Microparticles comprising PCL and uses thereof

Abstract

The invention relates to a process for preparing PCL-comprising microparticles, to microparticles obtainable by said process, to gel hence obtained and to several uses of the gel such as for the preparation of a medicament for treating a skin abnormality or disfigurement, and/or for controlling bladder function and/or controlling gastric reflux and/or for treating erectile dysfunction and/or for treating vocal cords. The gel may also be used for cosmetic applications.

Claims

1. A process for preparing polycaprolactone (PCL)-comprising microparticles wherein the process comprises the following steps: a1) solubilizing a PCL polymer, subsequently mixing the solubilized PCL polymer with a liquid comprising a surfactant, said liquid having a viscosity which is ranged between about 1 and about 400.000 cP, b) forming PCL-comprising microparticles from the solution obtained in a1).

2. A process preferably according to claim 1 for preparing PCL-comprising microparticles wherein the process comprises the following steps: a2) solubilizing a PCL polymer in substantially pure Tween and/or in dichloromethane, b) forming PCL-comprising microparticles from the solution obtained in a2).

3. A process according to claim 2, wherein substantially pure Tween is used as sole solvent in the solubilization step a2) and said step is carried out at a temperature above or close to the melting temperature of the PCL polymer.

4. A process according to claim 1 or 3, wherein in step b), the PCL-comprising microparticles are formed as a result of controlled cooling and stirring conditions.

5. A process according to claim 2, wherein DCM is used in the solubilization step a2) and wherein step b) comprises forming PCT-comprising microparticles by extracting the surfactant from these PCL-comprising microparticles dispersed in the gel by extraction evaporation.

6. A process according to claim 1 or 4, wherein the viscosity of the liquid is ranged between about 20 and about 200 cP.

7. A process according to claim 1 or 5 or 6, wherein the surfactant is methyl cellulose, preferably approximately 1% of MC having a Mn 63000.

8. A process according to any one of claims 1 to 7, wherein the PCL polymer is a linear polymer, a copolymer, a terpolymer or a blend of different types of homo/co/ter-polymers.

9. Microparticles obtainable by the process of any one of claims 1 to 8, preferably having at least one of the following characteristics: i) a diameter which is ranged between 5 and 100 μm, ii) homogenous density, form and content, iii) essential spherical microspheres.

10. A biodegradable injectable gel comprising a microparticle according to claim 9 and a carrier, optionally wherein an active ingredient is further present.

11. A gel according to claim 10, which is an implant or a filler.

12. A gel according to any one of claims 11 to 13 for use as a medicament, preferably for treating a skin abnormality or disfigurement, and/or for controlling bladder function and/or controlling gastric reflux and/or for treating erectile dysfunction and/or for treating vocal cords.

13. A gel according to claim 10 or 11 as a cosmetic gel.

14. Use of the gel as defined in any one of claims 10 to 12, for the preparation of a medicament for treating a skin abnormality or disfigurement, and/or for controlling bladder function and/or controlling gastric reflux and/or for treating erectile dysfunction and/or for treating vocal cords.

15. Use of the gel as defined in claim 13 in a cosmetic application.

Description

DESCRIPTION OF THE FIGURES

[0076] FIG. 1. Microscopic photography of microspheres as prepared in example 2.

[0077] FIG. 2. PCL microspheres prepared using a PCL (Mn=10000 g/mol) solution in DCM and an MC (Mn=63000 g/mol) solution in water. [PCL] is 20 g/100 g DCM, [MC] is 1.1 wt %, see Table 1. Light microscopy image, magnification 10×.

[0078] FIG. 3. PCL microspheres prepared using a 10 wt % PCL solution in DCM and a 1.0 wt % PVA solution in water while vigorously stirring at 1000 rpm. See Hunter, Table 4. Light microscopy image, magnification 10×.

[0079] FIG. 4. PCL microspheres prepared using a 10 wt % PCL solution in DCM and a 3.0 wt % PVA solution in water while vigorously stirring at 1000 rpm. See Emeta and Wu, Table 4 Light microscopy image, magnification 10×.

[0080] FIG. 5. PCL microspheres prepared using a 10 wt % PCL solution in DCM and a 0.1 wt % MC solution in water while vigorously stirring at 1000 rpm. See Iooss, Table 4. Light microscopy image, magnification 10×.

EXAMPLES

[0081] Protocol for the synthesis of the microspheres/microparticles and the suspension of these particles in a gel comprising a carrier which is ready for use is described below. [0082] 1. Microspheres are prepared using a classical solvent evaporation technique or by means of a solventless synthesis technique in order to obtain the desired properties. [0083] 2. The gel is prepared with a required viscosity using known preparation techniques, after which the microspheres are suspended in the gel by means of appropriate mixing. [0084] 3. Syringes are then filled with the sterilised gel suspension in a controlled atmosphere.

[0085] The following examples can be used or combined in order to obtain a suspension of microparticles comprised of polymers or blends mentioned afore in a ready for use application or (freeze-dried) vial application.

Example 1

[0086] 10 to 20 grams of Mn 10000 or Mn 42500 PCL is dissolved in DCM (10 to 20 w/w %). This solution is dispersed in 1000 ml water containing 0.1-5% MC. By means of ferocious stirring (1000 rpm) microparticles with an average diameter of 40 μm are obtained by solvent extraction as described in the publication cited in the description.

[0087] The microspheres obtained are filtrated, washed and dried. Subsequently, 10 to 50% of the microspheres are dispersed in the CMC (0.1 to 5%) or MC (0.1 to 5%) gel by moderate mixing and processed further.

Example 2

[0088] 10 to 20 grams of Mn 42500 PCL is dissolved in DCM (10 to 20 w/w %). This solution is dispersed in 1000 ml water containing 0,8% MC. By means of ferocious stirring (1000 rpm) microparticles with an average diameter of 60 μm are obtained by solvent extraction as in example 1. The microspheres obtained are filtrated, washed and dried. Subsequently, 10 to 50% of the microspheres are dispersed in the CMC (0.1 to 5%) or MC (0.1 to 5%) gel by moderate mixing and processed further. A microscopic photography of the microspheres prepared in example 2 is shown in FIG. 1.

Example 3

[0089] 40 g to 80 grams of Mn 10000 PCL is dissolved in pure Tween 20, 40, 60, or 80 by means of heating to 70-90 C and stirring (600-1000 rpm) after which the microspheres are obtained due to phase separation and controlled cooling towards 5 C within 30 min. The obtained microspheres obtained are filtrated, washed and dried. Average distribution 45 μm, yield 75% within the required range Subsequently, 10 to 50% of the microsheres are dispersed in the CMC (0.1 to 5%) or MC (0.1 to 5%) gel by moderate mixing and processed further.

Additional Examples

[0090] The invention relates to an efficient and effective process for the preparation of biodegradable microspheres. A key issue is the use of surfactant solutions with relatively high concentrations and viscosities. The inventions lead to the formation of homogeneous particles with smooth surfaces in a desired size range of approximately 38 to 75 μm.

[0091] A) In one process of the invention as extensively exemplified in example 4, the viscous polymer solution is rapidly added to a vigorously stirred solution of a surfactant in water. Particles are formed upon vigorous stirring of the mixture and evaporation of the solvent. A volatile solvent such as DCM is preferred. This rapid addition is possible due to the high viscositie of the vigorously stirred surfactant solution in water. Vigorous stirring also allows short solvent evaporation times before the particles can be collected and further processed. As this is efficient, this is advantageous and desired.

[0092] To be able to recover the polymer microspheres, essentially all (or at least the majority) of the polymer solvent needs to be removed. Only then will the spherical polymer particles harden (and in the case of crystallisable particles, will they be able to 30 crystallize).

[0093] The time required to evaporate and remove the solvent can be determined in several ways: [0094] the dispersion that had cooled due to evaporation of the solvent has warmed up again to ambient temperatures the dispersion of PCL microspheres turns white upon crystallization of the polymer [0095] with surfactants like MC, a foam on the surface of the surfactant solution is formed when DCM evaporates. This foam disappears as essentially all DCM has evaporated [0096] the microspheres do not coagulate upon standing

[0097] B) In another process of the invention as extensively exemplified in example 7, the PCL polymer is dissolved upon heating in a relatively viscous surfactant (solution) such as Tween. Here droplets of polymer form upon dispersion of the molten polymer in the surfactant solution due to vigorous stirring of the mixture. Particles form after upon continued stirring and (controlled) cooling of the mixture to room temperature. This process is very efficient, as no volatile solvents are required.

Characteristics of the processes: [0098] desired particle size range: we collected fractions of 38-75 μm [0099] efficient process: short evaporation times when using DCM and high yields in desired size range [0100] effective process: essentially spherical shape particles with smooth surfaces, leading to good injectability of the particles

[0101] Injectable gels could readily be formed from microspheres prepared according to the invention by mixing. Microsphere volumes of up to 50% could be homogeneously mixed into carboxymethyl cellulose gels (CMC, Aqulon from Hercules solutions in water or in phosphate buffered saline) by slow stirring.

Example 4: Experiments of the Invention

Preparation of Microspheres Using PCL Solutions in DCM and MC Solutions in Water

[0102] Poly(Σ-caprolactone) (PCL) microspheres were prepared by vigorously mixing PCL solutions in dichloromethane (DCM) into methylcellulose (MC) solutions in water, followed by evaporation of DCM.

[0103] Different amounts of PCL obtained from Sigma Aldrich with Mn=10000 g/mol were dissolved in DCM. Of these solutions 100 g was added to 1000 g of solutions of MC in water in a 2 liter beaker within 2 seconds while vigorously stirring at 1000 rpm. MC obtained from Colorcon Ltd. of different molecular weights (Mn=14000 g/mol, Mn=41000 g/mol and Mn=63000 g/mol) was employed.

[0104] Within three hours continuously stirring vigorously at room temperature, essentially all DCM had evaporated. Stirring was discontinued, and the microspheres that had formed were allowed to settle at the bottom of the beaker. The supernatant was removed and the microspheres were washed with water. Using stainless-steel sieves, the microspheres were sieved in the wet state and the fraction with diameters between 38 and 75 micrometer was collected.

[0105] The microspheres were vacuum-dried at room temperature, and the yield was 10 determined gravimetrically. Light microscopy (magnification 10×) was employed to analyze the morphology of the obtained microspheres.

[0106] A series of experiments, where the concentration of the PCL solution and the a characteristics of the MC solution were varied, was conducted. The results are presented in Table 1.

TABLE-US-00001 TABLE 1 Preparation of PCL microspheres upon mixing PCL solutions in DCM in MC solutions in water while vigorously stirring. PCL recovered as [PCL] Mn of MC [MC] Viscosity of MC particles of 38-75 μm PCL particle PCL particle (g/100 g DCM) (g/mol) (wt %) solution (cP) (wt %) shapes surfaces 10 14000 0.8 13 6.6 irregular rough 10 41000 0.8 56 35.2 irregular rough 10 63000 1.1 120 51.6 spherical smooth 20 63000 1.1 120 74.0 spherical .sup.a) smooth .sup.a) .sup.a) see light microscopy image presented in FIG. 2.

[0107] From Table 1 it can be seen that when using a relatively high viscosity MC solution, smooth spherical particles can be obtained efficiently. Furthermore, a high concentration of the PCL solution leads to high yields of PCL particles of the desired particle sizes.

Example 5: Experiments of the Invention

Preparation of Microspheres Using PLLA Solutions in DCM and MC Solutions in Water

[0108] Poly(L-lactide) (PLLA) microspheres were prepared by mixing a PLLA solution in dichloromethane (DCM) into a methylcellulose (MC) solution in water, followed by evaporation of DCM.

[0109] An amount of 10 g of PLLA obtained from Purac Biochem (with intrinsic viscosity in chloroform of 2.3 dl/g) was dissolved in 100 g of DCM. Of this solution 100 g was added to 1000 g of a solution of MC in water in a 2 liter beaker within 2 seconds, while vigorously stirring at 1000 rpm. MC with Mn=63000 g/mol was obtained from Colorcon Ltd.

[0110] Within three hours continuously stirring vigorously at room temperature, essentially all DCM had evaporated. Stirring was discontinued, and the microspheres that had formed were allowed to settle at the bottom of the beaker. The supernatant was removed and the microspheres were washed with water. Using stainless-steel sieves, the microspheres were sieved in the wet state and the fraction with diameters between 38 and 75 micrometer was collected.

[0111] The microspheres were vacuum-dried at room temperature, and the yield was determined gravimetrically. Light microscopy (magnification 10×) was employed to analyze the morphology of the obtained microspheres.

TABLE-US-00002 TABLE 2 Preparation of PLLA microspheres upon mixing a PLLA solution in DCM in an MC solution in water while vigorously stirring. PLLA recovered as [PLLA] Mn of MC [MC] Viscosity of MC particles of 38-75 μm PLLA particle PLLA particle (g/100 g DCM) (g/mol) (wt %) solution (cP) (wt %) shapes surfaces 10 63000 1.1 120 64.0 spherical smooth

[0112] This Table 2 shows that it is possible to also prepare PLLA microspheres efficiently. When using a high viscosity MC solution, smooth spherical PLLA microspheres of the desired particle sizes can be in high yields.

Example 6: Comparative Experiments

Preparation of Microspheres Using PCL and CL Copolymer Solutions in DCM and Surfactant Solutions in Water

[0113] I Typical Data from the Literature:

[0114] The preparation of PCL and CL copolymer microspheres has been described in the scientific and patent literature. For example in publications by Hunter (US2003/0157187A1), Erneta and Wu (EP1872803A1) and loos et al. (Biomaterials 22 (2001) 2785-2794) it is described that addition of PCL solutions in DCM to solutions of polyvinyl alcohol (PVA) or MC in water can lead to the formation of PCL microspheres.

[0115] To prevent coagulation of the PCL solution in the stirred aqueous medium, the conditions employed involve addition of the PCL solutions in DCM over relatively long periods of time, and long DCM evaporation times to allow the dispersed PCL particles to harden. Only then are the formed spherical PCL microspheres stable enough to be collected. PCL microspheres were prepared using various experimental setups, and different PCL and CL copolymers and surfactant concentrations, addition rates and solvent evaporation times.

Hunter (Example 41 in US2003/0157187A1):

[0116] PCL: Mn=25000-45000 g/mol; PCL concentration in DCM: 9.5 wt/vol %; PVA: Mn=12000-18000 g/mol; 2 ml of the polymer solution were poured into 100 ml of the aqueous surfactant solution at a stirring rate of 1000 rpm; addition time of polymer solution: 120 min; particles were centrifuged and washed with water; microspheres with sizes ranging from 30-100 micrometer were obtained. The particles were spherical, but had a rough or pitted morphology.

Erneta and Wu (Examples in EP1872803A1):

[0117] Semi-crystalline CL copolymers: molar masses between 5000 and 25000 g/mol; [0118] polymer concentration in DCM: 4 to 7.5 wt/vol %, PVA: Mn is not indicated; approximately 275 g of solution was poured into approximately 1500 ml of the aqueous surfactant solution while stirring at rates close to 250 rpm; addition times of polymer solution: up to 19 min; DCM evaporation times 14 to 16 hrs; fraction of recovered microspheres with sizes of 38-75 micrometer is up to 71%; surface morphology is not indicated.

Iooss (Biomaterials 22 (2001) 2785-2794):

[0119] PCL: Mw=150000; MC: Methocel A15LV with Mn=14000; PCL concentration in DCM: 6.7 to 9.1 wt/vol %; 15 ml of solution is poured into the aqueous MC solution while stirring at 400-600 rpm during 1 hour; DCM is removed by extraction in large volume (1000 ml) of water; fraction of recovered PCL particles with sizes smaller than 80 micrometer vary between approximately 1 and 40%.

[0120] An overview of this data is presented in Table 3.

TABLE-US-00003 TABLE 3 Overview of literature data on PCL and CL copolymers microspheres prepared by mixing polymer solutions in DCM in stirred surfactant solutions in water. [polymer] in [surfactant] in polymer solution DCM evaporation particle particle DCM water (wt %) adding time (min) time (hrs) shapes surfaces Hunter 9.5 wt/vol % PVA, 1.0 120 2 spherical rough Erneta, Wu 4 to 7.5 wt % PVA, 3.0 12 to 19 14 to 16 — — Iooss 9.1 wt/vol % MC, 0.1  60 1 no smooth
II Preparation of Microspheres Using PCL Solutions in DCM and Surfactant Solutions in Water. The Polymer and Surfactant Concentrations are as Described in 20 Literature.

[0121] An amount of 80 g of PCL (obtained from Sigma Aldrich with Mn=10000 g/mol) was dissolved in 800 g DCM. Of this solution 100 g were added to 1000 g of solutions of PVA or MC surfactants in water in a 2 liter beaker within 2 seconds while vigorously stirring at 1000 rpm. In the experiments PVA with Mn=9000-10000 g/mol obtained from Sigma Aldrich and MC obtained from Colorcon Ltd. with Mn=14000 g/mol were employed.

[0122] Within three to four hours continuously stirring vigorously at 1000 rpm at room temperature, essentially all DCM had evaporated. Stirring was discontinued, and the microspheres that had formed were allowed to settle at the bottom of the beaker. The supernatant was removed and the microspheres were washed with water. Using stainless-steel sieves, the microspheres were sieved in the wet state and the fraction with diameters between 38 and 75 micrometer was collected.

[0123] The microspheres were vacuum-dried at room temperature, and the yield was determined gravimetrically. Light microscopy (magnification 10×) was employed to analyze the morphology of the obtained microspheres.

[0124] A comparative experiment was conducted as described in the literature by Erneta and Wu (EP1872803A1). Here 270 g of a 7.5 wt % PCL solution in DCM was added over a period of 12 minutes to 1500 ml of solution of 3.0 wt % PVA in water while stirring at 240 rpm. Before collecting the microspheres, DCM was let to evaporate under continued stirring for 16 hrs.

[0125] The results are compiled in Table 4.

TABLE-US-00004 TABLE 4 Preparation of PCL microspheres upon mixing PCL solutions in DCM in MC solutions in water while stirring. The polymer and surfactant concentrations are typical of those used in experiments described in literature. Viscosity of PCL recovered as [PCL] in DCM [surfactant] in surfactant Stirring rate particles of 38-75 μm particle particle (wt %) water (wt %) solution (cP) (rpm) (wt %) shapes surfaces Hunter 10 PVA, 1.0 13 1000 44.0 irregular .sup.a) rough .sup.a) Erneta, Wu 10 PVA, 3.0 14 1000 23.2 irregular .sup.b) rough .sup.b) Erneta, Wu 7.5 PVA, 3.0 14 240 20.0 spherical smooth Iooss 10 MC, 0.1 15 1000 1.1 irregular .sup.c) rough .sup.c) .sup.a) see light microscopy images presented in FIGS. 3. .sup.b) see light microscopy images presented in FIGS. 4. .sup.c) see light microscopy images presented in FIGS. 5.

[0126] From Table 4 it follows that using typical polymer and surfactant concentrations described in literature it is not possible to efficiently prepare particles with the desired characteristics. At these low viscosities of the surfactant solutions, it apparently is necessary to add the polymer solution over longer periods of time, to stir at relatively low speeds and to evaporate DCM for longer time periods to efficiently prepare particles with the desired characteristics.

III Preparation of Microspheres Using PCL Solutions in DCM and Viscous Surfactant Solutions in Water.

[0127] PCL microspheres were prepared by vigorously mixing PCL solutions in dichloromethane (DCM) into surfactant solutions in water, followed by evaporation of DCM.

[0128] Different amounts of PCL obtained from Sigma Aldrich with Mn=10000 g/mol were dissolved in DCM. Of these solutions 100 g was added to 1000 g of solutions of MC in water in a 2 liter beaker within 2 seconds while vigorously stirring at 1000 rpm. PVA with Mn=9000-10000 g/mol obtained from Sigma Aldrich and MC with Mn=63000 g/mol obtained from Colorcon Ltd. were employed.

[0129] Within three to four hours continuously stirring vigorously at 1000 rpm at room temperature, essentially all DCM had evaporated. Stirring was discontinued, and the microspheres that had formed were allowed to settle at the bottom of the beaker. The supernatant was removed and the microspheres were washed with water. Using stainless-steel sieves, the microspheres were sieved in the wet state and the fraction with diameters between 38 and 75 micrometer was collected.

[0130] The microspheres were vacuum-dried at room temperature, and the yield was determined gravimetrically. Light microscopy (magnification 10×) was employed to analyze the morphology of the obtained microspheres.

[0131] A series of experiments, where the nature and the concentration of the surfactant solution were varied, was conducted. The results are presented in Table 5.

TABLE-US-00005 TABLE 5 Preparation of PCL microspheres upon mixing PCL solutions in DCM in viscous surfactant solutions in water while vigorously stirring. Viscosity of PCL recovered as [PCL] [surfactant] surfactant particles of 38-75 μm PCL particle PCL particle (g/100 g DCM) surfactant (wt %) solution (cP) (wt %) shapes surfaces 10 PVA 14.7 90 1.1 spherical smooth 10 MC 1.1 120 51.6 spherical smooth 20 MC 1.1 120 74.0 spherical .sup.a) smooth .sup.a) .sup.a) see light microscopy image presented in FIG. 2.

[0132] Table 5 indicates that using PVA at higher concentrations results in viscous solutions in water that can be used to prepare PCL microspheres by rapidly adding the PCL solution to the vigorously stirred surfactant solution. Very small particles are obtained, and although the morphology of the particles is adequate, the yield of particles in the desired size range that could be recovered is very low. When using MC as a surfactant, the efficiency of the process is significantly better.

Example 7: Experiments of the Invention: PCL Dissolution in Tween Mixtures at Elevated Temperatures Followed by Particle Formation Upon Cooling

[0133] Upon heating to approximately 80° C. and continuously stirring at 500 rpm, 15 g of PCL with Mn=10000 g/mol was dispersed in 100 ml of a 50/50 wt/wt mixture of Tween 60 and water in a 250 ml glass vessel. Tween 60 is obtained from Sigma Aldrich. The molten polymer droplets are maintained in this dispersed state by stirring for another 2 minutes. While still stirring, the liquid dispersion is then cooled overnight to room temperature. Upon solidification of the dispersed polymer droplets, microspheres are obtained that can be recovered by decantation.

[0134] After washing with water, the PCL microspheres were sieved in the wet state using stainless steel sieves and the fraction with sizes between 38 and 75 micrometer was collected. The microspheres were then vacuum dried at room temperature and the yield was determined gravimetrically. A total of 12.1 g of PCL microspheres was collected.

TABLE-US-00006 TABLE 6 Preparation of PCL microspheres by cooling PCL solutions in Tween 60 and water mixtures while vigorously stirring. Viscosity of a PCL recovered 50/50 mixture as particles PCL of Tween 60 and of 38-75 μm PCL particle PCL particle (g) water at 80° C. (cP) (wt %) shapes surfaces 15 125 80.7 spherical smooth

[0135] From this table it can be seen that using relatively viscous Tween 60 and water mixtures, PCL microspheres can be formed from stirred solutions of the polymer at elevated temperatures by cooling.