Process for producing nanoparticles laden with active ingredient

Abstract

The present invention relates to a novel process for the production of nanoparticles laden with active compounds and to the use thereof as medicaments. The process for the production of nanoparticles comprises the steps (a) dissolution of at least one active compound and at least one polymer in an organic solvent, (b) mixing of the solution prepared in step (a) with an aqueous phase, (c) evaporation of the organic solvent, (d) purification of the nanoparticles laden with active compound obtained in step (c) by means of dialysis against aqueous dialysis solution comprising the same active compound.

Claims

1. Process for the production of nanoparticles comprising the steps of (a) dissolution of at least one active compound and at least one polymer in an organic solvent, (b) mixing of the solution prepared in step (a) with an aqueous phase, (c) evaporation of the organic solvent, (d) purification of the nanoparticles laden with active compound obtained in step (c) by means of dialysis against aqueous dialysis solution comprising the same active compound.

2. Process according to claim 1, characterised in that the active compound has a saturation solubility in water <200 g/ml, measured at 25 C.

3. Process according to claim 2, characterised in that the active compound used is an active compound which is selected from the group consisting of chemotherapeutic agents, antirheumatics, anti-infective agents, antimycotic agents, lipid-lowering agents, antioxidants and vitamins, such as, for example, tocopherol derivatives, retinoic acid derivatives, cholecalciferol, antibiotics, cholesterol and fatty acids.

4. Process according to claim 1, characterised in that the polymer employed is an amphiphilic polymer.

5. Process according to claim 4, characterised in that the polymer employed is a block copolymer.

6. Process according to claim 4, characterised in that the block copolymer contains as hydrophilic component: polyethylene glycol, polypropylene glycol, polybutylene glycol, polyacrylamide, polyvinyl alcohol, polysaccharide or a copolymer thereof, and as hydrophobic component: polylactic acid, polyglycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid or a copolymer thereof, and as an additional component polyacrylic acid and derivatives thereof, or polysiloxane and derivatives thereof.

7. Process according to claim 6, characterised in that the block copolymer employed is polyethylene glycol-polylactic acid, polyethylene glycol-polyglycolic acid, polyethylene glycol-polylactic acid-co-glycolic acid, polyethylene glycol-polyhydroxyvaleric acid, polyethylene glycol-polysiloxane, polyethylene glycol-polysiloxane-co-acrylic acid, polyethylene glycol-polymethylmethacrylic acid, polyethylene glycol-polymethylethacrylic acid, polyethylene glycol-polyisoprylacrylic acid or polyethylene glycol-polystyrene.

8. Process according to claim 1, characterised in that the organic solvent used is a solvent which is at least partially miscible with water.

9. Process according to claim 8, characterised in that the organic solvent employed is methanol, ethanol, isopropanol, n-butanol or tert-butanol, acetone, dimethylformamide, tetrahydrofuran or dimethyl sulfoxide.

10. Process according to claim 1, characterised in that, in step (a) according to claim 1, an acid or base is dissolved in the organic solvent besides polymer and active compound, and/or in that an acid or base is dissolved in the aqueous solvent in step (b) of claim 1.

11. Process according to claim 10, characterised in that the acid is formic acid, acetic acid, trifluoroacetic acid, hydrochloric acid, nitric acid or sulfuric acid, and the base is dimethylamine or trimethylamine, sodium hydroxide, potassium hydroxide or ammonia.

12. Process according to claim 1, characterised in that the organic solvent used in the production of the nanoparticles in step (a) of claim 1 is the organic solvent with which the greatest proportion of aqueous solvent can be admixed without active compound precipitating out of the solution during preparation of a solution comprising the active compound in defined amount compared with solutions comprising this active compound in the same amount in each case in other organic solvents on successive admixing of aqueous solvent.

13. Process according to claim 12, characterised in that the organic solvent is determined by the following method: (a) preparation of solutions of the active compound having the same proportion of active compound in each case in various organic solvents, (b) addition of an in each case identical amount of aqueous solution to each of the solutions prepared in step (a), (c) checking whether the active compound is in each case fully dissolved in the solutions of step (b), (d) repeated performance of steps (b) and (c) with the solutions in which the active compound is fully dissolved in step (c), until the active compound is no longer fully dissolved in step (c), (e) identification of the organic solvent with which the greatest amount of aqueous solution can be admixed cumulatively in step (d) before the active compound is no longer fully dissolved.

14. Process according to claim 12, characterised in that the organic solvents employed are methanol, ethanol, isopropanol, n-butanol, tert-butanol, acetone, dimethylformamide, tetrahydrofuran and dimethyl sulfoxide.

15. Process according to claim 1, characterised in that the amount of aqueous phase is selected so that, after mixing of the solution of organic solvent and aqueous phase in step (b), the aqueous phase is present in an amount, in relation to the organic solvent, which is below the maximum amount which can be admixed with the organic solvent without the active compound no longer being fully dissolved.

16. Process according to claim 15, characterised in that the determination of the maximum amount of aqueous phase which can be admixed with the solution of organic solvent is carried out in accordance with steps (a) to (d) (a) preparation of solutions of the active compound having the same proportion of active compound in each case in various organic solvents, (b) addition of in each case an identical amount of aqueous solution to each of the solutions prepared in step (a), (c) checking whether the active compound is in each case fully dissolved in the solutions of step (b), (d) repeated performance of steps (b) and (c) with the solutions in which the active compound is fully dissolved in step (c), until the active compound is no longer fully dissolved in step (c).

17. Nanoparticles, produced by the process according to claim 1.

18. Process according to claim 1, characterized in that the active compound has a saturation solubility <100 g/ml, measured at 25 C.

19. Process according to claim 2, characterized in that the active compound used is selected from the group consisting of taxol derivatives, camptothecin derivatives, platinum complexes or N-mustard compounds, dexamethasone, mometasone, beclomethasone, prednisolone, ritonavir, ketoconazole, itraconazole, griseofulvin, fenofibrate, tocopherol derivatives, retinoic acid derivatives, cholecalciferol, vancomycin or teicomycin.

20. Process according to claim 4, characterized in that the block copolymer contains as hydrophilic component: polyethylene glycol-polypropylene glycol copolymer, polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer, and as hydrophobic component: polylactic-co-glycolic acid, and as an additional component hydroxypropylethylacrylic acid, or hydroxypropylmethylacrylic acid, or polysiloxane and derivatives thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagrammatic representation of the procedure.

(2) FIG. 2 shows the visual solubility of dexamethasone.

(3) FIG. 3 shows the visual solubility of active compound B.

(4) The examples, without being restricted thereto, explain the invention.

EXAMPLES

Example 1

(5) For loading experiments, the active compounds dexamethasone and 5-[2-(2-fluorophenyl)-1,8-naphthyridin-4-yl]-2,6-naphthyridin-1-ylamine (also called active compound B below) were used. For solvent selection, the active compounds were each dissolved in the following solvents with a concentration of 1 mg/100 l: tetrahydrofuran (THF), acetonitrile (ACN), acetone, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, ethanol. Additionally, 0.1% of trifluoroacetic acid (v/v) were added to each organic solution of active compound B in order to establish an apparent pH. 10 l of water were added successively to each of the solutions and mixed until the active compound began to precipitate (visual solubility). FIG. 2 shows the visual solubility of dexamethasone, FIG. 3 shows the visual solubility of active compound B.

(6) For dexamethasone, owing to its increased visual solubility in tetrahydrofuran, this solvent was selected as organic solvent for the production of the nanoparticles. 4:1 v/v (THF:water) was fixed as the starting ratio.

(7) For active compound B, owing to its increased visual solubility in, this solvent was selected as organic solvent for the production of the nanoparticles. 5:1 v/v (ACN:water) was fixed as the starting ratio.

Example 2

(8) The following polymers were used for the production and loading of nanoparticles: PEG-PDLLA [5-b-23], PEG-PCL [5-b-32.5], PEG-PVPy [5-b-20] from Polymersource Inc., Montreal, Canada. Furthermore, PEG-PLGA [5-b-28] (Resomer RGP 50155 d) from Boehringer Ingelheim, Ingelheim, Germany, was used. All polymers were in research quality.

(9) In order to be able to compare the other production processes with co-solvent evaporation and the encapsulation in various polymers with one another, the nanoparticles were produced as follows and loaded with dexamethasone: Direct dialysis from acetone: 10 mg or 20 mg of block copolymer and 1 mg or 2 mg respectively of dexamethasone were dissolved in 1 ml of acetone. This solution was introduced into a dialysis tube (MWCO 6-8 kDa, Spectrumlabs Inc., Breda, The Netherlands) and sealed. The dialysis was carried out against 5 l of water for 24 h; the water was replaced once after 4 h. The formulation formed was subsequently removed from the dialysis tube, passed through a 0.2 m filter and adjusted to a volume of 2 ml. O/W Emulsion: Pre-shaped micelles without active compound were firstly produced as described under 2.a. Direct dialysis. For the active-compound loading, 2 mg of dexamethasone were dissolved in 1 ml of dichloromethane (VWR, Darmstadt, Germany). This organic solution was injected into 5 ml of the aqueous micellar phase with constant stirring. An O/W emulsion was formed, which was stirred further at room temperature overnight. The filtration step through a 0.2 m filter and the volume adaptation to 5 ml was subsequently carried out. Co-Solvent Evaporation with Subsequent Dialysis: 10 mg of block copolymer and 2 mg of dexamethasone were dissolved in 6 ml of THF. 2 ml of water were added to this solution. This solution was evaporated in a round-bottomed flask at a temperature of at 25 C. and a pressure of 30 mbar for 10 min. The formulation obtained was introduced into a Float-A-Lyzer G2 dialysis tube (MWCO 8-10 kDa, Spectrumlabs Inc., Berda, The Netherlands), which, in the case of the subsequent dialysis, had been preequilibrated against saturated solution in dexamethasone-saturated water. The formulation was then dialysed against 5 l of water or dexamethasone-saturated water for 24 h. Finally, the formulation was passed through a 0.2 m filter, and the volume was adjusted to 2 ml.

(10) The nanoparticles produced by the various processes were characterised with respect to their active-compound loading and particle sizes and the size distributions thereof.

(11) Determination of the Active-Compound Loading Via HPLC

(12) 100 l of the resultant micellar formulation were dissolved in 900 l of acetonitrile. This solution was detected using an HPLC system (Merck Hitachi La Chrom Elite) via a UV detector (detection wavelength: 282 nm). The separation was carried out on an Agilent Eclipse Plus C18 column (particle size 3.5 m, length 5 cm) at 30 C. A gradient method was utilised for the separation. The mobile phase A here consisted of 90% of acetonitrile and 10% of ammonium acetate buffer pH 4.5 (v/v), the mobile phase B had the reverse composition. The dexamethasone sample concentration was determined via a calibration curve.

(13) The calculation of the active-compound loading was carried out via formula 1 below:

(14) $\begin{array}{cc}\mathrm{active}\mathrm{compound}\mathrm{loading}\left[\%\right]=\frac{\mathrm{active}\mathrm{compound}\mathrm{concentration}\left[\frac{\mathrm{mg}}{\mathrm{ml}}\right]}{\mathrm{polymer}\mathrm{concentration}\left[\frac{\mathrm{mg}}{\mathrm{ml}}\right]}.Math.100\%& \left(1\right)\end{array}$
Particle Size Determination by Means of Dynamic Light Scattering (DLS)

(15) The DLS technique determines the hydrodynamic particle radius or diameter. For this purpose, the samples are diluted 1:100 (v/v) with water and measured in a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) in back-scatter mode. Particle sizes were calculated via cumulate analysis. In addition, the polydispersity index (PdI) was calculated, which is regarded as a measure of the scattering of the particle-size distribution. The PdI can have values between 0 and 1 where 0 denotes monodisperse and 1 denotes (fully) polydisperse.

(16) The results are compiled in the following Table 1.

(17) TABLE-US-00001 Polymer compound/polymer Active dynamic concentration ratio Production Solvent and compound particle size Polymer [%] w/v initial method conditions loading [%] [nm] Pdl PEG- 0.5 1:5 Co- THF, <LOQ 50.41 2.47 0.120 0.047 PDLLA solv. dialysis [5-b-23] evaporation against water PEG- 0.5 1:5 Co- THF, 1.56 0.24 61.43 1.39 0.102 0.006 PDLLA solv. dialysis [5-b-23] evaporation against active- compound- saturated soln. PEG- 0.5 1:5 Co- THF, <LOQ 62.67 1.60 0.091 0.014 PLGA solv. dialysis [5-b-28] evaporation against water PEG- 0.5 1:5 Co- THF, 1.19 0.13 69.18 1.23 0.057 0.026 PLGA solv. dialysis [5-b-28] evaporation against active- compound- saturated soln. PEG-PCL 0.5 1:5 Co- THF, <LOQ 80.59 2.98 0.093 0.053 [5-b-32.5] solv. dialysis evaporation against water PEG-PCL 0.5 1:5 Co- THF, 1.39 0.36 87.69 2.70 0.126 0.034 [5-b-32.5] solv. dialysis evaporation against active- compound- saturated soln. PEG- 0.5 1:5 Co- THF, 10.74 1.8 33.97 1.50 0.204 0.016 PVPy solv. dialysis [5-b-20] evaporation against water PEG- 0.5 1:5 Co- THF, 18.67 0.21 36.73 0.95 0.213 0.006 PVPy solv. dialysis [5-b-20] evaporation against active- compound- saturated soln. PEG- 1.0 1:5 Co- THF, 19.25 0.54 52.13 1.34 0.258 0.011 PVPy solv. dialysis [5-b-20] evaporation against active- compound- saturated soln. PEG- 0.5 1:5 Co- Acetone, 12.07 1.21 41.09 2.80 0.136 0.011 PVPy solv. dialysis [5-b-20] evaporation against active- compound- saturated soln. PEG- 1.0 1:5 Co- Acetone, 10.84 2.64 44.73 4.65 0.118 0.011 PVPy solv. dialysis [5-b-20] evaporation against active- compound- saturated soln. PEG- 0.5 1:10 Direct Acetone 1.71 0.15 56.42 7.29 0.178 0.056 PVPy dialysis [5-b-20] PEG- 1.0 1:5 Direct Acetone 0.62 0.60 66.91 2.29 0.162 0.011 PVPy dialysis [5-b-20] PEG- 0.5 1:10 O/W Dichloro- 8.74 0.03 52.42 2.00 0.150 0.012 PVPy emulsion methane, [5-b-20] prefabricated particles from acetone PEG- 1.0 1:10 O/W Dichloro- 7.81 0.18 68.92 3.53 0.185 0.036 PVPy emulsion methane, [5-b-20] prefabricated particles from acetone PEG- 0.5 2:5 O/W Dichloro- 13.50 5.05 52.19 0.67 0.186 0.022 PVPy emulsion methane, [5-b-20] prefabricated particles from acetone Table 1, in which PEG-PDLLA denotes pegylated poly(D,L-lactic acid), PEG-PLGA denotes pegylated poly(lactic acid-co-glycolic acid), PEG-PCL denotes pegylated poly(caprolactone), PEG-PVPy denotes pegylated poly-4-(vinylpyridine), LOQ denotes limit of quantification (determination limit of the HPLC method), co-solv. evaporation denotes co-solvent evaporation and THF denotes tetrahydrofuran.

Example 3

(18) Production and Loading of Nanoparticles Laden with Active Compound B

(19) The production and loading of the nanoparticles was carried out by the process described in this invention as a combination between co-solvent evaporation and dialysis against active-compound-saturated solution.

(20) 10 mg of block copolymer and 1 mg of active compound were dissolved in 8 ml of acetonitrile/0.1% of trifluoroacetic acid (v/v) with ultrasound treatment. The solution obtained was mixed with 2 ml of water. The mixture was subsequently introduced into a round-bottomed flask, and the organic solvent was evaporated under reduced pressure (30 mbar) and at 25 C. (10 min). The nanoparticles obtained were introduced into a Float-A-Lyzer G2 dialysis tube (MWCO 8-10 kDa, Spectrumlabs Inc., Berda, The Netherlands) and dialysed for 24 h against 5 l of phosphate-buffered saline solution (PBS buffer), pH 7.4, saturated with active compound B. Finally, the formulation was passed through a 0.2 m filter, and the volume of the formulation was adjusted to 2 ml.

(21) Determination of the Active-Compound Loading

(22) 100 l of the formulation obtained were dissolved in 900 l of acetonitrile. This solution was detected using an HPLC system (Merck Hitachi La Chrom Elite) via a UV detector (detection wavelength: 254 nm). The separation was carried out on an Agilent Eclipse Plus C18 column (particle size 3.5 m, length 5 cm) at 30 C. A gradient method was utilised for the separation. The mobile phase A here consisted of 90% of acetonitrile and 10% of water with 0.1% of trifluoroacetic acid (v/v), the mobile phase B had the reverse composition. The active compound B sample concentration was determined via a calibration curve.

(23) The calculation of the active-compound loading was carried out here in accordance with formula 1. Particle sizes and size distributions were determined analogously to Example 2.

(24) The results with the optimum loading technique on use of various block copolymers are summarized in Table 2.

(25) TABLE-US-00002 Polymer compound/polymer Active concentration ratio Production Solvent and compound particle size Polymer [%] w/v initial method conditions loading [%] [nm] Pdl PEG- 0.5 1:10 Co-solv. ACN/0.1% <LOQ n.a. n.a. PDLLA evaporation TFA, [5-b-23] dialysis with saturation PEG- 0.5 1:10 Co-solv. ACN/0.1% 25.4 n.a. n.a. PLGA evaporation TFA, [5-b-28] dialysis with saturation PEG-PCL 0.5 1:10 Co-solv. ACN/0.1% 24.9 n.a. n.a. [5-b-32.5] evaporation TFA, dialysis with saturation PEG- 0.5 1:10 Co-solv. ACN/0.1% 40.6 n.a. n.a. PVPy evaporation TFA, [5-b-20] dialysis with saturation PEG- 5.0 1:10 Co-solv. ACN/0.1% 101.90 6.44 122 0.183 PLGA evaporation TFA, [5-b-28] dialysis with saturation Table 2, in which PEG-PDLLA denotes pegylated poly(D,L-lactic acid), PEG-PLGA denotes pegylated poly(lactic acid-co-glycolic acid), PEG-PCL denotes pegylated poly(caprolactone), PEG-PVPy denotes pegylated poly-4-(vinylpyridine), LOQ denotes limit of quantification (determination limit of the HPLC method), co-solv. evaporation denotes co-solvent evaporation, ACN denotes acetonitrile and TFA denotes trifluoroacetic acid.