WO2003043605A1

WO2003043605A1 - Method for preparing microparticles free of toxic solvent, resulting microparticles and pharmaceutical compositions

Info

Publication number: WO2003043605A1
Application number: PCT/FR2002/003817
Authority: WO
Inventors: Anne Aubert-Pouessel; Marie-Claire Venier-Julienne; Jean-Pierre Benoit
Original assignee: Institut National De La Sante Et De La Recherche Medicale
Priority date: 2001-11-21
Filing date: 2002-11-07
Publication date: 2003-05-30
Also published as: FR2832312A1; FR2832312B1; AU2002350884A1

Abstract

The invention concerns a method for preparing biodegradable microparticles comprising at least an active principle, the microparticles obtained by using said method and their use and pharmaceutical compositions containing them.

Description

PROCESS FOR THE PREPARATION OF SOLVENT-FREE MICROPARTICLES

TOXIC, MICROPARTICLES OBTAINED ACCORDING TO THIS PROCESS,

PHARMACEUTICAL USES AND COMPOSITIONS

The present invention relates to a process for the preparation of biodegradable microparticles comprising at least one active principle, to the microparticles obtained by implementing this process, to their use and to the pharmaceutical compositions comprising them.

At present, microparticles, and in particular microspheres and microcapsules for therapeutic purposes, that is to say microparticles comprising an active principle and intended in particular to be administered by injection, can be prepared by different types of preparation processes.

Thus, the processes for preparing the prior art of such microparticles can be schematically divided into three main categories called physico-chemical processes, mechanical processes and chemical processes. The physico-chemical preparation processes are based on phase separation techniques, solvent evaporation and extraction, simple coacervation and thermal gelation.

The mechanical preparation processes are based on nebulization / drying techniques, coating in a fluidized air bed, gelation and drop freezing ("prilling") and finally extrusion-spheronization.

The chemical preparation processes are based on interfacial polycondensation techniques.

Microencapsulation processes using a solvent evaporation step are based on the evaporation of the organic phase of an emulsion with stirring. Initially, the coating material, generally a hydropliobic polymer, is dissolved in a volatile organic solvent. The active principle to be encapsulated is then either dissolved or dispersed in the organic solution of the polymer. During the next step, the organic phase is emulsified with stirring in a non-solvent dispersing phase of the polymer, generally water, containing a surfactant such as a polyvinyl alcohol. Once the emulsion is formed, the organic solvent gradually diffuses into the continuous phase of the emulsion, with stirring, to evaporate, leaving the polymer to precipitate in the form microparticles.

These processes for preparing microspheres by evaporation of so-called "conventional" solvents use organic solvents, most often toxic, and consequently lead to microspheres containing residual contents of solvents.

The solvents used according to these methods are most often of halogenated nature, such as for example dichloromethane and chloroform. These so-called class 2 solvents are classified as "avoid" according to the "International Conference of Harmonization" (ICH)). However, when they are intended to be administered by injection, the microspheres or microparticles must meet very strict criteria from the point of view of their toxicity.

However, the various preparation methods currently used and which use such toxic solvents or surfactants are very effective in terms of encapsulation rate but they do not make it possible to meet these toxicological requirements, insofar as the microspheres obtained by implementing such methods contain significant residual contents of organic solvents requiring a long desorption step and without then guaranteeing compatibility of the microspheres with use by injection. Even if the risk-benefit ratio is taken into account when it comes to curative microspheres, potential toxicity cannot be tolerated when it is a question, for example, of microspheres intended for vaccination where the risk predominates over profit. This residual toxicity in the microspheres remains a major defect in the various techniques for encapsulating known active ingredients. ^' Thus, various attempts at improvement have been made to overcome the use of halogenated solvents.

In the particular case of the manufacture of polymeric microspheres prepared from polymers of the poly lactic-co-glycolic acid (PLGA) type, which are biocompatible polymers approved by the Food and Drug Administration (FDA), it has for example been proposed to replace halogenated solvents with different types of solvents such as: ethyl acetate (French patent application No. 2,797,784), ethyl formate (SAH H., Journal of Controlled Release, 1997, 47, 233-244), and the acetone / dichloromethane mixture (PEAN JM et al., International Journal of Phaπnaceutics, 1998, 166 (1), 105 -115). These solvents, although less toxic than halogenated solvents, are nevertheless not compatible with administration by the injectable route.

On the other hand, the processes for preparing microspheres used in the prior art, in addition to using toxic solvents, generally include strong stirring and / or sonication steps, making them incompatible with the encapsulation of fragile active ingredients such as, for example, peptides (insulin, luteinizing hormone-releasing hormone analogs: LH-RH), proteins (enzymes, antibodies, etc.) and nucleic acids (DNA, RNA, etc.).

The development of injectable implants has also made it possible to find non-toxic polymer solvents. Thus, the article by ELIAZ Rom E. et ai, J. of Biomed. Mater. Res., 2000, 50, 388-396, describes the preparation of an injectable implant system in which a biocompatible polymer of the PLGA type is dissolved at room temperature in a solvent (glycofurol). The active ingredient is dispersed there using a vortex and an ultrasonic probe. The suspension thus obtained is then injected into a patient, for example by the intramuscular, subcutaneous, intrathecal, epidural, intracerebral, etc. route, thus causing, in vivo, the formation of a solid implant by precipitation of the polymer in contact with the interstitial aqueous medium.

However, this type of technique involves preparing the suspension at the time of use, which necessarily has drawbacks from a practical point of view, in particular in terms of ease of use and intra and interindividual reproducibility.

At the present time, there is therefore no system for delivering active principles in the form of microparticles of variable size, which simultaneously makes it possible to dispense with the use of toxic solvents so that they can be administered without risk to patients. man or animal, easy to prepare, and allowing to encapsulate fragile active ingredients.

It is therefore in order to remedy these major problems that the Applicant has developed what is the subject of the invention.

The present invention therefore has for first object a process for the preparation of solid microparticles containing at least one hydrophilic or hydropliobic active principle, comprising the following steps: a) the preparation of a solution A of at least one biodegradable, biocompatible and insoluble polymer in water, in a biocompatible and water-soluble solvent; b) dissolving or suspending in the solution obtained above in step a), at least one hydrophilic or hydrophobic active principle, to obtain a composition B; c) the production, with or without surfactant, of an emulsion of composition B obtained above in step b) in a vegetable oil which constitutes the continuous hydrophobic phase of the emulsion, characterized in that it then comprises the following stages: d) extraction of the biocompatible solvent by adding at least one hydrophilic biocompatible oil, with gentle stirring, until solid microparticles of said polymer containing said active ingredient are obtained, e) recovery of the solid microparticles obtained in step d). The process for the preparation of solid microparticles in accordance with the invention has the advantages: of not denaturing the active principle or principles which it is desired to encapsulate in the microparticles. In fact, the process only requires two stages of gentle mechanical stirring, preferably at a speed of between 500 and 1000 revolutions per minute. It is therefore particularly suitable for the encapsulation of fragile active principles such as for example peptides, proteins (enzymes, antibodies, growth factors, cytokines, hormone analogs, colony stimulating factors, angiogenesis inhibitors, insulin, etc.) and nucleic acids (DNA, RNA, etc.), it makes it possible to encapsulate both hydrophilic and hydrophobic active principles, it allows, if desired, to work in completely anhydrous conditions thus allowing in particular the encapsulation of proteins and DNA or RNA which are particularly fragile active principles which require to be handled in an anhydrous medium, it avoids all use of toxic solvents which has the double advantage of not bringing the manipulator into contact with toxic products during the preparation and of leading to microparticles free of toxic solvent and therefore compatible with a method of administration by injectable route in humans or animals, the raw materials for implementing the process are inexpensive and the equipment is very simple, which makes it possible to transpose the process according to the invention on a large scale. According to the invention, the term microparticles includes both microspheres and microcapsules. By microsphere is meant any solid microparticle having a globally spherical shape and consisting of a single homogeneous phase (matrix system). The term “microcapsule” means any solid microparticle having a globally spherical shape and consisting of at least two phases, namely a solid external phase (envelope) trapping an internal phase (core) solid, semi-solid or liquid. Each capsule thus constitutes a reservoir system.

According to the invention, the term "biodegradable, biocompatible and water-insoluble polymer" means any polymer or copolymer which degrades in a biological medium, which is compatible from a pharmaceutical point of view (Food and Drug Administration (FDA) ), Standard ISO 10993-13 (1998)) to be injected in humans or animals, and which is not soluble in water.

Among these polymers, mention may in particular be made of polymers soluble in the biocompatible solvent such as, for example, polylactides, polylactide-co-glycolides, polyesters, poly (orthoesters), poly (anhydrides), polyphosphazenes, polyiminocarbonates, polycaprolactones, poly (bis (p-carboxyphenoxy) propane), and their mixtures. In an advantageous embodiment of the invention, said biodegradable polymer is chosen from polylactides (PLA) and polylactide-co-glycolides (PLGA).

These biodegradable polymers preferably have a molar mass of between 17,500 and 125,000.

PLGAs can contain varying proportions of polylactide and glycolide. PLGAs are preferably used in which the polylactide / glycolide ratio varies between 75/25 and 50/50.

The term "biocompatible and water soluble solvent" according to the present invention, any solvent which is compatible from a pharmaceutical point of view (according to the European Pharmacopoeia) for injection into humans or animals, and which is soluble in water.

Mention may in particular be made of glycofurol, ethanol, propylene glycol, ie poly (ethylene glycol) and their mixtures. In an advantageous embodiment according to the invention, the biocompatible and water-soluble solvent is glycofurol or α - [(tetrahydro-2-furanyl) methyl] -w-hydroxypoly (oxy-1,2-ethanediyl ), known under the brand name tetraglycol and sold by the company SIGMA.

The process according to the invention makes it possible to incorporate hydrophilic as well as hydrophobic active ingredients.

When the active principle is of hydrophilic nature, step a) is a dissolution step and when the active principle is of hydrophobic nature, step a) is a suspension step.

The active principles which can be encapsulated according to the process according to the invention are preferably fragile active principles such as peptides such as insulin or analogs of LH-RH, proteins such as enzymes, antibodies, growth factors, cytokines, hormone analogs, angiogenesis inhibitors, colony stimulating factors (GM-CSF: Granulocyte Macrophage-Colony Stimulating Factor), insulin, etc., vitamins, acids nucleic acids (DNA, RNA, etc ...), and in general, any active ingredient requiring immediate or prolonged release in microencapsulated form (cytostatics, etc.). Among the proteins which can be encapsulated according to the process in accordance with the invention, mention may be made most particularly of recombinant proteins whose administration is of therapeutic interest and such as, for example, erythropoietin (useful in the treatment of anemia in renal failure chronic), γ interferons (useful in the treatment of hepatitis B and C), β interferon (useful in the treatment of multiple sclerosis), growth hormones, neurotrophic factors, cytokines (useful in the treatment certain cancers), etc.

Among the vegetable oils which can be used in accordance with the present invention, mention may in particular be made of sunflower oil, corn oil, olive oil sold for example by the Pharmaceutical Cooperative (Melun, France) or else sesame oil sold for example under the name Super Refined oils® by the company Croda (Trappes, France).

The term "biocompatible hydrophilic oil" according to the present invention means any oil which has both a hydrophobic and hydrophilic character and which is injectable from a pharmaceutical point of view.

Among these biocompatible hydrophilic oils, there may be mentioned in particular the polyglycolized glycerides of fatty acids such as, for example, polyethylene glycol-6 glyceryl mono oleate sold under the brand Labrafil® M 1944 CS by the company Gattefossé, polyethylene glycol-6 glyceryl linoleate sold under the brand Labrafil® M 2125 CS, "corn oil PEG-6 esters" sold under the brand Labrafil® M 2735 CS by the company Gattefossé, caprylocapric macrogolglycerides such as, for example, the mixture of mono-, di- and triglycerides and mono- and polyethylene glycol diesters sold under the brand Labrasol®, medium chain triglycerides of caprylic, capric or lauric fatty acids such as "PEG-8 caprylic / capric glycerides" sold under the brand Labrafac® Hydro by the company Gattefossé, and their mixtures.

The use of ρolyéthylèneglyco-6 glyceryl linoleate sold under the brand Labrafil® M 2125 CS is particularly preferred according to the invention. The biocompatible hydrophilic oil or oils may optionally be used in a mixture with an aqueous medium such as water, and in particular water for injection. According to an advantageous embodiment of the process according to the invention, said biodegradable polymer preferably represents from 1 to 4% by weight of the total weight of solution A.

During step c) of forming the emulsion, the amount of composition B preferably represents from 1 to 20% by weight of the weight of the vegetable oil.

According to a preferred embodiment of the invention, step c) of forming the emulsion is carried out at a temperature between 22 and 26 ° C; a temperature of 24 ° C. being particularly preferred. According to an advantageous embodiment of the invention, steps c) and d) are preferably carried out with mechanical stirring, at a speed of between 500 and 1000 revolutions per minute.

The solid microparticles obtained at the end of step e) have sizes which can vary between a few nanometers to a few hundred micrometers. They are preferably substantially spherical (microspheres or microcapsules) and have a size between 10 nm and 500 μm, more particularly between 1 and 50 μm.

According to a particularly advantageous embodiment of the invention, the method described above further comprises, at the end of step d) and before step e), an additional step during which the microparticles obtained at the end of step d) are brought into contact with water, preferably water for injectable preparation (PPI) such as PPI quality water sold by the Pharmaceutical Cooperative (Melun, France) .

To do this, the mixture containing the microparticles obtained at the end of step d) is added with water or poured into water, preferably with stirring. The water used during this step preferably has a temperature between 22 and 26 ° C, and even more preferably a temperature of 24 ° C.

This additional step improves the quality of the microparticles; it allows in particular to obtain smoother and more spherical microparticles. This step of adding water also makes it easier to carry out the subsequent step of recovering the microparticles, in particular in the particular case where the latter is carried out by filtration. Stage e) of recovery can be carried out by any conventional recovery technique known from the state of the art such as by filtration.

At the end of step e), the solid microparticles are preferably washed, for example with water, then dried. According to a particularly advantageous embodiment of the process according to the invention, the microparticles are firstly washed with an aqueous solution containing an injectable surfactant, before being then washed with water.

The use of an aqueous washing solution containing an injectable surfactant peraiet to remove traces of oil possibly present on the surface of the microparticles and thus avoid their aggregation during drying.

Among the injectable surfactants which can be used during this washing step, mention may in particular be made of block polymers consisting of polyethylene glycol and polypropylene glycol (poloxamers) such as the product sold under the name Lutrol® F68 by the company BASF, polyoxyethylenated sorbitan esters (polysorbates) such as the product sold under the name LVlontanox® 80 DF by the company SEPPIC and polyethylene glycol hydroxystearates (macrogol hydroxystearates) such as the product sold under the name Solutol® HS 15 by BASF and lecithins such as for example those sold by the company LIPOID Gmbh (Ludswigshafen, Germany).

When used, these surfactants preferably represent from 0.5 to 5% by weight relative to the total weight of the aqueous washing solution.

The present invention also relates to microparticles and in particular microspheres and microcapsules, containing at least one active principle, capable of being obtained by the process according to the invention.

Within these microparticles, the amount by weight of active ingredients will obviously vary depending on the very nature of the active ingredient (s) and the amount used during the preparation process. Advantageously, this amount is preferably between 0.01% and 50% of the total weight of the microparticles.

These microparticles constitute delivery systems immediate or progressive and / or prolonged release systems of the active principles which they contain, these then being released progressively and in a prolonged manner, as and when "in vivo" degradation of the biodegradable polymer constituting them. These microparticles can be used for the preparation of pharmaceutical compositions, in particular for the preparation of pharmaceutical compositions injectable by intravenous, intramuscular, subcutaneous, intrathecal, epidural or intracerebral route.

Another object according to the invention is therefore a phamiaceutical composition comprising microparticles, in particular microspheres or microcapsule islands in accordance with the invention and optionally a pharmaceutically acceptable vehicle.

The amount of microparticles present in said pharmaceutical composition is variable depending on the amount of active ingredients present in each microparticle and the desired therapeutic effect.

Preferably, the microparticles represent from 2% to 35% by weight of the total weight of the pharmaceutical composition.

The pharmaceutical compositions according to the invention may in particular be in the form of an injectable suspension, in particular a vaccine.

They can in particular be used for the administration of active principles for which a progressive and / or prolonged release is desirable.

The speed of release of the active principle present in the microparticles according to the invention can be modulated by varying certain parameters such as: the percentage by weight of the biocompatible polymer relative to the biocompatible solvent in solution A prepared in step a) the process, • the polylactide / glycolide ratio of PLGA, the molecular weight of the biocompatible polymer, the polymer / active ingredient ratio, in the case of a suspension, the size of the particles of active principle, the size of the microparticles. For example, for the prolonged release of an active ingredient over several months, it is in particular possible to use microparticles obtained from a PLGA polymer containing 37.5% of polylactide and 25% of glycolide at a rate of 50 mg of said polymer for 250 μg of active ingredient.

In addition to the foregoing provisions, the invention also comprises other provisions which will emerge from the description which follows, which refers to two examples of preparation of microparticles containing lysozyme as well as an example of preparation of a pharmaceutical composition injectable. EXAMPLE 1 Preparation of microparticles containing -vsozvme.

1) Equipment and products used

- Active ingredient: lysozyme sold under the reference "chicken egg white" by the company Sigma Aldrich (Saint Quentin Fallavier, France);

- Biodegradable polymer: PLGA polymers containing 37.5% of polylactide and 25% of glycolide with an average molar mass of 21,000, sold by the company Phusis (Saint-Ismier, France);

- Solvent for the polymer: glycofurol sold under the name tetraglycol by the company Sigma Aldrich (Saint Quentin Fallavier, France);

- Vegetable oil: olive oil sold by the Pharmaceutical Cooperative (Melun, France);

- Hydrophilic oil: Labrafil M 1994 CS® (apricot kernel oil PEG-6 esters) sold by the company Gattefossé (Saint Priest, France); - paddle stirrer sold under the name Heidolph® RGH 500 by the company Merck Eurolab (Paris, France);

- Retsch® ultrasonic bath sold by the company Servilab (La Chapelle Saint Aubin, France);

- filtration system sold by the Millipore company (Saint Quentin en Yvelines, France);

2) Dissolution of the active ingredient

100 mg of PLGA polymer are dissolved in 2.7 g of tetraglycol, then 4 mg of lysozyme are added to obtain a solution A.

3) Preparation of the emulsion

250 mg of solution A obtained above in the previous step are dispersed in 8 g of olive oil by mechanical stirring at 500 revolutions per minute for 5 minutes and at a controlled temperature of 24 ± 2 ° C (bath thermostated). Composition B is obtained in the form of an emulsion.

4) Extraction of the solvent and formation of the solid microparticles The microparticles are formed by addition, in the composition

B obtained above in the previous step, drop by drop and with stirring at 500 revolutions per minute, of 1.75 g of Labrafil M1944 CS®.

5) Filtration of the microspheres

The microparticles obtained above in the previous step are filtered under vacuum on a 0.45 μm filter (HNLP type).

6) Washing and drying of the microparticles The microparticles are then washed with 500 ml of water and dried in the open air on filter paper.

The microparticles are observed by light microscopy. They are smooth and spherical, with an average size of around 10 μm.

The encapsulated lysozyme is assayed by an enzymatic method after dissolution of the microparticles in dimethylsulfoxide (DMSO). 20% of the lysozyme initially dissolved in the PLGA / tetraglycol mixture is encapsulated in the microparticles in biologically active form.

The encapsulation rate (ratio between the quantity of active principle and the weight of microspheres) is 0.75%, which corresponds to approximately 75 μg of active principle per 10 mg of microparticles.

These results show that the preparation process according to the invention allows to encapsulate a significant quantity of protein without causing their denaruration.

EXAMPLE 2 Preparation of microparticles incorporating lysozyme. 1) Equipment and products used

In this example, the material and the products described above are used in Example 1 but in different proportions and according to a method of preparation with some variations. A 1% aqueous solution of Lutrol® F68 is also used.

2) Dissolution of the active ingredient

160 mg of PLGA polymer are dissolved in 4 ml of tetraglycol and then 1.39 mg of lysozyme is added to obtain a solution A.

3) Preparation of the emulsion

1.25 g of solution A obtained above in the previous step are dispersed in 16 g of olive oil by mechanical stirring at 500 revolutions per minute for 5 minutes in a bath thermostatically controlled at 24 ° C. Composition B is obtained in the form of an emulsion.

B obtained above in the previous step, drop by drop and with stirring at 500 rpm, 2.6 ml of Labrafil M1944 CS®. Stirring of the mixture containing the microparticles is continued for 10 minutes, then the mixture is poured into 400 ml of deionized water and placed under magnetic stirring for 10 minutes.

5) Filtration of microparticles

The microparticles obtained above in the previous step are filtered under vacuum on a 0.45 μm filter (HVLP type).

6) Washing and drying of microparticles

The microparticles are then washed with 250 ml of a 1% aqueous solution of Lutrol® F68 and then with 500 ml of water.

They are then taken up in 2 ml of deionized water and frozen at -20 ° C to be lyophilized.

The microparticles are observed by light microscopy. They are spherical, with an average size of approximately 13 ± 4 μm. These microparticles are moreover smoother and more regular than those obtained above in Example 1 during which on the one hand the additional step of bringing the microparticles into contact with water was not carried out and on the other hand, Lutrol® F68 was not used during the washing step. These results therefore show that the additional step of bringing the microparticles into contact with water and the use of a surfactant during the washing step make it possible to improve the surface appearance and the homogeneity of the microparticles obtained by implementing the process according to the invention.

The degree of encapsulation of lysozyme, dosed according to the method described • above in Example 1, is 40%.

EXAMPLE 3 Pharmaceutical composition for injection

1) Equipment and products used

- Microparticles: 6 mg of microparticles obtained according to the process described above in Example 1, and prepared under sterile conditions, packaged in an Eppendorf® tube and stored at 4 ° C away from humidity;

- Excipients: sterile solution of sodium carboxymethylcellulose (CMC Na) at 1.2% sold Cooper Phannaceutique (Melun, France), containing 1% Tween 80 and 4% mam itol, stored at 4 ° C.

2) Extemporaneous preparation of the pharmaceutical composition 40 μl of sterile CMC Na solution are withdrawn using a sterile syringe and added to the Eppendorf® tube containing the microparticles. The suspension is carried out by simple agitation.

The suspension is ready to be injected.

Claims

1. A process for the preparation of solid microparticles containing at least one active principle, comprising the following steps: a) the preparation of a solution A of at least one biodegradable, biocompatible and water-insoluble polymer, in a biocompatible solvent and soluble in water; b) dissolving or suspending in the solution obtained above in step a), at least one hydrophilic or hydrophobic active principle, to obtain a composition B; c) the production, with or without surfactant, of an emulsion of the composition B obtained above in step b) in a vegetable oil which constitutes the continuous hydrophobic phase of the emulsion, characterized in that it then comprises the following stages: d) the extraction of the biocompatible solvent by adding at least one hydrophilic biocompatible oil, with gentle stirring, until solid microparticles of said polymer containing said active principle are obtained, e) recovery solid microparticles obtained in step d).

2. Method according to claim 1, characterized in that the biodegradable, biocompatible and water-insoluble polymer is chosen from polylactides and polylactide-co-glycolides.

3. Method according to claim 2, characterized in that the polylactide-co-glycolides are chosen from those in which the polylactide / glycolide ratio varies between 75/25 and 50/50.

4. Method according to any one of the preceding claims, characterized in that the biocompatible and water-soluble solvent is glycofurol.

5. Method according to any one of the preceding claims, characterized in that the active principle is chosen from peptides, proteins, vitamins and nucleic acids.

6. Method according to claim 5, characterized in that the proteins are recombinant proteins chosen from erythropoietin, γ interferons, β interferon, growth hormones, neurotrophic factors, and cytokines.

7. Method according to any one of the preceding claims, characterized in that the vegetable oil is chosen from sunflower oil, corn oil, olive oil or even sesame oil .

8. Method according to any one of the preceding claims, characterized in that the hydrophilic biocompatible oil is chosen from polyglycolized glycerides of fatty acids, macrogolglycerides caprylocapric, triglycerides with medium chains of caprylic, capric or lauric fatty acids and their mixtures.

9. Method according to claim 8, characterized in that the biocompatible hydrophilic oil is polyethylene glycol-6 glyceryl mono oleate.

10. Method according to any one of the preceding claims, characterized in that the said biodegradable polymer represents from 1 to 4% by weight of the total weight of the solution A.

11. Method according to any one of the preceding claims, characterized in that during step c) of forming the emulsion, the amount of composition B preferably represents from 1 to 20% by weight of the weight of the 'vegetable oil.

12. Method according to any one of the preceding claims, characterized in that step c) of forming the emulsion is carried out at a temperature between 22 and 26 ° C.

13. Method according to any one of the preceding claims, characterized in that steps c) and d) are carried out with mechanical stirring, at a speed of between 500 and 1000 revolutions per minute.

14. Method according to any one of the preceding claims, characterized in that the microparticles are spherical (microspheres or microcapsules) and have a size between 10 nm and 500 μm.

15. Method according to any one of the preceding claims, characterized in that it further comprises, at the end of step d) and before step e), an additional step during which the microparticles obtained at the end of step cl) are brought into contact with water.

16. Method according to any one of the preceding claims, characterized in that at the end of step e), the microparticles are washed with an aqueous solution containing an injectable surfactant, before being then washed with the water. 5

17. The method of claim 16, characterized in that the injectable surfactants are chosen from block polymers consisting of polyethylene glycol and polypropylene glycol, polyurethane sorbitan esters and polyethylene glycol hydroxystearates.

18. Microparticles containing at least one active principle, characterized in that they are capable of being obtained by the process as defined in any one of claims 1 to 17.

19. Microparticles according to claim 18, characterized in that they contain from 0.01 to 50% by weight of active principle (s) relative to the total weight of the microparticles.

20. Microparticles according to claim 18 or 19, characterized in that they constitute immediate release systems or Lie systems for progressive and / or prolonged release of the active ingredients which they contain.

21. Use of microparticles as defined in any one of claims 18 to 20, for the preparation of compositions

20 pharmaceuticals, in particular injectable pharmaceutical compositions.

22. Pharmaceutical composition characterized in that it comprises microparticles as defined in any one of claims 18 to 20 and optionally an acceptable pharmaceutical vehicle.

23. Pharmaceutical composition according to claim 22,

T characterized by the fact that the microparticles represent from 2% to 35% by weight of the total weight of the pharmaceutical composition.

24. Pharmaceutical composition according to claim 22 or 23, characterized in that it is in the form of an injectable suspension.

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