Chitosan Hydrogel Microbead

Abstract

The present invention relates to hydrogel microbeads comprising at least water, chitosan, at least one polyphosphate compound, water being present at a concentration of at least 85% by mass of hydrogel, said microbeads having an average number diameter ranging from 100 to 900 μm. The present invention also relates to their manufacturing method and their uses, in particular in pharmaceutical compositions or medical devices, more particularly for treating an articular pathology.

Claims

1. A plurality hydrogel microbeads, the hydrogel comprising water, chitosan, and at least one polyphosphate compound, the water is at a concentration of at least 85% by mass of the hydrogel, said microbeads having an average diameter in the range from 100 to 900 μm.

2. The hydrogel microbeads according to claim 1, wherein the microbeads have a D(0.9) that is in the range from 100 and 950 μm.

3. The hydrogel microbeads according to claim 1, wherein the chitosan is at a concentration in the range from 0.3 to 10% by mass of the hydrogel, and the polyphosphate compound(s) is/are at concentration sufficient for forming the hydrogel microbeads.

4. The hydrogel microbeads according to claim 1, wherein the chitosan is at a concentration in the range from 0.5 to 5% by mass of the hydrogel.

5. The hydrogel microbeads according to claim 1, wherein the polyphosphate compound(s) is/are selected from among phytic acid, tripolyphosphates, glycerophosphates, and combinations thereof.

6. The hydrogel microbeads according to claim 1, wherein the hydrogel microbeads are sterilized.

7. A method for preparing hydrogel microbeads comprising adding a chitosan solution to a cross-linking solution comprising at least one polyphosphate compound, and then gelling the chitosan in the presence of the cross-linking solution to form the hydrogel microbeads, wherein the hydrogel microbeads have a concentration of water that is at least 85% by made of the hydrogen, and wherein the hydrogel microbeads have an average diameter in the range from 100 to 900 μm.

8. (canceled)

9. A pharmaceutical composition or a medical device comprising a plurality of microbeads as defined according to claim 1.

10. The pharmaceutical composition or medical device according to claim 9, wherein it consists in an artificial synovial fluid.

11. A method of treatment of an articular pathology, said method comprising administering to a subject in need thereof the pharmaceutical composition or medical device according to claim 9.

12. The method of claim 11, wherein said method treats pain or discomfort associated with a pathology affecting a joint or for slowing down the progression of an articular pathology.

13. A medical device, optionally as one or several packaging kits, optionally physically separated, the medical device comprising a syringe, a needle and a pharmaceutical composition that comprises a plurality of microbeads as defined according to claim 1, said syringe comprising a reservoir optionally pre-filled with said pharmaceutical composition.

14. The method of claim 11, wherein said method comprises injecting the pharmaceutical composition or medical device according to claim 9 via an intra-articular route.

15. A method for modifying the properties of a viscosupplement, said method comprising adding the plurality of hydrogel microbeads of claim 1 to the viscosupplement of microbeads as defined according to any of.

16. The plurality hydrogel microbeads of claim 1, wherein the concentration of the chitosan is in the range from 1 to 3% by mass of the hydrogel.

17. The plurality hydrogel microbeads of claim 1, wherein the polyphosphate compound is selected from the group consisting of a sodium salt of phytic acid, a sodium tripolyphosphate, a sodium beta-glycerophosphate, and combinations thereof.

Description

[0143] In the figures:

[0144] FIG. 1 illustrates an observation by optical microscopy of hydrogel microbeads formed in the presence of the cross-linking solution containing sodium beta-glycerophosphate at the concentration of 5% and sodium hydroxide at the concentration of 0.1M (No. 7).

[0145] FIG. 2 represents an observation by optical microscopy of hydrogel microbeads formed in the presence of the cross-linking solution containing sodium tripolyphosphate at the concentration of 2%, the sodium glycerophosphate at the concentration of 5% and sodium hydroxide at the concentration of 0.1M (No. 8).

[0146] Other objects, features and advantages of the invention will become clearly apparent to one skilled in the art following the reading of the explanatory description which makes reference to examples which are only given as an illustration and cannot in anyway limit the scope of the invention.

[0147] The examples are an integral part of the present invention and any feature appearing to be novel relatively to any prior state of the art from the description taken as a whole, including the examples, is an integral part of the invention in its function and in its generality.

[0148] Thus, each example has a general scope.

[0149] On the other hand, in the examples, all the percentages are given by mass unless indicated otherwise, and the temperature is expressed in degree Celsius unless indicated otherwise, and the pressure is atmospheric pressure, unless indicated otherwise.

EXAMPLES

[0150]

TABLE-US-00001 TABLE 1 References for the needle size with view to injecting microbeads according to the ISO9626 (1991:Amd 1:2001) standard Outer Internal diameter (ID) diameter Extra- (OD) Normal/regular Fine/fine fine/ultrafine (min-max) walls ID walls ID walls ID Source Gauge (μm) (μm) (μm) (μm) 1 29 324-351 133 190 — 27 400-420 184 241 — 26 440-470 232 292 — 25 500-530 232 292 — 22 698-730 390 440 522 20 860-920 560 635 687 19 1030-1100 648 750 850 18 1200-1300 790 910 1041  2 23 ~620 ~325 — — 21 ~820 ~500 — — 1 - “The Gauge system for the medical use” in Anesthesia & Analgesia, 2002; the values are extracted from the ISO9626:1991/Amd 1:2001 standard. 2 - “Does needle size matter ?”, in J. Diab. Sci. Technol. 1, 725, 2007.

[0151] The outer diameter (OD) (min-max) designates the tolerance according to the aforementioned standard.

[0152] Unless indicated otherwise, the mentioned internal diameter of the needles is with normal/regular walls.

Example 1—Preparation of the Chitosan Solution

[0153] An ultrapure chitosan from a fungal source (Synolyne Pharma, Belgium), with an average viscosimetric molecular mass (Mv) of 180,000 (greater than 140,000) and with an acetylation degree (DA) of 27 mol % (greater than 20 mol %) is dispersed in a solution containing 1% (0.167M) of acetic acid, at a concentration of 1.5% (comprised between 1 and 2%) with magnetic or mechanical stirring. The solution is mixed for a period of 3 hours (1 to 12 hours). The solution is filtered on a filter with a pore diameter of 5 μm. At a concentration of 1.5%, the pH of the chitosan solution is of about 4, its osmolarity at 25° C. is of about 150 mOsm/kg, and its dynamic viscosity is of about 220 mPa.Math.s (measured by viscosimetry with a rotating mobile with a Brookfield equipment, at 5 rpm with the Spindle SC4-18).

[0154] It is possible to form droplets starting with this chitosan solution with nozzles of a small diameter up to the smallest size available for the piece of equipment VAR-D (Nisco), i.e. with the 100-μm diameter nozzle.

Example 2—Preparation of the Cross-Linking Solutions Based on Polyphosphate Compounds

[0155] The cross-linking solutions are mixtures of polyphosphate compounds alone or combined, with different concentrations, for which the pH is adjusted in the presence of a base like for example sodium hydroxide, or not. A compound selected from sodium tripolyphosphate (TPP, Sigma), sodium beta-glycerophosphate (GP, Safic Alcan), or phytic acid in the form of a anhydride sodium salt (or anhydrous sodium inositol hexakisphosphate, PA, Sigma) are used as a polyphosphate. The concentrations of the polyphosphates compounds and of the base (NaOH), as well as the pH of the cross-linking solutions are reported in table 2.

TABLE-US-00002 TABLE 2 Cross-linking solutions based on polyphosphate compounds Feasibility of the preparation of stable hydrogel microbeads No. Polyphosphate NaOH pH (Examples 3 and 4)  1 0 0.05M  12.8 No  2 0 0.1M 13.0 No  3 0 0.5M 13.2 No  4 TPP 5% 0 8.6 No  5 GP 5% 0 9.3 No  6a TPP 5% 0.1M 12.9 Yes  6b TPP 2.5% 0.05M  12.4 No  6c TPP 1.25% 0.075M  12.7 Yes  7 GP 5% 0.1M 13.0 Yes  8a* TPP 2% and GP 5%   0M 8.8 No  8b** TPP 2% and GP 5% 0.05M  12.5 Yes  8c TPP 2% and GP 5% 0.1M 12.9 Yes  9 PA 5% 0 3.0 No 10 PA 5% 0.1M 6.0 Yes 11 PA 5% 0.3M 9.0 Yes 12 PA 5% 0.5M 13.0 Yes 13 PA 2% 0.1M 6.0 No 14 PA 2% 0.3M 9.0 No 15 PA 2% 0.5M 13.0 No *8a: TPP 2% + GP 5% without NaOH => the beads form but are unstable: they break up after 5 to 10 minutes: the beads are not compliant with the microbeads of the invention; **8b: TPP 2% + GP 5% + NaOH 0.05M => the beads form and remain stable after one hour in the cross-linking solution: the beads are compliant with the invention.

Example 3—Preparation of Hydrogel Microbeads of Chitosan by Cross-Linking with Tripolyphosphate (TPP) Alone or Combined with Glycerophosphate (GP)

[0156] Droplets are formed initially from the solution of chitosan according to Example 1 with an electromagnetic process with a piece of equipment “Encapsulator VarD (Gen 2) (Nisco, Zürich, Switzerland), equipped with a 150-μm diameter nozzle.

[0157] The droplets are immersed in a 50-ml volume of one of the cross-linking solutions according to Example 2 (No. 1 at 8c of Table 1), and are stirred for a period of 3 hours by means of a magnetic bar at a velocity comprised between 100 and 1,000 rpm.

[0158] When microbeads form, they are then washed with water (about one liter for each wash) several times consecutively. Slight stirring is achieved with a magnetic bar for about one minute between each wash, at a velocity comprised between 100 and 1,000 rpm. The beads are left to settle between each wash. The beads are finally recovered by gravity. A container is obtained containing a known mass of hydrogel beads, as well as a known mass of water.

[0159] It emerges from this example that stable hydrogel beads may be formed in the presence of the cross-linking solutions Nos. 6, 6c, 7 and 8c, i.e. only in the presence of polyphosphate salts TPP and/or GP and of a sufficient amount of NaOH.

[0160] The stable hydrogel beads cannot be formed in the presence of NaOH alone (Nos. 1 to 3), in the absence of any polyphosphate. In the presence of TPP and/or GP polyphosphate and in the absence of NaOH, the beads formed are not sufficiently stable from a mechanical point of view and do not withstand successive washes.

[0161] The conditions of the cross-linking solution which give the best results in terms of cohesion and of stability of the hydrogel microbeads are the conditions Nos. 6c, 7 and 8c. The characteristics of the thereby obtained hydrogel beads are summarized in Table 3.

[0162] The microbeads of the invention do not have any solid trabeculae.

[0163] The solid trabeculae of chitosan may be sought by optical microscopy after staining the sample with hematoxylin-eosin. As the eosin is anionic with a tendency of binding onto the chitosan, positively charged. [0164] The beads are stained and observed as entire beads (and free) or in the form of sections made with a microtome or further with a bistoury. The sections are then included in paraffin: [0165] Attaching the beads and including them in paraffin: [0166] The beads are incubated for 4 hours at 4° C. in a buffer solution of 100 mM sodium cacodylate and 20 mM CaCl.sub.2 at pH 7.4 and 40 g/L of paraformaldehyde. [0167] The beads are washed 3× by means of a buffer solution of 100 mM sodium cacodylate and 50 mM BaCl.sub.2 at pH 7.4 in order to prevent their disintegration. [0168] The beads are then dehydrated by successive passages in the baths with increasing concentration baths of methanol, isopropanol and xylene. [0169] The beads are then included in the paraffin and the paraffin blocks are cut into lamellas with a thickness of 5 μm by means of a microtome (Leica RM 2145). [0170] Staining with hematoxylin-eosin: [0171] In order to be stained, the beads sections are de-paraffinated beforehand and rehydrated with successive baths of xylene, ethanol in decreasing concentration and of demineralized water. The entire beads are directly stained. [0172] The beads or beads sections are incubated for 15 minutes in a solution of Mayer hematoxylin solution. [0173] The beads or bead sections are rinsed 2× with water, and then by means of a 2.6% NH.sub.4OH solution. [0174] The beads or bead sections are incubated in a solution of 0.5% aqueous eosin Y and 0.5% acetic acid. [0175] The beads or bead sections are then rinsed with water and then dehydrated by means of a bath of increasing concentrations of ethanol and then of xylene. [0176] For observation with an optical microscope (Olympus CKX41), the bead sections are then mounted on slides and lamellas or entire beads are placed in a cup with water. [0177] The observation of the internal frame of the bead(s) with the optical microscope gives the possibility of appreciating the presence or the absence of directly visible trabeculae and exacerbated by staining with eosin. If the observed internal frame is homogeneous and without any filaments emerging from the contrast, the bead is described as not including any solid trabeculae.

TABLE-US-00003 TABLE 3 Characteristics of the hydrogel microbeadss formed in the presence of GP and of a GP/TPP mixture in the presence of NaOH (nozzle with a diameter of 150 μm) Diameter distribution of the hydrogel beads Cross-linking By laser Mechanical aspect and solution By optical microscopy diffraction strength No. 6c ND D(0.1) = 190 μm Good strength TPP 1.25% D(0.5) = 490 μm NaOH 0.075M D(0.9) = 740 μm pH = 12.7 No. 7 average diameter = 645 μm ND Beads not very resistant, GP 5% min diameter = 370 μm elastic, round on average NaOH 0.1M max diameter = 890 μm (FIG. 1) pH = 13.0 No. 8c average diameter = 560 μm ND More resistant beads, less GP 5% min diameter = 420 μm elastic, more round TPP 2% max diameter = 700 μm (FIG. 2) NaOH 0.1M pH = 12.9 ND: Not determined
In FIGS. 1 and 2 the substantially spherical shape of the hydrogel microbeads of the invention is observed.

[0178] From this example, it is concluded that it is necessary to attain a sufficiently high pH in order to obtain hydrogel beads of good stability and integrity and well elastic with polyphosphates TPP and GP.

[0179] It is also concluded that hydrogel beads formed by contact with the solution based on TPP and GP in combination are more stable and elastic than the beads formed starting with TPP or GP alone, with an equivalent basic pH.

[0180] It is also possible to produce stable hydrogel microbeads of a smaller size with the 100-μm diameter nozzle.

[0181] The microbeads of the invention are injectable through fine needles.

Example 4: Preparation of the Hydrogel Microbeads of Chitosan with Phytic Acid

[0182] Droplets are formed starting with the chitosan solution according to Example 1 by an electromagnetic process with a piece of equipment “Encapsulator VarD (Gen 2) (Nisco, Zürich, Suisse), fitted with a 100-μm diameter nozzle.

[0183] The droplets are immersed in a 50 ml volume of one of the cross-linking solutions based on phytic acid (PA) according to Example 2 (Nos. 9 to 15 of Table 1), and are stirred for a period of 3 hours by means of a magnetic bar, at a speed comprised between 100 and 1,000 rpm.

[0184] When microbeads form, they are then washed with water (about one liter of each wash) several times consecutively. Slight stirring is achieved with a magnetic bar lasting for about one minute between each wash, at a speed comprised between 100 and 1,000 rpm. The beads are left to settle between each wash. The beads are finally recovered by gravity. A container containing a known mass of hydrogel beads, as well as a known mass of water are obtained.

[0185] It emerges from this example that stable hydrogel beads may be formed in the presence of the cross-linking solutions Nos. 10, 11 and 12, i.e. only in the presence of polyphosphate PA and of NaOH simultaneously, both components having to be found in a sufficient amount of each other. The characteristics of the thereby obtained hydrogel beads are summarized in Table 4.

[0186] In the presence of phytic acid at a concentration of 5% and in the absence of NaOH, the formed beads are not stable overtime and do not resist to the successive washes with water. When NaOH is added, stable beads are formed as soon as the NaOH concentration is of 0.1M (pH 6.0), unlike the cases of the TPP and GP polyphosphates of Example 3 for which the pH should be higher (for example greater than 12.5) so that the beads are stable.

[0187] In the presence of phytic acid at a lower concentration of 2% regardless of the amount of NaOH (from 0.1 to 0.5M), beads are formed but they are not stable.

TABLE-US-00004 TABLE 4 Characteristics of the hydrogel microbeads formed in the presence of phytic acid and of NaOH Diameter distribution of the hydrogel beads (before Cross-linking sterilization) Mechanical aspect and solution By optical microscopy strength of the beads No. 10 Average diameter = 325 μm Opaque PA 5% Min diameter = 260 μm Soft, deformable, elastic NaOH 0.1M Max diameter = 360 μm and tender pH = 6.0 No. 11 Average number diameter Transparent with an PA 5% comprised between opaque core, NaOH 0.3M 100 and 700 μm Elastic on average, hard pH = 9.0 on average No. 12 Average number diameter Very transparent, with a PA 5% comprised between very dense core and a NaOH 0.5M 100 and 700 μm small diameter, pH = 13.0 not very deformable and elastic, hard Min/max diameter: Smallest/largest diameter measured from among the 20 observed beads.

[0188] It is concluded that with a sufficient amount of phytic acid (for example 5%) and in the presence of NaOH, it is possible to form hydrogel microbeads regardless of the NaOH concentration and the pH (above 6.0). On the other hand, the NaOH proportion strongly influences the mechanical aspect and strength of the hydrogel beads. It is thus possible to modulate the properties of the microbeads.

[0189] The beads are sterilized with humid heat (autoclave—model SYSTEC DX-23, Wettenberg, Germany). The autoclave parameters are the following: temperature of 121° C., duration of 15 minutes.

[0190] The thereby sterilized microbeads have an average number diameter comprised between 100 and 700 μm. Specifically, the beads prepared by means of the cross-linking solution No. 10 have an average number diameter of 200 μm.

They are injectable through fine needles.

Example 5—Rheological Properties

[0191] When the microbeads according to Examples 3 and 4 are added to a fluid, a viscous solution or a hydrogel, for example to a viscosupplement based on hyaluronic acid, an increase in the elastic modulus (G′) is obtained measured by rheology (detail of the method). This expresses the resistance to stress of the hydrogel microbeads, and therefore an improvement in the resistance to stress of the fluid containing the microbeads. By extension, the capability of the fluid of absorbing impacts, for example when it is injected into a joint and more specifically the knee joint, is improved in the presence of beads. The viscosity of the fluid at a physiological temperature is unchanged advantageously, so that it remains easy to inject through a needle with a diameter acceptable by a physician, and it keeps sufficient viscosity for acting as a viscosupplement.

Example 6—Elasticity of the Microbeads

[0192] In order to determine the elastic and absorption properties of the hydrogel microbeads, microbeads—prepared according to Examples 3 and 4 with cross-linking solutions Nos. 6a, 6c, 8b, 8c, 10, 11 and 12 of Example 2 are added to a viscous fluid, for example a solution of hyaluronic acid, at 37° C.

[0193] The rheological properties with oscillation of the viscous fluid alone and with the viscous fluid with microbeads are measured, by means of a rotary rheometer with shearing of plates, (ARES G2, TA Instruments). This analysis may give information on the time-dependent change of the variables G′, G″ and tan(δ) on a range of given shearing frequencies, for example 0.1 Hz to 100 rad.Math.s.sup.−1, and at a given temperature, for example 37° C., the amplitude is set in constant value, for example 1%.

[0194] It is shown that the elastic modulus (G′, also called a storage modulus) of the viscous solution comprising the microbeads is significantly greater than the modulus G′ of the solution of the viscous solution without the microbeads, and this regardless of the composition of the cross-linking solution. The difference in G′ is indicative of the elasticity of the microbeads. The value of the modulus G′ of the fluid is increased significantly in the presence of the microbeads, which indicates that the microbeads impart to the fluid a better capability of resisting to the stress and of absorbing impacts.

[0195] In parallel, the dynamic viscosity of the viscous solution is measured with and without the microbeads at 37° C., by means of the same piece of rheometry equipment, with continuous rotation, at increasing speed, over a determined shearing range. It is observed that the dynamic viscosity of the viscous fluid is not modified by addition of the microbeads. It thus remains easily injectable through a needle, and may for example act as a viscosupplement for relieving a joint after injection into the joint of the knee.

[0196] For example, a mixture of a viscosupplement (based on hyaluronic acid) available commercially (SynVisc®, Sanofi) with the microbeads prepared according to Example 3 with the cross-linking solution No. 10 (phytic acid 5%, NaOH 0.1M, pH 6) is prepared. The 2 solutions (with and without microbeads) containing the same concentration of hyaluronic acid. The elasticity modulus G′ and the dynamic viscosity of 2 solutions are measured at 37° C., according to the measurement parameters reported in tables 5 and 6.

[0197] The results are shown in tables 5 and 6. It emerges from this example that the addition of chitosan and phytic acid microbeads causes an increase in the elasticity modulus of the solution of 40% hyaluronic acid, without modifying its dynamic viscosity.

[0198] The microbeads are easily injectable through a needle with variable diameter, and in particular with a needle adapted to the intra-articular injection. The microbeads of the invention recovered after injection substantially retain the same size distribution.

[0199] Moreover, the same solution of hyaluronic acid with the microbeads (obtained with the solution No. 10 of table 3) is injectable through needles with variable diameter, for example a needle suitable for intra-articular injection. The beads recovered after injection substantially retain the same size distribution.

TABLE-US-00005 TABLE 5 Elasticity modulus G′ of commercial hyaluronic acid, with and without microbeads (No. 10), at 37° C. G′.sub.HA G′.sub.HA+MB Difference Difference Oscillation Without With G′.sub.HA+MB − (G′.sub.HA+MB − G′.sub.HA)/ frequency microbeads microbeads G′.sub.HA G′.sub.HA × 100 (Hz) (Pa) (Pa) (Pa) (%) 1 25 35 10 +40% 10 66 93 27 +41%

TABLE-US-00006 TABLE 6 Dynamic viscosity of commercial hyaluronic acid with and without microbeads (No. 10), at 37° C. Dynamic viscosity Dynamic viscosity Shear rate of HA without HA with (per second) microbeads (mPa .Math. s) microbeads (mPa .Math. s) 1 32 33 10 3.7 3.7 HA: hyaluronic acid; MB: Microbead

Example 6—Stability of the Beads Upon Storage and Injectability

[0200] The hydrogel microbeads prepared by means of the cross-linking solutions Nos. 6a, 6c, 8b, 8c, 10, 11 and 12 of Example 2 are stored at 4° C. in an aqueous solution. After storage duration of 3 and 6 months, their aspect, their average number diameter and their size distribution, measured according to the methods of the description, are unchanged. The microbeads are injectable without any difficulty through a fine needle, and substantially retain their size characteristics after injection.