion obtained from the 3.sup.rd stage is subsequently evaporated by magnetic stirring at a rate of 500 revolutions/minute for 4 h at 20° C. in a fume cupboard.

[0077] The polymer particles charged with hyaluronic acid are subsequently filtered on an SSWP 3 μm hydrophilic filter in order to remove the aqueous phase. The particles obtained are subsequently washed with ultrapure water in a proportion of 3 1 per 900 mg of particles.

[0078] The polymer particles are subsequently packaged in flasks made of amber glass with a height of 50 mm and a diameter of 24 mm (150 mg of particles per flask). The particles are subsequently covered with 1 ml of ultrapure water per 150 μg of polymer particles.

[0079] 5.sup.th Stage: Freeze-Drying and Sterilization

[0080] The polymer particles present in the flasks are subsequently freeze-dried in order to obtain a lyophilizate of dry particles. The freeze-drying is carried out according to the following cycle: decreasing the temperature to a temperature of −38° C. over 1 h, then increasing the temperature from −38° C. to +15° C. over 2 h, followed by drying at +15° C. for 5 h, then increasing the temperature from +15° C. to +25° C. over 30 min, followed by drying at +25° C. for 10 h.

[0081] The freeze-dried particles charged with hyaluronic acid are subsequently sterilized by irradiation with gamma radiation at 15 kGy.

[0082] 6.sup.th Stage:

[0083] 150 mg of polymer particles are dissolved in 1 ml of a sterile aqueous solution comprising 2 ml of NaCl in which 16 mg of hyaluronic acid are dissolved. Dissolution is carried out with a LuerLock® syringe system.

Analysis of the Polymer Particles of the Invention According to Example 1

[0084] The analyses were carried out on 10 batches of polymer particles according to the invention. The results obtained are as follows: [0085] size of the particles obtained by polydispersity according to the particle size analysis using a Multisizer® 3 Coulter counter (Beckman Coulter): 66.34 μm±3.6 μm (before irradiation) and 65.34 μm±7.09 μm (after irradiation), [0086] a content of hyaluronic acid charge of 13 μg of hyaluronic acid per mg of freeze-dried polymer particles, and [0087] a combination yield of the hyaluronic acid molecules with the polymer particles of approximately 40%.

[0088] The results obtained are summarized in table 2 (size of the particles charged with hyaluronic acid, combination yield of the hyaluronic acid molecules with the polymer particles and content of hyaluronic acid charge in the particles).

TABLE-US-00002 TABLE 2 Combination yield of the hyaluronic acid molecules Content of charge Mean size (μm) with the polymer particles (%) (μg/mg) Before After Before After Before After irradiation irradiation irradiation irradiation irradiation irradiation Batches 1 65.87 61.74 50 51 16.20 16.53 2 72.13 73.45 57 55 18.47 17.82 3 62.54 65.89 57 52 18.47 16.85 4 64.58 70.87 40 38 12.96 12.31 5 61.32 58.44 29 27 9.40 8.75 6 67.12 69.78 29 28 9.40 9.07 7 71.45 76.44 40 38 12.96 12.31 8 69.63 62.77 42 40 13.61 12.96 9 63.82 55.23 37 36 11.99 11.67 10 64.97 58.75 35 38 11.34 12.31 Mean 66.34 65.34 41.60 40.30 13.48 13.06 Standard 6.69 7.09 10.18 9.60 3.30 3.11 deviation

[0089] Images of polymer particles combined with hyaluronic acid molecules according to the invention prepared according to example 1 are represented in FIG. 1a.

Results

[0090] The release profile of the polymer particles bonded to hyaluronic acid molecules prepared according to example 1 is represented in FIG. 3. The measurements were carried out in a biomimetic medium, a medium similar to synovial fluid (medium devoid of proteins having a viscosity identical to that of synovial fluid) being reconstituted according to N. Gerwin et al., Advanced Drug Delivery Reviews, 2006, 58, 226-242 (assaying once per week for 12 weeks, with cumulative results on each occasion). The polymer particles of the invention were added to this medium at 37° C. with stirring and then centrifuged in order to recover the hyaluronic acid released and to assay the hyaluronic acid released by the Stain-All assaying method (colorimetric assaying of the Stain-All complex at 460 nm) (Fagnola M. et al., Contact Lens & Anterior Eye, 2009, 108-112; Langeslay D. J. et al., Proteoglycans: Methods and Protocols, Methods in Molecular Biology, Vol. 836, Chap. 9, pp. 131-143). In order to extract an assay with hyaluronic acid, 10 mg of polymer particles according to the invention are mixed for 30 seconds with 2 ml of CH.sub.2Cl.sub.2 under stirring of vortex type (Vortex-Genie 2, Scientific Industries, Bohemia, N.Y., USA), left to stand for 5 min, before the addition of 2 ml of water under stirring of vortex type, left to stand for 30 min and then centrifuged at a speed of 3500 revolutions/min at 4° C. for 5 min (Jouan Thermo CR3 centrifuge). 150 μl of the aqueous phase recovered are mixed for 30 seconds with 1.850 ml of water at a pH of 7.4 under stirring of vortex type (Vortex-Genie 2, Scientific Industries, Bohemia, N.Y., USA). A Stain-All solution (1 ml) is subsequently added to the medium and assaying is carried out at an absorbance of 640 nm with a UV-visible spectrophotometer using a hyaluronic acid calibration curve.

[0091] Evaluations of in vivo tolerance were also carried out on a pharmaceutical composition prepared according to example 1. This composition is injected into the intraarticular space of rabbits and the effect on the synovial membrane and on the synovial fluid was studied. Quantitative and semiquantitative analyses of the tissue effects were carried out according to Standard ISO 10993-6 on different circulating cells of the immune system. The results obtained with regard to the synovial membrane are presented in table 3 and those obtained with regards to the synovial fluid are presented in table 4.

TABLE-US-00003 TABLE 3 Score of the effect of the injection of the polymer particles according to the invention with regard to the synovial membrane 0: Absent; 1: Slight; 2: Moderate; 3: Marked; 4; Severe Polymer particles Polymer particles obtained according to obtained according to Injected product example 1 PBS Sinovial ® example 1 PBS Sinovial ® Injection volume 0.4 ml 0.4 ml Concentration of the 150 mg of 15 mg of — 8 mg of 150 mg of 15 mg of — 8 mg of product particles*/ml particles*/ml hyaluronic particles*/ml particles*/ml hyaluronic acid/ml acid/ml mg of hyaluronic acid 1.5 0.15 — 3.2 1.5 0.15 — 3.2 administered *(25 μg of hyaluronic acid/mg of particles) Day post-injections D3 D7 Polymorphonuclear cells 2 0.7 0 0 1 0.3 0 0 Lymphocytes 0.7 0 0 0 0.3 0 0 0 Plasmocytes 0 0 0 0 0 0 0 0 Macrophages 2 1.3 0 1 1.3 0.7 0 0 Giant multinucleated cells 1.7 1.3 0 0 1.3 0.3 0 0 Overall score/4 1.28 0.66 0 0.2 0.78 0.26 0 0 Visual appearance of 0.3 0 0 0 0 0 0 0 the membrane Necrosis 0.3 0 0 0 0 0 0 0 Fibrosis 2 1.3 1 0 1.3 1 0 0 Neovascularization 2 1.3 0 0 1.3 1 0 0 Lipid vesicle 0 0 0 0 0 0 0 0 Fibrin 2 1 0 0 1.3 0.3 0 0 Degeneration 1.3 0.7 0 0 1 0.3 0 0 Hyperplasia 1.7 1.7 0 0 2 1 0 0 Hypertrophy 2 1.7 0 1 2 1.3 0 1 Erosion 2 1.3 0 0 1.7 0.3 0 0

[0092] These results show that the injected composition according to the invention is well tolerated by the synovial membrane of the joint of rabbits. At the highest dose, the slight inflammatory effect observed on the 3.sup.rd day disappears on the 7.sup.th day.

TABLE-US-00004 TABLE 4 Score of the effect of the injection of the polymer particles according to the invention with regard to the synovial fluid 0: Absent; 1: Slight; 2: Moderate; 3: Marked; 4: Severe Polymer particles Polymer particles obtained according to obtained according to Injected product example 1 PBS Sinovial ® example 1 PBS Sinovial ® Injection volume 0.4 ml 0.4 ml Concentration of the 150 mg of 15 mg of — 8 mg of 150 mg of 15 mg of — 8 mg of product particles*/ml particles*/ml hyaluronic particles*/ml particles*/ml hyaluronic acid/ml acid/ml mg of hyaluronic acid 1.5 0.15 — 3.2 1.5 0.15 — 3.2 administered *(25 μg of hyaluronic acid/mg of particles) Day post-injections D3 D7 Polymorphonuclear cells 1 1.3 0 0 0.3 0 0 0 Lymphocytes 0.7 0.7 0 1 0.7 0 0 0 Plasmocytes 0 0 0 0 0 0 0 0 Macrophages 1.7 1.3 0 1 0.7 0.5 0 1 Giant multinucleated cells 0 0 0 0 0 0 0 0 Overall score/4 0.68 0.66 0 0.4 0.34 0.1 0 0.2 Fibrin 0 0 0 0 0 0 0 0 Red blood cells 1 1 0 0 0.3 0.5 0 1 Synovial cells 2 2 1 1 1 1 1 1

[0093] These results show that the injected composition according to the invention is well tolerated with regard to the synovial fluid of rabbits.

[0094] Macroscopic measurements of the inflammation of the knee joint of rabbits were subsequently carried out, at the point where the composition according to the invention was injected. The thickness of the joint with respect to day 0 (D0) was measured using a Vernier caliper. The “macroscopic edema measurements (%/D0)” presented in table 5 below represent the increase as % in the volume of the edema with respect to D0.

TABLE-US-00005 TABLE 5 Macroscopic edema measurement (%/D 0) Polymer particles Polymer particles obtained according to obtained according to Injected product example 1 PBS Sinovial ® example 1 PBS Sinovial ® Injection volume 0.4 ml 0.4 ml Concentration of the 150 mg of 15 mg of — 8 mg of 150 mg of 15 mg of — 8 mg of product particles*/ml particles*/ml hyaluronic particles*/ml particles*/ml hyaluronic acid/ml acid/ml mg of hyaluronic acid 1.5 0.15 — 3.2 1.5 0.15 — 3.2 administered *(25 μg of hyaluronic acid/mg of particles) Day post-injections D3 D7 Macroscopic edema 1.00 1.00 N.D. 1.02 1.02 1.02 N.D. 1.03 measurement (%/D 0) N.D.: not determined

[0095] These results show that the injected composition according to the invention is well tolerated with regard to the knee joint of rabbits and does not cause macroscopic edema.

Example 2

Preparation of the Polymer Particles According to the Invention

[0096] A similar process to that of example 1 is employed to prepare polymer particles according to the invention. The only variation which was made with respect to the process of example 1 is the preparation of a “secondary” emulsion during the 2.sup.nd stage using a Silverson stirrer at a speed of 2800 revolutions/minute, in place of magnetic stirring at 750 revolutions/minute in example 1.

Analysis of the Polymer Particles of the Invention According to Example 2

[0097] The analyses were carried out on 10 batches of polymer particles according to the invention. The results obtained are as follows: [0098] size of the particles obtained by polydispersity according to the particle size analysis using a Multisizer® 3 Coulter counter (Beckman Coulter): 34.98 μm±7.02 μm (before irradiation) and 34.12 μm±7.23 μm (after irradiation), [0099] a content of hyaluronic acid charge of 16 μg of hyaluronic acid per mg of freeze-dried polymer particles, and [0100] a combination yield of the hyaluronic acid molecules with the polymer particles of approximately 20%.

[0101] The results obtained are summarized in table 6 (size of the particles charged with hyaluronic acid, combination yield of the hyaluronic acid molecules with the polymer particles and content of hyaluronic acid charge in the particles).

TABLE-US-00006 TABLE 6 Combination yield of the hyaluronic acid molecules Content of charge Mean size (μm) with the polymer particles (%) (μg/mg) Before After Before After Before After irradiation irradiation irradiation irradiation irradiation irradiation Batches 1 35.54 31.45 29 27 23.72 22.09 2 37.62 32.56 20 21 16.36 17.18 3 39.83 41.25 18 18 14.72 14.72 4 34.68 30.63 24 26 19.63 21.27 5 44.25 41.57 17 18 13.91 14.72 6 20.19 25.74 15 14 12.27 11.45 7 25.22 22.12 20 21 16.36 17.18 8 32.47 35.89 18 17 14.72 13.91 9 41.56 47.34 19 19 15.54 15.54 10 38.47 32.69 20 22 16.36 18 Mean 34.98 34.12 20.00 20.30 16.36 16.61 Standard 7.02 7.23 3.74 3.80 3.06 3.11 deviation

[0102] Images of polymer particles combined with hyaluronic acid molecules according to the invention prepared according to example 2 are represented in FIG. 1b.

Results

[0103] The release profile of the hyaluronic acid molecules combined with polymer particles prepared according to example 2 is represented in FIG. 4.

Example 3

Preparation of the Polymer Particles According to the Invention

[0104] A similar process to that of example 2 was employed in order to prepare polymer particles according to the invention. The only variation which is made with respect to the process of example 2 is the addition of a pharmaceutically acceptable excipient also having anti-inflammatory properties (trapper of free radicals), namely 1 ml of sorbitol in the form of a 10% by weight solution of sorbitol in ultrapure water, in place of the ultrapure water used in example 2.

Analysis of the Polymer Particles of the Invention According to Example 3

[0105] The analyses were carried out on 6 batches of polymer particles according to the invention. The results obtained are as follows: [0106] size of the particles determined by polydispersity according to the particle size analysis using a Multisizer® 3 Coulter counter (Beckman Coulter): 31.15 μm±7.80 μm, and [0107] a content of hyaluronic acid charge of 13.94 μg of hyaluronic acid by mg of freeze-dried polymer particles.

TABLE-US-00007 TABLE 7 Mean size Content of charge (μm) (μg/mg) After irradiation After irradiation Batches 1 32.23 12.17 2 21.12 9.03 3 43.55 15.14 4 25.64 14.13 5 29.33 15.32 6 35.02 17.82 Mean 31.15 13.94 Standard deviation 7.80 3.02

Example 4

Preparation of the Polymer Particles According to the Invention

[0108] A similar process to that of example 1 was employed in order to prepare polymer particles according to the invention. The only variation which is made with respect to the process of example 1 is the addition of a pharmaceutically acceptable excipient also having anti-inflammatory properties (trapper of free radicals), namely 1 ml of sorbitol in the form of a 10% by weight solution of sorbitol in ultrapure water, in place of the ultrapure water used in example 1.

Analysis of the Polymer Particles of the Invention According to Example 4

[0109] The analyses were carried out on 6 batches of polymer particles according to the invention. The results obtained are as follows: [0110] size of the particles determined by polydispersity according to the particle size analysis using a Multisizer® 3 Coulter counter (Beckman Coulter): 71.39 μm±7.70 μm, and [0111] a content of hyaluronic acid charge of 23.67 μg of hyaluronic acid by mg of freeze-dried polymer particles.

TABLE-US-00008 TABLE 8 Mean size Content of charge (μm) (μg/mg) After irradiation After irradiation Batches 1 65.82 23.61 2 72.37 25.57 3 63.92 22.19 4 85.17 26.03 5 67.61 22.73 6 73.45 21.91 Mean 71.39 23.67 Standard deviation 7.70 1.75