MXPA00010501A - Novel poly(methylidene malonate) microspheres, preparation method and pharmaceutical compositions containing them - Google Patents

Abstract

The invention concerns novel microspheres particularly useful in pharmaceutics as particulate vectors for carrying biologically active substances, in particular hydrophilic substances, for oral administration. The invention is characterised in that said microspheres consist of a support material continuous lattice wherein is optionally dispersed a substance, said support material containing at least 70 wt.%of a homopolymer consisting of recurrent units corresponding to general formula (I) wherein:R1 represents an alkyl group containing 1 to 6 carbon atoms or a (CH2)m - COOR3 group wherein m is an integer ranging between 1 and 5 and R3 represents an alkyl group having 1 to 6 carbon atoms;R2 represents an alkyl group having 1 to 6 carbon atoms;and n is an integer ranging between 1 and 5. The invention is useful in pharmaceutics.

Description

NEW MICROSPHERES BASED ON POLY (METHYLIDEN MALONATE), ITS PROCEDURE FOR PREPARATION AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM Description of the Invention The present invention has as its object new microspheres especially useful in the pharmaceutical field, as particular vectors intended for the transport of biologically active substances, in particular hydrophilic substances (peptides or proteins), with a view to an oral administration. The subject of the invention is also a method of manufacturing those microspheres and pharmaceutical compositions containing them. Within the scope of the present description, the term "microspheres" means particles that are substantially spherical, with an average diameter between 1 μm and 100 μm, and preferably between 5 and 100 μm, formed by a continuous, more or less dense network , of a support material. These microspheres differ from the microcapsules, which are constituted by a wall that covers a cavity. However, it should be noted that microspheres prepared in multiple emulsion may include a set of dispersed globules in the network Ref: 124303 continuous that constitutes them. In the latter case, e.1 total volume of said globules will generally represent a fraction comprised between 1:20 and 1: 2 of the total volume of the microspheres. In the course of recent years, numerous studies have shown that particular systems based on polymers can be used to modify the release profile of a therapeutically active substance. This is how they have been prepared by means of various techniques, microspheres based on synthetic polymers, such as, for example, poly (lactic acid), poly (lactic acid co-cog 1 i co 1 i co), polystyrene, polyepsiloncaprolactone, polymethylmethacrylate, or also based on methylcellulose or ethylcellulose. However, the microspheres thus obtained are generally not biodegradable, and when they are, they are characterized by a degradation very delayed in time. Thus, in the case of microspheres based on poly (lactic acid), for example, the degradation is not progressive and occurs all at once after a significant interval of time. In addition, lactic polymers are degraded by releasing strongly acidic products that not only lead to the autocatalysis of polymer degradation, but also cause the induction of incompatibilities with the encapsulated substances. In the case of other polymers used, the microspheres exhibit an extremely low degradation rate, even zero. The time of permanence in the organism of said particles, can limit its repeated application in man. Finally, the known microspheres are characterized, for the most part, by an important hydrophobicity that favors strong interactions and often denature the substance to be encapsulated, in particular when the latter is of protein or peptide nature. It has been discovered, and this is the basis of the present invention, that it was possible to make new microspheres which allow to remedy the disadvantages of the microspheres of the state of the art. Thus, according to a first aspect, the present invention relates to microspheres constituted by a continuous network of a support material in which a substance is optionally dispersed, characterized in that said support material contains at least 70% by weight of a homopolymer constituted by recurring units that respond to the following general formula (I): COORi CH2 COO (CH- COOR2) wherein: - Rx represents an alkyl group having from 1 to 6 carbon atoms or a group (CH2) n -COOR3 in which m is an integer between 1 and 5 and R3 represents an alkyl group having 1 to 6 carbon atoms; - R2 represents an alkyl group having from 1 to 6 carbon atoms; and - n is an integer between 1 and 5. It has been shown that due to the chemical nature of the polymers that form its matrix, these new microspheres: - exhibit a kinetic of progressive and modulated degradation; - they allow to encapsulate with a high efficiency hydrophilic substances, especially of biological origin; It has also been observed, in a totally surprising and unexpected way, that these microspheres: - can induce a stimulation of the immune response, when they are associated with an antigen; they allow, in certain cases, the suppression of pathological reactions of hypersensitivity (induction of a tolerance), when they are administered orally.

Therefore, the nature of the polymer material forming the matrix of the microspheres is what constitutes the originality of the present invention. That polymeric material is essentially formed by a homopolymer constituted by recurring units of general formula (I). Said polymers have the remarkable property of being biocompatible and bioerodible, that is to say they are capable of being degraded by chemical or biochemical means, by cutting the lateral substituents. Since the erosion rate of the microspheres according to the invention is independent of the molecular weight of the support material, it can then be simply modulated, using a support material having a molecular weight adapted to the desired erosion rate. The microspheres according to the invention then have a modulated and progressive bioerosion which allows, for example, the transport of a biologically active substance, dispersed in the support material, to the place of the organism where its action will be more effective. The bioerosion of the microspheres also prevents their accumulation in the organism; therefore its use is no longer limited. According to a particular characteristic, the aforementioned homopolymer is constituted by recurring units corresponding to the general formula (I) in which: - R-L represents an alkyl group having from 1 to 6 carbon atoms; - R2 represents an alkyl group having from 1 to 6 carbon atoms; and - n is a number equal to 1; and preferably wherein Rx and R2 represent a CH2-CH3 group. These different types of polymers of the poly (methylidene malonate) family are particularly suitable for encapsulating hydrophilic substances, especially of biological origin and possibly biologically active substances. By "biologically active molecule" is meant in a non-limiting manner any molecule having a prophylactic or curative biological activity, in vi tro or in vi ve, especially an anti-infective agent, in particular an antiseptic agent, antiviral, antiparasitic or antifungal antibiotic , especially anticancer. The usable antibiotic or antiseptic agents may be, for example, rifampicin and colistin. As examples of antiviral agents, didanosine, ribavirin, zidovudine, acyclovir, ganciclovir, foscarnet, vidarabine and zalcitabine can be mentioned in a non-limiting manner. Cisplatin and taxol can be used, for example, as anticancer agents. According to a presently preferred embodiment of the invention, the support material of the microspheres contains: - 90% 99.5% by weight of a homopolymer as defined above; and - from 0.5% to 10% by weight of a copolymer comprising at least one sequence having a hydrophilic character and at least one sequence having a hydrophobic character, said hydrophobic character preferably comprising at least one recurring unit that responds to the general formula (I). Advantageously, the hydrophilic character sequence of the aforementioned copolymer is chosen from a poly (oxyethylene), a poly (vinyl alcohol), a poly (vinylpyrrolidone), a poly (N-2-hydroxypropyl methacrylamide), a poly (hydroxyethyl methacrylate) , a hydrophilic poly (amino acid) such as a polylysine, a polysaccharide and preferably it will be a poly (oxyethylene). The copolymer can have a block structure, preferably diblock or triblock, or a modified structure. The addition of said copolymers in the support material makes it possible to obtain a homogeneous dispersion of the substance to be encapsulated inside each of the microspheres. This addition also makes it possible to modulate the hydrophilic / hydrophobic relationship of the surface of the microspheres, which makes it possible to avoid or limit the important interactions and which often denature the substance to be encapsulated. further, those copolymers whose chemical nature of hydrophobic sequence is identical to that of the homopolymer which essentially constitutes the microspheres, are particularly advantageous for the use of the presently preferred method of preparation of microspheres, as will be explained in more detail below. In general, the microspheres according to the present invention can be obtained by the use of a process that includes: the preparation of a multiple emulsion of three phases, whose intermediate phase is constituted by a solution of the polymer (s) that constitute the support material in a volatile organic solvent, and the evaporation of said organic solvent, under conditions that allow to cause the precipitation of the polymer around the droplets that constitute the internal phase. This multiple emulsion can be obtained as usual by dispersing a water-in-oil primary emulsion in a second aqueous phase containing a stabilizing agent. This multiple emulsion can also be obtained by an "inverse" process consisting in pouring an aqueous solution into a water-in-oil primary emulsion. In a totally unexpected way, this "inverse" procedure allowed to obtain very remarkable results, sometimes even better than those obtained by the aforementioned classical technique. Thus, according to a second aspect, the present invention relates to a process for obtaining microspheres as described above, comprising: a) the preparation of a first solution of the polymer (s) above (s) constituting the support material, in a volatile organic solvent optionally containing a surfactant, b) the preparation of a second solution not miscible with the solution obtained in a), which optionally contains the substance it is desired to disperse and optionally a surfactant, c) the preparation of a primary emulsion by dispersing the second solution in the first solution, where the continuous phase is constituted by the polymer solution (s), d) the preparation of an emulsion secondary: either dispersing under agitation, the primary emulsion obtained in c) in a dispersion medium not miscible with said primary emulsion, possibly containing said means a stabilizing agent; - or by pouring under stirring in said primary emulsion, a solution constituted by a medium immiscible with said primary emulsion, said medium optionally containing a stabilizing agent; e) evaporation of said organic solvent under agitation. According to a particular characteristic of the invention, the above-mentioned process further comprises: f) the isolation of the microspheres by centrifugation g) one or several successive washes of said microspheres h) the lyophilization of said microspheres. The first stage of the preparation process of the microspheres according to the invention therefore includes the production of a water-oil type emulsion, preferably in the presence of an appropriate surfactant, the phase containing the oily or organic phase of the polymer (s) intended to constitute the support material of said microspheres. At first, a solution of the polymer (s) constituting the support material is prepared with the aid of an appropriate volatile organic solvent, optionally in the presence of a surfactant.

Advantageously, polymers preformed to the extent in. that the homopolymers constituting essentially the support material of the microspheres can be obtained under conditions which allow a good characterization in terms of molar mass and mass dispersity. The homopolymers constituted by recurring units corresponding to the general formula (I) can be prepared starting from monomers, for example, following the procedure described in the patent EP 283346 corresponding to the patents US 4 931 584 and US 5 142 098 incorporated herein as reference, said monomers being generally degassed in vacuum with a paddle pump until a constant weight is reached to remove the polymerization inhibitor (S02). However, these homopolymers will advantageously be prepared by the anionic route in an aprotic medium, for example by dispersing the monomer in acetone, followed by the addition of soda under stirring, also followed by evaporation of acetone and drying of the polymer thus obtained. Other aprotic organic solvents such as acetonitrile, dioxane and tetrahydrofuran can be used in place of acetone.

The molecular mass of the aprotic homopolymer capable of being obtained by the application of that process can be perfectly controlled by an appropriate choice of the application conditions, and in particular of the monomer concentration in the organic phase, of the pH and of the molarity of the polymerization initiator (soda). In general, homopolymers having an average molar mass of 1,000 to 100,000, and preferably 5,000 to 80,000, will be used within the framework of the present invention. The volatile organic solvent capable of being used for the preparation of the first solution contains the polymer (s) constituting the support material will generally be chosen in such a way that its boiling point is lower than that of water. That solvent may therefore be easily removed during the final stage of evaporation, allowing precipitation of the polymer. Ethyl acetate constitutes a volatile organic solvent particularly suitable for this effect. The surfactants that can be used for the stabilization of the primary emulsion can be of a varied nature and will be added to the organic phase containing the polymer (s) (first solution) and / or the aqueous phase (second solution) that constitutes the dispersed phase. It can be, for example, a poloxamer such as the product marketed under the name Pluronic® F68, or also a polyvinyl alcohol such as the product marketed under the name Mowiol® 40-88, or also a polysorbate, or even a surface-active copolymer whose sequence hydrophobic presents a chemical nature identical to that of the homopolymer constituted by recurring units that respond to the general formula (I). It has been shown that said surface-active copolymers and in particular the poly (methylidene malonate) and polyoxyethylene copolymers are particularly advantageous insofar as they allow, on the one hand, to obtain a very stable primary emulsion and, on the other hand, to obtain a good anchoring of the surfactant in the matrix after evaporation of the solvent. the above-mentioned surface-active copolymers can be prepared by conventional polymerization techniques well known to man by trade. Among these techniques, polymerization by anionic route, radical polymerization, or the coupling technique of copolymer precursor sequences will preferably be used, said sequences having been previously functionalized at the end of the chain in suitable forms. Anionic polymerization is more particularly convenient for the preparation of block copolymers. It includes the sequential addition of monomers and allows to obtain copolymers of perfectly defined structure, allowing the amounts of initiators and monomers used to control the degree of polymerization of each of the sequences. In this way, a copolymer can be obtained: either by anionic polymerization of a first monomer and reaction on the increasing chain of a second monomer; - either by activation of a precursor polymer that will serve as initiator for the polymerization of a second monomer. The initiators which can be used in the context of these anionic polymerizations will generally be: on the one hand, organometallic derivatives such as butyl lithium and in particular diphenylhexyl lithium; - on the other hand, the alcoholates and in particular the macromolecular alcoholates such as a POE alcoholate which can be generated by activation of a hydroxy function with the help of cumyl potassium, of diphenylmethylpotassium, of naphthalene potassium. The anionic polymerization will generally be carried out in a solvent compatible with the various copolymer sequences. In the case where the sequence of hydrophilic character is constituted by a poly (oxyethylene) and the sequence of hydrophobic character is constituted by a poly (methylidenemalonate), the block copolymers will preferably be prepared by successive anionic polymerization of the ethylene oxide, then of the methylidene malonate or by activation of a commercial monohydric polyoxyethylenated precursor and subsequent anionic polymerization of the poly (methylidene malonate) sequence. In general, tetrahydrofuran will be preferably used as the polymerization solvent, since said product allows to work in a homogeneous medium and favorably influences the polymerization kinetics. The monomers used for the preparation of the hydrophilic sequences will generally be commercial products. The coupling technique is also especially suitable for the preparation of block copolymers. This reaction is generally carried out starting from presynthesized and functionalized homopolymers, in the presence of a coupling agent and optionally of an activation agent, in a suitable solvent. In the case of the preparation of the preferred copolymers according to the invention, the hydrophilic sequence of which consists of a poly (oxyethylene) and the hydrophobic sequence consists of a poly (methylidene malonate), a poly (oxyethylene) homopolymer will advantageously be used. functionalized by an or-carboxy group and a poly (methylidene malonate) homopolymer functionalized by an α-hydroxy group. The homopolymer of pol i (oxie t i 1 ene) functionalized by an α-carboxy group can be obtained, for example, by transformation by the succinic anhydride of a poly (oxyethylene) functionalized by a commercial α-hydroxy group. The poly (methylidene malonate) homopolymer functionalized by an α-hydroxy group can be obtained directly by anionic synthesis in aqueous medium or by anionic synthesis in a solvent using an aqueous solution of soda as the polymerization initiator.

As a coupling agent particularly suitable for this polymerization, dicyclohexylcarbodiimide (DCCI) will be advantageously used. The coupling reaction can optionally be activated by basic catalysis and will generally be developed in a solvent compatible with the homopolymers, such as in particular dichloromethane in the particular case of the copolymers preferred by the invention. Radical polymerization is particularly convenient for the preparation of modified copolymers. This polymerization is generally carried out starting from a macromonomer, that is to say an oligomer bearing, at one of its ends, a radical-polymerizable etiienic group capable of reacting with a monomer to form a modified structure copolymer. This polymerization will generally be carried out in the presence of an initiator in an appropriate solvent. In the case of the preparation of the copolymers whose hydrophilic sequence is constituted by a poly (oxyethylene), several functionalized macro onomers may be used. The use of a poly (oxyethylene) macromonomer functionalized by an ethacryloyl group will be more precisely preferred. Said product can be commercial (Aldrich) and will be constituted for example by a poly (oxyethylene) chain of molar mass comprised between 308 and 440 g / mol, or it will be prepared starting from a commercial poly (ethylene glycol) monomethyl ether by coupling with acid methacrylic in dichloromethane to form a terminal methoxy function. It is also possible to prepare said macromonomer by activation of a poly (oxyethylene) and subsequent reaction on methacryloyl chloride. Modified structure copolymers can also be prepared by transesterification of a poly (oxyethylene) monomethyl ether onto side chains of a presynthesized poly (methylidene malonate) ester.

This transesterification will usually be carried out with alcohol, in the presence of a catalyst at elevated temperature. Copolymers whose total molar mass of the hydrophobic character sequences is between 1,000 and 80,000 g / mol, and preferably between 1,000 and 50,000 g / mol are particularly suitable in the context of the present invention. In general, the primary emulsion used for the preparation of the microspheres according to the invention can be obtained by means of a shear homogenizer, for example Ultraturrax (13, 500 r.p.m. -5 mm). The substance to be encapsulated is generally added to the dispersed aqueous phase of the primary emulsion. The second stage of the preparation process of the microspheres according to the invention comprises the preparation of a secondary emulsion: - either dispersing under stirring, the primary emulsion obtained in the first stage in a dispersion medium immiscible with said primary emulsion, said dispersing medium containing, optionally, a stabilizing agent; - by pouring under stirring in said primary emulsion, a solution constituted by a medium immiscible with said primary emulsion, said medium optionally containing a stabilizing agent. Generally, the dispersing medium which is not miscible with the primary emulsion is an aqueous phase in which the primary emulsion is preferably introduced dropwise, and the emulsion is also carried out, for example, with the aid of an Ultraturrax-type homogenizer (8,000 rpm; 5mm).

The polyvinyl alcohol constitutes a stabilizing agent particularly suitable for the preparation of the secondary emulsion. Eventually, this second stage can be followed by an additional step of displacing the organic solvent. . The third essential step of the process for preparing the microspheres according to the invention consists in evaporating the volatile organic solvent that was used for the preparation of the solution of the polymer (s). In the particular case in which said solvent is ethyl acetate, this evaporation is carried out for approximately 12 hours at room temperature, under mechanical agitation (1,400 r.p.m.). The man of office will appropriately choose the different conditions of use of those three first essential steps of the process according to the present invention, depending on the physicochemical and morphological characteristics of the microspheres sought. In general, these microspheres will have an average diameter comprised between 1 μm and 100 μm, preferably between 5 μm and 50 μm, for their application as vectors in the pharmaceutical field.

In general, the microspheres obtained as a result of the third stage will be isolated by centrifugation, washed and eventually lyophilized.

According to a third aspect, the present invention also relates to pharmaceutical compositions, which contain the microspheres that have just been described. These compositions will generally be suitable for oral administration and will be presented, for example, in the form of tablets, capsules, powders or granules. The present invention will be illustrated below by means of the following non-limiting examples: In those examples, the following abbreviations have been used: OE: ethylene oxide POE: poly (oxyethylene) MM 2.1.2 .: methylidenemalonate that responds to the formula: O O Also called: 1 - e t ox i c a r bon i 1 - 1-ethoxycarbonylmethylene oxycarbonylethene. PMM 2.1.2 .: polymer consisting of recurring monomer units that respond to the formula: COOCH2CH3 CO- -O .CH, COOCH, CH3 On the other hand, in these examples: the size of the microspheres has been measured by the Coulter counter technique and the morphological examination performed in scanning electron microscopy, either on the raw microspheres of manufacture, or after the cryofracture; - The molecular mass of the polymers has been determined by gel permeation chromatography (CPG).

EXAMPLE 1 100 mg of methylidene malonate 2.1.2 are dissolved. in 10 ml of acetone under magnetic stirring. The polymerization is maintained for 5 minutes, then 100 microliters of 0.1 N HCl are added, always under magnetic stirring. The acetone is evaporated completely under vacuum. The polymer obtained is then washed with the aid of approximately 100 ml of distilled water and then dried under vacuum. The molecular mass of that polymer is 30,000. 280 mg of polymethylidene malonate are dissolved in 10 ml of ethyl acetate. 1 ml of aqueous phase containing 60 mg of ovalbumin is emulsified in the organic phase under stirring with the aid of an Ultraturrax at a rate of 13,500 rpm. for 5 minutes. This emulsion is then added to 100 ml of an aqueous solution of polyvinyl alcohol at 2%, stirring with the aid of an Ultraturrax at a speed of 8,000 r.p.m. for 5 minutes. The evaporation of the ethyl acetate is carried out at room temperature overnight, under mechanical agitation (rotating vane) at a speed of 1,400 rpm. The microspheres are collected by centrifugation at 4,000 r.p.m. for 10 minutes, then washed 6 times with distilled water and each time they undergo a new centrifugation. After the last centrifugation, the microspheres are resuspended in a volume of 3 ml of distilled water and then lyophilized. The microspheres thus obtained have an average diameter of 6 microns and 14.2% of the ovalbumin used in the preparation is encapsulated in the microspheres of PPM 2.1.2, which corresponds to an encapsulation of 2.5% (w / w). This preparation is administered orally to C3H mice in a dose of 100 micrograms of encapsulated ovalbumin (per mouse per day) for 5 consecutive days. The last forced feeding takes place 7 days before the sensitization of the animals to ovalbumin, which is carried out by subcutaneous injection of free ovalbumin (100 micrograms per mouse) on days DO and D14. 90% of the mice survive the second ovalbumin injection while less than 30% of the mice forced to eat the microspheres without ovalbumin or with the same dose of unencapsulated ovalbumin survive.

EXAMPLE 2 Proceed according to example 1 but add Pluronic F 68 in the aqueous phase containing the ovalbumin in a concentration of 2%.

EXAMPLE 3 The procedure is as in Example 1, but 20 bg of POE-PMM copolymer is added to the organic phase containing the polymer. In this example, a block copolymer POE-PMM 2.1.2 has been used. That copolymer has been obtained by successive polymerization of the two monomers beginning with the preparation of the POE block, by use of the following experimental protocol. The reactor in which the polymerization has been carried out (250 ml) is connected to a vacuum ramp that allows to work with high vacuum and free of protic impurities. The solvent (THF, 150 ml) purified from any trace of moisture is cryodistillated in the reactor at -70 ° C.

Then add the initiator (potassium terbutanolate (0.1 N / THF), 10 ml) with the help of a syringe through a septum. Ethylene oxide (5 g) is then introduced by cryo-distillation. The polymerization is carried out at room temperature for 48 hours. After this time, a sample allows to control by gel permeation chromatography, the molar mass (4,000 g / mol and the polymolecularity index (1.13) of the first sequence.) Then the MM 2.1.2 (0.5 ml) is added. recently degassed to remove the S02 used as a polymerization inhibitor, quickly and once at room temperature, after 5 hours the copolymer is deactivated by the addition of methanol and precipitated in diethyl ether. 2.1.2 to the POE, which corresponds to a molar mass for the PMM 2.1.2 of 1, 150 g / mol The thermal analysis of the copolymer reveals a glassy transition temperature of -16 ° C as well as a peak fusion temperature of 45 ° C (? H = 117 D / g).

EXAMPLE 4 The procedure described in Example 1 is carried out, but ovalbumin (60 mg) is replaced by 2 g of peptide V3 28 of the BRU loop of HIV gp 120 (sequence NNTRKSIHI GPGRAFYATGDIIGDIRQA). The obtained microspheres have an average size of 5.8 microns and 70% of the peptide V3 28 used are encapsulated in the microspheres which corresponds to an encapsulation of 0.48% w / w. The study carried out in scanning electron microscopy reveals smooth and spherical particles.

EXAMPLE 5 The procedure is as in Example 4, but Pluronic F 68 is added in the aqueous phase containing the peptide in the 2% concentration. The microspheres obtained have a size of 7.0 microns and 70% of the peptide V3 28 used are encapsulated in the microspheres.

EXAMPLE 6 The procedure is as in Example 4, but 20 mg of POE-PMM copolymer are added in the aqueous phase containing the peptide.

EXAMPLE 7 The procedure is as in Example 4, but 20 mg of POE-PMM copolymer is added to the organic phase containing the polymer.

EXAMPLE 8 The procedure is according to Example 1, but the internal aqueous phase is constituted by 1 ml of 0.5 M acetic acid containing 3 mg of type II collagen. The microspheres thus obtained have an average diameter of 6 microns and 66.6% of the collagen used in the preparation are encapsulated in the microspheres of PMM 2.1.2, which corresponds to an encapsulation of 0.7% (w / w).

EXAMPLE 9 The procedure is as in Example 1, but the internal aqueous phase consists of 1 ml of distilled water. The microspheres thus obtained do not contain biologically active substance. Its average diameter is 7.0 microns.

EXAMPLE 10 The procedure is carried out according to the technique described in Example 1, but the ovalbumin (60 mg) is replaced by the plasmid pCDNA3 (5 mg). The obtained microspheres have an average size of 7 μm and 9.8% of plasmid used are encapsulated in the microspheres which corresponds to an encapsulation of 0.17% (w / w). The study carried out in scanning electron microscopy reveals smooth and spherical particles.

EXAMPLE 11 The procedure is as in Example 10, but Pluronic® is added in the aqueous phase containing the plasmid, in the concentration of 2%. The microspheres thus obtained have an average diameter of 7 μm and 12% of the plasmid used are encapsulated in the microspheres which corresponds to an encapsulation of 0.22% (w / w).

EXAMPLE 12 The procedure is carried out according to the technique described in Example 1, but the ovalbumin (60 mg) is replaced by the oligonucleotide (pdT16) (2 mg). The obtained microspheres have an average size of 4.8 μm and 20.6% of oligonucleotide used is encapsulated in the microspheres which corresponds to an encapsulation of 0.19% (w / w).

EXAMPLE 13 The procedure is as in Example 12, but Pluronic® is added at the concentration of 2% in the phase containing the oligonucleotide. The obtained microspheres have an average diameter of 5.7 μm and 23% of oligonucleotide used are encapsulated in the microspheres which corresponds to an encapsulation of 0.21% (w / w).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. Microspheres consisting of a continuous network of a support material in which a substance is optionally dispersed, characterized in that the network is dense or incorporates a dispersed aqueous phase and in that the support material contains at least 70% by weight of a homopolymer consisting of recurring units that respond to the following general formula (I):

COORi

CH,

COO (CH2) n COOR, wherein: - Rx represents an alkyl group having 1 to 6 carbon atoms or a group (CH2) m-COOR3 in which m is an integer between 1 and 5 and R3 represents an alkyl group having 1 to 6 carbon atoms; - R2 represents an alkyl group having from 1 to 6 carbon atoms; and - n is an integer between 1 and 5. 2. Microspheres according to claim 1, characterized in that the aforementioned homopolymer is constituted by recurring units corresponding to the general formula (I) in which: Rx represents a group alkyl having from 1 to 6 carbon atoms; R2 represents an alkyl group having from 1 to 6 carbon atoms; and n is a number equal to 1. 3. Microspheres according to claim 1, characterized in that the aforementioned homopolymer consists of recurring units corresponding to the general formula (I) in which Rx and R2 represent a group CH2-CH3. 4. Microspheres according to any of claims 1 to 3, characterized in that the support material contains: - 90% 99.5% by weight of a homopolymer as defined in claim 1, 2 or 3; and - from 0.5% to 10% by weight of a copolymer comprising at least one sequence having a hydrophilic character and at least one sequence having a hydrophobic character, the hydrophobic character sequence comprises at least one recurring unit that responds to the general formula (I).

5. Microspheres. according to claim 1, characterized in that the hydrophilic character sequence of the aforementioned copolymer is chosen from a poly (oxyethylene), a poly (vinyl alcohol), a poly (vinylpyrrolidone), a poly (N-2-hydroxypropyl methacrylamide) , a poly (hydroxyethyl methacrylate), a hydrophilic poly (amino acid) such as a polylysine, a polysaccharide.

6. Microspheres according to claim 1, characterized in that the copolymer has a block structure, preferably diblock or triblock, or an inserted structure.

7. Microspheres according to any of claims 1 to 3, characterized in that a substance is effectively dispersed in the support material, optionally the substance is biologically active.

8. Microspheres according to any of claims 1 to 7, characterized in that the dispersed substance is a peptide.

9. Microspheres according to any of claims 1 to 8, characterized in that the dispersed substance is a protein. Process for preparing microspheres according to any of claims 1 to 9, which contains a network incorporating a dispersed aqueous phase, characterized in that it comprises: a) the preparation of a first solution of the above polymer (s) (s) constituting the support material, in a volatile organic solvent optionally containing a surfactant, b) preparing a second solution not miscible with the solution obtained in a), optionally containing the substance to be dispersed and optionally a surfactant, c) the preparation of a primary emulsion by dispersion of the second solution in the first solution, the continuous phase is constituted by the polymer solution (s), d) the preparation of a secondary emulsion: either dispersing, under agitation, the primary emulsion obtained in c) in a dispersion medium not miscible with said primary emulsion, the dispersion medium optionally contains a stizing agent; - by pouring under stirring, in the primary emulsion, a solution constituted of a medium not miscible with the primary emulsion, the medium optionally contains a stabilizing agent; e) evaporation of said organic solvent under agitation. 11. Method according to claim 10, characterized in that it further includes a step e ') of displacement of the organic solvent, step e') is carried out between step d) and step e). Method according to claim 10, characterized in that it further comprises: f) the isolation of the microspheres by centrifugation g) one or more successive washes of the microspheres h) lyophilization of the microspheres. 13. Method according to any of claims 10 to 12, characterized in that the surfactant used for the preparation of the primary emulsion is chosen from poloxamers, polysorbates, polyvinyl alcohols and copolymers, as defined in any 14. Process according to any of claims 10 to 13, characterized in that the stabilizing agent used for the preparation of the secondary emulsion is a polyvinyl alcohol. 15. Pharmaceutical compositions intended for oral administration, characterized in that it contains microspheres as defined in any of claims 1 to 9, or obtained by using the method according to any of claims 10 to 14.