PRODUCT AND PROCESS FOR THE PREPARATION OF AN INJECTABLE COMPOSITION BASED ON CHITOSAN AND BIOGLASSES, INTENDED FOR THE REGENERATION OF BONE TISSUE

Abstract

The invention relates to a product intended for the preparation of an injectable composition containing, separated from one another, at least one first and one second composition(s), and in which: the first composition, referred to as A, is an aqueous solution of chitosan with a pH in the range extending from 3 to 7, and the second composition, referred to as B1, corresponds to particles of one or more bioactive glass(es), said product not containing glycerophosphate; and also a process for preparing an injectable composition, the corresponding injectable compositions and devices for packaging a product according to the invention.

Claims

1-38. (canceled)

39. A product intended for the preparation of an injectable composition containing, separately from each other, at least one first composition A and one second composition B1, and optionally a third composition C, wherein: composition A is a chitosan aqueous solution with a pH in the range of 3 to 7, the chitosan having an average molecular weight M.sub.w in the range of 80 to 1000 kg/mol and a degree of acetylation in the range of 0 to 60%, composition B1 consists in particles of one or more bioactive glass(es), composition C, when present, is water or an aqueous solution with a pH in the range of 5.5 to 8; said product not containing glycerophosphate and wherein the weight of composition B1 relative to the total weight of compositions A and B1, or the weight of compositions B1 and C relative to the total weight of compositions A, B1 and C, is in the range of 20% to 90%.

40. The product according to claim 39, wherein it does not contain genipin, glutaraldehyde or formaldehyde.

41. The product according to claim 39, wherein the pH of composition A is in the range of 4 to 6.5.

42. The product according to claim 39, wherein composition A comprises a chitosan concentration of 0.5 to 6% (w/w).

43. The product according to claim 39, wherein the chitosan has an average molecular weight M.sub.w in the range of 100 to 700 kg/mol.

44. The product according to claim 39, wherein the chitosan has a degree of acetylation in the range of 0 to 20%.

45. The product according to claim 39, wherein composition B1 comprises particles of a bioactive glass that contains at least SiO.sub.2, Na.sub.2O, CaO and P.sub.2O.sub.5.

46. The product according to claim 39, wherein said bioactive glass contains 30 to 70 wt % SiO.sub.2, 10 to 50 wt % CaO, 5 to 40 wt % Na.sub.2O and 2 to 20 wt % P.sub.2O.sub.5.

47. The product according to claim 39, wherein composition B1 comprises particles of a first bioactive glass containing 40 to 60 wt % SiO.sub.2, 10 to 30 wt % CaO, 10 to 35 wt % Na.sub.2O and 2 to 8 wt % P.sub.2O.sub.5 and particles of a second bioactive glass containing 60 to 75 wt % P.sub.2O.sub.5, 6 to 35 wt % CaO, 5 to 12 wt % Na.sub.2O, 0 to 18 wt % K.sub.2O and 0 to 8 wt % Al.sub.2O.sub.3.

48. The product according to claim 39, wherein the bioactive glass particles have a size in the range of 3 to 500 m.

49. The product according to claim 39, wherein it also comprises a composition C wherein composition C is present in an amount such that composition B1 represents at least 15% (w/w) of the total weight of compositions B1 and C.

50. The product according to claim 39, wherein the weight of composition B1 relative to the total weight of compositions A and B1, or of compositions B1 and C relative to the total weight of compositions A, B1 and C is in the range of 40% to 60%.

51. The product according to claim 39, wherein compositions A and B1 are packaged separately from each other in two compartments of a single packaging assembly.

52. The product according to claim 39, wherein compositions A and B1 are packaged separately from each other in two separate packaging units.

53. An injectable composition corresponding to a suspension of one or more bioactive glass(es) in a chitosan aqueous solution, obtained by mixing compositions A and B1, by mixing compositions A, B1 and C, or by mixing compositions B1 and C, to form an intermediate composition B2, then B2 and C, of the product as defined in claim 39.

54. The injectable composition according to claim 53, wherein it has a viscosity at a shear rate of 10.sup.4 s.sup.1 in the range of 0.005 to 200 Pa.Math.s.

55. A process for preparing an injectable, glycerophosphate-free composition containing particles of one or more bioactive glass(es) suspended in a chitosan aqueous solution, said process comprising the mixing of composition A and composition B1 as defined in claim 39 in the absence of glycerophosphate, wherein the weight of composition B1 relative to the total weight of compositions A and B1 is in the range of 20% to 90%.

56. A process for preparing an injectable, glycerophosphate-free composition containing particles of one or more bioactive glass(es) suspended in a chitosan aqueous solution, comprising the following successive steps of: providing composition A as defined in claim 39, providing composition B1 as defined in claim 39, providing water or an aqueous solution with a pH in the range of 5.5 to 8, mixing composition B1 with water or with the aqueous solution to form an aqueous suspension of particles of at least one bioactive glass, and immediately after mixing composition A aqueous suspension, the various compositions and solutions used being glycerophosphate-free, and wherein the weight of the aqueous suspension to the total weight of composition A and the aqueous suspension is in the range of 20% to 90%.

57. The process according to claim 56, wherein the aqueous suspension of particles of at least one bioactive glass has a pH in the range of 5.5 to 8.

Description

[0121] The following examples, with reference to the appended Figures, are intended to illustrate the invention but are in no way limiting.

[0122] FIG. 1 shows changes in G and G moduli as a function of frequency for a chitosan solution with a concentration of 0.375% w/v (imposed deformation: 0.5%).

[0123] FIG. 2 shows changes in G and G as a function of angular frequency, 2 minutes 30 seconds after mixing (imposed deformation: 0.5%).

[0124] FIG. 3 shows the X-ray diffractogram obtained after immersion of a hydrogel formed from a suspension containing bioglasses and chitosan for 4 days in SBF at 37 C.

METHOD FOR DETERMINING PH

[0125] The pH value of the mixture of compositions is advantageously measured using a contact pH meter (Mettler-Toledo AG, Switzerland).

[0126] Method for Determining Viscosity

[0127] The viscosity measurement is performed at high shear rate (typically 10.sup.4 s.sup.1) and is obtained by capillary rheometry according to the method described in the publication Chitosan solutions as injectable systems for dermal filler applications: rheological characterization and biological evidence by C. Halimi, A. Monternbault, A. Guerry, T. Delair, E. Viguier, R. Fulchiron, L. David. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. 2015; 2596-9.

[0128] Method for Adjusting the Granulometry (Particle Size) of the Bioglasses

[0129] The resulting glasses are ground using an agate mortar/pestle or a tungsten carbide ball mill.

[0130] The resulting powders are then dry-sieved through a column of vibrating sieves of variable mesh diameters (63 m, 90 m, 125 m, 150 m, 200 m, 250 m, 315 m and/or 500 m).

[0131] In order to remove the agglomerated fine particles not corresponding to the desired particle sizes, the various batches obtained after separation by sieving are each immersed in a beaker containing 200 ml of acetone and placed in an ultrasonic tank. Large particles fall to the bottom of the beaker, while fines are removed with acetone. This procedure is repeated until the acetone is dear (free of fine particles). The batches of powder of defined particle size are then dried in an oven at 92 C. for 20 to 30 minutes.

[0132] Method for Obtaining the Diffractogram

[0133] The measurement of diffractograms is performed using a laboratory X-ray diffractometer, using a Cu K X-ray source and diffraction angles comprised between 3 and 60 degrees 2 (i.e. moduli of the scattering vector q between 0.2 and 4), and in Bragg-Brentano geometry.

[0134] 1. Demonstration of Gel Formation

[0135] A 0.75 wt % chitosan aqueous solution is prepared. To that end, chitosan is dispersed in a volume of deionized water. Acetic acid is added so as to allow stoichiometric protonation of the amine functions of the chitosan. The chitosan used in this example has the following structural characteristics: degree of acetylation of 5%, molecular weight M.sub.w of 550 000 g/mol and a polydispersity index I.sub.p of 1.4.

[0136] At the same time, a bioactive glass powder is dispersed in deionized water, the volume of which corresponds to the volume of the chitosan solution. More precisely, it is 45S5 glass with the following weight composition: 45% SiO.sub.2, 24.5% CaO, 24.5% Na.sub.2O and 6% P.sub.2O.sub.5. The weight of glass incorporated is equal to half the weight corresponding to the chitosan solution.

[0137] Thus, the amounts used in this example are as follows: [0138] 750 mg of 0.75% (w/w) chitosan solution, [0139] 375 mg of 45S5 glass particles dispersed in 750 mg of deionized water.

[0140] The glasses used and presented in the context of this invention were prepared at the Laboratoire Charles Coulomb of the Universite de Montpellier. The bioglasses were elaborated from a mixture of the following raw materials: Al(OH).sub.3, H.sub.2NaPO.sub.4, CaCO.sub.3, H.sub.2(NH).sub.4PO.sub.4, SiO.sub.2 and K.sub.2CO.sub.3 in appropriate proportions as a function of the desired composition. In the case of elaboration of 45S5 glass, the raw materials used are Na.sub.2CO.sub.3 (Prolabo), CaCO.sub.3 (MerckKGaA), H.sub.2(NH).sub.4PO.sub.4 (Prolabo), and SiO.sub.2 (Prolabo). Each mixture was melted in a platinum crucible. The resulting melt was cast in a steel mould and the glass obtained after air cooling was annealed 1 h at a temperature slightly above its glass transition temperature and then cooled very slowly to room temperature.

[0141] Each glass ingot formed was then ground and sieved to obtain powders of different particle sizes. The particle size of the glass powder used in the context of this example is comprised between 200 and 315 microns and is selected by means of the sieves used.

[0142] The formulation combining the dispersion of bioglass and the chitosan solution was stirred to obtain a homogeneous system. In the end, the chitosan concentration in the mixture is 0.375% (w/v).

[0143] When the mixture is prepared under stirring, a spontaneous gelation mechanism occurs leading to the formation of a physical hydrogel containing a glass mineral part.

[0144] Fifteen minutes after mixing, the pH of the composite material stabilizes at 7.4.

[0145] 2. Rheological Characteristics

[0146] This section presents the rheological characterizations that were carried out in order to demonstrate the gelling capacity of the bioglasses when mixed with a chitosan solution.

[0147] A 0.75 wt % chitosan aqueous solution was prepared, as before.

[0148] At the same time, bioactive glasses in powder form are dispersed in deionized water whose volume is equal to the volume of the chitosan solution. More precisely, it is a mixture of glasses composed of 45S5 and Na.sub.16Ca.sub.30P.sub.50Al.sub.4 glass (in weight proportions). Na.sub.16Ca.sub.30P.sub.50Al.sub.4 glass (referred to here as phosphate glass) is composed of 9.8% Na.sub.2O, 16.5% CaO, 69.7% P.sub.2O.sub.5 and 4% Al.sub.2O.sub.3 (% w/w). This glass was elaborated by following the same procedure as that described above in the paragraph 1. Demonstration of gel formation, i.e. from a mixture of the following raw materials: AKOH).sub.3 (FlukaBioChemika), H.sub.2NaPO.sub.4 (FlukaChemie) CaCO.sub.3 (MerckKGaA), H.sub.2(NH).sub.4PO.sub.4 (Prolabo) in appropriate proportions, melted in a platinum crucible. The glass obtained by casting the melt followed by annealing and slow cooling was then ground and sieved, as was described for composition A in the paragraph Method for adjusting the granulometry (particle size) of the bioglasses. The particle size of the 45S5 and Na.sub.16Ca.sub.30P.sub.50Al.sub.4 glass powders used in the context of this example is between 90 and 150 microns.

[0149] The weight of the glasses incorporated is equal to half the weight of the chitosan solution.

[0150] Thus, the amounts used in this example are as follows: [0151] 750 mg of 0.75% (w/w) chitosan solution, [0152] 375 mg of glasses dispersed in 750 mg of deionized water.

[0153] This 375 mg is made up of 250 mg of 45S5 glass and 125 mg of phosphate glass.

[0154] The particle size of the glasses used in the context of this example is comprised between 90 and 150 microns and is selected by means of the sieves used.

[0155] The formulation combining the dispersion of bioglasses and the chitosan solution is stirred so as to obtain a homogeneous system. In the end, the chitosan concentration in the mixture is 0.375% (w/v).

[0156] In order to facilitate homogenization of the formulation, a system of two connected syringes is used. A connector connects the two syringes, one containing the chitosan solution, the other the suspension of bioglasses. Mixing is thus achieved by sending the contents back and forth between the two syringes several times.

[0157] During the homogeneous mixing process, a spontaneous gelation mechanism occurs leading to the formation of a physical hydrogel containing a glass mineral part.

[0158] The corresponding sol-gel transition was studied by rheometry.

[0159] These tests were performed on an AR 2000 rheometer (TA Instruments), using a plate-plate geometry with a 25 mm diameter plate and a sample containment system to maintain a water-saturated atmosphere and thus limit evaporation of the water present in the sample. All tests were performed at room temperature (25 C.).

[0160] A rheological characterization was first performed on a 0.375% (w/v) concentrated chitosan solution not containing glass. The curves obtained for a glass-free formulation are presented in FIG. 1 and show rheological behaviour corresponding to the solution state. The shape of the curves is characteristic of viscoelastic solutions with G<G at low frequencies and G>G at high frequencies, G and G being defined respectively as the elastic and viscous moduli of the sample.

[0161] A rheological characterization was then performed on the same solution, but into which a suspension of bioglass particles dispersed in deionized water was incorporated (suspension of 45S5 glass/phosphate glass and introduced in the proportion described above).

[0162] FIG. 2 shows the rheological curves obtained 2 minutes 30 seconds after mixing. These curves show rheological behaviour corresponding to a gel state, with G and G moduli values such that G>10 G, regardless of the angular frequency.

[0163] This gelation property is fully compatible with the development of injectable systems.

[0164] The presence of bioglasses within the solution causes an increase in pH in the medium and induces deprotonation of the amine sites of the chitosan, which allows gelation of the polymer solution.

[0165] 3. Study of Changes in the Resulting Gels Upon Contact with a Solution Having a Composition Close to that of Human Physiological Fluids (Simulated Body Fluid Solution)

[0166] A study was carried out to verify that when the bioglass particles are present in the hydrogel, the bioglasses retain their bioactivity properties.

[0167] In order to evaluate this bioactivity, the elaborated gels were immersed in a Simulated Body Fluid (SBF) solution. This SBF solution has an ion concentration dose to that of human physiological fluids maintained under physiological temperature and pH conditions (37 C. and 7.5) [T. Kokubo, H. K., S. Sakka, T. Kitsugi and T. Yamamuro. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W. J. Biomed. Mater. Res. 1990, vol. 24, 721-734].

[0168] The composites studied here had the following composition: [0169] 750 mg of 0.75% (w/w) chitosan solution in deionized water, [0170] 375 mg of glasses dispersed in 750 mg of deionized water, this 375 mg comprising 250 mg of 45S5 glass and 125 mg of phosphate glass described in section 2 above.

[0171] The particle size of the glasses used in the context of this example was comprised between 90 and 150 microns.

[0172] Thus, after immersing the composite materials in an SBF solution for two days, the water from the resulting sample was removed by freeze-drying and analysed by X-ray diffraction. The X-ray diffractogram presented in FIG. 3 shows the formation of a crystalline phase, calcite. The arrows point to the diffraction peaks characteristic of calcite. In the case of the bioglasses alone, it is generally observed that the calcite that precipitates initially is then transformed into hydroxyapatite: this phase may thus be considered under these conditions as a precursor of carbonated hydroxyapatite [Lefebvre, L., Dveloppement de bioverres poreu pour application I'ortopddie et I'ingnienre tissulaire. 2007, INSA Lyon].

[0173] In the case of the bioglasses incorporated into the hydrogel, the phenomena of ion exchanges with the environment are therefore comparable to those observed in the case of bioglasses alone. These experiments show that the behaviour of the bioglasses has not been modified when incorporated into a chitosan hydrogel and that these bioglasses have retained their bioactivity properties.