ES2358703A1 - Use of microparticles for use as vaccines and the liberation of biologically active molecules. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

The invention relates to microparticles based on a biodegradable polymer of plga type and an alginate polymer encapsulating peptides or immunologically active/active proteins. Said microparticles, as well as the pharmaceutical compositions derived therefrom and their uses, have application in the field of human and animal health as a vaccine and as a release system for biologically active molecules. (Machine-translation by Google Translate, not legally binding)

Description

Use of microparticles for use as vaccines and the release of biologically active molecules.

Field of the Invention

The invention relates to microparticles a base of a biodegradable polymer of the PLGA type and a polymer of alginate that encapsulate peptides or proteins immunologically active / active. Said microparticles, as well as the compositions pharmaceuticals derived from them and their uses, have application in the field of human and animal health as a vaccine and as a system of release of biologically active molecules.

Background

The development of effective vaccines against infectious diseases represents a unique challenge for the community scientific We must not forget, however, that it is necessary use new adjuvants and release systems that improve the immunogenic capacity of the antigens.

Vaccines (from Latin vaccinus-a-um , beef; from vacca-ae , cow) are antigen preparations that once inside the body cause an attack response, called an antibody (which eliminates the antigen). This response generates immunological memory, producing, in most cases, permanent immunity against the disease.

In general, there are considered to be four types Traditional vaccines:

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Inactivated : harmful microorganisms that have been treated with chemicals or heat and have lost their danger.

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Live attenuated : microorganisms that have been cultivated under specific conditions so that they lose their harmful properties.

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Toxoids : they are inactivated toxic components from microorganisms, in cases where those components are those that cause the disease instead of the microorganism itself.

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Subunit : they are fragments of a microorganism that can generate an immune response.

Immune adjuvants were originally described as substances that used in combination with a given antigen produce a more intense immune response than when the antigen is administered alone. The only adjuvant approved for use in humans is aluminum salts, generically referred to as Alum; This substance has a good safety profile but several studies conducted with different vaccines of a protein nature show that its adjuvant capacity is quite limited and it is also a fact that the induced response is fundamentally humoral, that is Th2 (Gupta et al . 1998, Adv. Drug Del, Rev. 32: 155-172). In addition, Alum is not effective in inducing an immunological response at the mucosal level. Finally it has been observed that it can also induce IgE, which has been associated with allergic reactions.

Several studies have shown that PLGA microparticles represent an interesting strategy as a vehicle for vaccine administration, since they control the release rate of the encapsulated antigen, which allows simulating repeated injections of conventional calendars with a single dose of immunization (Men et al . 1996, Vaccine 14: 1442-1450; Igartua et al . 1998, J. Control. Rel. 56: 63-73).

On the other hand, the microparticles confer antigen stability, protecting it against hydrolysis enzymatic and gastric acid pH, and allowing the presence of intact antigen at the place of deposit, as well as its administration Mucosal pathways. Finally, antigen particle favors its uptake by antigen presenting cells (APCs) such as dendritic cells, from which starts the immune response. Immunization studies carried out with PLGA microparticles have shown that they are able to get a good antibody response, activity T-cytotoxic cell (CTL), and induce responses Th1 / Th2 mixed.

On the other hand are the alginates. Alginates they are natural copolymers of polysaccharides constituted by the manuronic and glucuronic acids. Although synthetic polymers outperform the natives in purity and reproducibility, the Ultrapurification of alginates gives them low toxicity, being considered biocompatible. In addition to the important role that alginates represent in the textile and food industry, their similarity with the extracellular matrix (ECM) along with its biodegradability and biocompatibility allows them to be widely used as biomaterials in tissue engineering, cell therapy by immobilizing them and as systems of controlled release of various therapeutic agents. Like For the PLGA, numerous works have described the recurrence of alginates as polymeric matrices for microencapsulation of peptides and proteins Examples of these works are included in the European patent EP1079811.

The latest trends in the development of vaccines indicate that the uptake of antigens by the dendritic cells is more efficient when the characteristics Surface microparticular systems are designed to according to the so-called molecular patterns associated with pathogens which are sequences of small molecules that usually have surface pathogens such as lipopolysaccharide bacterial (LPS), peptidoglycans, nucleic acids, CpG sequences and RGD. RGD sequences are amino acid sequences (arginine, glycine and aspartic acid) that act as specific receptors for integrins and are capable of inducing phagocytosis mediated by receivers

At present there is a wide range of synthetic peptide or protein antigens that they have as a limitation their low immunogenicity, being necessary its joint administration with adjuvant substances and in a way repeated to obtain an effective immune response.

In recent decades microparticles biodegradable polymers have become a promising peptide and protein antigen release system for Obtain vaccines against various infectious diseases:

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 WO9511010 describes methods and compositions for encapsulation of antigens in PLGA microspheres for use as vaccines.

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 The document ES2190350A1 describes a vaccine composition against brucellosis that comprises polyecaprolactone microparticles as adjuvant

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 WO00 / 50050 describes multiparticular non-parenteral polymer formulations as a prophylactic therapeutic agent release system and Diagnostics through mucous membranes.

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The Jin document, Xiao Bing; et al . Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2. Biomaterials 2007, Vol. 28 (19), 2994-3003, presents alginate microspheres embedded in PLGA films for application in tissue engineering.

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The document Liu, Xiaohua; et al . Novel polymeric microspheres containing norcantharidin for chemoembolization. J. Control. Rel. 2006, Vol. 116 (1), 35-41, describes microspheres of PLGA-alginate for application in chemoembolization for the treatment of tumors.

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The Wang, Ye document; et al . Biodegradable and complexed microspheres used for sustained delivery and activity protection of SOD. J. Biomed. Mat. Res., Part B: Appl. Biomat 2006, Vol. 79B (1), 74-78, shows microspheres formed by at least one biodegradable polymer (PLGA; alginate or chitosan) and a protein (superoxide dismutase).

Compendium of the invention

The present invention also contemplates the possibility of obtaining microparticles comprising alginate and PLGA Thus, in one aspect, the invention relates to a procedure for the preparation of a microparticle that understand the stages of

to.

prepare a solution of PLGA in a organic solvent,

b.

prepare an aqueous solution of alginate,

C.

emulsify the solutions with the immunologically active / active peptide or protein,

d.

add the emulsion obtained in the section c) on a solution containing calcium chloride Y

and.

Isolate the microparticles.

The microparticle obtainable by the method previously described, where the alginate is not bound to a specific ligand for a surface receptor, constitutes a further aspect of the present invention, as well as all applications and uses derived from it, such as, its use as vaccine, in the preparation of a drug to stimulate and / or induce the immune response in an individual, as a system of release of immunologically peptides and proteins active / active, and forming part of pharmaceutical compositions and kit, including the uses of said compositions and kits.

Brief description of the figures

Figure 1 are microscopy photographs scanning electronics of the different formulations of elaborated microparticles.

Figure 2 is a graph showing the profile antigen release for each microparticle formulation loaded with SPf66 or S-3.

Figure 3 is a bar chart showing the profile of specific IgG antibody titers against SPf66.

Figure 4 is a bar chart showing IgG antibody profile against S-3.

Figure 5 is a bar chart that represents the ratio IgG2a / IgG1 versus SPf66 at 9 weeks of immunization.

Figure 6 is a bar chart that represents the ratio IgG2a / IgG1 versus S-3 at 9 immunization weeks

Figure 7 is a bar chart showing IFN-γ secretion at 48 hours of in vitro stimulation of cell culture of lymph nodes of animals immunized with SPf66.

Figure 8 is a bar chart showing IFN-γ secretion at 48 hours of in vitro stimulation of cell culture of lymph nodes of animals immunized with S-3.

Detailed description of the invention

The authors of the present invention have noted that, surprisingly, polymer microparticles biocompatible and biodegradable, such as microparticles formed by PLGA [acid poly (lactic-glycolic)] plus bound alginate to a specific ligand for a cell surface receptor, they are able not only to increase the immune response against antigen encapsulated in the microparticle, but also allow administration of immunologically peptides and proteins active / active by less invasive routes, such as the nasal route or intradermal, than the usual administration routes.

Without intending to be bound by no theory, this effect is thought to be due to the presence of alginate in the microparticle which, in combination with PLGA, allows for greater encapsulation of low weight synthetic peptides molecular and a lower initial release of the peptide, getting also an increase in the immune response induced against encapsulated peptide or protein (antigen), in addition to promoting administration of antigens by non-invasive routes thanks to the alginate mucoadhesivity.

Therefore, in a first aspect, the invention is relates to a microparticle, henceforth microparticle [I] of the invention, comprising (i) a first polymer biodegradable PLGA type [acid poly (lactic-glycolic)] and (ii) a second alginate polymer bound to a specific ligand for a receptor cell surface, which encapsulates a peptide or protein immunologically active / active.

In the present invention it is understood by "microparticle" to that particle that comprises a diameter less than 1 mm, preferably between 1 and 500 micrometers. Between 1 and 0.9, between 0.9 and 0.8, between 0.8 and 0.7, between 0.7 and 0.6, between 0.6 and 0.5, between 0.5 and 0.4, between 0.4 and 0.3, between 0.3 and 0.2, between 0.2 and 0.1 or less of 0.1 mm in diameter.

The average size of the microparticles is seen mainly influenced by different technological factors of production process of said microparticles, such as the biodegradable polymer concentration, agent concentration surfactant present in the external phase of the double emulsion and stirring rate.

The first polymer that is part of the microparticle [I] of the invention is a biodegradable polymer of the PLGA type. In the present invention, "biodegradable polymer" is understood to mean polymers that dissolve or degrade in a period of time that is acceptable for the desired application, in this case in vivo therapy, once they are exposed to a physiological pH solution. between 4 and 9, at a temperature between 25 ° C and 40 ° C.

Among this type of polymers is the poly (lactic-glycolic acid) or PLGA, a polymer formed from lactic acid and glycolic acid with the structural formula

one

The PLGA may have a different proportion of lactic acid and glycolic acid. So, the PLGAs that can used in the present invention preferably include PLGA 50:50 (50% lactic acid: 50% glycolic acid), PLGA 75:25 (75% lactic acid: 25% glycolic acid) or combinations of both.

The second polymer that is part of the microparticle [I] of the invention is an alginate polymer bound to a specific ligand for a cell surface receptor. In principle, any alginate capable of forming a hydrogel is suitable for use in the microparticles of the invention. Thus, the microparticles [I] of the invention may contain alginate formed mainly by regions of manuronic acid (MM blocks), by regions of guluronic acid (GG blocks) and by regions of mixed sequence (MG blocks). The percentage and distribution of uronic acids differ according to the origin of the alginate and contribute to the properties of the alginate such as its viscosity. The person skilled in the art knows the percentages of each of the different blocks that appear in the different biological sources of alginates. Thus, the invention contemplates the use of alginates from Laminaria hyperborea, Latvia nigrescens, Lessonia trabeculata, Durvillaea antarctica, Laminaria digitata, Eclonia maxima, Macrocystis pyrifera, Ascophyllum nodosum and / or Laminaria japanica as well as mixtures of alginates of different species until the desired content in GG, MM or GM blocks. By using a suitable combination of alginate polymers, a mixture whose modulus of elasticity has an adequate value can be obtained while the viscosity of the pre-gel solution is maintained at sufficiently low levels to allow adequate encapsulation / release of the immunologically active / active peptide or protein. Thus, the alginates that can be used in the present invention include GG alginates, MM alginates or combinations of both in a ratio of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30 : 70, 20:80 or 10:90. The results shown in the present invention have been obtained with medium viscosity alginate having more than 60% of
G. blocks

The invention also contemplates the use of alginates derived from the treatment of natural alginates with enzymes that are capable of modifying the integrating blocks to give rise to alginates with improved properties. Thus, alginates resulting from the treatment of alginates with C5-epimerases, which convert M blocks into G blocks, as well as with the AlgE4 enzyme of the bacterium Azotobacter vinelandii which is capable of converting the relatively rigid M blocks into MG blocks. Alternatively, the invention contemplates the use of alginates that have been modified by different physical treatments, in particular, gamma rays, irradiation with ultrasound or with ultraviolet light as described by Wasikiewicz et al . (Rad. Phys. Chem. 2005, 73: 287-295).

Additionally, the alginate that is part of the microparticle [I] of the invention is linked to a ligand specific for a cell surface receptor. At the moment invention is understood as "specific ligand for a receptor of cell surface "to the molecule or peptide that is capable of recognize a cell surface receptor and join it of specific form This ligand allows the microparticle [I] of the invention is recognized by the surface receptors of concrete cells, so you can join them and interact with them In principle, any peptide it possesses specific binding sites that can be recognized by cell surface receptors can be used in the present invention Thus, the specific ligand for a receptor of cell surface can come from cell adhesion molecules that interact with the extracellular matrix such as fibronectin, the different members of the families of the selectinas, the caderinas, lectins, integrins, immunoglobulins, collective and galectinas.

Thus, the specific ligand for a receptor of cell surface employed in the present invention may be a peptide derived from a region selected from the regions of the fibronectin that are involved in the union with the integrins that are found in the cell membrane. For example, and without limiting the invention, said peptides are derived from the tenth region Type III repeat of fibronectin containing the RGD peptide, from the fourteenth type III fibronectin repeat region containing the IDAPS peptide, from the CSI region of fibronectin that contains the LDV peptide and the CS5 region of fibrinectin that contains the REDV peptide. These peptides may consist of fragments of the corresponding regions that retain their adhesive capacity, such as the QAGDV peptide of the fibrinogen, the fibronectin LDV peptide and the IDSP peptide of VCAM-I.

The present invention also contemplates the use of integrin binding peptides as a specific ligand for a cell surface receptor, which are derived from the region of the tenth type III fibronectin repeat comprising the RGD sequence, such as, for example, a peptide selected from the group of RGD, RGDS, GRGD, RGDV, RGDT, GRGDG, GRGDS, GRGDY, GRGDF, YRGDS, YRGDDG, GRGDSP, GRGDSG, GRGDSY, GRGDVY, GRGDSPK, CGRGDSPK, CGRGDGRGDG -NH2, AcGCGY
GRGDSPG and RGDPASSKP, cyclic variants of said peptides, both linear and branched multivalent variants (see for example Dettin et al . 2002, J. Biomed. Mater. Res. 60: 466-471; Monaghan et al . 2001, Arkivoc, 2: U42-49; Thumshirn et al . 2003, Chemistry 9: 2717-2725; Scott et al. , 2001, J. Gene Med. 3: 125-134) as well as combinations of two or more of said peptides.

Therefore, in yet another embodiment particular of the microparticle [I] of the invention, the ligand specific for a cell surface receptor is a peptide It comprises the GDPR sequence.

Also, the specific ligand for a receptor cell surface may be linked to the alginate polymer with different degrees of substitution, so that the concentrations of both components may vary and thus control the number of specific ligands for a cell surface receptor that are bound to the alginate polymer. Thus, the invention contemplates alginates containing between 1 and 100, between 100 and 200, between 200 and 300, between 300 and 400, between 400 and 500, between 500 and 600, between 600 and 700, between 700 and 800, between 800 and 900 and between 900 and 1000 molecules of said specific ligand for each polymer molecule.

As mentioned above, the microparticle [I] of the invention can encapsulate a peptide or immunologically active / active protein. In the present invention, it is understood by "immunologically peptide or protein active / active "to that peptide or protein that stimulates immune response (humoral, cellular or both) when introduces into an organism other than the organism from which it comes said peptide or protein. Generically, to said peptide or Immunologically active / active protein is called antigen.

Therefore, in the present invention, the term antigen refers to any substance capable of being recognized by the immune system of a subject and / or capable of inducing in a subject a humoral immune response or a cellular immune response that leads to the activation of B and / or T lymphocytes when introduced into a subject; by way of illustration, said term includes any immunogenic product, native or recombinant, obtained from a superior organism or a microorganism, for example, a bacteria, a virus, a parasite, a protozoan, a fungus, etc., that contains one or more antigenic determinants, for example, structural components of said organisms; toxins, for example, exotoxins, etc. Virtually any antigen can be used in the preparation of microparticles of the invention loaded with antigen.

Examples of peptides and proteins immunologically active / active, without serving to limit the invention, include:

• Peptides or proteins capable of (appropriate or designed to) induce an immune response against an infectious disease , such as an infectious disease in animals caused by pathogenic microorganisms of animals, including humans, for example, viruses, bacteria, fungi and infectious parasites, relevant in human or animal health.

Proteins or Peptides capable of inducing an immune response may be recombinant proteins or peptides, identical or similar to those natural antigens of a specific microorganism.

Examples Illustrative, non-limiting, infectious viruses include virus Families: Arteriviridae, Retroviridae, Picornaviridae, Calciviridae, Togaviridae, Flaviridae, Coronoviridae, Rhabdoviradae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Reoviridae, Birnaviridae, Hepadnaviridae, Parvoviridae (parvovirus), Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae, etc. Examples of antigens that can be used according to the present invention include, but is not limited to, HIV antigens, gp120 antigen, surface antigen of the hepatitis B, rotavirus antigens such as VP4 and VP7, antigens of influenza viruses such as hemagglutinin or nucleoprotein, herpes simplex thymidine kinase antigen, etc.

Illustrative, non-limiting examples of bacteria include both Gram positive bacteria, eg, Pasteurella sp., Staphylococcus sp., Streptococcus sp. , etc., such as Gram negative bacteria, eg, Escherichia coli, Pseudomonas sp., Salmonella sp ., etc. Specific examples of infectious bacteria include: Helicobacter pylori, Borelia burgdorferi, Legionella pneumoplailia, Mycobacteria sp. (eg, M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae ), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogefaes (Group A of Streptococcus), Streptococcus a Group Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus aratracis, Corynebacteriumdiphtlzeriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers Clostridium tetani Enterobacter aerogenes Klebsiella pyzeunaoniae Pasturella multocida Bacteroides sp. Fusobacterium nucleatum Streptobacillus moniliformis Treponemapallidium Treponema pertenue Leptospira Rickettsia Actinornyces israelli etc.

Illustrative, non-limiting examples of infectious fungi include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis and Candida albicans . Infectious parasites include, by way of illustration, not limitation, protozoa, such as Plasmodium sp ., Causes of malaria, eg P. falciparum, P. malariae, P. ovale, P. vivax , etc., Leishmania sp . , causes of leishmaniasis, eg, L. major, L. donovani, L. infantum, L. braziliensis, L. panamensis, L. mexicana , etc., Toxoplasma gondii, Schistosoma sp ., etc., as well as parasitic nematodes, such as Dirofilaria immitis , etc. Examples of antigens for these parasites include the plasmodium spp . Circumsporozoite antigen; the merozoite surface antigen of Plasmodium spp .; SPf66 antigen from P. falciparum ; the S-3 antigen, a molecule that includes four epitopes corresponding to different stages of the life cycle of P. falciparum ; the gp63 of Leishmania spp ., etc.

• Peptides or proteins associated with tumors or cancers ("tumor markers") capable of (appropriate or designed to) induce an immune response against a tumor or cancerous cell , whereby the peptide or protein can be used in the treatment of cancers by stimulating an antigen specific immune response against a tumor antigen.

Examples illustrative, not limiting, of cancers that could be potentially treated in accordance with the teachings of this invention include biliary tract cancer, brain cancer, cancer breast, cervical cancer, choriocarcinoma, colon cancer, cancer endometrium, esophageal cancer, stomach cancer, neoplasms intraepithelial, lymphomas, liver cancer, lung cancer (e.g., small cell and non-cell lung cancer small), melanoma, neuroblastomas, mouth cancer, cancer ovaries, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, skin cancer, testicular cancer, thyroid cancer and renal cancer, as well as other carcinomas and sarcomas.

The selection of tumor antigens or antigenic determinants for the treatment of cancers can be carried out by a person skilled in the art in view of the state of the art (Renkvist et al . 2001, Cancer Immunol. Immunother. 50: 3-15), being said antigens and antigenic determinants included within the scope of the present invention. Representative examples of such antigens or antigenic determinants include: Her2 (breast cancer); GD2 (neuroblastoma); EGF-R (malignant glioblastoma); CEA (medullary thyroid cancer); CD52 (leukemia); human melanoma gp100 protein; melanoma-A / MART-1 human melanoma protein; tyrosinase; NA17-A nt protein; MAGE-3 protein; p53 protein; HPV16E7 protein; and antigenic fragments of said peptides or proteins.

• Peptides or proteins capable of (appropriate or designed to) induce an immune response against an allergen.

As used in this description, the term "allergen" refers to a peptide or protein to which a subject is sensitive and causes an immune reaction, for example, allergenic extracts of pollens, allergenic extracts of insects, allergenic extracts of food. or food products, components present in saliva, tweezers or stingers of insects that induce a sensitivity reaction in a subject, components present in plants that induce a sensitivity reaction in a subject, etc. Illustrative, non-limiting examples of allergens include protein extracts from pollens, eg, from Loliurn perennial, Poa pratense, Phleum pratense, Cynodon dactylon, Festuca pratensis, Dactylis glomerata, Secale cereale, Hordeum vulgare, Avena sativa, Triticum sativa, Chemismispopodium album, Plantago lanceolata, Taraxacum vulgare, Parietaria judaica, Salsola kali, Urtica dioica, Olea Europea, Platanus sp., Cupressus sp ., etc .; Protein extracts of insects, eg, from Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acarus siro, Blomia tropicalis, Euroglyphus maynei, Glyciphagus domesticus, Lepidoglyphus destructor, Tyrophagus putrescentiae , etc .; Protein extracts from fungi or animal epithelia, eg, Penicillium sp., Alternaria alternata, Cladosporium herbarum , dog epithelium, cat epithelium, horse epithelium, etc .; Protein extracts of food or food products, etc.

• Peptides or proteins capable of (appropriate or designed to) induce an improved response to a self-antigen . The term "auto-antigen", as used herein, refers to peptides or proteins encoded by the subject's DNA and products generated by proteins or RNA encoded by the subject's DNA. Examples of auto-antigens are described in WO 02/56905.

Thanks to the technical characteristics of the microparticle [I] of the invention and explained at the beginning of the present description, this is suitable for release in the organism of immunologically active / active peptides or proteins and thereby stimulate the body's immune response against said peptides and proteins. Therefore, in another aspect, the invention relates to the microparticle [I] of the invention For use as a vaccine.

In another aspect, the invention relates to the use of the microparticle [I] of the invention in the preparation of a medication to stimulate and / or induce the immune response in a individual.

In the present invention it is understood by "individual" or "subject" to a member of a kind of animal, preferably mammal and includes, but is not limited to, a pet, a primate and a human; in the context of the present invention, the individual is preferably a human being, male or female, of any race or age.

Due to the microparticle's capacity of the invention of releasing the peptides and proteins encapsulated in their inside, the microparticle is very useful as device for the release of compounds of a peptide nature With biological activity. Thus, in another aspect, the invention is relates to the use of the microparticle [I] of the invention for the release of peptides and proteins immunologically active / active.

The release of peptides and proteins can be done in a controlled manner by an adequate selection of the proportion of monomers (lactic acid and glycolic acid) that form the PLGA used in the preparation of the microparticle. The degradation rate of these polymers depends fundamentally on the proportion of both monomers. Thus, by proper selection of said composition it is possible to achieve that the degradation takes place over periods of time that may range from a few days to several months. The molecular weight of the polymer also influences the degradation rate, since the lower the molecular weight, the less time will be required to obtain small oligomers or hydrophilic monomers (Lewis, et al . Controlled release of bioactive agents from lactide / glycolide Polyesters In: Chasin, M. et al . Biodegradable polymers as drug delivery systems. Ed. Marcel Decker, Inc. New York, 1990).

The microparticle [I] of the invention can be administered to the individual alone or as part of pharmaceutical compositions together with a pharmaceutically carrier acceptable and, optionally, together with simultaneous administration or separated from a therapeutic agent. Therefore, in another aspect, the invention relates to a pharmaceutical composition, from here on hereinafter, pharmaceutical composition [I] of the invention, which comprises the microparticle [I] of the invention together with a pharmaceutically acceptable vehicle. Pharmaceutical vehicles Acceptable employees in the preparation of compositions pharmaceuticals, can be found in the book "Rowe, R, Sheskey, P. and Owen, S. Handbook of Pharmaceutical Excipients. 5th Ed. APhA and Pharmaceutical Press London, 2004. "

The microparticles [I] of the invention, after their administration to a subject, are they able to release the peptide or Immunologically active / active protein stably and gradually for prolonged times, so that they act as medications of sustained release. Therefore, the microparticle [I] of the invention allows to avoid the continuous and repeated administration of therapeutic agent in those patients that require levels baseline of said agent for a long time.

The microparticle [I] of the invention or the pharmaceutical composition [I] of the invention can be administered to the patient parenterally, including intravascular, intraperitoneal, subcutaneous, intradermal, etc. and non-parenteral, for example, topically, through the mucous membranes, that is, oral, buccal, sublingual, nasal (by inhalation), vaginal, etc., and will be formulated in a pharmaceutical form appropriate to the route of administration chosen, for example, in the form of sterile solutions, suspensions or lyophilized products in an appropriate unit dosage form. Suitable excipients, such as buffering agents, surfactants, preservatives, etc. may be used. Information on said excipients and pharmaceutical administration forms of the microparticle [I] of the invention or the pharmaceutical composition [I] of the invention can be found in the book "Rowe, R, Sheskey, P. and Owen, S. Handbook of Pharmaceutical Excipients. 5th Ed. APhA and Pharmaceutical Press. London, 2004 ", cited at supra .

The dose of microparticles [I] of the invention to administer to an individual may vary over a wide range, for example, between approximately 0.01 and 100 mg per kg of weight bodily.

The production of said pharmaceutical forms of administration by any of the chosen routes can lead to carried out by conventional methods known to experts in The matter. By way of illustration, information about the procedures for the production of said pharmaceutical administration forms of the protein of the invention can be found in the book "Swarbrick. J., Boylan, J.C. Encyclopedia of Pharmaceutical technology. 2nd Ed. Marcel Dekker Inc. New York, 2002-2004 ".

In another particular embodiment, the composition pharmaceutical of the invention comprises the microparticle [I] of the invention, a pharmaceutically acceptable carrier and an agent therapeutic.

The term "therapeutic agent" includes any compound that can be used to treat, prevent or cure diseases in a subject or one used to improve the physical and mental well-being in humans and animals. "Agents Therapeutics "include, but are not limited to, an agent chemotherapeutic, allergens, analgesic, antitumor, a steroid, a retinoid, an antibiotic compound (antimicrobial, antiviral or antifungal), an antioxidant, an anti-inflammatory compound, neuroprotective, anti-asthmatic, pulmonary surfactants, analogues of vitamins, salicylic acid, etc. A therapeutic agent includes the pharmaceutically acceptable salts thereof, prodrugs and pharmaceutical derivatives thereof.

In another aspect, the invention relates to the pharmaceutical composition [I] of the invention for use as vaccine.

In another aspect, the invention relates to the use of the pharmaceutical composition [I] of the invention in the development of a medicine to stimulate and / or induce Immune response of an individual.

In another aspect, the invention relates to the use of the pharmaceutical composition [I] of the invention as immunologically peptide or protein release system active / active.

As the expert in the field understands, the microparticle [I] of the invention may be contained in a kit which, optionally, may comprise a therapeutic agent such as It has been defined above.

Therefore, in another aspect, the invention is relates to a kit, hereinafter kit [I] of the invention, comprising the microparticle [I] of the invention and which, in a particular embodiment further comprises an agent therapeutic.

In the present invention it is understood by "kit", a product that contains the microparticles [I] of the invention or the various components of the pharmaceutical composition [I] of the invention packaged to allow transport, storage and administration simultaneously or sequentially. Thus, the kits of the invention may contain one or more suspensions, tablets, capsules, inhalers, syringes, patches and the like containing the microparticles [I] of the invention or the various components of the pharmaceutical composition [I] of the invention, and which can be found prepared in a single dose or as multiple doses. Additionally, the kit can contain a vehicle suitable for resuspension of microparticles [I] of the invention or the various components of the pharmaceutical composition [I] of the invention, such as means aqueous, e.g. saline solution, Ringer solution, Ringer solution lactate, dextrose and sodium chloride, water soluble media, such such as alcohol, polyethylene glycol, propyl ethylene glycol and vehicles Water insoluble like corn oil, seed oil cotton, peanut oil, sesame oil, ethyl oleate, Isopropyl myristate and benzyl benzoate. Another component that may be present in the kit is a packaging that allows keep the formulations of the invention within limits determined. Suitable materials for the preparation of such Packaging includes glass, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, Envelopes and the like.

The uses of the kit [I] of the invention constitute Additional inventive aspects. Thus, in another aspect, the invention relates to the kit [I] of the invention for use as vaccine.

In another aspect, the invention relates to the kit [I] of the invention in the preparation of a medicament for stimulate and / or induce the immune response in an individual.

In another aspect, the invention relates to the use of the kit [I] of the invention as a delivery system for immunologically active / active peptides and proteins.

In general, the microparticles can be obtained from biodegradable polymers which, in the case of the present invention are PLGA and alginate, by a process comprising dissolving said polymers in a suitable solvent (eg, dichloromethane, ethyl acetate, acetone , water etc.) and subsequent emulsification with an aqueous phase. This first emulsion is then added onto a solution of a surfactant (eg polyvinyl alcohol) to form a double emulsion. Finally, the desolvation or extraction of the solvent in which the PLGA was solubilized is carried out after the addition of an appropriate solvent, for example, a solvent or a mixture of solvents miscible with the dissolution of the biodegradable polymer, such as an isopropanol-water mixture , obtaining an aqueous suspension of microparticles from which the microparticles are isolated by conventional methods, for example, by vacuum filtration or centrifugation. The ratio, by volume, organic phase: hydroalcoholic solution (isopropanol / water) can vary within a wide range, for example, between 1:10 and
1: 100 (v: v).

The proportion of the peptide or protein immunologically active / active incorporated into the microparticle of the invention may vary within a wide range, by example, it can be up to 25% by weight with respect to the total weight of The microparticles However, the appropriate proportion will depend in each case of the incorporated peptide or protein.

The incorporation of the peptide or protein immunologically active / active to the microparticles of the invention can be done as described in WO 02/069938, by incorporation into the solution of the biodegradable polymer (in the phase internal of the first emulsion) before the formation of microparticles, or subsequently add it to the suspension aqueous of the microparticles already formed.

Therefore, in another aspect the invention is relates to a procedure for the preparation of the microparticle [I] of the invention, comprising the steps of

to.

prepare a solution of PLGA in a organic solvent,

b.

prepare an aqueous solution of alginate bound to a specific ligand for a receptor cell surface,

C.

emulsify the solutions with the immunologically active / active peptide or protein,

d.

add the emulsion obtained in the section c) on a solution containing calcium chloride Y

and.

Isolate the microparticles.

In a preferred embodiment of the procedure for the preparation of the microparticle [i] of the invention, the emulsion obtained in section c) can be added on a solution of a surfactant (e.g. polyvinyl alcohol) for obtain a double emulsion and then add said double emulsion on a hydroalcoholic solution containing chloride calcium to ensure total alginate gelation.

As the expert in the field understands, said procedure may comprise additional steps that involve preferred embodiments of the process, such as a evaporation / solvent extraction step to obtain the PLGA-alginate microparticles containing said immunologically active / active peptides and proteins, followed by a stage of isolation of the microparticles and, optionally lyophilize or dry the microparticles obtained.

The microparticles [I] of the invention can purified or isolated by conventional methods, for example, by centrifugation, ultracentrifugation, tangential filtration, vacuum filtration, or evaporation, including the use of empty.

In a particular embodiment of the procedure for the preparation of the microparticle [I] of the invention, the specific ligand for a cell surface receptor is a peptide comprises the RGD sequence.

Also, if desired, the microparticles [I] of the invention can be lyophilized by conventional methods. From a pharmacological point of view, it is important to have of microparticles in lyophilized form since this improves their long-term storage and preservation stability, In addition to reducing the volume of the product to be handled. The microparticles [I] of the invention can be lyophilized in presence of a usual cryoprotective agent such as glucose, sucrose, mannitol, trehalose, glycerol, lactose, sorbitol, polyvinyl pyrridone, etc., preferably, sucrose or mannitol; to a concentration within a wide range, preferably, between 0.1% and 20% by weight.

Optionally, an agent can be added crosslinker to improve the stability of the microparticles [I] of the invention, as described in WO 02/069938. Examples illustrative, not limiting, of crosslinking agents that can be use include diamine compounds, for example 1.3 diaminopropane, polysaccharides or simple saccharides, proteins and, in In general, any molecule that has capable functional groups to react with the functional groups present in the polymer biodegradable.

Additionally, the invention also relates to a process for the preparation of a microparticle in which the alginate is not bound to a specific ligand for a cell surface receptor but which, due to the characteristics of the alginate in combination with the PLGA, preserves the properties of the microparticle [I] of the invention. Therefore, in another aspect, the invention relates to a process for the preparation of a microparticle, hereinafter, microparticle [II] of the invention, comprising the steps
from

to.

prepare a solution of PLGA in a organic solvent,

b.

prepare an aqueous solution of alginate,

C.

emulsify the solutions with the immunologically active / active peptide or protein,

d.

add the emulsion obtained in the section c) on a solution containing calcium chloride Y

and.

Isolate the microparticles.

In a preferred embodiment of the procedure for the preparation of the microparticle [II] of the invention, the emulsion obtained in section c) can be added on a solution of a surfactant (e.g. polyvinyl alcohol) for obtain a double emulsion and then add said double emulsion on a hydroalcoholic solution containing chlorara calcium to ensure total alginate gelation.

As the expert in the field understands, said procedure may comprise additional steps that involve preferred embodiments of the process, such as a evaporation / solvent extraction step to obtain the PLGA-alginate microparticles containing said immunologically active / active peptides and proteins, followed by a stage of isolation of the microparticles and, optionally lyophilize or dry the microparticles obtained.

As mentioned above, the microparticles [II] of the invention can be purified or isolated by conventional methods, for example, by centrifugation, ultracentrifugation, tangential filtration, vacuum filtration, or evaporation, including the use of vacuum.

In a particular embodiment, the alginate linked or not to a specific ligand can be included in the phase external double emulsion and the rest of the procedure elaboration would be the same as described above.

Thus, in another aspect the invention relates with a microparticle obtainable by a procedure as has been described above, where the microparticle alginate resulting that is not bound to a specific ligand for a cell surface receptor, that is, a microparticle, of hereinafter microparticle [II] of the invention, comprising (i) a first biodegradable polymer of type PLGA [acid poly (lactic-glycolic)] and (ii) a second alginate polymer, which encapsulates a peptide or protein immunologically active / active.

The different uses of the microparticle [II] of The invention constitutes additional inventive aspects.

Therefore, in another aspect, the invention is relates to the microparticle [II] of the invention for use as a vaccine

In another aspect, the invention relates to the use of the microparticle [II] of the invention in the preparation of a medication to stimulate and / or induce the immune response in a individual.

In another aspect, the invention relates to the use of the microparticle [II] of the invention as a system of release of immunologically peptides or proteins active / active.

In another aspect, the invention relates to a pharmaceutical composition comprising the microparticle [II] of the invention, hereinafter, pharmaceutical composition [II] of the invention, and a pharmaceutically acceptable carrier.

In a particular embodiment, the composition Pharmaceutical [II] of the invention further comprises an agent therapeutic.

In another aspect, the invention relates to the pharmaceutical composition [II] of the invention for use as vaccine.

In another aspect, the invention relates to the use of the pharmaceutical composition [II] of the invention in the development of a medicine to stimulate and / or induce Immune response of an individual.

In another aspect, the invention relates to the use of the pharmaceutical composition [II] of the invention as Immunologically protein or peptide release system active / active.

In another aspect, the invention relates to a kit, hereinafter, kit [II] of the invention, comprising the microparticle [II] of the invention.

In another aspect, the invention relates to the kit [II] of the invention for use as a vaccine.

In another aspect, the invention relates to the use of the kit [II] of the invention in the preparation of a medication to stimulate and / or induce the immune response in a individual.

In another aspect, the invention relates to the use of kit [II] of the invention in the release of peptides or immunologically active / active proteins.

In a final aspect, the invention relates to with the use of alginate as an adjuvant of a peptide or protein immunologically active / active.

The invention is illustrated below based on the following examples that are provided by way of illustration and not limiting the scope of the invention.

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Example one

I. Preparation of the microparticles of the invention

The 50:50 PLGA, marketed by Boehringer Ingelheim GK, Ingelheim, Germany, was used to make the microparticles. The alginate used was an MVG alginate (medium viscosity, rich in G blocks) from NovaMatrix, FMC Biopolymers Philadelphia, PA, USA and the RGD alginate was obtained by modifying the MVG alginate following the procedure described by Rowley et al 1999. Biomaterials 20 : 45-53.

SPf66 and S3 peptides were obtained by solid phase synthesis following the methodology described by Merrifield (Merrifield, B. Solid phase peptide synthesis. I. Synthesis of Thetrapeptide. J. Am. Chem. Soc. 2149-2154. 1963) and modified by Houghten (Houghten R. General method for the rapid solid-phase synthesis of large number of peptides. Specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Nat. Acad. Sci, 82: 5131-5135. 1985.), under conditions of Correct Manufacturing (GMP) at the Institute of Immunology Foundation from Colombia. The amino acid sequence of the SPf66 peptide, in One letter code is as follows: CGDELEAETQNVYAAPNANPYSLFQKEKMVLPNANPPANKKNAGC (SEQ ID NO: 1). The amino acid sequence of the S3 peptide, is as follows: DELEAETQNVYAAPNANPYSLFQKEKMVLPNANPPANKKNA (SEQ ID NO: 2).

The remaining reagents are analytical grade and are obtained through regular distributors of products Chemicals

The microparticles (MP) were made by The double emulsion evaporation / solvent extraction technique. For each antigen, 3 different MP formulations were prepared: PLGA MP (PLGA MP), PLGA MP and 2% alginate (w / w) (PLGA-alg MP), and PLGA MP and 2% bound alginate with RGD (PLGA-alg RGD MP).

Preparation of PLGA MPs

A 10% aqueous solution of the peptide (w / v) is added another dichloromethane with 5% PLGA polymer (w / v) emulsifying them by sonication for 30 s at 50 W (Branson® sonifier 250). This emulsion w / o was poured onto a 8% aqueous solution of polyvinyl alcohol (w / v) homogenizing them for 5 minutes at 8,000 rpm to favor the formation of a second emulsion w / o / w by turbine homogenizer (Ultaturrax ® T-25). Then the double emulsion is added over another 2% aqueous solution of isopropanol (v / v) and let stir with a paddle shaker (HEIDOLPH. Mod.RZR1) to promote evaporation / extraction of organic solvent and induce thus the formation of the microparticles. After 1 hour, it collected the MPs by centrifugation 10,000 rpm and washed 3 times with distilled water by centrifugation and resuspension successive Finally the PLGA microparticles (PLGA MP) are they were resuspended in milliQ water and lyophilized.

Preparation of the MPs of PLGA-alginate

The preparation of alginate MPs (PLGA-alg MP) was carried out in a manner similar to those of PLGA but with certain modifications. For the formation of the first emulsion w / o, the aqueous phase, in addition to the 10% peptide, it also contained alginate in a 2% (w / w) proportion with respect to PLGA This first emulsion was formed by sonication, as well as the second by a turbine homogenizer. Once I know obtained the w / o / w, this was added on 2% isopropanol and CaCl2 0.6 mM to ensure total alginate gelation while dichloromethane is evaporated. The following steps are the same as for The previous formulation.

Preparation of the MPs of PLGA-alginate

As for the microparticles of PLGA-alginate linked to an RGD peptide (PLGA-algRGD MP), these were prepared exactly same as PLGA-alg MP but using an alginate which has been linked or linked with RGD (alg RGD).

II. Characterization of the microparticles

Once the microparticles were made, they were completely characterized by determining their morphology, size, Z potential, percentage of encapsulated and adsorbed peptide to the surface and in vitro release profile.

Morphological characterization was performed using the scanning electron microscopy technique (Jeol JSM-35 CF). The particle size was determined by the laser beam diffraction technique using a COULTER® COUNTER LS 130 particle analyzer. To determine the percentage of encapsulated peptide (w / w) the modified Lowry test known as the bicinconinic acid method was used (micro-BCA) (Smith et al . 1985, Anal. Biochem. 150: 76-85.). The zeta potential will be determined by laser doppler velocimetry (Malvern® Zetasizer 3000). The encapsulated total peptide was determined after complete digestion of the microspheres with a 0.2 M NaOH solution. The peptide adsorbed or associated to the surface of the microparticles, was determined by resuspending the microparticles, in a 20 mM phosphate buffer solution, pH 7, 4 and incubating at 37 ° C, under continuous orbital agitation for 30 min. The microspheres were subsequently sedimented by centrifugation and in the supernatant the content of SPf66 was analyzed by the micro-BCA technique. The surface-associated peptide corresponds to the percentage of the total encapsulated peptide that is present on the surface of the microparticles and consequently available for rapid release.

The analysis of the microparticles by scanning electron microscopy allowed us to observe its morphological characteristics, being spherical particles of smooth surface (figure 1).

The different parameters characterized in the microparticles are listed in Table 1. As can be seen in the table, the different types of MP developed they presented an average particle size around 1 micrometer. However, the net surface charge of the microparticles is seen influenced by the presence of alginate, since they were observed Notable differences in the Zeta potentials of the MPs depending of the presence or not of alginate in the formulation. For both peptides (SPf66 and S-3), MP with alginate presented zeta potential values more negative than their respective PLGA MP. These results show that the alginate (polyanion) is incorporated into the microparticles, and at least partly it does it at the superficial level, to which alginate is a polyanion Regarding the efficiency of encapsulation (EE) and peptide adsorbed to the surface (SAP), an increase in EE is observed when introducing the alginate while the SAP was reduced notably, with respect to the prepared microparticles only with PLGA (PLGA MP). The inclusion of alginate in the phase Internal aqueous double emulsion, together with the peptide, allows increase the viscosity of the first emulsion making it difficult to diffusion of the peptide to the aqueous external phase, which, together with the possibility of gelling the alginate by crosslinking with the calcium chloride, prevents the loss of the peptide during the phase of microparticle formation.

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TABLE 1

2

3

In vitro release studies

In order to obtain the profile of release of the peptide from the PLGA microparticles, it weighed exactly 20 mg of microspheres in test tubes and resuspended in 20 mM phosphate buffer, pH 7.4 incubating at 37 ° C in continuous orbital agitation. At preset time intervals, the samples were centrifuged at 10,000 rpm for 10 min. He supernatant was extracted and analyzed to quantify the peptide, by the micro-BCA technique described previously. The removed supernatant is replaced with a equivalent volume of fresh buffer solution to continue with the release test The release profile of the peptide obtained for each microparticle formulation it was expressed in terms of cumulative percentage of peptide released as a function of time. Release tests were performed in triplicate for each of the formulations elaborated.

As can be seen in figure 2 the release profiles of the peptides from the Microparticles exhibit a three-phase behavior. I only know find differences in peptide release over time initials (surface associated peptide), while the rest of the profile obtained during the 112 day duration of the trial shows very similar release rates between PLGA and 2 PLGA-alginate formulations, for each of the peptides.

III. Immunization studies

The immune response induced by microparticles were evaluated in Balb / c mice weighing 25 g and 6-8 weeks divided into 6 groups of 7 mice. He group 1 received an intradermal dose of 100 µg of the antigen SPf66 microencapsulated in 50:50 PLGA microspheres (PLGA MP). He group 2 received the same dose of microencapsulated SPf66 antigen in microspheres of PLGA and 2% alginate (PLGA-alg MP). Group 3 received 100 µg of the SPf66 antigen antigen microencapsulated in PLGA microspheres and 2% alginate bound with RGD (PLGA-alg RGD MP). Groups 4, 5 and 6 they received the same dose of microencapsulated S3 antigen in the three types of microparticles described above, PLGA MP, PLGA-alg MP and PLGA-alg RGD MP, respectively.

Antibody determination

All animals were bleeding from the plexus retro-orbital under anesthesia, before the first immunization and at periodic intervals until week 12. The blood samples were centrifuged at 5,000 rpm for 5 minutes, the serum was recovered and stored at -80 ° C for subsequent immunochemical characterization. Antibody levels Total anti-peptide IgG IgG2a and IgG1 were determined by a conventional ELISA technique.

Figures 3 and 4 show the levels of Total antibodies (IgG) obtained for the different groups of animals immunized with 100 µg of the SPf66 antigen or S-3 As it had been observed in studies Previous, PLGA microparticles (PLGA MP) loaded with both SPf66 as with S-3 caused a high response humoral administered intradermally. It should also be noted that the microparticles with alginate (PLGA-alg MP) significantly improved the response for both antigens peptides On the other hand, the incorporation of the RGD sequences in alginate (PLGA-alg RGD MP), were also observed differences between particles formulated without alginate (PLGA MP), with unbound alginate (PLGA-alg MP) or algRGD (PLGA-alg RGD MP), especially in the times initials, 3 and 6 weeks, where the PLGA-algRGD MP induced IgG titers higher than those of PLGA-alg MP. RGD sequences induce a specific recognition by the presenting cells of antigens (APCs), which can capture MPs in a more efficient.

The production of antibody isotypes (IgG1 e IgG2a) by immunized mice was studied at 9 weeks. All formulations induced high and similar levels of IgG1. As for IgG2a, although all groups showed detectable levels, differences between formulations were observed managed. For both peptides, the MPs that incorporate alginate induced levels of IgG2a higher than those of MP PLGA. Further, MPs containing alginate modified with RGD sequences increased secretion of this cytophilic isotype (IgG2a for mice) with respect to the MPs of unbound alginate, being said greater increase for the SPf66 peptide than for the S-3 This is of great importance, since in the Malaria infection cytophilic antibodies play a role important when generating protection against the parasite. So and as can be seen in figures 5 and 6 the ratio IgG2a / IgG1 was significantly higher for MP with alginate than for those made only with PLGA, for both peptides. The higher ratios correspond in both cases for formulations PLGA-alg RGD MP.

IFN-? Secretion assays

IFN-? Secretion of cell culture of lymph nodes extracted from mice 14 weeks after immunization, it was determined by capture ELISA (OptEIA IFN-? ELISA kit (Pharmingen, Becton Dickinson, San Diego, CA).

As Figures 7 and 8 show, all cell cultures of the lymph nodes of the groups immunized with the SPf66 or S-3 microparticles produced detectable levels of the IFN-γ cytokine after in vitro stimulation with the peptide corresponding to immunization. . The groups that received the PLGA MP induced the lowest levels of IFN-γ, while the highest were achieved by the PLGA-alg RGD MP and the PLGA-alg MP secreted intermediate levels to those of the other 2 formulations.

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13

Claims (14)

1. A procedure for the preparation of a microparticle comprising the stages of

to.

prepare a solution of PLGA in a organic solvent,

b.

prepare an aqueous solution of alginate,

C.

emulsify the solutions with the immunologically active / active peptide or protein,

d.

add the emulsion obtained in the section c) on a solution containing calcium chloride Y

and.

Isolate the microparticles.

2. A microparticle obtainable by a method according to claim 1. 3. A microparticle according to claim 2 For use as a vaccine. 4. Use of a microparticle according to the claim 2 in the preparation of a medicament for stimulating and / or induce an individual's immune response. 5. Use of a microparticle according to the claim 2 as delivery system of said peptides or immunologically active / active proteins. 6. A composition comprising a microparticle according to claim 2 and a vehicle pharmaceutically acceptable. 7. Composition according to claim 6 that It also comprises a therapeutic agent. 8. A composition according to claim 6 or 7 For use as a vaccine. 9. Use of a composition according to claim 6 or 7 in the preparation of a medicament for stimulate and / or induce the immune response of an individual. 10. Use of a pharmaceutical composition according to the claim 6 or 7 as a peptide release system or immunologically active / active proteins. 11. A kit comprising a microparticle according to claim 2. 12. A kit according to claim 11 for its Use as a vaccine. 13. Use of a kit according to claim 11 in the development of a medicine to stimulate and / or induce immune response in an individual. 14. Use of a kit according to claim 11 in the release of peptides or proteins immunologically active / active.