BIOCOMPATIBLE COMPOSITIONS COMPRISING A BIOCOMPATIBLE THICKENING POLYMER AND A CHITOSAN DERIVATIVE

Abstract

Biocompatible compositions, in particular for the preparation of a biodegradable coating for medical articles, include a biocompatible thickening polymer and a chitosan derivative comprising D-glucosamine units of the following formula (I):

##STR00001##

wherein X is an alditolic or aldonic polyol residue of the following formula (II):

##STR00002##

wherein: R is CH.sub.2 or CO; R1 is hydrogen, a monosaccharide moiety or an oligosaccharide moiety; R2 is OH or NHCOCH.sub.3.

The present invention also relates to uses of the disclosed compositions, to a kit of parts including a composition in powder form and to a method for the preparation of a biocompatible composition in gel form.

The biodegradable coating shows a good and long-lasting adhesion to the surface of a medical article and allows to improve both the coating operations and the effectiveness in preventing any biofilm formation on the medical article.

Claims

1-50. (canceled)

51. A biocompatible composition in powder form comprising a biocompatible thickening polymer and a chitosan derivative comprising D-glucosamine units of the following formula (I): ##STR00008## wherein X is an alditolic or aldonic polyol residue of the following formula (II): ##STR00009## wherein: R is CH.sub.2 or CO; R1 is hydrogen, a monosaccharide moiety, or an oligosaccharide moiety; and R2 is OH or NHCOCH.sub.3.

52. The composition according to claim 51, wherein said biocompatible thickening polymer is selected from non-ionic cellulosic or non-cellulosic polysaccharides, proteins, or mixtures thereof.

53. The composition according to claim 51, wherein said non-ionic cellulosic polysaccharide is selected from hydroxypropyl methyl cellulose (HPMC), methyl cellulose (MC), hydroxypropyl cellulose (HPC), ethyl cellulose (EC) and ethyl methyl cellulose (EMC), or mixtures thereof.

54. The composition according to claim 51, wherein the degree of derivatization of said chitosan derivative is between 10% and 95%.

55. The composition according to claim 51, wherein the degree of derivatization of said chitosan derivative is between 20% and 80%.

56. The composition according to claim 51, wherein said alditolic or aldonic polyol residue X is: (a) a residue of a monosaccharide selected from the group consisting of galactose, glucose, mannose, N-acetylglucosamine, and N-acetyl galactosamine; or (b) a residue of an oligosaccharide comprising from 2 to 4 glycosidic units.

57. The composition according to claim 56, wherein said alditolic or aldonic polyol residue X is a residue of an oligosaccharide selected from the group consisting of lactose, cellobiose, cellotriose, maltose, maltotriose, maltotetraose, chitobiose, chitotriose, mannobiose, melibiose, and aldonic acids thereof.

58. The composition according to claim 57, wherein said alditolic or aldonic polyol residue X is a lactose residue.

59. The composition according to claim 51, comprising an amount of the biocompatible thickening polymer equal to or higher than 25% by weight and equal to or lower than 50% by weight of the overall weight of the composition.

60. The composition according to claim 51, comprising an amount of the biocompatible thickening polymer equal to or higher than 34% by weight and equal to or lower than 48% by weight of the overall weight of the composition.

61. The composition according to claim 51, comprising an amount of the chitosan derivative equal to or higher than 10% by weight and equal to or lower than 40% by weight of the overall weight of the composition.

62. The composition according to claim 51, comprising an amount of the chitosan derivative equal to or higher than 20% by weight and equal to or lower than 35% by weight of the overall weight of the composition.

63. A method of preparing a biocompatible composition in gel form, comprising the steps of: a) providing a first container housing a composition in powder form according to claim 51; b) providing a second container housing an aqueous reconstituting solution; c) mixing the composition in powder form and the aqueous reconstituting solution to obtain a composition in gel form.

64. The method according to claim 63, further comprising the step of: d) allowing the composition in gel form obtained by step (c) to rest over a predetermined period of time.

65. The method according to claim 63, wherein said mixing step c) comprises: c1) transferring said aqueous reconstituting solution from the second container to the first container to dissolve the composition in powder form, thereby providing a reconstituted composition; and c2) transferring the reconstituted composition back into the second container.

66. The method according to claim 65, further comprising the step of: c3) repeating steps c1) and c2) so as to increase homogeneity of the reconstituted composition in gel form.

67. The method according to claim 63, wherein said aqueous reconstituting solution comprises water and a biologically-active substance.

68. A kit of parts for use in the preparation of a biocompatible coating in gel form for a medical article, comprising: a first container housing a composition in powder form according to claim 51; a second container configured to house an aqueous reconstituting solution.

69. The kit of parts according to claim 68, wherein said second container comprises an aqueous reconstituting solution comprising water and a biologically-active substance.

70. A method for applying a biocompatible coating onto a medical article, comprising the steps of: providing a first container housing the composition in powder form according to claim 51; providing a second container housing an aqueous reconstituting solution; mixing the composition in powder form and the aqueous reconstituting solution to obtain a composition in gel form; and applying said composition in gel form onto the medical article.

71. The method according to claim 70, wherein said medical article is an implantable biomedical article.

72. The method according to claim 71, wherein said implantable biomedical article is an articular prosthesis made at least in part of a metal alloy.

73. The method according to claim 72, wherein said articular prosthesis is a hip prosthesis or a knee prosthesis.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0155] Further characteristics and advantages of the invention will become clearer from the following description of some preferred embodiments thereof, made hereinafter, for indicating and not limiting purposes, with reference to the attached drawings. In such drawings:

[0156] FIG. 1 shows the results of rheological tests performed according to Example 11 on the biodegradable coating prepared according to Example 9A;

[0157] FIG. 2 shows the diffusion of an antimicrobial drug (vancomycin), evaluated according to Example 12, from the biodegradable coating prepared according to Example 10A;

[0158] FIG. 3 shows the diffusion of silver nanoparticles, evaluated according to Example 13, from the composition in gel form prepared according to Example 9 by resuspending in aqueous solution the composition containing silver nanoparticles obtained according to Example 8;

[0159] FIG. 4 shows the residual composition amount (%), evaluated according to Example 16, of the composition in gel form according to the invention prepared according to Example 9A and of the comparative compositions in gel form according to Examples 14 and 15, in terms of substrate surface coverage and composition mass, after being subjected to a water flow in standard conditions;

[0160] FIG. 5 shows the cell viability, evaluated according to Example 17, of MG-63 osteoblasts cultured in the presence of a composition prepared according to Example 9A and of a composition with silver nanoparticles prepared according to Examples 1 and 8, respectively;

[0161] FIG. 6 shows the antimicrobial effect on S. aureus bacteria, evaluated according to Example 17, of the composition comprising silver nanoparticles prepared by reconstituting the composition in powder form according to Example 8 on S. aureus bacteria.

DETAILED DESCRIPTION

[0162] In order to assess the performance of biocompatible compositions according to the invention, various experiments have been carried out, some of which are reported below, to be intended for illustrative and non-limiting purpose of the present invention.

Example 1—Preparation of a Composition in Powder Form

[0163] 150 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) with a degree of derivatization of 60% and according to the procedure described by International patent application WO 2018/116224 A1, 250 mg of HPMC, 100 mg of mannitol and 45 mg of Na.sub.2HPO.sub.4 were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

Example 2—Preparation of a Composition in Powder Form

[0164] 150 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 60%, 200 mg of HPMC, 100 mg of mannitol and 45 mg of Na.sub.2HPO.sub.4 were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

Example 3—Preparation of a Composition in Powder Form

[0165] 200 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 60%, 150 mg of HPMC, 100 mg of mannitol and 45 mg of Na.sub.2HPO.sub.4 were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

Example 4—Preparation of a Composition in Powder Form

[0166] 150 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 47%, 100 mg of HPMC and 150 mg of mannitol were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

Example 5—Preparation of a Composition in Powder Form

[0167] 150 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 60%, 100 mg of HPMC and 150 mg of mannitol were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

Example 6—Preparation of a Composition in Powder Form

[0168] 150 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 77%, 100 mg of HPMC and 150 mg of mannitol were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

Example 7—Exemplary Scale-Up of a Composition in Powder Form: Preparation of the Composition of Example 1 for a Batch of 100 Containers

[0169] 15 g of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 60%, 25 g of HPMC, 10 g of mannitol, 4.5 g of Na.sub.2HPO.sub.4 were mechanically mixed by means of a V-type blender Multigel Junior, manually weighted and aliquoted into 100 syringes, each syringe containing 545 mg of the mixed powders, to obtain a composition in powder form.

[0170] A scaled-up production of the composition according to the invention appears to be feasible in terms of performing a mixing procedure for the components at the solid state, in line with similar processes employed in the pharmaceutical/biomedical field, thereby allowing a possible transfer of the production of the medical devices to the industrial site.

[0171] This approach allows rapid operations of preparation and aliquoting into syringes of the composition, which can be easily transferred to the industrial site.

Example 8—Preparation of a Composition in Powder Form Comprising Silver Nanoparticles

[0172] 123 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 60%, 27 mg of the same chitosan derivative bearing silver nanoparticles (CTL-nAg, in which silver amount is 0.2% w/w), 250 mg of HPMC, 100 mg of mannitol and 45 mg of Na.sub.2HPO.sub.4 were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

[0173] The chitosan derivative bearing silver nanoparticles (CTL-nAg) was prepared as described by Travan A et. Al., Non-cytotoxic Silver Nanoparticle-Polysaccharide Nanocomposites with Antimicrobial Activity, Biomacromolecules 2009 10 (6), 1429-1435, DOI: 10.1021/bm900039x.

[0174] EXAMPLES 9A-9G—Preparation of compositions in gel form Seven compositions in gel form were obtained by resuspending in aqueous solution, by means of inter-syringe mixing using two containers as disclosed above, a composition in powder form prepared according to Examples 1-6 and 8.

[0175] Seven syringes (first containers) each containing one of said compositions in powder form were coupled with other seven syringes (second containers) each containing 5 ml of water and then the compositions in powder form were gradually rehydrated by inter-syringe mixing as known in the art by feeding the materials back and forth between the coupled syringes until a total rehydration of the powder compositions occurred and substantially homogeneous gels, as determined by visual inspection, were obtained.

[0176] The compositions in gel form thus obtained were allowed to rest for about 10 minutes so as to make the hydrogels settle.

[0177] In the following Table 1, the amounts of chitosan derivative and thickening polymer in the composition in gel form prepared are reported:

TABLE-US-00001 TABLE 1 Amount Total of chitosan amount of Amount of derivative Amount of chitosan chitosan bearing silver thickening derivative plus derivative nanoparticles polymer thickening (w/V) (w/V) (w/V) polymer (w/V) Example 9A 3 — 5 8 Example 9B 3 — 4 7 Example 9C 4 — 3 7 Example 9D 3 — 2 5 Example 9E 3 — 2 5 Example 9F 3 — 2 5 Example 9G 2.46 0.54 5 8

Examples 10A-10F—Preparation of Compositions in Gel Form Having Antimicrobial Activity

[0178] Six compositions in gel form containing antimicrobial molecules were obtained by resuspending in aqueous solution containing vancomycin (20 mg/ml), by means of inter-syringe mixing, compositions in powder form prepared according to Examples 1-6.

[0179] Six syringes (first containers) each containing one of said compositions in powder form were coupled with other six syringes (second containers) each containing 5 ml of a vancomycin aqueous solution (20 mg/ml), and then the compositions in powder form were gradually rehydrated by inter-syringe mixing as known in the art by feeding the materials back and forth between the coupled syringes until a total rehydration of the powder compositions occurred and substantially homogeneous gels, as determined by visual inspection, were obtained.

[0180] The reconstituted compositions in gel form were allowed to rest for about 10 minutes so as to make the hydrogels settle.

Example 11—Determination of the Rheological Behavior of the Compositions in Gel Form

[0181] Mechanical spectroscopy of a composition in gel form prepared according to Example 9A (and sterilized by beta irradiation) was carried out in order to determine the elastic modulus (G′), the viscous modulus (G′) and the complex viscosity (η*) of said composition in gel form.

[0182] The rheological properties of the hydrogel were studied with a controlled stress rheometer Haake Mars III and the values of storage (elastic, G′) and loss (viscous, G″) moduli were measured at 2.5 Hz, while the complex viscosity (η*) was evaluated at 1 Hz. All measurements were performed at 25° C. using a cone-plate geometry (ϕ=60 mm, 1°)

[0183] The results obtained are shown in FIG. 1, and showed a dependency of the elastic modulus G′, the viscous modulus G″ and complex viscosity η* on the frequency; notably for a wide range of frequencies the elastic modulus G′ is higher than the viscous one G″, showing a good compactness on the hydrogel

Example 12—Diffusion of an Antimicrobial Drug from the Composition in Gel Form with Antimicrobial Molecules

[0184] Antimicrobial drug release tests were performed using a composition in gel form according to Example 10A obtained by reconstituting a composition in powder form according to Example 1 (and sterilized by beta irradiation), with an aqueous solution containing vancomycin (20 mg/ml), and by measuring the vancomycin release over time.

[0185] The composition in gel form (500 mg per sample) was spread on titanium cylinders and immersed in PBS (7 ml) at 37° C. At selected time-points (1, 4, 8 and 24 hours), the supernatant solution was collected and analysed to measure the amount released. Three samples were used for each selected time points and the results obtained were averaged.

[0186] The quantification of vancomycin in the supernatant solutions was performed by means of UV-Vis spectrometry, after having obtained a calibration curve.

[0187] FIG. 2 shows the release profiles of vancomycin from the composition in gel form according to Example 10A (black squares).

[0188] The graph shows that vancomycin could be gradually released from the hydrogel during the first immersion hours, while the total release appears to be reached after 24 hours.

Example 13—Diffusion of Silver from the Composition in Gel Form with Silver Nanoparticles

[0189] Silver release tests were performed using a composition in gel form prepared according to Example 9G, by rehydrating the composition in powder form according to Example 8 (beta-sterilized at medium dose), with aqueous solution, and by measuring the silver release over time. The silver concentration of the final composition in gel form was 0.1 mM.

[0190] The rehydrated composition in gel form (500 mg per sample) was spread on titanium cylinders and immersed in PBS (7 ml) at 37° C. At selected time-points (1, 4, 8 and 24 hours), the supernatant solution was collected and analyzed to measure the amount released. Three samples were used for each selected time points and the results obtained were averaged.

[0191] The quantification of silver in the supernatant solutions was performed by means of Atomic Emission Spectroscopy (AES).

[0192] FIG. 3 shows the release profile of silver from the hydrogel (black squares).

[0193] The graph shows that silver could be gradually released from the hydrogel during the first immersion hours, while the total release appears to be reached after 24 hours.

Example 14 (Comparative)—Preparation of a Composition in Gel Form not Containing any Chitosan Derivative

[0194] 150 mg of mannitol and 100 mg of HPMC were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a comparative composition in powder form.

[0195] The composition in powder form was used to obtain a comparative composition in gel form by means of inter-syringe mixing according to the procedure disclosed in any one of Examples 9A-9F.

Example 15 (Comparative)—Preparation of a Composition in Gel Form not Containing any Biocompatible Thickening Polymer

[0196] 150 mg of a chitosan derivative obtained from the reaction between chitosan and lactose (CTL) as described in Example 1 and having a degree of derivatization of 60% and 150 mg of mannitol were mixed by means of a spatula and loaded into a 5 ml syringe to obtain a composition in powder form.

[0197] The composition in powder form was used to obtain a comparative composition in gel form by means of inter-syringe mixing according to the procedure disclosed in any one of Examples 9A-9F.

Example 16—Quantification of the Adhesive Properties of a Composition in Gel Form According to the Invention and of Comparative Compositions

[0198] A quantification of the adhesive properties of a composition in gel form according to the invention (Example 9A) and of the comparative compositions of Examples 14 and 15 was carried out with the following method.

[0199] Each composition in gel form was spread on a titanium plate having the following approximate dimensions: [0200] length: 80 mm [0201] width: 20 mm [0202] thickness: 1 mm

[0203] The volume of hydrogel used for each test was 1.5 ml and the material was rubbed on the metal surface with a spatula in order to achieve a uniform coating layer having a thickness of about 1 mm

[0204] A water nozzle was placed 10 cm above the higher extremity of the hydrogel spread on the titanium plate inclined at 30° with respect to the water stream which was directed onto the upper end of the coating layer at a flow rate of 1.5 l/min for 30 seconds.

[0205] The amount of each coating layer was quantified before and after water rinsing in terms of surface coverage (computerized image analyses carried out by the software Image J on digital pictures of the top views of the coated plates) and weight.

[0206] The weight of the residual coating was measured after freeze-drying so as to convert the compositions in gel form back in powder form.

[0207] FIG. 4 shows the results of the comparative tests carried out on the compositions of Examples 9A (invention) and 14 and 15 (comparative).

[0208] FIG. 4 clearly shows that the composition in gel form according to the invention, comprising both the chitosan derivative disclosed herein and a biocompatible thickening polymer, has a much better adhesion performance in terms of a higher residual amount of the coating composition left on the substrate after the rinsing procedure with respect to the comparative gel compositions without the chitosan derivative or the biocompatible thickening polymer.

[0209] The results of the comparative testing reported in FIG. 4 also show an unexpected synergistic effect of the combination of the chitosan derivative and of a biocompatible thickening polymer disclosed herein.

[0210] The compositions according to the invention therefore achieve the desired properties of imparting to a biocompatible coating prepared from the same a good and long-lasting adhesion to the surface of a medical article such as an implantable medical article as disclosed herein.

[0211] Such an improved adhesion not only facilitates the spreading operations of the coating composition carried out on site in a sterile operating room, but also achieves the very important advantage of remaining in substantial amounts on the surface of the implantable medical article after insertion of the same into the tissues (for example, the bone tissues) of a patient.

[0212] This technical effect is particularly remarkable as it allows to prevent biofilm formation on the implanted medical article effectively hindering the development of implant-related infections until such time that the coating composition is completely absorbed by the body, i.e. until such time that the possibility of the onset of possible implant-related infections becomes acceptably low.

[0213] According to the invention, this technical effect may be further enhanced by providing the composition disclosed herein with antimicrobial substances, such as silver and/or an antibiotic, which may be advantageously released on-site as an additional measure to prevent or substantially hinder the development of implant-related infections.

[0214] According to the invention and as disclosed herein, other biologically-active substances such as Antimicrobial Peptides (AMPs), Platelet-rich plasma (PRP), phages, and any combination thereof, may be included in the biocompatible compositions disclosed herein and in the coating composition obtained therefrom thereby achieving other desired therapeutic effects such as avoiding antibiotic resistance (AMPs and phages) or promoting tissue regeneration (PRP).

Example 17—Cytocompatibility

[0215] The viability of cells after treatment with two compositions in gel form prepared according to Examples 9A and, respectively, 9G by reconstituting the composition in powder form of Example 8 as disclosed in any one of Examples 9A-9F, was evaluated by the colorimetric assay MTS (CellTiter 96® Aqueous One Solution Cell Proliferation Assay; Promega).

[0216] For this test, osteoblast (MG-63) cell lines were seeded on 24-well plates at the density of 25.000 cells/well. The day after seeding, the syringes containing the compositions in powder form of Examples 1 and 8 were rehydrated by syringe mixing as disclosed above, and the reconstituted compositions in gel form were employed for the treatment of cells. The compositions in gel form were weighed on filter paper (60 mg for each paper) and added to the wells. Incubation of cells with the formulations was allowed for 24 and 72 hours at 37° C.

[0217] As negative control, cells cultured in the presence of filter paper with no composition in gel form were considered. Cells treated with Triton X-100 (a compound that induces cellular lysis) at the concentration of 0.01% w/V was employed as positive control of cell death. Cells cultured in plain medium were considered as growth control.

[0218] At each time point, the MTS assay was performed: the cell medium was removed and the MTS was added to each well. The incubation of MTS with the cells was allowed for 4 hours at 37° C. in dark and the absorbance values of the samples, that correlate with the amount of viable cells, were read at 485 nm with a spectrophotometer. The cell viability of the growth control was considered as 100% and relative viability was calculated for all samples. For each series, four replicates were considered.

[0219] The results obtained are illustrated in FIG. 5 and show that the viability of the osteoblasts in contact with both the compositions in gel form according to the invention was comparable with that of the positive controls at both time points (24 and 72 hours); as expected, cells treated with Triton displayed a time-dependent decrease of cell viability.

[0220] These results show that the compositions according to the invention do not display a measurable cytotoxic activities towards MG-63 osteoblasts in the experimental conditions adopted. These quantitative data were qualitatively supported by optical investigations of cultured cells, which showed that the cells treated with both the compositions according to the invention retain their stretched morphology without any visible sign of cell suffering.

Example 18—Antimicrobial Effect of a Composition Including Silver on S. aureus Bacteria

[0221] A hydrogel formulation prepared by reconstituting the composition in powder form of Examples 1 and 8 as disclosed in Examples 9A and 9G, was employed to evaluate the antimicrobial effect of the material towards S. aureus bacteria. The formulation was transferred in a falcon tube and bacteria suspension (0.5 mL) was added at the final concentration of 1*10.sup.7 bacteria/mL. Untreated bacteria grown in Luria-Bertani medium (LB) and phosphate buffered saline (PBS)—LB:PBS (10:90)—were considered as growth control. The samples were incubated at 37° C. for 24 hours under shaking before plating on LB agar plates for colony counting units.

[0222] The results obtained are shown in FIG. 6, and showed that compositions including silver nanoparticles, achieved an effective and measurable antimicrobial activity, as the number of colony-forming units of S. Aureus was significantly lower than in compositions without silver nanoparticles.