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Title:
NANOPARTICLES
Document Type and Number:
WIPO Patent Application WO/2010/046681
Kind Code:
A1
Abstract:
A process for the preparation of nanoparticles comprising an active hydrophobic compound, the process comprising the steps of: a) providing a first stream comprising the compound, a hydrophobic polymer, an amphiphilic polymer and a solvent; b) providing a second stream comprising an antisolvent; c) rapidly intermixing the first and second streams in a precipitation chamber thereby forming the nanoparticles; and d) rapidly intermixing the intermixed first and second streams with a third steam comprising a stabilising agent.

Inventors:
VAN BOXTEL HUIBERT (NL)
Application Number:
PCT/GB2009/051316
Publication Date:
April 29, 2010
Filing Date:
October 06, 2009
Export Citation:
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Assignee:
FUJIFILM MFG EUROPE BV (NL)
FUJIFILM IMAGING COLORANTS LTD (GB)
VAN BOXTEL HUIBERT (NL)
International Classes:
B01D9/00; A61K9/16; A61K9/51; B01J13/06
Domestic Patent References:
WO2008035028A12008-03-27
Other References:
LIGGINS AND BURT: "Paclitaxel-loaded poly(l-lactic acid) microspheres 3: blending low and high molecular weight polymers to control morphology and drug release", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 282, 20 April 2004 (2004-04-20), pages 61 - 71, XP002521950
JACKSON ET AL.: "The characterization of paclitaxel-loaded microspheres manufactured from blends of poly(lactic-co-glycolic acid) (PLGA) and low molecular weight diblock copolymers", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 342, 3 May 2007 (2007-05-03), pages 6 - 17, XP002521952
QUINTANAR-GUERRERO ET AL.: "Preparation Techniques and Mechanisms of Formation of Biodegradable Nanoparticles from Preformed Polymers", DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, vol. 24, no. 12, 1998, pages 1113 - 1128, XP009114665
PATRICK COUVREUR ET AL: "Nanocapsule Technology: A Review", CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, BEGELL HOUSE PUBLISHING INC, vol. 19, no. 2, 1 January 2002 (2002-01-01), pages 99 - 134, XP009114641, ISSN: 0743-4863
DATABASE COMPENDEX [online] ENGINEERING INFORMATION, INC., NEW YORK, NY, US; DENG L-D ET AL: "Studies on methoxy poly(ethylene glycol)-poly(D, L-lactic acid) block copolymers", XP002567274, Database accession no. E2006089713124
Attorney, Agent or Firm:
MAYALL, John (Hexagon TowerBlackley,Manchester, Greater Manchester M9 8ZS, GB)
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Claims:

CLAIMS

1. A process for the preparation of nanoparticles comprising an active hydrophobic compound, the process comprising the steps of: a) providing a first stream comprising the compound, a hydrophobic polymer, an amphiphilic polymer and a solvent; b) providing a second stream comprising an antisolvent; c) rapidly intermixing the first and second streams in a precipitation chamber thereby forming the nanoparticles; and d) rapidly intermixing the intermixed first and second streams with a third steam comprising a stabilising agent.

2. A process according to claim 1 wherein the nanoparticles have a median particle size D50 of less than 500nm.

3. A process according to any one of the preceding claims, wherein the weight ratio of active hydrophobic compound to hydrophobic polymer is from 1 :99 to 95:5.

4. A process according to any one of the preceding claims wherein the hydrophobic polymer comprises a poly(D,L-lactide) and the amphiphilic polymer comprises a poly(ethylene glycol) alkyl ether-block-poly(D,L-lactide).

5. A process according to claim 4 wherein the amphiphilic polymer has a number average molecular weight below 10,000 Dalton.

6. A process according to any one of the preceding claims wherein the first and second streams are continuously fed into a precipitation chamber where they rapidly intermix to form the nanoparticles and then exit the precipitation chamber.

7. A process according to any one of the preceding claims wherein the first stream comprises 0.1 to 50wt% of hydrophobic polymer, relative to the weight of solvent.

8. A process according to any one of the preceding claims wherein the second stream comprises citric acid in free acid or salt form.

9. A process according to any one of the preceding claims wherein the stabilising agent is or comprises a gelatine.

10. A process according to any one of the preceding claims wherein the nanoparticles have a median particle size D50 below 220nm.

11. A process according to any one of the preceding claims wherein the hydrophobic polymer has a water solubility at 20 0 C of less than 1wt%.

12. A process according to any one of the preceding claims wherein the solvent and the antisolvent are partly or completely miscible.

13. A process according to any one of the preceding claims wherein said intermixing is performed in a mixing chamber fitted with at least one mechanical stirring means having a diameter of 70% to 99% of the smallest diameter of the mixing chamber.

14. Nanoparticles having a median particle size D50 of less than 500nm which comprise at least 10wt% of a pharmaceutically active hydrophobic compound.

15. Nanoparticles according to claim 14 which further comprise a hydrophobic polymer and an amphiphilic polymer.

16. Nanoparticles according to claim 15 wherein the hydrophobic polymer comprises poly(D,L-lactide) and the amphiphilic polymer comprises poly(ethylene glycol) alkyl ether-block-poly(D,L-lactide) with a number average molecular weight below 10,000 Dalton.

17. A process for the manufacture of medicament comprising the mixing of the nanoparticles according to claim 14 or 15 with a pharmaceutically acceptable carrier or excipient to give the medicament.

18. A process according to claim 17 wherein the medicament is in the form of a tablet, troche, powder, syrup, patch, liposome, dispersion, suspension, capsule, cream, ointment or aerosol.

19. A medicament obtained by a process according to claim 17 or 18.

Description:

NANOPARTICLES

This invention relates to nanoparticles comprising active hydrophobic compounds, to processes for their preparation and to their uses.

A number of injectable drug delivery systems have been investigated over the years, including microcapsules, microparticles, liposomes and emulsions. A significant obstacle to the use of these systems is the rapid clearance of the materials from the blood stream by the macrophages of the reticuloendothelial system.

International patent application No. WO03/077887 describes the preparation of microparticles comprising an anti-cancer drug (goserelin acetate) as the active ingredient, a hydrophobic polymer (Resomer ® 756) and a surfactant (Pluronic ® F68). The microparticles resulting from the Examples were rather large, typically having a diameter of 1 -40 micrometres.

International patent application No. WO98/14174 mentions the manufacture of drug-containing nanoparticles comprising the steps of dissolving polylactic acid in a water-immiscible solvent (e.g. methylene chloride), dissolving the pharmaceutically active agent in the polymer solution, adding a surfactant to the oil phase or the aqueous phase, forming an oil-in-water emulsion by suitable means, and evaporating the emulsion slowly under vacuum.

International patent application No. WO98/14174 describes the formation of paclitaxel-containing nanoparticles by emulsification techniques.

French Patent 2 660 556 discloses the formation of injectable microspheres by dissolving a biodegradable, biocompatible polymer and a biologically active compound in a water-immiscible solvent more volatile than water, then mixing using a high pressure homogeniser with an aqueous solution of a biocompatible surface active agent, followed by evaporation of the solvent.

International patent application WO2008035028 describes a process for the precipitation of a biologically active compound wherein streams of solvents and antisolvents interact with the compound to form nanoparticles without the need for potentially wasteful and damaging milling. This process was developed further in International patent application WO2008035962. Both of these documents form nanoparticles from amphiphilic polymers, typically containing PEG groups, in the absence of a hydrophobic polymer.

Liggins and Burt described the preparation of paclitaxel-loaded microspheres in the International Journal of Pharmaceutics, vol. 282, 20 April 2004, pages 61 -71. The microspheres could contain up to 30% paclitaxel loading and they were quite large at 1 ,000 to 105,000nm. The microspheres lacked a hydrophobic polymer.

Jackson et al also described the preparation of paclitaxel-loaded microspheres in the International Journal of Pharmaceutics, vol. 342, 3 May 2007, pages 6-17. The preparation involved pipetting a solution of paclitaxel and diblock copolymer in DCM into a 'bulk' 2.5% PVA solution with stirring at 600rpm. This process did not use a first and second stream. Furthermore, there was no third stream comprising a stabilising agent. The resultant microparticles were very large, with sizes of 30,000-120,000nm.

While the processes described in the preceding documents are useful, there is a need for a process for preparing nanoparticles comprising biologically active materials with good half lives. The nanoparticles should be stable under biological conditions (aqueous conditions at 37 0 C) and be readily redispersible in water. Ideally the nanoparticles release their contents steadily rather than rapidly such that their payload is continuously available for extended periods.

Producing nano-sized particles is much more difficult than producing microparticles because they are less stable than microparticles and tend to agglomerate and/or ripen by recrystallisation processes. For anticancer drug delivery there is a need for nano-sized particles.

Surprisingly, the present process provides nanoparticles with good stability in aqueous solutions. The active hydrophobic compound contained in these nanoparticles can survive in an aqueous environment for a long time and, by varying the process parameters and ingredients, one can tune the release rate of the active hydrophobic compound without adversely affecting the size of the nanoparticles.

According to the present invention there is provided a process for the preparation of nanoparticles comprising an active hydrophobic compound, the process comprising the steps of:

(a) providing a first stream comprising the compound, a hydrophobic polymer, an amphiphilic polymer and a solvent;

(b) providing a second stream comprising an antisolvent;

(c) rapidly intermixing the first and second streams in a precipitation chamber thereby forming the nanoparticles; and

(d) rapidly intermixing the intermixed first and second streams with a third steam comprising a stabilising agent.

In this document (including its claims), the verb "comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly

requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Preferably the nanoparticles have a median particle size (D50) of less than 500nm, more preferably less than 400nm, especially less than 350nm, more especially less than 220nm. The D50 may be measured by techniques known in the art, for example by laser diffraction using the method according to ISO 13320- 1 , e.g. using a Malvern Mastersizer 2000 particle size analyser.

For slow-release nanoparticles wherein the active hydrophobic compound is an anti-cancer drug intended for infusion the preferred median particle size (D50) is less than 400nm, especially 100 to 200nm. The vascular system in solid tumors shows artefacts and openings with a size that allows particles smaller than approx. 400nm to leave the blood stream and get trapped inside these openings. Such trapping is usually referred to as the Enhanced Permeability and Retention (EPR) effect. It is a mechanism that localizes the cytostatic drugs at the tumor site, enhancing the specific killing of cancer cells and reducing the non-wanted killing of healthy tissue. A second advantage of such trapping is a potentially longer duration of drug exposure for the cancerous cells than is possible with a standard infusion of an immediate release drug formulation. Normally the drug is eliminated from the body by various mechanisms such as enzymatic degradation(metabolism) and elimination by the kidneys causing a rapid drop in drug concentration in the blood. There is a need for drug formulations that show a slower than instant release in order to maintain effective drug concentrations for extended periods of time.

Preferably the nanoparticles have a unimodal particle size distribution. The nanoparticles can be in any form, with spherical particles being preferred.

The hydrophobic polymer preferably comprises a polylactide (especially poly(DL-lactide) or poly(L-lactide)), a polycaprolactone and/or a polylactic-co- glycolic acid polymer.

In a preferred embodiment the hydrophobic polymer has a number average molecular weight of more than 2,000 Daltons, preferably 2,000 to 150,000 Daltons, especially 10,000 to 150,000 Daltons, more especially 50,000 to 150,000 Daltons.

In another preferred embodiment the hydrophobic polymer has a number average molecular weight of 5,000 to 100,000 Daltons, especially 25,000 to 50,000 Daltons.

Preferred hydrophobic polymers have a solubility in water at 2O 0 C of less than 1wt%, more preferably less than 0.1 wt%, especially less than 0.01 wt%.

Preferred hydrophobic polymers have a solubility in tetrahydrofuran at 2O 0 C of greater than 1wt%, more preferably greater than 5wt%, especially greater than 10wt%.

Preferably the hydrophobic polymer is biocompatible.

Preferred active hydrophobic compounds have a solubility in water at 2O 0 C of less than 1wt%, more preferably less than 0.2wt%, especially less than 0.1 wt%.

Preferably the water-solubility of the active hydrophobic compound is higher than the water-solubility of the hydrophobic polymer.

Preferably the hydrophobic polymer comprises a poly(D,L-lactide) and the amphiphilic polymer comprises a poly(ethylene glycol) alkyl ether-block-poly(D,L- lactide).

Preferred active hydrophobic compounds have a solubility in tetrahydrofuran at 2O 0 C of greater than 1wt%, more preferably greater than 5wt%, especially greater than 10wt%.

The active hydrophobic compound is preferably a colorant or, more preferably, a biologically active compound, especially a pharmaceutically active compound or an agrochemical. Examples of biologically active compounds include hormones, lipids, vitamins and medicaments.

The hydrophobic biologically active compounds which can be nanoencapsulated by the process of this invention are preferably pharmaceutically active compounds. Classes of such compounds include anabolic steroids, analeptics, analgesics, anaesthetics, antacids, anti-arrythmics, anti-asthmatics, antibiotics, anti-carcinogenics and anti cancer drugs, to name but a few. A particularly preferred biologically active compound from the class of anti-cancer drugs is paclitaxel (also known as Taxol ® ).

When the active hydrophobic compound is or comprises paclitaxel the rapid intermixing is preferably performed at a pH of 3 to 5. The use of such a pH can result in particularly good chemical stability.

The amphiphilic polymers preferably have an affinity for both the hydrophobic compound and the anti-solvent e.g. water. When the hydrophobic compound has a low solubility in water, the amphiphilic polymer will generally possess a hydrophilic part which has an affinity for water and a less hydrophilic part, e.g. a relatively hydrophobic part, which has an affinity for the hydrophobic compounds. The relatively hydrophilic part of the amphiphilic polymers are often non-ionic (e.g. poly(alkylene)oxide units) and/or ionic (e.g. they have anionic or cationically charged groups) while the less hydrophilic or hydrophobic parts are often electrically neutral and relatively non-polar (e.g. polylactide groups). The amphiphilic polymers used in the first stream are preferably amphiphilic block copolymers.

The preferred amphiphilic block copolymers are those that do not exhibit an extreme tendency to form micelles. This preference arises because such

copolymers are more efficient in stabilising the active hydrophobic compound particle surfaces during the rapid intermixing.

Preferred amphiphilic polymers which are not block copolymers include gelatines, especially gelatines having a molecular weight of at least 2kD. Many gelatine solutions form gels at or below room temperature. The suspension stability of particles prepared by the process of the present invention is in some cases greatly enhanced at temperatures below room temperature. Therefore, in one embodiment the use of non-gelling gelatines is preferred over the use of gelling gelatines. Examples of such gelatines include fish gelatines, non-gelling recombinant gelatines, e.g. recombinant gelatines which are free from hydroxyproline, and hydrolysed gelatines with very low molecular weight. In one embodiment, the gelatine shows no gelling properties when stored as a 2wt% solution in water at 1 O 0 C for 4 hours. Particularly preferred gelatines are recombinant gelatines, especially where the particles are intended for use in vivo (e.g. in pharmaceutical applications) due to the absence of BSE or viral risks.

Preferably, the amphiphilic polymer is an amphiphilic block copolymer, for example comprising hydrophilic and relatively hydrophobic segments. Preferably, the amphiphilic polymers are triblock or diblock copolymers, especially diblock copolymers. Typically such copolymers comprise at least one hydrophilic block and at least one relatively hydrophobic block. The preferred block-type and block- lengths in amphiphilic polymers varies depending on the chemical composition of the streams and on the preferred average particle size of the nanoparticles.

Considering the aforementioned, preferred hydrophilic blocks are poly(ethylene glycol) ("PEG") and/or poly(ethylene glycol) monoether ("PEG ether") blocks. One of the reasons for preferring PEG and PEG ether hydrophilic blocks is because stabilisation agents containing these blocks tend to be excreted from the body less quickly than stabilisation agents lacking these blocks. So where the hydrophobic compound is, for example, an anti-cancer drug, the presence of PEG or PEG ether hydrophilic blocks in the stabilisation agent can increase the time the drug is in the body making it more effective.

Furthermore, amphiphilic block copolymers having a number average molecular weight (M n ) below 10,000 generally have better stabilising properties than those of higher M n .

The preferred ethers have from 1 to 4 carbon atoms, with methyl ether being most preferred. Preferred hydrophobic blocks are poly (lactic-co- glycolic)acid ("PLGA"), poly(styrene) ("PS"), poly(butyl acrylate), poly(ε- caprolactone) and especially polylactide ("PLA") blocks. Polylactides are polyesters formed from the polymerisation of lactic acid or lactide. Polylactides exist as poly(L-lactide), poly(D-lactide) and poly(D,L- lactide). Among these

polylactides the poly(D,L-lactide) is preferred because it is soluble in some water- miscible solvents like THF, whereas poly(L-lactide) is soluble in very few solvents, like dichloromethane which is not water miscible.

Preferred biocompatible amphiphilic block copolymers include copolymers comprising one or more PEG and/or PEG ether blocks and one or more PLA blocks. The PEG blocks are water-soluble and relatively hydrophilic compared to the PLA blocks.

In one embodiment it is preferred that the PEG and PEG ether block(s) have a number weighted average molecular weight (M n ) of 250 to 5000, more preferably 500 to 4000, especially 1000 to 3000. Very good results were obtained with a PEG and PEG ether blocks having an Mn of about 2000.

In a preferred embodiment the PLA block(s) have an M n of 250 to 5000, more preferably 500 to 4000, especially 1000 to 3000. Very good results were obtained with a PLA block having an Mn of about 2000.

A particularly preferred amphiphilic block copolymer is a diblock copolymer of a PEG ether and a PLA, especially having the M n mentioned above, with the preferences for M n in each block being as mentioned above. Bearing in mind the above, one preferred category of amphiphilic diblock copolymers are poly(ethylene glycol) M n 350-5000 (Ci -4 -alkyl) ether-block-polylactide M n 1000-5000. Thus preferred amphiphilic block copolymers are of the formula [poly(ethylene glycol) alkyl ether] x -block-[poly(D,L-lactide)]γ (MPEGχ-PLA γ ) wherein x and y are integers such that the resultant copolymer has a molecular weight below 10,000 Dalton.

Examples of valuable subsets of such amphiphilic diblock copolymers include: poly(ethylene glycol) M n 350-1500 (Ci -4 -alkyl) ether-block-polylactide M n 500-2000; polyethylene glycol) M n 500-1100 (Ci -4 -alkyl) ether-block-polylactide M n 600-1600; poly(ethylene glycol) M n 600-900 (Ci -4 -alkyl) ether-block-polylactide M n 800-1200; polyethylene glycol) M n 700-900(Ci -4 -alkyl) ether-block-polylactide M n 800-1200; polyethylene glycol) M n 700-900 methyl ether-block-polylactide M n 800- 1200; poly(ethylene glycol) M n 750 (Ci -4 -alkyl) ether-block-polylactide M n 1000; and polyethylene glycol) M n 750 methyl ether-block-polylactide M n 1000.

Examples of amphiphilic block copolymers include: polyethylene glycol) M n 750 methyl ether-block-polylactide M n 1000; polyethylene glycol) M n 2000 methyl ether-block-polylactide M n 2000; polyethylene glycol) M n 3000 methyl ether-block- polylactide M n 2000; polyethylene glycol) M n 2000 methyl ether-block-polylactide M n 3000; polyethylene glycol) M n 350 methyl ether-block-polylactide M n 1000; and polyethylene glycol) M n 5000 methyl ether-block-poly(lactone) M n -5,000.

Further amphiphilic block copolymers which can be used include: polyethylene glycol) M n 5000 methyl ether-block-poly(ε-caprolactone) M n 5,000;

poly(ethylene glycol) M n 5000 methyl ether-block-poly(ε-caprolactone) M n 13,000; and poly(ethylene glycol) M n 5,000 methyl ether-block-poly(ε-caprolactone) M n 32,000; all of which are commercially available from Sigma-AIdrich Co. As will be readily understood by those skilled in the art, "methyl ether" refers to a methyl group on one end of the PEG chain (not both ends because this would prevent the PLA from attaching to the PEG). Also the Mn values for the PEG, such in "PEG mono methyl ether Mn 750" refer to the Mn of the PEG per se, not including the extra CH 2 group of the methyl group.

Amphiphilic polymers are available from commercial sources or they may be synthesised ad hoc for use in the process. The preparation of the preferred amphiphilic diblock copolymers with poly(alkylene glycol) (PAG) blocks (e.g. poly(ethylene glycol) (PEG) blocks) can be performed in a number of ways. Methods include: (i) reacting a hydrophobic polymer with methoxy poly(alkylene glycol), e.g. methoxy PEG or PEG protected with another oxygen protecting group (such that one terminal hydroxyl group is protected and the other is free to react with the hydrophobic polymer); or (ii) polymerizing the hydrophobic polymer onto methoxy or otherwise monoprotected PAG, such as monoprotected PEG. Several publications teach how to carry out the latter type of reaction.

In an alternative embodiment, the hydrophobic block can be reacted with a poly(alkylene glycol) that is terminated with an amino function (available from Shearwater Polymers, Inc.) to form an amide linkage, which is in general stronger than an ester linkage.

Triblock or other types of block amphiphilic copolymers terminated with poly(alkylene glycol), and in particular, poly(ethylene glycol), can be prepared using the reactions described above, using a branched or other suitable poly(alkylene glycol) and protecting the terminal groups that are not to be reacted. Shearwater Polymers, Inc., provides a wide variety of poly(alkylene glycol) derivatives. Examples are the triblock PEG-PLGA-PEG.

In one embodiment, a multiblock amphiphilic copolymer is used and this may be prepared by reacting the terminal group of the hydrophobic polymeric block such as PLA or PLGA with a suitable polycarboxylic acid monomer, for example 1 ,3,5-benzenethcarboxylic acid, butane-1 ,1 ,4-thcarboxylic acid, tricarballylic acid (propane-1 ,2,3-tricarboxylic acid), and butane-1 ,2,3,4- tetracarboxylic acid, wherein the carboxylic acid groups not intended for reaction are protected by means known to those skilled in the art. The protecting groups are then removed, and the remaining carboxylic acid groups reacted with poly(alkylene glycol). In another alternative embodiment, a di, tri, or polyamine is similarly used as a branching agent.

The first stream may of course comprise one or more than one of the aforementioned amphiphilic polymers, for example two or more amphiphilic block copolymers.

The process of the present invention is particularly useful for preparing nanoparticles having a high content of active hydrophobic compound (e.g. a pharmaceutically active hydrophobic compound). Nanoparticles having a median particle size D50 of less than 500nm which comprise at least 10wt% of a pharmaceutically active hydrophobic compound form a further feature of the present invention, as do medicaments comprising such nanoparticles.

While prior processes may be useful for achieving a content of up to about 10wt% of active hydrophobic compound, the present process can be used to provide nanoparticles having a content of active hydrophobic compound of over 10wt%, over 20wt%, over 25wt%, over 35wt%, over 45wt% and even over 50wt%. In a preferred embodiment the nanoparticles comprise from 25wt% to 90wt% of active hydrophobic compound, especially 35wt% to 65wt% of active hydrophobic compound. In another preferred embodiment the nanoparticles comprise from 5wt% to 90wt% of active hydrophobic compound, especially 10wt% to 65wt% of active hydrophobic compound.

The nanoparticles of this further feature of the present invention preferably comprise a hydrophobic polymer and an amphiphilic polymer. Particularly preferred nanoparticles are where the hydrophobic polymer comprises poly(D,L- lactide) and the amphiphilic polymer comprises poly(ethylene glycol) alkyl ether- block-poly(D,L-lactide) with a number average molecular weight below 10,000 Dalton.

The present invention also provides a process for the manufacture of medicament comprising the mixing of the abovementioned nanoparticles of the present invention with a pharmaceutically acceptable carrier or excipient to give the medicament. Preferred medicaments are in the form of a tablet, troche, powder, syrup, patch, liposome, dispersion, suspension, capsule, cream, ointment or aerosol. Medicaments obtained by this mixing process form a still further feature of the present invention.

The combination of high active hydrophobic compound content and the controlled release provided by the nanoparticle construction makes the present invention particularly valuable in the pharmaceutical field, leading to less frequent patient dosings and even drug deliver rates.

The first stream may of course comprise one or more than one of the aforementioned hydrophobic polymers. The resultant nanoparticles preferably comprise 1 to 95wt%, more preferably 2 to 95wt%, especially 10 or 25 to 90wt% of the hydrophobic polymer.

The resultant nanoparticles preferably comprise 1 to 80wt%, more preferably 5 to 75wt%, especially 10 to 75wt% of the amphiphilic polymer.

The invention also provides medicaments comprising the nanoparticles of the invention and a pharmaceutically acceptable carrier.

The amounts of each component in the first stream may be varied between wide limits and depend to some extent on the properties of the components and the properties of the other streams they come into contact with.

Typically however the first stream will comprise 0.1 to 20wt%, more preferably 0.2 to 15wt%, especially 0.3 to 10wt% of the active hydrophobic compound, relative to the weight of solvent.

The amount of hydrophobic polymer included in the first stream is typically 0.1 to 50wt%, more preferably 0.2 to 20wt%, relative to the weight of solvent.

In one embodiment the composition of the streams is such that the weight ratio of active hydrophobic compound to hydrophobic polymer is greater than 1 :99, preferably greater than 5:95, especially greater than 10:90, e.g. greater than 15:85 or 20:80. Preferably the composition of the streams is such that the weight ratio of active hydrophobic compound to hydrophobic polymer is from 1 :99 to 95:5, more preferably from 10:90 to 90:10.

In another embodiment the composition of the streams is such that the weight ratio of active hydrophobic compound to hydrophobic polymer is greater than 5:95, preferably greater than 10:90 and especially greater than 20:80.

In yet another embodiment the composition of the streams is such that the weight ratio of active hydrophobic compound to hydrophobic polymer is greater than 10:90, preferably greater than 25:75, especially greater than 45:55, e.g. about 50:50.

The preferred weight ratio of active hydrophobic compound to the combined weight of hydrophobic polymer and amphiphilic polymer is 1 :20 to 10:1 , more preferably 1 :10 to 2:1 , especially 1 :10 to 1 :1.

The solvent may be any liquid in which the hydrophobic compound and the hydrophobic polymer are soluble or dispersible. The solvent may comprise, for example, polar or non-polar solvents, protic or aprotic solvents, ionic or non-ionic solvents, or a mixture of two or more solvents of any of the aforementioned types. Preferably the solvent comprises one or more than one water-miscible organic solvents. Preferably the solvent is such that the hydrophobic compound has a high solubility therein, preferably a solubility when measured at 2O 0 C of at least 10g/l.

Preferably the solvent and the antisolvent are partly or completely miscible. Examples of solvents include alcohols, especially Ci -4 -alkyl alcohols (e.g. ethanol, methanol, propanol and ethylene glycol; ketones, e.g. acetone; esters, e.g. ethyl

acetate; dimethyl sulfoxide; acetonitrile; 1 ,4-dioxane; C 2-4 - carboxylic acids e.g. acetic acid; tetrahydrofuran; and combinations of 2 or more thereof.

The first stream comprising the hydrophobic compound may comprise a single solvent or a mixture of solvents.

The first stream comprising the solvent, active hydrophobic compound, amphiphilic polymer and the hydrophobic polymer may be prepared in any convenient manner, typically by a process comprising mixing the compound, the hydrophobic polymer, the amphiphilic polymer and the solvent. This mixing may be performed inside or outside of the precipitation chamber.

In one embodiment the mixing to prepare the first stream is performed by a process comprising combining two or more streams each comprising a solvent and one or more of the ingredients selected from the hydrophobic compound, the hydrophobic polymer and the amphiphilic polymer.

So, for example, the first stream may be prepared by combining a stream comprising a solvent and the active hydrophobic compound with a stream comprising a solvent, the hydrophobic polymer and the amphiphilic polymer. Alternatively the first stream may be prepared by combining a stream comprising a solvent, the active hydrophobic compound and the amphiphilic polymer with a stream comprising a solvent and the hydrophobic polymer. Furthermore one may combine three streams, each containing a solvent and either the active hydrophobic compound, the amphiphilic polymer or the hydrophobic polymer. The amphiphilic polymer may be included in more than one of the streams used to make the first stream if desired. Other permutations will also be apparent to those of ordinary skill in the art.

The anti-solvent may be any liquid in which the hydrophobic compound and the hydrophobic polymer have low solubility, e.g. a solubility of less than 1wt%, more preferably less than 0.1 wt%, at a temperature of 20 0 C and a pressure of 1 bar. Preferably the anti-solvent comprises water (e.g. at least 50% water by weight).

The anti-solvent used in the second stream may be chosen to suit the components of the first stream and bring about the formation of nanoparticles during rapid intermixing of the first and second streams. The particular conditions used for the process may also influence the decision on which anti-solvent to use. The second stream comprising the anti-solvent may, for example, be a liquid having a lower temperature (in case of low temperature precipitation), different ionic strength or different pH than the first stream. The second stream may comprise one antisolvent or more than one antisolvent for the hydrophobic polymer and the hydrophobic compound. Examples of anti-solvents for hydrophobic species include water, alcohols, aqueous organic solvents and liquid

alkanes. Whether or not the specific liquid is an anti-solvent depends on the hydrophobic compound and hydrophobic polymer that need to be precipitated.

In addition the second stream may also contain a solvent for the hydrophobic compound and/or the hydrophobic polymer, although this would generally be present in only small amounts so as not to adversely affect the ability of the second stream to cause the hydrophobic compound and hydrophobic polymer to precipitate when the first and second streams are mixed.

In one embodiment the first and/or second stream comprises a wetting agent.

In step (c) the first stream may be fed into the flow of the second stream or the second stream may be fed into the flow of the first stream. In other words the terms "first stream" and "second stream" are not intended to imply any particular order but merely identify the particular two streams being referred to.

In a preferred process according the present invention the second stream further comprises a peptising agent, especially citric acid. The citric acid may be in the free acid or salt form. We have found that the addition of citric acid in the second stream exerts a stabilising effect on the nanoparticles. The amounts of peptising agent in the second stream is typically 0 to 5wt%, more preferably 0.1 to 2wt%, relative to the weight of antisolvent.

The preferred weight ratio of peptising agent to the combined weight of active hydrophobic compound, hydrophobic polymer and amphiphilic polymer is 1 :100, more preferably 1 :25.

Preferably the second stream has a faster flow rate than the first stream. In one embodiment the second stream has a flow rate of 1.5 to 10, more preferably 2 to 7 times the flow rate of the first stream. Preferably however the second stream has a flow rate of 1.5 to 50, more preferably 2 to 20, especially preferably 3 to 8 and more especially about 5 times the flow rate of the first stream.

While not wishing to be limited to any particular theory, the stabilisation agents are believed to be useful in the present process in a number of ways. For example, a stabilisation agent may inhibit particle agglomeration by surface adsorption and steric repulsion of particles. A stabilisation agent may lower the particle growth rate by polymer adsorption to the particle surface. The stabilisation agents may even increase viscosity due to the polymer presence, thereby limiting the agglomeration rates. These effects are mostly observed for polymeric stabilizers.

The third stream may of course comprise one or more than one of the aforementioned stabilising agents.

The amount of stabilising agent in the third stream is typically 0.1 to 50wt%, more preferably 1 to 25wt%, relative to the weight of the third stream.

The stabilising agent is preferably biocompatible, especially where the hydrophobic compound is a pharmaceutical compound.

Stabilisation agents optionally comprise a synthetic or natural polymer or they may comprise non-polymeric compounds. Stabilising agents which may be included in a third stream include amphiphilic polymers.

The stabilising agent preferably does not form micelles in the anti-solvent. Thus the stabilising agent preferably has no CMC value or a very high CMC value e.g. higher than 10wt% in the anti-solvent.

Good results are often obtained when a gelatine is used as stabilisation agent. Other polymeric stabilisation agents may also be used, for example methylcellulose, poly(vinylpyrrolidone), polyvinyl alcohol, poly(ethyleneoxide) and/or poly(ethyleneglycol) derivatives. We have found gelatines to be preferred stabilizing agents. Preferred gelatines are as described in the section on amphiphilic polymers.

The third stream comprises a stabilising agent and preferably an anti- solvent for the hydrophobic compound and hydrophobic polymer. Examples of anti-solvents and stabilising agents are mentioned above, especially water and aqueous media. The anti-solvent in the third stream may be different from the anti-solvent used in the second stream, although preferably they are the same. The stabilising agent in the third stream may be the same as the amphiphilic polymer used in the first stream, although preferably they are different. For example the amphiphilic polymer in the first stream is preferably an amphiphilic block copolymer and the stabilising agent in the third stream is preferably other than a block copolymer, e.g. a gelatine.

The preferred weight ratio of stabilising agent to the combined weight of active hydrophobic compound, hydrophobic polymer and amphiphilic polymer is from 1 :2 to 10:1.

In one embodiment the weight ratio of stabilising agent to the combined weight of active hydrophobic compound, hydrophobic polymer and amphiphilic polymer is from 1 :1 to 6:1 , more preferably 1 :1 to 3:1 and especially from 2:1 to 3:1.

In another embodiment the weight ratio of stabilising agent to the combined weight of active hydrophobic compound, hydrophobic polymer and amphiphilic polymer is from 1 :2 to 3:1 , more preferably from 1 :2 to 2:1 and especially from 2:3 to 3:2.

The third stream can have a lower, a similar or a faster flow rate than the intermixed first and second streams. Preferably however the third stream has a flow rate similar to or somewhat higher than that of the intermixed first and second streams. For example, good results are obtained with a flow rate ratio of third

stream of 0.1 to 10, more preferably 0.5 to 10, especially 0.5 to 5 times, e.g. between 0.9 and 1.1 times the flow rate of the intermixed first and second streams.

One or more of the first, second and third stream may contain a wetting agent if desired. In one embodiment the wetting agent is biocompatible. This is especially useful when the hydrophobic compound is a pharmaceutically active compound. Preferred wetting agents include sodium dodecylsulphate, Tween 80, Cremophor A25, Cremophor EL, Pluronic F68, Pluronic L62, Pluronic F88, Span 20, Tween 20, Cetomacrogol 1000, Sodium Lauryl Sulphate, Pluronic F127, Brij 78, Klucel, Plasdone K90, Methocel E5, PEG, Triton X100, Witconol-14F and Enthos D70-30C.

Biocompatible wetting agents include polyethoxylated castor oils, for example Cremophor EL.

The intermixing may be achieved by a number of means, for example turbulent flow, sonication and/or mixing using a mechanical stirring means. Preferably the intermixing comprises the use of a mechanical stirring means, for example as described in more detail below.

The resultant nanoparticles may be collected in a continuous or batch wise manner.

If desired the process may also include the step of drying the nanoparticles, for example using a spray drier and/or freeze drying. Drying is often useful to provide good storage stability and typically entails removal of solvents and anti- solvents.

During freeze drying, the nanoparticles together with any solvent and anti- solvent are cooled and subjected to a reduced pressure. As a result the solvent and anti-solvent are removed from the nanoparticles by evaporation under very mild conditions which do not adversely effect hydrophobic compound.

Still further the process optionally further comprises the step of re- dispersing the dried nanoparticles in a liquid medium.

The process of the present invention may be performed on any scale and steps (a) to (d) may be performed in a continuous manner. In this way large quantities of the desired nanoparticles may be prepared, including on an industrial scale. There is no need to include jets in the process which have to be carefully aligned. The conditions may be tailored to give small particles which can be isolated and redispersed without difficulty.

The first and second streams may be continuously fed into a precipitation chamber where they rapidly intermix to form the nanoparticles and then exit the precipitation chamber. In one embodiment the first and second streams are fed into the precipitation chamber through separate inlet ports.

In one embodiment, step (c) is performed in a precipitation chamber and step (d) is performed in a stabilisation chamber. In another embodiment, step (c) and step (d) are performed in different parts of the same chamber.

Each mixing chamber (i.e. collectively the precipitation and stabilisation chamber) may have one or more than one outlet. Additionally, in one embodiment, there are no other openings in the mixing chamber or stabilisation chamber besides the inlet(s) and the outlet(s). This means that no solvents, liquids, solutions, particles and the like can enter or exit the mixing chamber or stabilisation chamber except via the specific inlet(s) and the outlet(s). Such chambers are often referred to as "closed type" mixing chambers because they are not open to the air, e.g. in contrast to a beaker which would be an "open type" mixing vessel because it is open to the air.

In another preferred embodiment, the precipitation chamber is a closed type mixing chamber. Preferably step (d) is also performed in a closed type mixing chamber which is the same chamber or a different chamber from that used in step

(C).

In a particularly preferred embodiment of the process: i. the first stream comprises an hydrophobic compound, a hydrophobic polymer, a water-miscible organic solvent for the hydrophobic compound and hydrophobic polymer and an amphiphilic polymer; ii. the second stream comprises water and optionally citric acid; iii. the third stream comprises water and a stabilising agent which is not identical to the amphiphilic polymer used in the first stream.

After the process has begun, a steady state may be reached in which the first and second streams are continuously fed into a precipitation chamber where they rapidly intermix to form the nanoparticles and then exit the precipitation chamber and are fed continuously into a stabilisation chamber where they are intermixed with the third stream comprising the stabilising agent.

The process is preferably a continuous process.

Preferably, the residence time in each of the precipitation and stabilisation chambers is more than 0.0001 second and less than 5 seconds, preferably more than 0.001 second and less than 3 seconds, more preferably between 0.05 and 1 second. The residence time of the streams in the precipitation and stabilisation chambers can be varied by, amongst other things, changing e.g. the inflow speeds of the streams, the chamber internal volume or the choice of the type, e.g. shape and size, of the mechanical stirring means.

The intensity of intermixing and the positions of the inlets and outlets determine the chances of stirring means being bypassed, causing unmixed fluids to leave the chamber before becoming thoroughly intermixed. A too short

residence time of mixed streams in the mixing chambers is undesirable as it may result in uncontrolled nucleation outside the mixing chamber. A too long residence time before coming into contact with the third stream is also undesirable as it may result in excessive agglomeration and growth. The optimum residence time will vary from one hydrophobic compound to another and may be optimised by simple trial and error. Solvent and anti-solvent, together with, for example, the temperature, can be selected to control the precipitation rate. The nucleation time can for example range from 10 ~7 to 10 ~2 seconds. Also for compounds not having such a fast nucleation time, the residence times in the precipitation chamber should not be too long, because the efficiency of the precipitation process will be lowered. Furthermore, a long residence time may result in a wide average particle size distribution and larger particles. In practice, the residence time in each of the precipitation and stabilisation chambers (or areas of a combined precipitation and stabilisation chamber) is preferably from 0.1 to 3 seconds. In cases where nucleation proceeds only slowly, e.g. from 10 ~2 until 10 ~3 seconds, the conditions are preferably chosen such that the residence time is from 0.1 to 5 seconds, more preferably below 3 seconds and even more preferably below 1 second.

The residence time may be calculated as described in International patent application WO2008/035028, page 23, line 23 to line 30.

As mentioned above, the intermixing of the first and second streams may be performed in various manners. The preferred method for intermixing comprises mixing using a mechanical stirring means, which can be driven in any way, for example by a drive shaft or by a rotating magnet.

Preferably the mechanical stirring means is rotatable within a mixing chamber, for example it may comprise a rotatable blade. When a "mixing chamber" is referred to here this is used generically to encompass the precipitation chamber and a stabilisation chamber, unless the context implies otherwise. The mechanical stirring means may be in any form and have any aspect ratio, for example it may be in the form of a paddle where the ratio of its height to width are similar, or it may be in the form of disc, e.g. its height is very much smaller than its width. By width we mean twice the diametric distance from the central axis of rotation of the paddle to its outermost edge. It is preferred that the volume of the mechanical stirring means is at least 10% and not more than 99%, more preferably at least 15% and not more than 95%, even more preferably at least 25% and not more than 95%, e.g. about 50%, of the volume of the relevant mixing chamber. In this way it is easier to ensure the first and second streams rapidly intermix before coming into contact with the third stream. These same preferences apply to the stabilisation chamber.

The mechanical stirring means preferably comprises a shaft and stirrer blade which may be rotated by the shaft. Preferably the diameter of the mechanical stirring means is at least 50%, more preferably at least 70% and most preferably from 70 to 99% of the smallest diameter of the relevant mixing chamber. Very good results were obtained with a mechanical stirring means which had a diameter of around 90% to 95% of the smallest diameter of the mixing chamber. In another embodiment, very good results were obtained with a mechanical stirring means which had a diameter of 80% to 90% of the smallest diameter of the mixing chamber.

In the present invention, when opposite mechanical stirring means are driven in a mixing chamber (i.e. the shafts rotate in opposite directions), it is preferable to rotate the stirring means at high speed to obtain rapid intermixing. The rotation speed is preferably 1 ,000rpm or more, more preferably 3,000 or more, and especially 5,000rpm or more. A pair of conversely rotating stirring means may be rotated at the same rotating speed or at different rotating speeds. In case of a mechanical stirring means which is symmetrical around an axis, the stirrer speed is preferably at least 500rpm, for example at least 1 ,000rpm or at least 5,000 or even more than 10,000rpm. Nowadays, mechanical stirrers are commercially available having a stirrer speed of 20,000rpm and higher. In general, the higher the stirring speed the better the rapid intermixing and therefore there is no particular upper limit for the stirring speed. At very high stirring speeds there is a risk of suspension or solution overheating due to the mixing shear forces and this might cause thermal damage to the precipitated particles or the fluid medium. Such negative effects would set the upper limit of stirring speed for a particular compound or chemical composition unless cooling is applied.

To assist with the intermixing in step (c) and step (d) it is preferred that the precipitate of the nanoparticles are discharged from the mixing chamber(s) through an outlet which is towards the opposite end of the mixing chamber from the inlets and not directly in line with the inlets. For example, the inlets may be positioned at the bottom part of the mixing chamber and the outlet(s) may be positioned at the top part of the mixing chamber. In one embodiment the inlets are below the middle line of the mixing chamber (e.g. below 30% height or 20% height). The outlet(s) may be above 70% height. In another embodiment, the outlet(s) is or are approximately at a right angle (e.g. 80 to 100 ° angle, especially 90 ° angle) relative to the flow of streams through the inlets. In this way the streams entering through the inlets do not immediately exit through the outlet without proper intermixing.

In one embodiment the mixing chamber(s) have more than one outlet.

The nanoparticles arising from step (d) are preferably discharged into a collecting vessel. The collecting vessel may comprise a second liquid phase comprising one or more of stabilisation agents, wetting agents, non-solvents, solvents or mixtures thereof.

In another embodiment, ripening of the nanoparticles is performed in a collecting vessel until the preferred median particle size and/or particle size distribution is achieved. This modification or ripening can be achieved by stirring the product of step (d) in a collecting vessel. During modification or ripening, the median particle size may increase, but the particle size distribution usually becomes narrower which is sometimes advantageous. Modification or ripening can be controlled by various parameters, e.g. temperature, pH or ionic strength. Consequently, according to this preferred embodiment, the process according to the present invention comprises a further step (e), wherein the product of step (d) is fed into a collecting vessel and subjected to a ripening step.

During an induction period of the process according to the present invention, the second stream comprising the anti-solvent may be introduced with a continuous flow into a precipitation chamber and may travel from there to a stabilisation chamber via fluid communication means (e.g. a pipe) and thereafter to a collecting vessel. Subsequently, the first stream may be introduced with a continuous flow into the precipitation chamber where it is intermixed with the second stream which results in a super saturation of the hydrophobic compound and hydrophobic polymer thereby initiating the formation of a precipitate and a liquid phase. The term "super saturation" refers to a concentration of an hydrophobic compound and hydrophobic polymer that is in excess of saturation under the given conditions, i.e. solvent or solvent mixture, temperature, pH, ionic strength etc. In the liquid phase, the super saturation may be reduced to such a level that essentially no precipitation will occur outside the precipitation chamber. Then the precipitated nanoparticles may be transferred to the stabilisation chamber if desired where they are intermixed with the third stream. In this embodiment the initial output of the stabilisation chamber may be discarded because it may not contain any precipitate until the steady state has been reached and all streams are flowing. Since in this embodiment the streams are fed continuously, a continuous outflow of the nanoparticles and the liquid phase is eventually achieved. After the induction period, a steady state is reached in the mixing chamber(s) meaning that basically the composition of the mixture within each mixing chamber is stable and essentially does not change over time. Additionally, the composition of the outflow of the mixing chamber(s) is stable and essentially does not change over time either.

The velocities of the inflow of the various streams do not need to be identical. If multiple inlets are used, the velocity of one stream may differ from the velocity of another stream. However, in general the feed velocity of the streams may be, for example, 0.01 m/s, 0.1 m/s or 1 m/s. Even velocities of 10m/s or more than 50m/s can be used. The ratio of feed velocities of first stream to second stream can be 1 :99 to 99:1. The ratio of feed velocities of third stream to the intermixed first and second stream can also be 1 :99 to 99:1. During the induction period, the outflow from the chamber(s) is collected until the composition of the outflow is essentially constant. As soon as a steady state is reached, the nanoparticles and the liquid phase may be collected, for example in a collecting vessel.

Apparatus suitable for producing the nanoparticles of the present invention are described in WO2009/013466, page 14-21 , the content of which is hereby expressly incorporated by reference.

Figure 1 shows an example of an apparatus which may be used to perform the process of the present invention.

Key to the symbols used in the drawing:

1 P-a, 1 P-b, 1S-a, 1S-b: Mechanical stirring means

2P-a, 2P-b, 2S-a, 2S-b: Axis or shaft

3P, 3S: Mixing chamber

4P: First inlet to the precipitation chamber for the first stream

5P: Second inlet to the precipitation chamber for the second stream

5S: Second inlet to the stabilisation chamber for the third stream

6P: Outlet of the precipitation chamber

6S: Outlet of the stabilisation chamber

7P, 7S: Mixing or precipitation chamber wall

8P, 8S: Seal plate

9P-a, 9P-b, 9S-a, 9S-b: Outer magnets

10: Fluid communication means

In a typical process according to the present invention, the first stream is provided which may be fed with a continuous flow via a first inlet into the precipitation chamber. Simultaneously, the second stream may be fed, also with a continuous flow, via a second inlet into the precipitation chamber. The precipitation chamber may be provided with more than one first inlet for this first stream and more than one second inlet for this second stream. In a next step, the first stream and the second stream are intermixed and said mixture provides a super saturation and the nanoparticles precipitate. The mixture of the nanoparticles and the liquid phase is discharged from the precipitation chamber to

a stabilisation chamber, preferably also with a continuous flow. A third stream which contains a stabilising agent is also fed into the stabilisation chamber where it mixes with the output of the precipitation chamber. The contents of the stabilisation chamber exit through its outlet, preferably into a collecting (or receiving) vessel.

The size of the mixing chamber(s) is dependent on the scale at which the process is performed. On a small scale one typically would use mixing chamber(s) of volume 0.15 to 100cm 3 , for medium scale mixing chamber(s) of 101 to 250cm 3 and for large scale mixing chamber(s) of more than 250cm 3 may be used. Preferably, the size of the mixing chamber(s) is 1 cm 3 to 1 litre. As will be understood, the volume of the mixing chamber(s) is volume without the mechanical stirring means being present. In a preferred embodiment the mixing chamber(s) are closed type mixing chamber(s).

Preferably at least one stirrer blade is positioned between mixing chamber inlets such that it acts as a physical barrier between the incoming streams. In this way the stirrer blade reduces the chance of nanoparticle formation at the inlets which could otherwise block these inlets. Instead the streams come into contact in a circumferential instead of 'head-on' manner.

An apparatus which may be used to perform the process of the present invention is shown schematically in Figure 1. This apparatus comprises a precipitation chamber and a stabilisation chamber, as shown on the left and right respectively in Figure 1 , with the outlet of the precipitation chamber connected to the inlet of the stabilisation chamber by a fluid communication means. This apparatus is essentially two of the apparatus disclosed in US Patent No. 5,985,535, the disclosure in which is expressly incorporated by reference herein, connected by a pipe or hose. If desired the stabilisation chamber and the fluid communication means may be omitted.

The apparatus is preferably provided with or may be connected to a collecting vessel. The collecting vessel preferably comprises a stirring means. Optionally, one or more of the mixing chambers may be surrounded by the collecting vessel. Alternatively, the mixing chambers may be positioned adjacent to or remote from the collecting vessel, depending on user preference. The apparatus and/or the collecting vessel can be provided with a means to control temperature in e.g. the mixing chambers and/or the collecting vessel, respectively. Such control means can for example be used to control the temperature of the streams.

The apparatus may comprise supply tanks (not shown) comprising the fluids which are used to make the streams. The supply tanks may be connected to the relevant chamber by feed lines which can be, for example, hoses or fixed

pipes. The transportation of the liquids to the mixing chamber can be done with a continuous flow provided by a pump. The shape of the chambers can in principle be chosen freely. Preferably the chambers are rotationally symmetric around a central axis. The chambers can be of the same shape or different shapes. The chambers can be of the same size or different sizes.

In a preferred embodiment the process according to the invention comprises the following steps: i) the first stream passes through a first inlet to the precipitation chamber; ii) the second stream passes through a second inlet to the precipitation chamber; iii) the first and second streams are rapidly intermixed using a mechanical stirring means occupying 70% to 90% of the smallest diameter of the precipitation chamber; iv) the nanoparticles exit the precipitation chamber through the outlet; v) the outlet of the precipitation chamber releases the nanoparticles via a fluid communication means to a stabilisation chamber comprising a mechanical stirring means, a first inlet, a second inlet and an outlet, wherein: i) the first inlet of the stabilisation chamber receives the nanoparticles from the precipitation chamber via the fluid communication means; ii) the second inlet of the stabilisation chamber receives a stream comprising a stabilising agent for the nanoparticles; iii) the mechanical stirring means intermixes the nanoparticles and the stabilising agent to stabilise the nanoparticles; and iv) the nanoparticles exit the stabilisation chamber through the outlet. Preferably, all parts of the chambers that are in contact with one or more of the streams are coated with a layer of a material that prevents adhering, fouling, incrustation and the like.

The process according to the present invention is very suitable for preparation of active pharmaceutical compounds in nanoparticulate form with a narrow particle size distribution. Small pharmaceutical particles are very suitable to be used in a medicament.

In another aspect the present invention also provides a process for the manufacture of a medicament comprising performing the process of the present invention wherein the hydrophobic compound is a pharmaceutically active compound.

Preferably this process further comprises the step of mixing the product of the process, i.e. the nanoparticles of the invention, with a pharmaceutically acceptable carrier or excipient to give the medicament.

The identity of the carrier or excipient is not crucial provided it is pharmaceutically acceptable. Examples of such carriers and excipients include the diluents, additives, fillers, lubricants and binders commonly used in the pharmaceutical industry.

In a preferred aspect the medicament is in the form of a tablet, troche, powder, syrup, patch, liposome, injectable dispersion, suspension, capsule, cream, ointment or aerosol.

Thus, medicaments intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents in addition to the product of the presently claimed process (the product of the presently claimed process often being abbreviated herein as simply as "the active ingredient").

If desired the process may further comprise the step of sterilising the resultant nanoparticles. The object of the sterilisation is to kill any undesirable bacteria which may cause harm to a patient, particularly if their immune system has been compromised. Typical sterilisation methods include irradiation, filtration through a 0.22 micron sterile filter, heating and treatment with a biocide.

The pharmaceutically active compound referred to in the above further aspects of the present invention may be any of the pharmaceutically active hydrophobic compounds mentioned earlier in this specification or others, especially paclitaxel, fenofibrate or a cyclosporin (e.g. cyclosporin A).

Also the invention provides a medicament obtained by the process of the present invention.

Also the invention provides a method for the treatment of a human or animal comprising administration of a medicament obtained by the process of the present invention. Also the invention provides use of a pharmaceutically active hydrophobic compound obtained by the process of the present invention for the manufacture of a medicament for the treatment of cancer.

The invention is now illustrated by the following non-limiting examples in which all parts and percentages are by weight unless otherwise specified.

In the following Examples the following chemicals were used:

The chemicals were obtained from Sigma-Aldrich Co., Zwijndrecht, The Netherlands unless specified otherwise:

Paclitaxel from taxus brevifolia, >95% by HPLC, poly(ethylene glycol) Mn 2000 methyl ether-block-polylactide Mn 2000, poly(ethylene glycol) Mn 2000 methyl ether-block-polylactide Mn 3000, poly(D,L-lactide), mol wt 75,000-120,000

Tetrahydrofuran (THF), biotech grade >99.9%, inhibitor-free,

Citric acid, USP grade,

The water used was purified by demineralization and filtration techniques on-site.

In all of the Examples of the invention the measured particle size parameters D50, D90 and D[4,3] were stable for at least 15 minutes unless noted otherwise. D[4,3] means the weight averaged size, D50 means the median size and D90 means the size which 90% of the particles are below. D50, D90 and D[4,3] were measured using a Malvern Mastersizer 2000 particle size analyser. During measurement cycles the internal stirrer was set to the maximum speed of 3500 rpm to avoid particle aggregation.

Freshly prepared product batches were freeze-dhed by allowing the suspensions to cool down under gentle stirring to just above freezing point. The suspensions were then slowly added dropwise into liquid nitrogen to shock-freeze them. The resultant frozen (near)-spherical particles (ca. 3 to 5mm diameter) were then transferred into a freeze-drying chamber and dried under a vacuum.

Drug release rates were determined in a simplified blood plasma medium at 37°C. The medium consisted of 40g/l bovine serum albumin, pH-buffered at 7.4 using a phosphate buffer at a temperature of 37°C. A 20nm pore size AI2O3 Anotop™ syringe filter is used to separate the medium from any particles when sampling the medium to check the amount of released drug. The following procedure was used. A dose of dried product amount corresponding to 30mg/l paclitaxel was added to a 40g/l bovine albumin solution buffered to pH 7.4 at 37.0 0 C with gentle stirring. The dosage level was fixed for reference purposes to somewhat above the maximum detected paclitaxel plasma concentration in a standard Abraxane ® 30 minute 260mg/m 2 infusion in humans. Nanoparticles were separated from the medium after sampling using the Anotop syringe filter, followed by adding an equimass amount of 100% ethanol to inhibit any further drug precipitation. Detection was done by UPLC chromatography using UV-detection.

Example 1

(A) First Stream - Preparation of First Stock Solution

A first stock solution was prepared comprising an amphiphilic diblock copolymer (poly(ethylene glycol) Mn 2000 methyl ether-block-polylactide Mn 2000, 60.0g/l), a hydrophobic polymer (poly(DL-lactide) molecular weight 75,000 to 120,000, from Sigma-Aldrich,15g/I) and the hydrophobic compound paclitaxel (45.0g/l) in tetrahydrofuran solvent. The temperature of the solution was adjusted to 293K.

(B) Second Stream - Preparation of Anti-solvent

A second stock solution, of anti-solvent, was prepared by dissolving citric acid in purified water (5g/l). The temperature of this anti-solvent was adjusted to 273K.

(C) Third Stream - Preparation of a Stabilising Agent Solution

A third stock solution of a stabilising agent was prepared by dissolving deep-sea fish gelatine (non-hydrolysed high molecular weight (~150kD) from Norland Products Inc.) in water (45.8g/l). The solution temperature was adjusted to 293K.

(D) Apparatus

An apparatus for performing the claimed process was assembled by connecting two magnetically stirred chambers by a hose, each constructed as described in US Patent No. 5,985,535, Figure 1. This construction is illustrated in International Patent application WO2009/013466. The first chamber was a precipitation chamber and the second chamber was a stabilisation chamber, with the outlet of the first chamber connected to an inlet of the second chamber using a pipe as fluid communication means. Each chamber was of the closed type, cylindrical, and had an internal volume of 1.5cm 3 (prior to incorporation of the mechanical stirring means), two spaced inlets, a pair of magnetically driven stirrer blades as mechanical stirring means and one outlet. The mechanical stirring means took the form of stirrer blades having diameters of 83% of the chamber diameters. The volume of the magnetically driven stirrer blades was about 55% of the volume of the (empty) mixing chamber. The average residence time in the precipitation chamber was about 0.35 seconds.

(E) The Process

The assembled apparatus described in (D) above was filled with liquid by pumping a stream of the second stock solution (anti-solvent) through it at a rate of 100 cm 3 /min. The stirrer blades in the precipitation and stabilisation chambers were operated at 6,000 RPM in opposite directions.

When the apparatus was full, the first stream (containing the hydrophobic compound and the hydrophobic polymer) was pumped into the precipitation chamber at a rate of 20cm 3 /min and the third stream (containing the second stabilising agent) was pumped into the stabilisation chamber at a rate of 120cm 3 /min. The temperature in the precipitation chamber was 1 0 C.

The initial output of the apparatus was discarded until its composition became constant, after which the three streams were continuously pumped into the apparatus and the output from the stabilisation chamber was collected in a batch wise manner and worked-up by freeze-drying to give a dried product. At the end of the precipitation process the apparatus was flushed through with solvent and the washings retained for recycling.

The weight ratio of active hydrophobic compound (paclitaxel) to hydrophobic polymer is thus 3 to 1. The weight ratio of active hydrophobic compound (paclitaxel) to the combined weight of hydrophobic polymer and amphiphilic polymer was 0.6:1.

(F) Results - Particle Size Distribution

The resultant nanoparticles comprising paclitaxel, the hydrophobic polymer and the amphiphilic polymer were found to have an almost unimodal particle size distribution, a D[4,3] of 321 nm, a D50 of 126nm and a D90 of 233nm.

The average hydrodynamic particle size as measured by a Coulter ® N4 Plus Submicron Particle Sizer was 143nm.

(G) Results - Particle shape

The physical shape of the nanoparticles was inspected using a scanning electron microscope (SEM) and they were found to have a spherical shape.

(H) Results - Redispersibility

After freeze drying the nanoparticles were redispersed in water at room temperature and subjected to 120 seconds of ultrasound treatment. The resulting suspension was inspected visually and by means of a size measurement using the Coulter ® N4 Plus Submicron Particle Sizer. The result showed that the nanoparticles had readily redispersed in water to an average hydrodynamic particle size of 134nm.

(I) Results - Particle stability in aqueous solution at 37°C

The freeze dried nanoparticles obtained from step (E) remained intact in water at 37 ° C for at least 1 hour at a dose of 30mg paclitaxel per litre. In addition to the nanoparticles numerous paclitaxel crystals were observed by Scanning Electron Microscope, indicating still a certain quantity of drug had released from the nanoparticles.

Example 2

Example 1 was repeated except that the weight ratio of active hydrophobic compound (paclitaxel) to hydrophobic polymer was decreased by a factor of three.

The weight ratio of active hydrophobic compound (paclitaxel) to hydrophobic polymer is thus 1 to 1. The weight ratio of active hydrophobic compound (paclitaxel) to the combined weight of hydrophobic polymer and amphiphilic polymer was 0.33 to 1.

The concentration of components in water in the first stream was as follows: active hydrophobic compound (paclitaxel), 15g/l; poly(DL-lactide), 15g/l; polyethylene glycol) Mn 2000 methyl ether-block-polylactide Mn 2000, 30g/l.

The temperature of the first stream was adjusted to 293K. The second stream consisted of citric acid in water at a concentration of 2.5g/l at 273K. The third stream consisted of gelatine in water at a concentration of 22.9g/l at 293K.

(F) Results - Particle Size Distribution

The resultant nanoparticles were found to have an almost unimodal particle size distribution, a D[4,3] of 198nm, a D50 of 124nm and a D90 of 242nm. The average hydrodynamic particle size as measured by a Coulter ® N4 Plus Submicron Particle Sizer was 213nm.

(G) Results - Particle shape

The shape of the nanoparticles was inspected using a scanning electron microscope and they were found to be spherical.

(H) Results - Redispersibility

After freeze drying the nanoparticles were redispersed in water at room temperature and subjected to ultrasound for 120 seconds. The resulting suspension was inspected visually and by means of a size measurement using the Coulter ® N4 Plus Submicron Particle Sizer. The inspection showed that the nanoparticles had readily redispersed in water to an average hydrodynamic particle size of 295nm.

(I) Results - Particle stability in aqueous solution at 37°C

The freeze dried nanoparticles obtained from step (E) remained intact in water at 37 ° C for at least 1 hour at a dose of 30mg paclitaxel per litre. In addition to the nanoparticles some paclitaxel crystals were observed by Scanning Electron Microscope, indicating that some drug had released from the nanoparticles.

Example 3

(A) First Stream - Preparation of First Stock Solution

A first stock solution was prepared comprising an amphiphilic diblock copolymer (poly(ethylene glycol) Mn 2000 methyl ether-block-polylactide Mn 2000, 30.0g/l), a hydrophobic polymer (poly(DL-lactide) molecular weight 75,000 to 120,000, from Sigma-Aldrich,15g/I) and the hydrophobic compound paclitaxel (5.0g/l) in tetrahydrofuran solvent. The solution temperature was adjusted to 293K.

(B) Second Stream - Preparation of Anti-solvent

A second stock solution, of anti-solvent, was prepared by dissolving citric acid in purified water (2.5g/l). The temperature of this anti-solvent was adjusted to 273K.

(C) Third Stream - Preparation of a Stabilising Agent Solution

A third stock solution of a stabilising agent was prepared by dissolving deep-sea fish gelatine (15OkD molecular weight, Norland) in water (22.9g/l). The solution temperature was adjusted to 293

(E) The Process

The weight ratio of active hydrophobic compound (paclitaxel) to hydrophobic polymer is thus 1 to 3. The weight ratio of active hydrophobic compound (paclitaxel) to the combined weight of hydrophobic polymer and amphiphilic polymer was 1 :9.

(F) Results - Particle Size Distribution

The resultant nanoparticles comprising paclitaxel, the hydrophobic polymer and the amphiphilic polymer were found to have an almost unimodal particle size distribution, a D[4,3] of 200nm, a D50 of 126nm and a D90 of 254nm.

The average hydrodynamic particle size as measured by a Coulter ® N4 Plus Submicron Particle Sizer was 179nm.

(G) Results - Particle shape

The physical shape of the nanoparticles was inspected using an scanning electron microscope and they were found to be spherical.

(H) Results - Redispersibility

After freeze drying the nanoparticles were redispersed in water at room temperature and subjected to 120 seconds of ultrasound treatment. The resulting

suspension was inspected visually and by means of a size measurement using the Coulter ® N4 Plus Submicron Particle Sizer. The result showed that the nanoparticles had readily redispersed in water to an average hydrodynamic particle size of 235nm.

(I) Results - Particle stability in aqueous solution at 37°C

The freeze dried nanoparticles obtained from step (E) remained intact in water at 37 ° C for at least 1 hour at a dose of 30mg paclitaxel per litre. Only very few drug crystals were observed by Scanning Electron Microscope.

Example 4

(A) First Stream - Preparation of First Stock Solution

A first stock solution was prepared comprising an amphiphilic diblock copolymer (poly(ethylene glycol) Mn 2000 methyl ether-block-polylactide Mn 3000, 30.0g/l), a hydrophobic polymer (poly(DL-lactide) molecular weight 75,000 to 120,000, from Sigma-Aldrich,15g/I) and the hydrophobic compound paclitaxel (0.789g/l) in tetrahydrofuran solvent. The solution temperature was adjusted to 293K.

(B) Second Stream - Preparation of Anti-solvent

A second stock solution, of anti-solvent, was prepared by dissolving citric acid in purified water (2.5g/l). The temperature of this anti-solvent was adjusted to 273K.

(C) Third Stream - Preparation of a Stabilising Agent Solution

A third stock solution of a stabilising agent was prepared by dissolving deep-sea fish gelatine (15OkD molecular weight, Norland) in water (22.9g/l). The solution temperature was adjusted to 293K.

(E) The Process

The weight ratio of active hydrophobic compound (paclitaxel) to hydrophobic polymer is thus 1 to 19. The weight ratio of active hydrophobic compound (paclitaxel) to the combined weight of hydrophobic polymer and amphiphilic polymer was 1 :57.

(F) Results - Particle Size Distribution

The resultant nanoparticles comprising paclitaxel, the hydrophobic polymer and the amphiphilic polymer were found to have an almost unimodal particle size distribution, a D[4,3] of 146nm, a D50 of 128nm and a D90 of 220nm.

The average hydrodynamic particle size as measured by a Coulter N4 Plus Submicron Particle Sizer was 175nm.

(G) Results - Particle shape

The physical shape of the nanoparticles was inspected using an scanning electron microscope and they were found to be spherical.

(H) Results - Redispersibilitv

After freeze drying the nanoparticles were redispersed in water at room temperature and subjected to 120 seconds of ultrasound treatment. The resulting suspension was inspected visually and by means of a size measurement using the Coulter ® N4 Plus Submicron Particle Sizer. The result showed that the nanoparticles had readily redispersed in water to an average hydrodynamic particle size of 245nm.

(I) Results - Particle stability in aqueous solution at 37°C

The freeze dried nanoparticles obtained from step (E) remained intact in water at 37 ° C for at least 1 hour at a dose of 30mg paclitaxel per litre. No drug crystals were observed by Scanning Electron Microscope.

Comparative Example 1 - Hydrophobic Polymer Omitted

Example 1 was repeated except that the hydrophobic polymer was replaced by the same mass of paclitaxel. Therefore the concentration paclitaxel was 60g/l instead of 45g/l.

(F) Results - Particle Size Distribution

The nanoparticles were found to have a D[4,3] of 284nm, a D50 of 129nm and a D90 of 244nm. The average hydrodynamic particle size as measured by a Coulter ® N4 Plus Submicron Particle Sizer was 170nm.

(G) Results - Particle shape

The physical shape of the nanoparticles was inspected using a scanning electron microscope and they were found to have a spherical shape.

(H) Results - Redispersibilitv

After freeze drying the nanoparticles were dispersed in water at room temperature and subjected to 30 seconds ultrasound. The resulting suspension was inspected visually and by means of a size measurement using the Coulter ®

N4 Plus Submicron Particle Sizer. The result showed that the nanoparticles readily redispersed in water to an average size of 185nm.

(I) Results - Particle stability in aqueous solution at 37°C

The freeze dried nanoparticles were not stable in water at 37 ° C at a dose of 30mg paclitaxel per litre. After 1 hour they had completely recrystallised into small rods as observed by scanning electron microscope.

Comparative Example 2: Commercially available Abraxane ® (F) Results - Particle Size Distribution

The mean particle size (130nm) was taken from literature as given by Sparreboom et al (Clinical Cancer Research Vol. 11 , 4136-4143, June 1 , 2005).

(I) Results - Particle stability in aqueous solution at 37°C

The percentage of drug released from commercially available Abraxane ® , which comprises paclitaxel particles and albumin in a ratio of 1 :9, was determined according the method described above. The particles were not stable in time, many crystals were observed by scanning electron microscope a few minutes after mixing the particles in water at 37°C.

Table 1 : Percentage of paclitaxel released from the particles as determined by UPLC.

o

C1 is Comparative Example 1. C2 is Comparative Example 2.

The results in Table 1 indicate a strong downward trend in release rate upon lowering the ratio paclitaxel/hydrophobic polymer to values lower than 0.33 (or increasing the ratio of hydrophobic polymer/pad itaxel to values higher than 3).




 
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