WO2014057203A1

WO2014057203A1 - Pharmaceutical compositions comprising capsules obtained by coacervation without the use of toxic cross-linking agents

Info

Publication number: WO2014057203A1
Application number: PCT/FR2013/052386
Authority: WO
Inventors: Jean-Noël OLLAGNIER
Original assignee: Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic
Priority date: 2012-10-09
Filing date: 2013-10-08
Publication date: 2014-04-17
Also published as: FR2996455A1

Abstract

The invention relates to a double-walled capsule comprising a lipophilic core surrounded by a first layer of polymer coacervate and a second layer comprising a hydrogel, characterised in that it contains no trace of cross-linking agents.

Description

Pharmaceutical compositions comprising capsules obtained by coacervation not using a toxic crosslinking agent

The present invention relates to new double-walled capsules obtained by coacervation without the use of crosslinking agent and their process of obtaining, as well as the use of these capsules for the preparation of pharmaceutical compositions.

Coacervation describes the phenomenon of desolvation of macromolecules, such as polymers, leading to phase separation within a solution. Simple coacervation refers to processes involving the desolvation of a single polymer by one of the following factors: lowering of temperature, addition of a non-solvent, addition of electrolytes, addition of a second incompatible polymer. When the simultaneous desolvation of two water-soluble polyelectrolytes bearing opposite charges is caused by a change in pH of the aqueous medium, it is called complex coacervation.

Complex coacervation is a well-known and industrialized encapsulation technique since the 1950s. It encapsulates ingredients that are insoluble in water. US Pat. No. 2,800,457 of July 23, 1957 describes, for example, the process for encapsulating oils in a coacervate of two organic polymers: gelatin and gum arabic. Canadian patent CA88263 of October 5, 1971 describes a similar process using an organic polymer and a mineral polymer.

The method of manufacturing microcapsules by this technique generally takes place in five successive steps. In a first step, the product to be encapsulated (in liquid or solid form, pure or in oily solution) is dispersed in an aqueous solution containing two polymers of opposite charges (step a). In a second step, the coacervation is induced by an adjustment of the pH of the solution, so that the positive charges of the first polymer balance the negative charges of the second (step b). The electrostatic attraction of the two polyelectrolytes causes the appearance of a mixed coacervate. In a third step, the formed coacervate droplets are adsorbed (step c) on the surface of the active ingredient to be encapsulated and form a continuous coating (step d). In the fourth step, this coating is consolidated by crosslinking (step e) of the macromolecules constituting the coacervate to form stable microcapsules. Finally, the microcapsules are separated from the reaction medium by decantation and filtration, before undergoing washing or purification operations to remove the unreacted products, in particular the excess of crosslinking agents, and optionally drying.

Among the macromolecules or organic polymers of cationic nature that can be used in the coacervation technology, mention may be made, without limitation, of animal proteins such as pork or fish gelatin, albumin, plant proteins derived for example from soybean, potato or wheat, chitosan and its derivatives, synthetic polymers derived from the combination of amino acids such as polylysine or polymers of plant origin such as guar gum and its derivatives.

Among the anionic organic polymers that can be used are natural polymers such as gum arabic, alginates, carrageenates, cellulose derivatives such as carboxymethyl cellulose, starch derivatives such as carboxymethyl starch, or synthetic polymers. such as acrylic, methacrylic, polylactic, polyglycolic polymers or combinations thereof.

The encapsulated ingredients may be cosmetic active ingredients such as sunscreens, essential oils, vitamins A, D, E or their derivatives, lipoamino acids or even pharmaceuticals.

To obtain sufficiently resistant capsules mechanically, the crosslinking of step (e) is essential. This operation involves a crosslinking agent. Among the most effective crosslinking agents include formaldehyde or glutaraldehyde. Other crosslinkers have also been proposed, such as carbodiimides, isocyanates (HDI or Hexamethylene diisocyanate, TDI or Toluene Di Isocyanate, IPDI or Isopropyl Di Isocyanate), proanthocyanidines ... All these crosslinkers have either a significant toxicity or are unstable in an aqueous medium and must be used under conditions that complicate the crosslinking step. Other authors have described cross-linking methods with enzymes such as transglutaminases or genepine. The costs of these products are such that only a few applications with very high added value can be envisaged. A crosslinking agent is a chemical compound that makes it possible to link one polymer chain to another by forming covalent bonds. In the state of the art, it is in particular a reaction between the aldehyde functions of the crosslinking agent and the residual amine functions of the proteins, in particular with the amine functions of the lysine units to form covalent bonds -N = CH- .

Glutaraldehyde is the most commonly used crosslinking agent. It is efficient and inexpensive. But it must be used at high doses, close to 1 to 5 mol / kg of gelatin (ie 100 to 500 g / kg of protein) and has a significant toxicity for both the manipulator and the user. The elimination of excess glutaraldehyde is essential, especially for all pharmaceutical, food or cosmetic applications; it requires many successive washes that consume water and time to obtain microcapsules containing an acceptable residual glutaraldehyde, less than a hundred ppm.

A first problem to be solved for the inventors of the present invention is therefore to produce sufficiently resistant microcapsules by a complex coacervation technique which does not use a crosslinking agent.

Given the very principle of the coacervation process, only water-insoluble lipophilic active agents can be incorporated in the microcapsules obtained by this technology. This undoubtedly constitutes a limitation of the process whereas a large number of water-soluble ingredients must also be incorporated in microcapsules.

An object of the invention is therefore to develop an improved coacervation technique, without the use of toxic or expensive crosslinkers, and to encapsulate both soluble and insoluble ingredients in water.

To encapsulate water-soluble ingredients, other techniques have been described such as, for example, granulation by means of hydrophilic polymers, emulsification in oils in the form of water-in-oil emulsions, absorption on resins. ion exchangers to form resinates. One of the most widely used technologies for encapsulating water-soluble ingredients is the incorporation into microbeads of water-soluble polymers such as chitosans, polyvinyl alcohols or alginates. These are implemented in many applications pharmaceutical or food to obtain microbeads by a simple coacervation process, also called dripping. By way of example, a description of such a method of cell encapsulation can be found in the patent application WO91091 19 of 27 June 1991.

Dripping consists of preparing an aqueous solution containing the water-soluble ingredient to be encapsulated and a polymer such as sodium alginate. This solution is pressurized through nozzles calibrated to form drops collected in an aqueous solution of divalent salts such as calcium chloride, magnesium or manganese. Calcium ions react with sodium alginate to form solid, insoluble, calcium alginate beads immediately. The beads obtained are separated by filtration or sieving and then generally washed with water to remove the excess calcium chloride.

In this context, the inventors of the present invention have developed new double-walled capsules that may contain a lipophilic active ingredient in the primary capsules and optionally a hydrophilic active agent included in the second with a coacervation method without chemical crosslinking.

These original microcapsules consist of a lipophilic core surrounded by a first layer of polymeric coacervate and a second layer comprising a hydrogel. They have good tensile strength at break and are particularly distinguished by their non-toxicity since no toxic crosslinking agent is used. They are also flexible since it is conceivable to encapsulate both a lipophilic active in the heart and a hydrophilic active in a hydrogel matrix. They can be presented either in wet form or in dry form with a reasonable cost price.

Thus, the subject of the invention is a method for preparing double-walled capsules comprising the following steps:

step a) dispersing a lipophilic active principle in an aqueous solution, said solution containing at least one anionic polymer and at least one cationic polymer;

step b) adjusting the pH of the solution obtained in step a) so that the positive charges of the cationic polymer balance the negative charges of the anionic polymer in order to induce coacervation;

step c) adsorbing the coacervate droplets from step b) on the surface of the active ingredient to form capsules; step d) introducing a solution of anionic polymers into the reaction medium containing the capsules obtained in step c);

step e) introducing the mixture from step d) into a means for forming drops;

step f) mixing the drops from step e) with a solution of divalent salts and forming the double-walled capsules;

characterized in that no crosslinking agent is used and that the active pharmaceutical ingredient is selected from proteins, glycoproteins, proteins covalently bound to one or more polyalkylene glycol chains, preferably polyethylene glycol or PEG, peptides, polysaccharides, liposaccharides, oligonucleotides, polynucleotides, synthetic pharmaceutical substances and mixtures thereof, taxanes and their derivatives, taxols and their derivatives, retinoids, flavonoids, alkaloids, steroids, polyphenols, antioxidants, anti-inflammatory agents, erythropoietins, growth factors, blood factors, hemoglobin, cytochromes, prolactin albumins, luliberin or luteinizing hormone releasing hormone (or LHRH) or the like, such as leuprolide , gosereline, triptoreline, busereline, nafareline, LHRH antagonists, con LHRH currents, human growth hormones (GH), porcine or bovine hormones, growth hormone releasing hormone, insulin, somatostatin, interleukins or their mixtures, interferons, such as interferon alpha, alpha-2b, beta, betaine, or gamma, gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, thyrotropin (TRH), tumor necrosis factor (TNF), nerve growth factor (NGF), growth factors such as beclapermine, trafermine, ancestim, keratinocyte growth factor, stimulating factor granulocyte colonies (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) heparinase, bone morphogenetic protein (BMP), l hANP, glucagon-like peptide (GLP-I), VEG -F, recombinant hepatitis B antigen (rHBsAg), renin, cytokines, cyclosporins and synthetic analogs, pharmaceutically active modifications and fragments of enzymes, cytokines, antibodies, antigens and vaccines, antibodies, polysaccharides as for example, heparin, oligo- or polynucleotides, DNA, RNA, RNA, antibiotics, psychotropic agents, one or more molecules for therapeutic purposes, such as antibacterials, antiparasitics, antihelminthics, including cestocides, nematocytes, fasciolicides; tenicides, anti-coccidials and anti-cryptosporidials, antiparamphistomas, antiprotozoans, anti-mycotic agents, especially against actinomycosis and candidiasis; anti-inflammatories, antiallergics and immunomodulators; central analgesics, and antihistamines.

According to other particular aspects, the subject of the invention is:

- A process as described above, characterized in that the anionic polymer is chosen from natural polymers such as gum arabic, alginates, carrageenates, cellulose derivatives such as carboxymethyl cellulose, starch derivatives such as carboxymethyl starch, or synthetic polymers such as acrylic, methacrylic, polylactic, polyglycolic polymers or combinations thereof.

- A process as described above, characterized in that the cationic polymer is chosen from animal proteins such as pork or fish gelatin, albumin, vegetable proteins derived for example from soya, potato or wheat, chitosan and its derivatives, synthetic polymers derived from the combination of amino acids such as polylysine or polymers of plant origin such as guar gum and its derivatives.

- A process as described above, characterized in that the solution of anionic polymers of step d) is a solution of sodium alginate.

- A method as described above, characterized in that said means for forming the drops implemented in step e) is a nozzle or a needle.

- A process as described above, characterized in that the divalent salt solution of step f) is selected from solutions of calcium chloride, barium chloride, manganese chloride.

- A method as described above, characterized in that the solution of anionic polymers of step d) contains an active ingredient to be encapsulated, hydrophilic; and optionally at least one additive selected from insoluble insoluble solids of mineral nature, such as, for example, silicas, laponites, alumino-silicates, titanium dioxide, calcium sulphate or of organic nature, such as micronized waxes such as the wax of carnauba or beeswax, cationic polymers such as chitosan or polylysine, stearic acid or its micronized derivatives, microcrystalline cellulose, starches.

A process as described above comprising the step g) of filtration of the capsules obtained in step f); optionally a step h) washing with water; and optionally a drying step.

The capsules may finally be dried by any drying method known to those skilled in the art, such as in an oven, a lyophilizer or a fluidized bed. They can also be resuspended in a suitable solution to be stored, transported and used in liquid form.

The invention also relates to a double-walled capsule comprising a lipophilic core surrounded by a first layer of polymeric coacervate and a second layer comprising a hydrogel characterized in that it contains no trace of crosslinking agent.

According to other particular aspects, the subject of the invention is:

- A capsule as described above characterized in that the lipophilic core comprises an active ingredient selected from proteins, glycoproteins, proteins covalently bound to one or more polyalkylene glycol chains, preferably polyethylene glycol or PEG, peptides, polysaccharides, liposaccharides, oligonucleotides, polynucleotides, synthetic pharmaceutical substances and mixtures thereof, taxanes and their derivatives, taxols and their derivatives, retinoids, flavonoids, alkaloids, steroids, polyphenols, antioxidants antiinflammatory agents, erythropoietins, growth factors, blood factors, hemoglobin, cytochromes, prolactin albumin, luliberin or luteinizing hormone releasing hormone (or LHRH) or the like, such as leuprolide, goserelin, triptoreline, busereline, nafareline, LHRH antagonists, competitors of LHRH, human growth hormones (GH), porcine or bovine hormones, growth hormone releasing hormone, insulin, somatostatin, interleukins or their mixtures, interferons, such as interferon alpha, alpha-2b, beta, betaine, or gamma, gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, thyrotropin (TRH), tumor necrosis factor (TNF), nerve growth factor (NGF), growth factors such as beclapermine, trafermine, ancestime, keratinocyte growth factor, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM- CSF), macrophage colony stimulating factor (M-CSF) heparinase, bone morphogenetic protein (BMP), hANP, glucagon-like peptide (GLP-I), VEG-F, recombinant hepatitis B antigen (rHBsAg), renin, cytokines, cyclosporins and synthetic analogues, pharmaceutically active modifications and fragments of enzymes, cytokines, antibodies, antigens and vaccines, antibodies polysaccharides such as, for example, heparin, oligo- or polynucleotides, DNA, RNA, RNA, antibiotics, psychotropic agents, one or more molecules for therapeutic purposes, such as antibacterials, antiparasitic agents, antihelminthics, whose cestocides, nemato of, fasciolicides; tenicides, anti-coccidials and anti-cryptosporidials, antiparamphistomas, antiprotozoans, anti-mycotic agents, especially against actinomycosis and candidiasis; anti-inflammatory, anti-allergic and immunomodulatory agents; central analgesics, and antihistamines.

- A capsule as described above, characterized in that the hydrogel comprises a pharmaceutical hydrophilic active ingredient.

- A capsule as described above, having a diameter of between 100 μιτι and 3000 μιτι and preferably between 500 μιτι and 2000 μιτι.

- A capsule as described above, obtainable by the method as defined above.

- A capsule as described above characterized in that it comprises from 0.5% to 40% by weight of lipophilic active agent, more particularly from 1% to 30%, and even more particularly from 1% to 20% from 0% to 20% by weight of hydrophilic active agent, more particularly from 0.5% to 10% and even more particularly from 0.5% to 5%, from 0.1% to 5% by weight of anionic polymer more particularly from 0.5% to 5%, and even more particularly from 1% to 3%.

The invention finally relates to the use of at least one capsule as defined above for the preparation of a pharmaceutical composition. As well as a pharmaceutical composition comprising from 0.01% to 20% by weight, more particularly from 1% to 10% by weight of at least one capsule according to the invention. The encapsulated ingredients may be pharmaceutical active ingredients such as substances of therapeutic, prophylactic or imaging interest.

In the field of digestive and hepatology, we can mention:

- antiulcer and anti-emetic,

- anti-diarrheal and antispasmodic,

- digestive enzymes and flora, laxatives, purgatives,

- the meteorifers,

- modifiers and regulators of liver function.

In the field of cardiology and angiology, we can mention:

vasodilators,

- Vascular active principles.

In the field of the immune system, there may be mentioned the active ingredients used in immunotherapy, in the supply of immunoglobulin, interferons, and immunomodulators.

In the field of metabolism or nutrition, there may be mentioned the active ingredients used for the supply of anabolic, anti-anemic, anti-ketonic, oral rehydration agents.

In the field of metabolism, mention may be made of the active ingredients used for the supply of mineral salts, in particular calcium, magnesium or phosphorus salts, in the form in particular of calcium glycerophosphate, calcium gluconate, manganese gluconate.

In the field of lipotropic factors and diseases of the liver and kidneys, the products are used for the supply of vitamins and trace elements.

In the field of hormonology, endocrinology and reproduction, there may be mentioned the active ingredients used for the delivery of fertility hormones, anti-hyperthyroid drugs, anti-galactogens, abortives, hormone inhibitors.

In the field of the muscles and the musculoskeletal system, mention may be made of the active principles used for the supply of anti-rheumatic drugs, anti-osteoarthritis drugs, muscle stimulants and the musculoskeletal system, anti-myopathic molecules.

In the field of the respiratory system, there may be mentioned the active ingredients used for the supply of respiratory analeptics, bronchodilators, mucoregulators and expectorants, antitussives, anti-infectives. In the field of the urinary system, there may be mentioned the active ingredients used for the intake of diuretics, urinary acidifiers, urinary antispasmodics.

In the field of the neurological system, mention may be made of the active principles used for the supply of general anesthetics, analgesics, anticonvulsants, sedatives and tranquilizers.

We can cite more particularly:

- the proteins,

glycoproteins,

proteins which are covalently bound to one or more polyalkylene glycol chains, preferably polyethylene glycol or PEG chains,

peptides,

polysaccharides,

liposaccharides,

the oligonucleotides,

polynucleotides,

- synthetic pharmaceutical substances and mixtures thereof,

- taxanes and their derivatives, taxols and their derivatives

- retinoids

- flavonoids,

alkaloids,

- steroids,

- polyphenols

the antioxidants,

anti-inflammatory agents,

erythropoietins,

- growth factors,

- blood factors,

- hemoglobin,

- cytochromes,

prolactin albumins, luliberin or luteinizing hormone releasing hormone (or LHRH) or the like, such as leuprolide, goserelin, triptorelin, buserelin, nafareline, LHRH antagonists, LHRH competitors, - human growth hormones (GH), porcine or bovine,

- the growth hormone releasing hormone,

- insulin,

somatostatin,

interleukins or their mixtures,

interferons, such as interferon alpha, alpha-2b, beta, beta-la, or gamma,

- gastrin,

- tetragastrin,

- pentagastrin,

- urogastrone,

- secretin,

calcitonin,

- enkephalins,

- endomorphins,

angiotensins,

the thyrotropin releasing factor (TRH),

tumor necrosis factor (TNF),

nerve growth factor (NGF),

growth factors such as beclapermine, trafermine, ancestim,

the growth factor of keratinocytes,

granulocyte colony stimulating factor (G-CSF),

Granulocyte macrophage colony stimulating factor (GM-CSF),

macrophage colony stimulating factor (M-CSF),

heparinase,

the morphogenic protein of bone (BMP),

- the hANP,

glucagon-like peptide (GLP-I), VEG-F,

recombinant hepatitis B antigen (rHBsAg),

- renin,

cytokines,

cyclosporins and synthetic analogues, pharmaceutically active modifications and fragments of enzymes, cytokines, antibodies, antigens and vaccines,

the antibodies,

polysaccharides, for example heparin,

the oligo- or polynucleotides, DNA, RNA, iRNA,

- antibiotics,

- psychotropic agents,

one or more molecules for therapeutic purposes, such as:

- antibacterials,

- antiparasitics,

- antihelminthics, including cestocides, nematocides, fasciolicides,

- the tenicides,

- anti-coccidians and anti-cryptosporidians,

- antiparamphistomas,

- the anti-protozoa,

anti-mycotic agents, especially against actinomycosis and candidiasis,

anti-inflammatories, antiallergics and immunomodulators,

central analgesics, and

- antihistamines.

Surprisingly, the non-crosslinked microcapsules obtained in step (d) are stable in the presence of the solution of added anionic polymers and do not break when steps (e) and (f) of the process according to the invention are carried out. invention. The lipophilic ingredient remains confined in the oily heart of the new microcapsule and the hydrophilic ingredient is encapsulated in the second alginate shell.

The anionic polymer solution implemented in step (e) may also contain technological additives intended to enhance the mechanical strength of the microbeads, to adjust their density or to modulate the kinetics of release of the hydrophilic ingredient. These additives may be finely divided insoluble solids of mineral nature, such as, for example, silicas, laponites, alumino-silicates, titanium dioxide, calcium sulphate or of organic nature such as micronized waxes (carnauba wax, waxes). bee ...), cationic polymers such as chitosan or polylysine, stearic acid or its micronized derivatives, microcrystalline cellulose, starches. The technological additives can also be soluble products such as mineral salts, glycols or surfactants allowing a better dispersion of the microcapsules or facilitating the drying operations.

The following examples describe an implementation of the process according to the invention and the microcapsules obtained.

EXAMPLE Ν

Protocol of double encapsulation by coacervation with potato protein and gum arabic then insertion into alginate / Laponite beads.

1.1 Preparation of microcapsules by coacervation

# Oil phase

0.1 g of red oil and 100 g of TCM oil are introduced into a beaker and stirred with a magnetic stirrer for 20 minutes at 40 ° C. Filter and let cool.

# Aqueous phase

15g of gum arabic are introduced into a beaker containing 400g of water. Stir with a magnetic stirrer until dissolution (5 minutes) then introduce the mixture into the reactor and shake at 200 revolutions per minute (rpm). # Incorporation of the oil phase

Increase agitation at 350 rpm from the reactor and slowly introduce the oil. Leave to shake for 15 minutes.

* Potato isolate

10 g of potato isolate and 225 g of water are introduced into a beaker and stirred with a magnetic stirrer. When the dissolution is complete (5 minutes), very slowly introduce the solution into the reactor by controlling the pH (about 4 after all the addition).

# Lowering pH

Decrease the pH of the medium to 3.65 with 10% acetic acid. So that coacervation is formed.

* Temperature increase

Increase the temperature to 50 ° C for 1 hour to harden the potato isolate. Then let cool and decant.

1.2 Preparation of microbeads by double encapsulation

1. Preparation of the alginate solution

4 g of alginate and 1 g of Laponite are slowly introduced into a beaker containing 200 g of water with vigorous stirring for 30 minutes.

2. Microcapsules / alginate mixture

Weigh 100 g of microcapsules obtained in step 1 .1 and introduce them into 150 g of a 2% solution of sodium alginate.

3. Formation of microbeads

Introduce the mixture into a syringe and make drops in the calcium chloride solution. Leave a contact time of 15 minutes. Finally, rinse with water.

1.3. Characterization of the microbeads obtained by the process.

The undried balls obtained by the process are colored spheres having an average size of 1000 μιτι.

They contain about 20% of an oily heart, 5% of a coacervate of gelatin / gum arabic, 1% of alginate, 0.5% of laponite, the rest being water.

When mechanical pressure is exerted on the microbeads, they burst while releasing red oil. EXAMPLE 2

Protocol of double encapsulation by coacervation with gelatin and gum arabic then insertion into alginate beads

2.1 Preparation of microcapsules by coacervation

fi Oil phase

0.1 g of red oil (used as a model lipophilic ingredient to be encapsulated) and 100 g of TCM oil are introduced into a beaker and stirred with a magnetic stirrer for 20 minutes at 40 ° C. Filter and leave at 40 ° C.

ϋ Aqueous phase

300 g of water are introduced into the reactor thermostated at 40 ° C.

12.5 g of gum arabic, 12.5 g of gelatin are introduced into a beaker containing 250 g of water. Stir at 40 ° C until dissolved (15 minutes). Then add the mixture to the reactor and shake at 200 rpm.

§1 Incorporation of the oil phase

Increase agitation to 350 rpm then slowly introduce hot oil. Leave to shake for 15 minutes.

Lowering pH

Stop the heating and reduce the pH of the medium to 4.10 with 10% acetic acid. So that coacervation is formed. Lowering the temperature

Reduce the temperature to 10 ° C to harden the gelatin. Leave for 15 minutes then stop stirring. Then let it settle.

2.2. Preparation of microbeads by double encapsulation

Preparation of the alginate solution

4 g of alginate and 1 g of Laponite are slowly introduced into a beaker containing 200 g of water with vigorous stirring for 30 minutes,

ϋ Mixture microcapsules / alginate (50/50)

Weigh 100g of microcapsules and introduce them into the same amount of the sodium alginate solution. Homogenize with magnetic stirring.

Introduce the mixture into a syringe and make drops in the calcium chloride solution. Leave a contact time of 15 minutes, then rinse with water.

2.3. Characterization of the microbeads obtained by the process.

The microbeads obtained are translucent, of average size approximately δθθμιτι. The primary microcapsules of oil and gelatin are clearly visible inside the microbeads.

The microbeads contain about 20% of an oily heart, 5% of a gelatin / gum arabic coacervate, 1% alginate, 0.5% laponite, the balance being water.

It has been verified that the microbeads according to the invention are stable and leakproof. For this, they were dispersed in a mineral oil and subjected to magnetic stirring for two hours. The coloring of the mineral oil was observed after two hours.

Any coloring of this oil reflects diffusion of the dye or rupture of the microcapsules. As a control, microcapsules made by the non-crosslinking coacervation method obtained as described in paragraph 2.1 and microbeads obtained by the same method but crosslinked with glutaraldehyde were subjected to the same test. Microcapsules Microcapsules Non-crosslinked microbeads crosslinked according to the invention glutaraldehyde

Bright Red stain No staining No oil after 2 hours staining

These results demonstrate the good stability of the microbeads according to the invention.

EXAMPLE No. 3

Protocol for double encapsulation of microcapsules (by coacervation with gelatin) in alginate beads.

3.1 Preparation of microcapsules by coacervation

The same protocol as that described in Example 2 is implemented.

3.2 Preparation of microbeads by double encapsulation

He Preparation of alginate solution and caffeine

4 g of alginate and 1 g of caffeine are slowly introduced into a beaker containing 200 g of water with vigorous stirring for 30 minutes,

ϋ Mixture microcapsules / alginates (50/50)

Weigh 100 g of microbeads obtained in step 3.1, and introduce the same amount of alginate at 2%. Mix to obtain a homogeneous suspension. Introduce the mixture into the tank of the NISCO VAR D drop generator equipped with a nozzle of diameter δΟΌμιτι and switch on the device with the following parameters:

Flow rate: 12ml / min, vibration frequency of the vibrating nozzle: 0.22khz, amplitude 79%.

The drops generated are collected in the calcium chloride solution where they form solid microbeads. Leave a contact time of 15 minutes, filter the beads and rinse with water.

3.3. Characterization of the microbeads obtained by the process

Microbeads with a mean diameter of 150Όμηη are obtained, containing two encapsulated ingredients: caffeine in the external matrix of alginate and red oil in the oily heart. Their quantitative composition is about 20% oil, 5% gelatine / gum arabic coacervate, 1% alginate, 0.5% caffeine, the balance being water.

Claims

1. A process for preparing double-walled capsules comprising the steps of:

step c) adsorbing the coacervate droplets from step b) on the surface of the active ingredient to form capsules;

step d) introducing a solution of anionic polymers into the reaction medium containing the capsules obtained in step c);

step e) introducing the mixture from step d) into a means for forming drops;

characterized in that no crosslinking agent is used and that the active pharmaceutical ingredient is selected from proteins, glycoproteins, proteins covalently bound to one or more polyalkylene glycol chains, preferably polyethylene glycol or PEG, peptides, polysaccharides, liposaccharides, oligonucleotides, polynucleotides, synthetic pharmaceutical substances and mixtures thereof, taxanes and their derivatives, taxols and their derivatives, retinoids, flavonoids, alkaloids, steroids, polyphenols, antioxidants, anti-inflammatory agents, erythropoietins, growth factors, blood factors, hemoglobin, cytochromes, prolactin albumins, luliberin or luteinizing hormone releasing hormone (or LHRH) or the like, such as leuprolide , gosereline, triptoreline, busereline, nafareline, antagonists of the LHRH, the concu rrents of LHRH, human growth hormones (GH), porcine or bovine, the growth hormone releasing hormone, insulin, somatostatin, interleukins or their mixtures, interferons, such as interferon alpha, alpha-2b, beta, betaine, or gamma, gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, thyrotropin releasing factor (TRH), tumor necrosis factor (TNF), nerve growth factor (NGF), growth factors such as beclapermine, trafermine, ancestim , keratinocyte growth factor, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) heparinase, bone morphogenetic protein (BMP), hANP, glucagon-like peptide (GLP-I), VEG-F, recombinant hepatitis B antigen (rHBsAg), renin, cytokines, cyclosporins and synthetic analogues, modifications and fragments pharmace utically active enzymes, cytokines, antibodies, antigens and vaccines, antibodies, polysaccharides such as, for example, heparin, oligo- or polynucleotides, DNA, RNA, iRNA, antibiotics, psychotropic agents, one or more molecules for therapeutic purposes, such as antibacterials, antiparasitics, antihelminthics, including cestocides, nematocytes, fasciolicides; tenicides, anti-coccidials and anti-cryptosporidials, antiparamphistomas, antiprotozoans, anti-mycotic agents, especially against actinomycosis and candidiasis; anti-inflammatory, anti-allergic and immunomodulatory agents; central analgesics, and antihistamines, characterized in that the anionic polymer is chosen from natural polymers such as gum arabic, alginates, carrageenates, cellulose derivatives such as carboxymethyl cellulose, starch derivatives such as carboxymethyl starch, or synthetic polymers such as acrylic, methacrylic, polylactic, polyglycolic polymers or combinations thereof.

2. Method according to claim 1, characterized in that the cationic polymer is chosen from animal proteins such as pork or fish gelatin, albumin, vegetable proteins derived for example from soybeans, potatoes or cereals. wheat, chitosan and its derivatives, synthetic polymers derived from the combination of amino acids such as polylysine or polymers of plant origin such as guar gum and its derivatives.

3. Method according to one of claims 1 to 2, characterized in that the solution of anionic polymers of step d) is a solution of sodium alginate.

4. Method according to one of claims 1 to 3, characterized in that said means for forming the drops implemented in step e) is a nozzle or a needle.

5. Method according to one of claims 1 to 4, characterized in that the divalent salt solution of step f) is selected from solutions of calcium chloride, barium chloride, manganese chloride.

6. Method according to one of claims 1 to 5, characterized in that the solution of anionic polymers of step d) contains an active ingredient to be encapsulated, hydrophilic.

7. Process according to claim 6, characterized in that the solution of anionic polymers of step d) additionally contains at least one additive chosen from insoluble solids which are finely divided and of a mineral nature, such as, for example, silicas or laponites. alumino-silicates, titanium dioxide, calcium sulphate or organic nature such as micronized waxes such as carnauba wax or beeswax, cationic polymers such as chitosan or polylysine, stearic acid or its micronized derivatives, microcrystalline cellulose, starches.

8. Method according to one of claims 1 to 7 comprising step g) filtration of the capsules obtained in step f).

9. The method of claim 8 comprising a step h) washing with water and a drying step.

10. Double-walled capsule comprising a lipophilic core surrounded by a first layer of polymeric coacervate and a second layer comprising a hydrogel characterized in that it contains no trace of crosslinking agent.

1 1. Capsule according to the preceding claim characterized in that the lipophilic core comprises an active ingredient selected from proteins, glycoproteins, proteins covalently bound to one or more polyalkylene glycol chains, preferably polyethylene glycol or PEG, peptides, polysaccharides, liposaccharides, oligonucleotides, polynucleotides, synthetic pharmaceutical substances and mixtures thereof, taxanes and their derivatives, taxols and their derivatives, retinoids, flavonoids, alkaloids, steroids, polyphenols, antioxidants, anti-inflammatory agents , erythropoietins, growth factors, blood factors, hemoglobin, cytochromes, prolactin albumin, luliberin or luteinizing hormone releasing hormone (or LHRH) or the like, such as leuprolide, goserelin, triptorelin, buserelin, nafareline, LHRH antagonists, l competitors of LHRH, human growth hormones (GH), porcine or bovine hormones, growth hormone releasing hormone, insulin, somatostatin, interleukins or their mixtures, interferons, such as interferon alpha, alpha-2b, beta, betaine, or gamma, gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, thyrotropin (TRH), tumor necrosis factor (TNF), nerve growth factor (NGF), growth factors such as beclapermine, trafermine, ancestim, keratinocyte growth factor, stimulating factor granulocyte colonies (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) heparinase, bone morphogenetic protein (BMP), hANP, the glucagon-like peptide (GLP-I), V EG-F, recombinant hepatitis B antigen (rHBsAg), renin, cytokines, cyclosporins and synthetic analogs, pharmaceutically active modifications and fragments of enzymes, cytokines, antibodies, antigens and vaccines, antibodies, polysaccharides such as, for example, heparin, oligo- or polynucleotides, DNA, RNA, RNA, antibiotics, psychotropic agents, one or more therapeutically oriented molecules, such as antibacterials, antiparasitics, antihelminthics, including cestocides, nematocytes, fasciolicides; tenicides, anti-coccidials and anti-cryptosporidials, antiparamphistomas, antiprotozoans, anti-mycotic agents, especially against actinomycosis and candidiasis, anti-inflammatory, anti-allergic and immunomodulatory agents; central analgesics, and antihistamines.

12. Capsule according to claim 10 or 1 1, characterized in that the hydrogel comprises a pharmaceutical hydrophilic active ingredient.

13. Capsule according to one of claims 10 to 12 having a diameter between 100 μιτι and 3000 μιτι and preferably between 500 μιτι and 2000 μιτι.

14. Capsule according to one of claims 10 to 13 obtainable by the method as defined in one of claims 1 to 8.

15. Capsule according to one of claims 10 to 14 characterized in that it comprises from 0.5% to 40% by weight of lipophilic active agent, more particularly from 1% to 30%, and more particularly 1% at 20%, from 0% to 20% by weight of hydrophilic active agent, more particularly from 0.5% to 10% and even more particularly from 0.5% to 5%, from 0.1% to 5% by weight anionic polymer, more particularly from 0.5% to 5%, and even more particularly from 1% to 3%.

16. Use of at least one capsule as defined in one of claims 10 to 15 for the preparation of a pharmaceutical composition.

17. A pharmaceutical composition comprising from 0.01% to 20% by weight, more particularly from 1% to 10% by weight of at least one capsule as defined in one of claims 10 to 15.