NO322106B1 - Process for Encapsulation of Active Substances by Phase Separation of Polymers in Non-Chlorinated Organic Solvents - Google Patents

Abstract

The present invention relates to a process for a microencapsulation of an active principle by co-accumulation, which consists in a controlled solution or co-accumulation of a polymer dissolved in an organic solvent containing said active principle, and wherein said co-accumulation is induced by adding a non-solvent which makes the polymer being deposited on the surface of the active principle, and then curing the deposited polymer by adding a curing agent, and said curing leading to the formation of a continuous film coating on the active principle. The invention is the solvent selected from ethyl acetate, N-methylpyrrolidone, methyl ethyl ketone and acetic acid. The non-solvent is advantageously an alcohol having from 2 to 5 carbon atoms. while the curing agent, for example, is selected from water,. alcohols having up to 4 carbon atoms and mixtures thereof.

Description

The present invention relates to a method for microencapsulation of a

active principle in phase separation, especially for producing pharmaceutical forms with long-term release of active substances.

Microencapsulation techniques are commonly used to separate incompatible chemical substances, for example to convert liquids into powders, to improve the bioavailability of active substances, to mask the unpleasant taste or smell of certain compounds and to prepare pharmaceutical forms with sustained release of active substances.

Pharmaceutical forms for prolonged and sustained release of active substances can be administered subcutaneously or intramuscularly, and can be found directly in the blood stream or close to the organ to be treated, and therefore biodegradable polymers are often chosen as part of their composition.

Systems for sustained release of active substances are based on biodegradable polymers, can be administered parenterally without subsequent removal by surgical intervention, as the biodegradable polymers are converted in the body into metabolites that are eliminated naturally. Active substances are released according to kinetics which are modulated by the diffusion of active substances and the degradation process of the polymer. Furthermore, the patient's reaction to the treatment improves, as the administrations become fewer with this method.

Among biodegradable polymers that are often used in the encapsulation of active substances are poly(α-hydroxy acids), especially polylactic acids (PLAs) and polylactic acid-glycolic acid (PLAGAs), poly-8-caprolactone, polyorthoesters, for example Chronomer<®> and Alzamer <®>, polyanhydrides, then especially the copolymer of

sebacic acid and (carboxyphenoxy)propane, and biodegradable natural polypeptides and polymers, such as albumin, bovine serum albumin, collagen and chitosan.

There are two main types of microencapsulation technique:

solvent-free techniques, for example atomization-solidification, extrusion (co-extrusion/spheronification), gelatinization, spray crystallization (prilling) and precipitation from supercritical solutions (RESS), and solvent techniques, for example so-called fogging, phase separation,

emulsion evaporation, emulsion extraction and variants of these starting with water/oil/water double emulsions.

The prolonged contact between the sustained-release pharmaceutical forms and an aqueous medium justifies the use of polymers of a more or less hydrophobic nature which are essentially soluble in an organic medium. However, biodegradable polymers based on controlled release systems are very poorly soluble in solvents with low possible toxicity (category 3 solvents according to the ICH standard).

The most common standard microencapsulation techniques (phase separation and emulsion evaporation) therefore essentially use chlorinated solvents, for example dichloromethane (category 2 solvent according to the ICH standard), i.e. a solvent whose use should be limited, as a solvent for the polymer. However, said compound is a chlorinated solvent known for its neurotoxicity. The permitted residual level of dichloromethane in the finished product is 600 ppm according to standard ICH4.

Regardless of which microencapsulation technique is used, the produced microparticles will contain residual amounts of solvents. It therefore seems necessary to be able to develop new microencapsulation methods that do not use chlorinated solvents. To achieve this, two general solutions have been developed.

A solution to achieve encapsulation without chlorinated solvents is based on methods where no solvent is used, but certain polymers cannot be used by these methods. Furthermore, the properties of the particles produced using these methods will not necessarily meet the requirement for long-term treatment.

Another solution consists in replacing the chlorinated solvents with non-toxic solvents. Microencapsulation methods using chlorinated solvents have been thoroughly investigated, and the variables of the methods are known. However, a replacement of the chlorinated solvents with non-chlorinated solvents modifies the physiochemical interactions between the different components of the composition or preparation. Biodegradable polymers in the replacement solvent behave in a completely different way than in the chlorinated solvents. Thus, poly(L-lactide) and poly(D,L-lactide) are insoluble in ethyl acetate or acetone, and neither polymer is soluble in ethanol, which is, however, a preferred solvent due to its low toxicity.

Thomasin, C. et al. (J. Pharmaceutical Sciences, US, American Pharmaceutical Association, Washington, Vol. 87, No. 3, March 1, 1998, pages 259-268) describes microencapsulation of drugs by PLA/PLGA phase separation. An overview is given of solvents, non-solvents and curing agents known to be used in this technique. However, it does not describe the same combination of solvent/non-solvent/hardening agent as the method according to claim 1 in the present application.

It is therefore an aim to provide a method for microencapsulation of an active substance without the above-mentioned disadvantages. This purpose has been achieved by the present invention characterized by what appears from the attached claims.

The present invention proposes a method for microencapsulation with phase separation that does not use a chlorinated solvent. More specifically, the invention relates to a method for phase separation by adding a non-solvent. Phase separation by addition of a non-solvent requires the use of three miscible solvents; one of these three solvents is a solvent for polymers, while the other two are non-solvents for the polymer.

The principle of phase separation is based on the controlled precipitation or desolvation of a polymer dissolved in an organic solvent containing an active substance, usually in particulate form, induced by adding a non-solvent or a polymer phase separating agent. The solubility of the polymer in the organic solvent is lowered, and two immiscible phases are formed: The separate phase will slowly deposit on the surface of the active substance. The addition of a curing agent causes a continuous polymer film to form around the active ingredient.

The active ingredient particles can be a liquid or a solid. The active substance may initially be dissolved in the solvent for the polymer. In this case, it will precipitate in particulate form when the phase separation agent is added, or may form a homogeneous solid solution in the polymer particles formed by the phase separation.

Investigations of the interaction between the polymer, the solvent and the phase separator for each combination make it possible to produce a phase diagram that defines the ideal polymer/solvent/phase separator ratio necessary to obtain an effective encapsulation. However, it is difficult to predict the encapsulation of an active substance, as the interphase properties in relation to the molecular interactions between the polymer, the solvent and the phase separating agent change constantly with the composition of the separated phase (Thomassin C, Merkle H.P., Gander B.A., Physico-chemical parameters governing protein microencapsulation into biodegradable polyester by coacervation, Int. J. Pharm., 1997, 147, 173-186).

The main problem with the phase separation technique is a possible aggregation of particles. In an attempt to solve this problem, it has been proposed to reduce the temperature of the system, essentially in the curing step. The walls are then sufficiently firm to prevent sticking. Solutions such as using chlorofluorocarbons (CFCs) or temperature reduction cannot be transferred to an industrial scale. On the other hand, silicone oil can be used to stabilize the system due to the viscosity of the oil (Ruiz J.M., Tissier B., Benoit J.P., Microencapsulation of peptide: a study of the phase separation of poly(D,L-lactic acid-co-glycolic acid) copolymers 50/50 by silicone oil, Int. J. Pharm., 1989, 49, 69-77).

Despite high residual contents of solvents, the phase separation technique has been the commonly chosen technique for the encapsulation of brittle active substances, and especially water-soluble active substances in a non-aqueous medium.

The choice of solvent/phase separating agent/curing agent combinations is governed by different criteria: the solvent must be able to dissolve the polymer; and it is preferred that it does not solve the active ingredient, although the process can even be used with an active ingredient that is soluble in the solvent for the polymer;

the phase separating agent must be miscible with the solvent for the polymer.

There must not be a solvent for the polymer, because otherwise there would be a fairly simple transfer of the polymer from the solvent to the phase separating agent. Furthermore, it must not dissolve the active substance in order to limit the loss during encapsulation;

the curing agent must be partially miscible with the solvent for the polymer to thereby facilitate extraction. It must not dissolve either the polymer or the active ingredient, otherwise the encapsulation yield would be greatly reduced.

Previously known phase separation techniques have used dichloromethane or chloroform as a solvent for the polymer, a silicone oil as a phase separation agent and heptane as a curing agent.

The present invention relates to a method for microencapsulating an active substance by phase separation, where the method includes controlled precipitation or desolvation or phase separation of a polymer dissolved in an organic solvent containing said active substance, and where said phase separation is induced by adding a non-solvent, something which causes the polymer to be deposited on the surface of said active substance, and then to harden the polymer deposit by adding a hardener, and where the hardening

occurs by the formation of a continuous film coating on the said active substance,

characterized by that

the solvent for the polymer is a non-chlorinated organic solvent with a boiling point of between 30°C and 240°C and a relative dielectric permittivity (absolute dielectric constant) of between 4 and 60, advantageously selected from

ethyl acetate, N-methylpyrrolidone, methyl ethyl ketone, acetic acid and propylene carbonate and mixtures thereof,

the non-solvent is an alcohol or a ketone with from 2 to 5 carbon atoms,

preferably 2 or 3 carbon atoms, then especially ethanol

(6 ■ 24), 2-propanol (s = 18), 1,2-propanediol (e between 18 and 24) and glycerol (e = 40) or methyl ethyl ketone (e = 18),

the curing agent is selected from water, alcohols having from 1 to 4 carbon atoms, under the condition that the curing agent is an alcohol different from said non-solvent, and mixtures thereof.

Although N-methylpyrrolidone is Category 2 in the same way as dichloromethane, its limit concentration is markedly higher (4840 ppm as opposed to 600 ppm for dichloromethane).

Advantageously, the non-solvent and curing agent are respectively selected from the following pairs: 1,2-propanediol and 2-propanol, glycerol and 1,2-propanediol, glycerol and 2-propanol and 2-propanol and 1,2-propanediol.

According to a preferred embodiment, the polymer is a biodegradable polymer having an average molecular weight mass (Mw) of between 10,000 and 90,000 g/mol, preferably between 15,000 and 50,000 g/mol and with a polydispersity index (lp) of between 1 and 3.5, preferably between 1.5 and 2.5.

A number of other additional features will be set forth hereinafter to illustrate various preferred embodiments of the invention.

According to these additional properties, the polymer is a lactic acid polymer (PLA) or a copolymer between lactic acid and glycolic acid (PLAGA).

The polymer is a PLAGA with a Mw of between 15000 and 20000 g/mol, preferably equal to about 17500. Ip is between 1 and 2, preferably equal to 1.6, and the percentage of glycolic acid is less than 30%, preferably equal to 25%.

The polymer concentration in the solvent is between 1 and 10% (weight/volume), preferably about 4% (weight/volume).

The non-solvent/solvent ratio per volume is between Vi and 1/1.

The phase separation temperature is less than the glass transition temperature of the polymer, preferably less than or equal to 25°C, preferably less than 4°C and most preferably less than or equal to -4°C.

The non-solvent is added in successive doses from 200 µl to 1 ml.

The phase separation is carried out under stirring, for example with magnetic stirring at a speed of between 200 and 1000 rpm.

The hardener also contains a surfactant, and the concentration of this in the hardener is between 0.5 and 10% (volume/volume).

The surfactant is a sorbitan ester, for example Tween<®> 80 or polyvinyl alcohol.

The ratio per volume between the curing agent and the solvent is between 5/1 and 180/1, preferably between 15/1 and 120/1;

The microspheres are hardened with stirring, for example by magnetic stirring at a speed of between 500 and 1500 rpm.

The curing temperature is less than or equal to 25°C, preferably less than 4°C and more preferably less than or equal to 0.5°C.

The hardener is added in several portions, preferably in at least four portions.

The curing lasts between 2 and 4 hours.

The microparticles produced after curing are filtered through a Millipore <®> system by centrifugation or through fluted paper.

When the active substance forms a dispersion in the solution for the polymer, the solvent and non-solvent should have sufficiently high viscosity for the active substance to be stabilized.

The particle size of the active principle is between 1 and 50 microns, preferably between 5 µm and 30 nm.

According to a preferred embodiment, the solvent is N-methylpyrrolidone, the non-solvent is ethanol while the curing agent is water.

According to a second preferred embodiment, the solvent is ethyl acetate, the non-solvent is 2-propanol and the curing agent is water. The polymer is a 75:25 PLAGA, so that Mw is between 15,000 and 20,000, preferably equal to 17,500, and lp is between 1 and 2, preferably equal to 1.6.

According to a third embodiment, the solvent is acetic acid, the curing agent is water and the polymer is a 50:50 PLAGA.

When the active principle is insoluble in the solvent for the polymer, then in the present invention either a suspension or an emulsion is produced.

To prepare the suspension, the active ingredient is ground in a mortar and then placed in suspension in the solvent. The suspension can be homogenized using magnetic stirring: After which the phase separation is also carried out with magnetic stirring. The suspension can also be homogenized by mechanical stirring at variable speed (propeller stirrer, Heidolph RGL500 Prolabo, Paris, France) or by using an Ultra-Turrax<®> T25 mixer (Prolabo, Paris, France). In the two latter cases, the phase separation is carried out with mechanical stirring.

The dispersion of the active ingredient in the polymer solution can also be conveniently carried out using ultrasonic stirring.

To prepare the emulsion when the active substance is water-soluble, this can be dissolved in water, after which the water/solvent emulsion of the polymer is prepared with mechanical stirring. The phase separation will then take place with mechanical stirring.

When the active substance is soluble in the solvent for the polymer, the phase separation is carried out with mechanical stirring.

The polymer used in the present invention is a biodegradable polymer of the type usually used for encapsulating an active substance, preferably a PLA or a PLAGA, with a weight-average molecular mass (Mw) of between 10,000 and 90,000 and a number-average molar mass (Mn) of between 4000 and 40000, a polydispersity index (lp) of between 1 and 3.5, and where the amount of glycolide is between 10 and 60%. Polymers with weight-average molar mass equal to or greater than 15,000 g/mol will be preferred, as they enable the yield to be increased by increasing the volume of the separated phase. It is also preferred to use polymers with a low polydispersion index (lp 2.6), as polymer fractions with low molecular weight will remain in solution and give a yield reduction or may produce an aggregation of the microparticles by the polymers sticking to the surface of the particles.

PLAGA is for example Resomer<®> RG 502 (Boehringer Ingelheim, Mw = 14300 g/mol, Mn 6900 g/mol, lp 2.5, 50% glycolide), Resomer<®> RG 756 (Mw 89800 g/mol, Mn = 35200 g/mol, lp 2.6, 25% glycolide), Resomer<®> RG 858 (Mw = 87000 g/mol, Mn 22000 g/mol, lp 3.9,15% glycolide), Phusilin purchased from Phusis (Mw = 17500 g/mol, Mn 10940 g/mol, lp 1.6,25% glycolide).

The polymer concentration in the solvent must be sufficient to increase the viscosity of the medium, which makes it possible to stabilize the dispersed phase separation droplets and limit their aggregation on the one hand, and reduce the formation of too small microparticles on the other hand.

The polymer concentration is preferably between 1 and 10% (weight/volume) and is most preferably equal to about 4% (weight/volume).

The viscosities of the solvent and non-solvent must be sufficient to stabilize the phase separation droplets.

The volume of non-solvent to be added must be sufficient for the system to come within a stability range and at the same time achieve a stable separated phase. However, the volume of the non-solvent also depends on the concentration of the crystals of the active substance in the suspension of the organic polymer solution.

An addition of an excess of the non-solvent makes it possible to accelerate the hardening of the walls of the microparticles, preventing their aggregation and also improving the extraction of the solvent.

The rate of addition of the non-solvent must be sufficiently low to prevent the formation of an excessive number of microparticles, ie between 1 and 2 µm in size. Also, the slower the phase separation, the more even the particle size distribution will be for the microparticles and the smoother their surface will be. The addition of the non-solvent should preferably take place in doses from 200 (il to 1 ml, with an interval of at least one minute between each dose.

A reduction of the stirring speed during the phase separation step increases the size of the phase separation droplets and thereby the final microparticles. However, below a certain limiting speed, which varies depending on the systems, the kinetics with regard to deposition of the separated phase will become too slow and/or the phase separation droplets will become too large and not sufficiently stable. A mechanical or magnetic stirring between 200 rpm and 1000 rpm usually gives good results.

The temperature is an important parameter during the phase separation; it must be lower than the glass transition temperature of the polymer. The lower the temperature, the more viscous the medium will be and the less chance there is for the microparticles to aggregate.

The ideal curing agent should not dissolve either the active ingredient or the polymer. It should also be able to easily extract the solvent from the polymer. The curing agent used is water, optionally supplemented with a surface-active agent or an alcohol. Water is very beneficial, as it very easily extracts the solvent from the polymer. It also has the advantage that it is cheap and does not require treatment of the waste liquids. However, water is not the ideal curing agent in compounds with water-soluble active substances, as prolonged contact will then cause a diffusion of the active substance, which will result in a low degree of encapsulation.

When the encapsulated active ingredient is hydrophilic during curing, it will quickly be dissolved by water that penetrates into the microparticles and then back-diffuses out of them. By using a low temperature, the diffusion phenomenon can be reduced, and thereby the loss of the active substance in relation to the aqueous phase, in addition to improving the encapsulation yield.

Other possibilities to reduce the diffusion of the active substance are, for example, to saturate the outer phase with an electrolyte or the active substance itself if this is cheap, or to mix the water with another solvent that has a strong affinity for the solvent for the polymer, so that it happens an extraction of microspheres. The volume of water used should therefore be reduced and the contact with the water weakened.

Said surfactant or alcohol makes it possible to limit self-aggregation of the microparticles so that a homogeneous dispersion is formed. They should therefore preferably be selected on the basis that they are harmless. Surfactants can be selected from those commonly used for preparations for injection, such as polyoxyethylene sorbitan esters, for example Tween<®> 20 (hydrophilic surfactants).

Montanox<®> 80 (polyoxyethylene sorbitan monooleate) is a hydrophilic emulsifier that can be used in the preparation of an emulsion of the oil/water type.

Montane<®> 80 (sorbitan oleate) is its equivalent in the group of lipophilic surfactants. Solutol<®> HS 15 (polyethylene glycol 660 hydroxystearate) is a nonionic surfactant of hydrophilic nature used in injectable solutions.

Synperonic<®> PE/F 68 (Poloxamer 188) is a block copolymer of polyoxyethylene and polyoxypropylene.

Furthermore, polyvinyl alcohol has been used in two different grades, Mowiol<®> 4/88 and Rhodoviol<®> 4/125.

The volume of the hardener is based on a compromise. It must be sufficient to quickly remove the solvent from the microspheres, but will, on the other hand, have to limit the diffusion of the active substance out of the microspheres. The volume is defined based on solubility criteria for the solvent in the outer phase, so that the final concentration of the solvent in the curing agent is less than the saturation concentration of the solvent in the outer phase. By quickly removing the solvent, it will be possible to prevent the diffusion of the active ingredient by forming a polymer barrier. Furthermore, the microspheres will be hardened, thus preventing their aggregation.

The increase in the volume of the curing agent, which occurs gradually by adding the curing agent at regular intervals, makes it possible to obtain a better extraction of the solvent from the microparticles.

The ratio between the volume of the curing agent and the volume of the solvent is between 5/1 and 180/1, preferably between 15/1 and 120/1.

Combinations using acetic acid or N-methylpyrrolidone as solvent for the polymer require little curing agent to produce solid microspheres, so that the ratio between the volume of the curing agent and the volume of the solvent in this case is advantageously about 5/1.

The procedure for drying the microparticles depends on their stiffness, the size of the microparticles and the volume to be treated.

The tendency of the microparticles to aggregate after drying will depend on their degree of dryness and on the remaining amounts of solvent.

If it is not possible to dry the particles in open air, then it will be possible to place them under vacuum and/or increase the temperature to obtain a complete drying, under the condition that the microparticles can withstand the applied vacuum. However, it is necessary to ensure that the drying speed and temperature are not too high to prevent an aggregation of the microparticles.

In those combinations where ethyl acetate is used as a solvent for the polymer, it will be a problem to choose a suitable method for separating the microspheres. More specifically, the microspheres in such a case will still be filled with solvent after stirring for 1 hour in the curing agent. Filtration in a Millipore<®->system through a filter with a porosity of 0.5 [im] gives rise to a solid cake which is again very difficult to disperse: Furthermore, the filter is so quickly clogged with the microspheres that at this point constantly can be deformed. Separation by centrifugation of a portion of the solution did not give very good hope of a good result. In this case, it was also found that the microspheres aggregated and formed a pellet that did not allow itself to be redispersed, even at relatively low centrifugation speeds. Another filtration technique using a fluted filter paper proved to be a solution. This method has the advantage of having a large filtration surface and is carried out at normal atmospheric pressure. However, it turned out that the very smallest microspheres tended to be absorbed into the pores and gradually clog the filter. It was also difficult to recover all the microspheres. With regard to drying, this was carried out with a stream of compressed air, after which the microspheres were placed in air at normal room temperature.

According to one embodiment of the invention, the solvent for the polymer is ethyl acetate, the non-solvent is 2-propanol, while the curing agent is a mixture of water and surfactant, possibly a mixture of water, surfactant and alcohol.

In this embodiment, the polymer is preferably a 75:25 PLAGA. The polymer concentration is between 1 and 5% (weight/volume), and is preferably equal to about 4% (weight/volume). The phase separation can be carried out at room temperature, but preferably at a temperature below 4°C, and most preferably less than or equal to -4°C with mechanical stirring, preferably at 300 rpm.

The concentration of the surfactant is between 1 and 10%

(volume/volume). The surfactant is Tween<®> 80. When an alcohol is mixed with water, it is advantageous that the concentration of the surfactant is between 1 and 10%, while the alcohol concentration should be between 2.5 and 5%. The alcohol can advantageously be 2-propanol or 1,2-propanediol. The aqueous solution with the hardener is added in at least four portions. The curing itself lasts at least 2 hours and 30 minutes and no more than 4 hours and is carried out at room temperature, but preferably at temperatures below 4°C, most preferably at 0.5°C with mechanical stirring (500 rpm).

The volume ratio of the non-solvent to the solvent is equal to 1/2. The volume ratio between the curing agent and the solvent is equal to 120/1.

The lower the curing temperature, the shorter the actual curing time will be. When the temperature is thus below 4°C, 4 hours will be sufficient. When the temperature is 0.5°C, the curing time will be 2 hours and 30 minutes.

According to another embodiment, the solvent is N-methylpyrrolidone, the non-solvent is ethanol while the curing agent is a mixture of water and surfactant. The polymer concentration is between 4 and 10% (weight/volume). The phase separation and curing is carried out at room temperature with magnetic stirring. The concentration of the surfactant is between 0.5 and 10% (volume/volume). The curing time is between 2 and 4 hours. The volume ratio between hardener and solvent is 40/1. The polymer is preferably Resomer RG<®> 502 or Resomer RG<®> 756.

The present invention is illustrated by means of the following examples without this limiting the invention as such.

Example 1: Evaluation of polymer/solvent/phase separating agent/curing agent combinations

In this investigation, poly(oc-hydroxy acids) were evaluated. Three copolymers of lactic acid and glycolic acid were used, where the ratios between L- and

The D-lactides and glycolides were variable. These were the following polymers supplied by Boehringer Ingelheim: Resomer<®> RG 502 (Mw = 14300 g/mol, Mn = 6900 g/mol) comprising 25% L-lactide, 25% D-lactide and 50% glycolide ,

Resomer<®> RG 756 (Mw = 89800 g/mol, Mn = 35200 g/mol) which includes

37.5% L-lactide, 37.5% D-lactide and 25% glycolide,

Resomer<®> RG 858 (Mw = 87000 g/mol, Mn = 22000 g/mol) which includes

42.5% L-lactide, 42.5% D-lactide and 15% glycolide.

With regard to the respective solvents for the polymers, the least toxic ones were chosen. They belong to category 2 and 3 solvents defined by the classification given in the ICH guidelines.

The miscibility of the solvent/non-solvent pairs for those polymers was then determined. Similarly, the non-solvents for the polymers were chosen on the basis of their low toxicity.

Screening for solvent/non-solvent/curing agent combinations was carried out in scintillation flasks in small volumes of organic solutions where

the polymer concentration was 1 to 4% (exclusively for N-methylpyrrolidone and 50:50 PLAGA) (m/v), and where 5 ml of the organic solution of the polymer was placed in the scintillation flask. The phase separating agent was then added until a persistent turbidity was observed upon stirring, which is characteristic of the formation of

the phase separation. At this stage, the phase separation was investigated using optical microscopy. Then 1 ml of this mixture was poured into 10 ml of an aqueous solution of the surfactant. The presence or absence of microspheres was then observed by optical microscopy.

a) Phase separation tests with 50:50 PLAGA (Resomer<®> RG 502)

The solvents in which 50:50 PLAGA is soluble are ethyl acetate, acetone, acetonitrile,

acetic acid, dimethylacetamide, dimethylformamide, ethyl lactate, N-methylpyrrolidone and propylene carbonate. 50:50 PLAGA is insoluble in toluene, 2-propanol, glycerol, dioctyl adipate, 1,2-propanediol, xylene, diethyl carbonate and methyl ethyl ketone.

The combinations where a formation of separated phases and microspheres was observed are the following:

ethyl acetate/2-propanol/water + Tween<®> 80,

acetic acid/1,2-propanediol or 2-propanol/water + Tween<®> 80,

N-methylpyrrolidone/ethanol/water + Tween<®> 80 or 2-propanol,

N-methylpyrrolidone/methylethyl ketone or 2-propanol/water + Tween<®> 80,

propylene carbonate/2-propanol/water optionally with Tween<®> 80, ethanol or

NaCl.

Acetic acid can be used as a solvent for the polymer and gives rise to the formation of a separated phase and correctly individualized microspheres under the condition that water is used as a curing agent. Small droplets of separated phase form effectively in the combination of acetic acid and 2-propanol, but in the presence of methyl ethyl ketone or 1,2-propanediol as curing agent, the phase separation droplets will not retain their spherical shape and lumps of polymer are formed.

The ethyl acetate/2-propanol/water + Tween<®> 80 combination gives good results, as does the N-methylpyrrolidone/ethanol/water + Tween<®> 80 combination. The investigation was concluded by examining curing agents. It turned out that 1,2-propanediol and 2-propane were good candidates. However, a reduced capacity for extraction of the solvent with 1,2-propanediol in the presence of 2-propanol was observed.

Propylene carbonate is an advantageous solvent because of its low toxicity and also because it is partially soluble in water. The propylene carbonate/2-propanol combination makes it possible to omit the water due to the possibility of replacing the external phase with a solvent, or at least limiting the diffusion of that active substance, by adding an electrolyte to the external phase or by mixing water and a solvent.

b) Phase separation tests with 75:25 PLAGA (Resomer<®> RG 756)

The solvents in which 75:25 PLAGA is soluble are ethyl acetate, acetone, acetonitrile,

acetic acid, dimethylacetamide, dimethylformamide, diethyl ether, methyl ethyl ketone and N-methylpyrrolidone.

75:25 PLAGA is insoluble in toluene, 2-propanol, glycerol, 1,2-propanediol, propylene carbonate, dioctyl adipate and triethyl citrate.

The two solvents that give the best results are acetic acid and ethyl acetate. The investigations were therefore focused on investigating acetic acid/glycerol and acetic acid/1,2-propanediol combinations, and also the ethyl acetate/2-propanol and ethyl acetate/1,2-propanediol pairs.

The combinations where the formation of individualized microspheres was observed are the following: ethyl acetate/1,2-propanediol/water + Tween<®> 80 or 2-propanol,

acetic acid/glycerol or 1,2-propanediol/water + Tween<®> 80.

c) Phase separation tests with 85:15 PLAGA (Resomer<®> RG 858)

The solvents in which 85:15 PLAGA is soluble are ethyl acetate, acetone, acetonitrile,

acetic acid, dimethylformamide, ethanolamine, ethylenediamine, methyl ethyl ketone, N-methylpyrrolidone, toluene and triethyl citrate. 85:15 PLAGA is insoluble in 2-propanol, glycerol, 1,2-propanediol, dioctyl adipate and xylene.

The combinations where the formation of a separated phase and microspheres were observed are the following: ethyl acetate/2-propanol or 1,2-propanediol/water + Tween<®> 80,

ethyl acetate/2-propanol/1,2-propanediol,

ethyl acetate/1,2-propanediol/2-propanol,

acetic acid/glycerol (water + Tween 80) or 1,2-propanediol or 2-propanol,

acetic acid or methyl ethyl ketone/l,2-propanediol/water + Tween<®> 80,

acetic acid/2-propanol/l,2-propanediol,

N-methylpyrrolidone or methyl ethyl ketone/2-propanol/water + Tween<®> 80,

methyl ethyl ketone/1,2-propanediol/2-propanol.

Example 2: Production of microspheres without active substance by varying the phase separation parameters

In a first step, operating conditions were established for the production of microspheres without active substance, in order to thereby be able to produce particles of the desired size. What was investigated was the effect of the various factors, for example the volume of the added phase separating agent, the volume of curing agent, type of stirring and speed as well as the method of collecting the microspheres.

The polymer was dissolved in 50 ml of organic solvent (beaker 1) to a 1% solution (w/v). The polymer concentration was adjusted to 4% (w/v) when the solvent was N-methylpyrrolidone. While stirring, the phase separation agent was added until a stable and visible phase separation was achieved. The mixture was then poured into a solution of the hardener with a surfactant added (beaker 2) while stirring. The microspheres were then recovered by filtration. The combinations that were tested are those chosen in example 1.-,

a) With 50:50 PLAGA (Resomer<®> RG 502)

The different protocols corresponding to each of the selected combinations

are indicated in Table 1.

b) Application of 75:25 PLAGA (Resomer<®> RG 756)

Microspheres were prepared under the conditions indicated in Table 2 with ethyl acetate

as a solvent. Three stirring options were used for the ethyl acetate/1,2-propanediol/water + Montanox<®> 80 or Tween<®> 80 combination: ultrasonic stirring, mechanical paddle stirring and magnetic stirring. There are no striking differences with regard to the morphology and size of the microspheres that would justify a choice of one of these methods.

Example 3: Production of microspheres containing 5-fluorouracil (5-FU) as active ingredient

The tests were carried out with combinations of examples la) and b), which led to the formation of a phase separation.

a) Effect of active substance on the phase separation

The active substance in low concentration (5% by weight) forms a homogeneous dispersion with

the polymer solution. The stability of this depends on the solvents; N-methylpyrrolidone, which is thus a viscous solvent, leads to better stability of the dispersion, and there are reduced losses of antimitotic agent by shedding and absorption on the walls of the beaker.

The use of 1,2-propanediol as a phase separating agent further stabilizes the system by increasing the viscosity of the medium.

b) Production yield

This varies from 15 to 100% depending on the combinations. Combinations like

using ethyl acetate as solvent for the polymer gives the best yields, close to 100%.

c) Yield and degree of encapsulation

When the theoretical contents of active principle are increased, the encapsulation yield becomes

very low.

By changing to ultrasound, it is possible to individualize the active principle particles. When low concentrations are used, the dispersion becomes homogeneous, and the encapsulation yield increases to 70%.

Example 4: Examination of the combination of N-methylpyrrolidone, ethanol, water and surfactant

Among the N-methylpyrrolidone-soluble polymers tested, the following two were used: Resomer<®> RG 502 (Boehringer Ingelheim) which is a 50:50 D,L-PLAGA,

Resomer<®> RG 756 (Boehringer Ingelheim) and Phusiline (Saint-Ismier), which

is 75:25 D,L-PLAGAs.

The first preparation of the microparticles uses an organic solution of polymer at 4 or 10% (w/v) for a solvent volume of 5 ml. The phase separation is carried out with magnetic stirring. The non-solvent for the polymer is added using a micropipette, milliliter by milliliter. As soon as a cloud appears in the solution, a sample is taken and this is examined using optical microscopy. One minute after the turbidity occurred, the phase separation medium is poured dropwise into 200 ml curing agent (water + Tween<®> 80 or PV A). The hardening of the microparticles is carried out with mechanical stirring. A further observation of the microparticles by optical microscopy is carried out during curing. The microparticles are then filtered under vacuum or atmospheric pressure on filter paper that will be impossible to filter under vacuum (in other words, immediate clogging of the filter or too long filtration times). Finally, the microparticles are observed immediately after filtration and after freeze-drying.

The microparticles produced with the combination of NMP, ethanol, water and surfactant were examined by optical microscopy and were round, smooth and uniform, regardless of the stage of preparation and regardless of the type of polymer.

For all the prepared portions, the filtration after curing was carried out on a filter paper and at atmospheric pressure. The size of the microparticles is between 30 and 50 µm. Numerous microparticles with a diameter of approx

5 Jim.

Effect of polymer type and its concentration

By optical microscopy, it was demonstrated that all the produced microparticles had the same appearance.

The best yields were obtained with Resomer<®> RG 502 and Resomer<®> RG 756 under the following conditions (Table 3).

The effect of grouse of curing agent and curing time

The type of curing agent (water, water + Tween<®> 80 or water + PV A) for the same polymer, 50:50 PLAGA (RG 502) and at room temperature, has no influence either on the filtration or on the actual yield of microparticles, which was between 17.5 and 23.8%.

The curing time for the same polymer, i.e. 50:50 PLAGA (RG 502) makes it possible to increase the manufacturing yield by approx. 10% at room temperature.

Effect of temperature

a) During curing

Curing at a temperature below 4°C made it possible to double the manufacturing yield

of microparticles for 50:50 PLAGA (RG 502), that is to 35.9 as opposed to 17.5%.

b) During phase separation

The tests which were carried out in their entirety at temperatures below 4°C have a maximum

manufacturing yield of 10.5%.

The combination of N-methylpyrrolidone, ethanol, water and surfactant leads to the formation of microparticles of satisfactory appearance as this could be demonstrated by optical microscopy.

Example 5; Microspheres containing progesterone or budesonide Progesterone and budesonide are hydrophobic active substances that are soluble in ethyl acetate.

The combination of ethyl acetate, 2-propanol, water and Tween<®> 80 was used, while the polymer was 75:25 D,L-PLAGA (Phusis, Saint-Ismier).

The production yields were in all cases above 90%.

The results obtained are shown in table 4.

The yields and degrees of encapsulation of the active principles are given in the following table, and were calculated as follows: Ttheo. = [weight of active substance/(weight of polymer + weight of active substance)] x

100,

Exp. = [weight of active substance/weight of dry microparticles] x 100.

This allows the encapsulation yield Y to be calculated:

Y = [Texp./Ttheo.] x 100

The combination of ethyl acetate, 2-propanol, water and surfactant leads to the formation of microparticles with a satisfactory appearance as this could be demonstrated by optical microscopy. On the other hand, the microparticles obtained using this solvent/non-solvent pair were more brittle and dried in open air, i.e. at room temperature and atmospheric pressure, and they were not irreversibly aggregated. Manufacturing yields are close to 100%. Aggregation is the main problem encountered with this combination of this solvent, non-solvent and curing agent. It can be reduced by modifying the various parameters during manufacture. The temperature at which the phase separation is carried out is particularly a very important factor, and when it is lowered to -4 °C it is possible to harden the polymer and give the microparticles stiffness, so that these can subsequently be individualized and no longer stick together.

Claims (21)

1. Procedure for microencapsulation of active substance by phase separation which consists of controlled dissolution and phase separation of a polymer dissolved in an organic solvent containing said active principle, and wherein said phase separation is induced by adding a non-solvent, after which the polymer is deposited on the surface of the active principle, and then curing the deposited polymer by adding a curing agent, and wherein the curing leads to the formation of a continuous film coating on said active principle, characterized by that the solvent for the polymer is a non-chlorinated organic solvent with a boiling point of between 30 and 240°C and a relative dielectric permittivity of between 4 and 60, advantageously selected from ethyl acetate, N-methylpyrrolidone, methyl ethyl ketone, acetic acid and propylene carbonate and mixtures thereof, the non-solvent is an alcohol or a ketone with from 2 to 5 carbon atoms, preferably 2 or 3 carbon atoms, then especially ethanol (e = 24), 2-propanol (e = 18), 1,2-propanediol (e = between 18 and 24) and glycerol (e = 40) or methyl ethyl ketone (s = 18 ), the curing agent is selected from water, alcohols with from 1 to 4 carbon atoms, on the condition that when the hardener is an alcohol, this is different from said non-solvent, and mixtures of these 2. Method according to claim 1, characterized in that the non-solvent and the curing agent are respectively selected from the following pairs: 1,2-propanediol and 2-propanol, glycerol and 1,2-propanediol, glycerol and 2-propanol, and 2-propanol and 1,2-propanediol. 3. Method according to claim 1 or 2, characterized in that the polymer is a biodegradable polymer with a weight average molecular mass (Mw) of between 10,000 and 90,000 g/mol, preferably between 15,000 and 50,000 g/mol, and with a polydispersity index (lp) of between 1 and 3.5, preferably between 1.5 and 2.5. 4. Method according to claim 3, characterized in that the polymer is a lactic acid polymer (PLA) or a polymer of lactic acid and glycolic acid (PLAGA). 5. Method according to claim 4, characterized in that the polymer is a PLAGA with a Mw of between 15,000 and 25,000, preferably equal to 17,500, lp is between 1 and 2, preferably equal to 1.6, and the percentage of glycolic acid is less than 30%, preferably equal to 25%. 6. Method according to any one of claims 1 to 5, characterized in that the polymer concentration in the solvent is between 1 and 10% (weight/volume), preferably about 4% (weight/volume). 7. Method according to any of the preceding claims, characterized in that the volume ratio between the non-solvent and the solvent is between 1/2 and 1/1. 8. Method according to any of the preceding claims, characterized in that the phase separation temperature is lower than the glass transition temperature of the polymer, preferably equal to or lower than 25°C, preferably lower than 4°C and most preferably equal to -4°C. 9. Method according to any of the preceding claims, characterized in that the curing agent also contains a surfactant and that the concentration of the latter in the curing agent is between 0.1 and 10% (v/v). 10. Method according to any of the preceding claims, characterized in that the surfactant is a sorbitan ester, for example Tween<®> 80 or polyvinyl alcohol. 11. Method according to any of the preceding claims, characterized in that the volume ratio between the curing agent and the solvent is between 5/1 and 180/1, preferably between 15/1 and 120/1. 12. Method according to any of the preceding claims, characterized in that the microspheres are hardened by stirring at a speed of between 500 and 1500 rpm. 13. Method according to any of the preceding claims, characterized in that the curing temperature is lower than or equal to 25°C, preferably lower than 4°C and most preferably less than or equal to 0.5°C. 14. Method according to any of the preceding claims, characterized in that when the active principle forms a dispersion in the polymer solution, the solvent and the non-solvent have a sufficiently high viscosity to stabilize the active principle. 15. Method according to any of the preceding claims, characterized in that the active principle is dispersed by means of ultrasound to form a dispersion in the polymer solution, and that the phase separation is carried out by gentle stirring, preferably of a magnetic or mechanical type. 16. Method according to any of the preceding claims, characterized in that the particle size of the active principle is between 1 and 50 micrometres, preferably between 5 and 30 micrometres. 17. Method according to any of the preceding claims, characterized in that the solvent is N-methylpyrrolidone, the non-solvent is ethanol and the curing agent is water. 18. Method according to any one of claims 1 to 16, characterized in that the solvent is ethyl acetate. 19. Method according to claim 18, characterized in that the solvent is ethyl acetate, the non-solvent is 2-propanol and the curing agent is water. 20. Method according to claim 18 or 19, characterized in that the polymer is a 75:25 PLAGA so that Mw is between 15,000 and 20,000, preferably equal to 17,500, and lp is between 1 and 2, preferably equal to 1.6. 21. Method according to any one of claims 1 to 16, characterized in that the solvent is acetic acid, the curing agent is water and the polymer is a 50:50 PLAGA.