Composition, stimulating immune response, which contains nanoparticles based on copolymer of methylvinyl ether of maleic anhydride

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and concerns composition, which stimulates immune answer, containing nanoparticles, based on copolymer of methylvinyl ether of maleic anhydride. Mentioned nanoparticles can additionally contain allergen or antigen and/or immunostimulating agent, which can be inside of said nanoparticles and/or at least partially cover surface of mentioned nanoparticles and optionally crossing-linking agent. Composition, which stimulates immune response, can be applied as adjuvant in immunotherapy and vaccines.

EFFECT: increase of activity.

32 cl, 7 ex, 2 tbl, 13 dwg

 

The technical field to which the invention relates

The present invention relates to the use of nanoparticle-based copolymer metilfenidato ether maleic anhydride, optionally containing an allergen or antigen and/or an immunostimulating agent, adjuvant in immunotherapy and vaccines. Also the present invention relates to compositions that stimulate the immune response containing these nanoparticles.

The prior art prior to the invention of

As you know, there are highly immunogenic antigens that are able to induce a protective immune response in a patient, although there are other antigens that do not induce the specified protective response or which induce very weak immune response. Typically, the immune response of the host to weak immunogenic antigen can be stimulated through co-injection with adjuvant.

Adjuvants.

An adjuvant is any substance that enhances the immune response to an antigen with which it is mixed. Adjuvants mainly operate through three mechanisms: (i) the formation of antigenic or allergenic Deposit in place of vaccines, from which biologically active product will be released during the different periods of time; (ii) delivery of the antigen or allergen by antigen-pre is enthuasim cells; iii) inducing the secretion of interleukin.

Some classic examples of adjuvants are aluminium salts (Alhydrogel) and catecholamines (which amplify the Th2 response) and the lipopolysaccharide of gram-negative bacteria and certain sequences (CpG to enhance Th1 response). On the other hand, numerous studies show that certain non-biological vectors, such as particles (spherical particles of a polymeric nature, covered with a substance) or liposomes (spherical bubbles with the water of the Central cavity, covered with various bimolecular phospholipid and cholesterol films), can also act as adjuvants [Eldridge et al., Infect Immun, 59 (1991) 2978-2986; O Hagan et al., Vaccine, 18 (2000) 1793-1801; Murillo et al., Vaccine, 30 (2001) 4099-4106].

Another type of non-biological vectors, which can be considered for use as adjuvants are solid particles of colloidal systems with size less than one micrometer, also called nanoparticles, which are divided into the matrix of the nanospheres and vesicular nanocapsules [Orecchioni and Irache, Formes pharmaceutiques pour application locale. Lavoisier Tech and Doc., Paris, (1996) 441-457]. Nanocapsules are vesicular systems, educated internal cavities that are surrounded by a polymeric membrane or wall. Nanospheres are matrix forms, educated prostranstve.naprimer net. In both cases, the molecules are biologically active substance can be dissolved, captured or linked to a macromolecular structure (nanospheres) or encapsulated by a polymeric membrane (nanocapsules) and can even be adsorbed on the surface of the nanoparticles.

The distribution of nanoparticles in the body, as a rule, depends on its physical and chemical characteristics (mainly, size, and surface properties)that define its interaction with the biological environment. Thus, nanoparticles are pharmacological forms, which are a particular interest for immunotherapy or as vaccine adjuvants for injection of antigens and/or allergens.

Typically, the most significant opportunities provided by such vectors non-biological nature, are (i) they protect the encapsulated material from chemical, enzymatic or immunological inactivation in the introduction and at the place of action; (ii) they improve the transport of biologically active molecules to inaccessible places and its penetration into the cell; (iii) they prolong the residence time of drugs in the body and control its release; (iv) they increase the specific activity through selectivity, efficiency and regulation of the concentration of encapsulated materials what s in target cells and/or target molecules; and (v) they increase the stability of the material into which they are introduced in the manufacturing process, transportation and storage of the drug.

The use of adjuvants in vaccination

Application of particle adjuvants in the form of emulsions, microparticles, ISCOMS or liposomes have been evaluated previously by several research groups [see: Singh et al., Int J Parasitology 33 (2003) 469-478].

The capture antigen antigen-presenting cells" is raised if these antigens are associated with the polymer particles or inside them. For many years, as controlled antigen-producing systems in humans and animals was used biodegradable and biocompatible polyesters [Okad et al., J Pharm Sci, 12 (1995) 1-99; Putney et al., Nat Biotechnol, 16 (1998) 153-157]. Unlike aluminievyh adjuvants microparticles are effective to induce cellular and cytotoxic immune responses in mice [Nixon et al., Vaccine 14 (1996) 1523-1530; Maloy et al., Immunology 81 (1994)661-667; Moore et al., Vaccine 13 (1995) 1741-1749]. Oral immunization microparticles in mice induces strong immune responses in mucosal and systemic level compared to the encapsulated antigens [Chalacombe et al., Immunology 176 (1992) 164-168; Eldrige et al., J Control Rel 11 (1990) 205-214; O Hagan et al., Novel Delivery Systems for Oral Vaccines (1990) 175-205]. This ability is the result of internalization of antigen by specialized cells of lymphoid tissue slithis what's membranes [O Hagan, J Anat, 189 (1996) 477-482]. It has been shown that mucosal immunization with various particulate systems effective against various pathogens, such asBordetella pertussis[Chaill et al., Vaccine 13 (1995) 455-462; Jones et al., Vaccine 15 (1997) 814-817; Shahin et al., Infect Immun, 63 919950 1195-1200; Conway et al., Vaccine 19 (2001) 1940-1950],Chlamydia trachomatis[Whittum-Hudson et al., Nat Med 2 (1996) 1116-1121],Salmonella Typhimurium[Allaoui-Attarki et al., Infect Immun 65 (1997) 853-857] and Brucella [Murillo et al., Vaccine, 19 (2001) 4099-4106].

The use of adjuvants in immunotherapy

Allergic diseases are pathological manifestation,caused by the negative immune response (hypersensitivity reaction), in fact, harmless macromolecules, called allergens. This hypersensitivity reaction is evident in approximately 30% of the world population, mainly in industrialized countries. Hypersensitivity reaction is the cause of diseases such as allergic rhinitis, bronchial asthma, food allergies, and allergies to medications and insects [Settipane et al., Allergy Proc, 15 (1994) 21-25].

In Spain this type of diseases typical for the age group from 4 to 17 years and is 13.3%; among them, 6.4% occurs bronchial asthma, the death rate from asthma in Spain is 1.5/100,000 inhabitants.

According to the mechanistic theory of allergic diseases result from violations of the ball the sa between two main types of responses which may occur after the activation of helper T cells: Th1 and Th2. Cytokines found in the external environment of the cell, act in a certain way, directing the differentiation of immature T cells (Th0) so that the presence of interleukin 12 (IL-12), interferon gamma (INF-γ), inteleukin 18 (IL-18) and interferon-alpha (INF-α) induces differentiation into Th1, which is mainly characterized by the production of a large number of INF-γ and to a lesser extent interleukin 2 (IL-2) and beta-interferon (INF-β). Subsequent stimulation of b-cells in this type of response leads to increased production of IgG2a, IgG2b, IgG3. On the other hand, if Th0 cell is in the environment, which is dominated by interleukin 4 (IL-4) and prostaglandin E2 (PGE2), there will be differentiation into Th2, which is characterized by the synthesis of a large number of IL-4, interleukin 5 (IL-5) and interleukin 13 (IL-13), and the synthesis of IgG1and IgE, and biotype directly involved in triggering the process [Hannah et al., Ann Rev Immunol, 21 (2003) 579-628].

The importance of the prevalence of allergen specific Th2-type response in allergic diseases has been confirmed by a large number of studies [Romagnani, Ann Rev Immunol, 12 (1994) 227; Bousquet et al., Allergy, 53 (1998) 1-42; Majori et al., Clin Exp Allergy, 30 (2000) 341-347]. Cells with a Th2 phenotype cells are only able to directly recognize antigenic peptides, and take part in about the products IgE by b-cells, activation and production of mastocytes, activation and maturation of eosinophils, which was shown as in animal models and in humans [Cohn et al., Pharmacology and Therapeutics, 88 (2000) 187-196].

Thus, the functional predominance of Th2 cells over Th1 cells leads to allergic response, whereas the functional predominance of Th1 cells over Th2 cells can inhibit the allergic response [Martin et al., Alergol Clin Immunol, 17 (2002) 104-110].

Based on other studies have made the claim that the inhibition of the Th2 response with a predominance of Th1 may lead to the development of autoimmune diseases, so it would be good to strengthen the immune regulation of the balance of Th1/Th2 through increased populations of regulatory T cells (Tr) and IL-10, and growth factor β T cells (TGF-β). This could lead to the synthesis of IgG4and IgA antibodies (non-inflammatory mediators of the response) and the suppression of IgE production by b-cells [Akids et al., Immunology, 103 (2001) 131-136; Akids et al., J Clin Invest, 102 (1998) 98-106; Blaser et al., Int Arch Allergy Immunol, 117 (1998) 1-10]. Recent research has confirmed the importance of IL-10 in inactivation of Th2 cells (Gruning et al., J Exp Med, 185 (1997) 1089-1099; Adachi et al., Int Arch Allergy Immunol, 118 (1999) 391-394] and has also been found that the introduction of IL-10in vivohas a beneficial effect on allergies in animals [Zuany-Amorim et al., J Clin Invest, 95 (2003) 2644-2651; Stampfli et al., Am J Respir Cell Mol Biol, 21 (1999) 586-596; Hall et al., Vaccine, 21 (2003) 549-561]. On this basis it was made FAV is the situation, that IL-10 plays an important regulatory role in hyperactive characteristic of Th2 cells in patients with allergies.

IL-10 may play an important physiopathological role in combating inflammatory diseases (Crohn's disease, rheumatoid arthritis, psoriasis etc), some viral infections (hepatitis C, infection caused by the human immunodeficiency virus (HIV), and so on) and even in the suppression of side effects in organ transplantation. Thus, direct application of IL-10 or, alternatively, the use of adjuvants that stimulate the production of IL-10, can have a huge impact on the treatment of these diseases [sadullah et al., Pharmacol Rev, 55, (2003) 241-269]. Currently there are research opportunities such treatment of autoimmune diseases such as rheumatoid arthritis [Feldman et al., Annu Rev Immunol (1996) 397-440; Katsikis et al., J Exp Med (1994) 1517-1527; Chomarat et al., J Immunol (1995) 1432-1439]. Anti-inflammatory and regulatory role of the cytokine, therefore, is significant in the case of Th1 (autoimmune disease) in the case of Th2 (allergic) hyperactive responses.

There are mainly three approaches in the treatment of allergic diseases (i) avoiding any contact with the allergen; (ii) the use of antihistamines; and (iii) treatment by immunotherapy. Taking into account that the first two methods can in some cases be the ü inappropriate, immunotherapy could be the most appropriate method of control.

Specific immunotherapy with allergens is a re-introduction of allergens to patients with IgE-mediated disorders in order to protect against allergic symptoms and inflammatory reactions associated with natural exposure to these allergens [Jutel, M., J Immunol, 154 (1995) 4178-4194].

This alternative treatment is aimed at strengthening the functional predominance of Th1 response compared with the Th2 response, which will inhibit the allergic symptoms. Modulation of Th1 can also be used in other processes, such as control through vaccination against intracellular bacterial parasites (such asBrucellaandSalmonella).

Although it has been described the use of various non-biological vectors, such as nanoparticles, such as adjuvants, immunotherapy or vaccine for administration of antigens and/or allergens, there is still a need to create alternative adjuvants to replace the existing ones with the aim of increasing the opportunities for the production of vaccines and compositions for immunotherapy. Mostly these adjuvants should be effective for immunization or immune therapy by oral administration without the necessity to use very high doses of the allergen and antigen the century As you know, despite the potential benefits of oral immunization therapeutic or prophylactic purposes should be resolved some of the difficulties associated with the fact that required for the beneficial clinical effect dose immunogenic or allergenic active ingredient is excessively high due to loss of immunogenic activity. Thus, as a rule, due to the low stability of the allergen or antigen in the gastrointestinal tract (pH and the presence of hydrolytic enzymes) dose should always be higher (up to 200 fold increase)than after subcutaneous administration [Taudorf et al., J Allergy Clin Immunol (1987) 153-161; Creticos et al., J Allergy Clin Immunol (1990) 165]. In addition, the mucosa of the gastrointestinal tract is a formidable barrier to the absorption of these macromolecules.

Unexpectedly it was found that the nanoparticles-based copolymer metilfenidato ether maleic anhydride, optionally containing an allergen or antigen and/or an immunostimulating agent that can stimulate or enhance the immune response when administered to a patient, which allows their use in immunotherapy and vaccines. These nanoparticles are stable in oral administration, have satisfactory bioadhesive characteristics and, thus, can be used immunizat and or immunotherapy using various routes of administration, including oral, without the necessity to use such high doses of allergen or antigen, as that described in the section on prior art. In addition, these nanoparticles are of lower toxicity, biodegradable and easy to retrieve.

Nanoparticles copolymer metilfenidato ether maleic anhydride

In the patent application WO 02/069938 owned by the same applicant, describes a copolymer nanoparticles metilfenidato ether maleic anhydride (PVM/MA), method of their production and use as a carrier of drugs. The specified copolymer (PVM/MA consists of two differentiated functional groups having different solubility characteristics: hydrophobic ether group and anhydrite group. The carboxyl group is solubilizers agent as capable of dissolving the polymer, if it is ionized, and the ether group is hydrophobic, that is, prevents the penetration of water into the polymer [Heller et al., J Appl Polym Sci, 22 (1978) 1991-2009]. Synthetic copolymer (PVM/MA have a very different application. Gantrez® AN is widely used as thickener and flocculonodular agent, dental adhesive, filler tablets for oral administration, filler for transdermal patches, etc. on the other hand, was about what isano the use of these copolymers for controlled release of drugs [Heller et al.,J Appl Polym Sci, 22 (1978) 1991-2009] and matrix forms for the local release of drugs for ophthalmic use [Finne et al., J Pharm Sci, 80 (1991) 670-673; Finne et al., Int J Pharm, 78 (1992) 237-241].

Nanoparticles based on PVM/MA have bioadhesive characteristics [Arbs et al., Int J Pharm, (2002)129-136] and, thus, when ingested they can interact with peyrovani plaques, which contain 20% of all lymphocytes in the body and trigger an augmented immune response to those antigens and/or allergens, which were introduced with the water solution.

Nanoparticles based on PVM/MA are colloidal systems, which is capable of holding a biologically active substance by: (i) dissolution or capture macromolecular structures or matrix, (ii) covalent binding of drugs with anhydrite groups of the copolymer and (iii) absorption processes, mediated by weak ties. Excessive reactivity of the copolymer (PVM/MA because of anhydrous cyclic groups also contributes to the capture molecules, drugs or other substances. In the patent application WO 02/069938 describes the use of nanoparticles copolymer (PVM/MA as carriers of drugs, in particular 5-pteridine, ganciclovir and of antisense oligonucleotide ISIS 2922.

Brief description of the invention

The object of this izobreteny what is the composition, stimulating an immune response in a patient, used as adjuvant in immunotherapy and vaccines stable when administered orally, with satisfactory bioadhesive characteristics in respect of interaction with the mucosa, not necessarily able to carry the allergen or antigen and/or an immunostimulating agent and release the specified agent in a controlled manner and, thus, used for immunization or immune therapy by introducing a variety of ways drugs, including oral.

Unexpectedly it was found that the nanoparticles-based copolymer metilfenidato ether maleic anhydride, optionally containing an allergen or antigen and/or an immunostimulating agent that can stimulate or enhance the immune response when administered to a patient, which makes possible their use in immunotherapy and vaccines. In particular, it was found that these nanoparticles is easy to obtain, possess satisfactory bioadhesive characteristics are of lower toxicity and biodegradable materials (e.g., they dissolve or decompose over a period of time acceptable to the desired form of application, in the case of therapy,in vivoif they are available in a physiological solution with a pH of 6-9 and a temperature in promesed is from 25 to 40°C).

In addition, one aspect of the present invention is a composition for stimulating the immune response containing nanoparticles-based copolymer metilfenidato ether maleic anhydride. These nanoparticles may optionally contain an allergen or antigen and/or an immunostimulating agent that can be contained within these nanoparticles and/or at least partially cover the surface of these nanoparticles. If necessary, these metabolic pathways may also contain a cross-linking agent. This composition, optionally, may be in lyophilized form.

In another aspect, the present invention relates to a vaccine or immunotherapy compositions containing the specified composition for stimulating an immune response. In one of specific embodiments of the present invention specified immunotherapy vaccine or composition is a composition suitable for oral administration, whereas in another specific embodiment, the present invention specified immunotherapy vaccine or composition is a composition suitable for parenteral administration.

In another aspect, the present invention relates to the use of specified composition for stimulating an immune response, the production of WACC the us or immunotherapy composition.

In another aspect, the present invention relates to the use of specified composition for stimulating an immune response, when the manufacture of a pharmaceutical composition for the selective stimulation of the Th1 immune response, or in the manufacture of a pharmaceutical composition for the selective stimulation of the Th2 immune response, or in the manufacture of a pharmaceutical composition for the balanced stimulation of the immune Th1 and Th2 responses.

In another aspect, the present invention relates to a method for producing a composition for stimulating the immune response containing these nanoparticles based on PVM/MA and the allergen or antigen and/or an immunostimulating agent, in which you will add the specified allergen or antigen and/or immunostimulating agent in an organic solution containing a specified copolymer (PVM/MA before desolately in aqueous-alcoholic solution, or, alternatively, incubation specified allergens or specific antigens and/or immunostimulatory agent with the specified nanoparticles PVM/MA. In the specified way, in addition, may be additionally provided for the elimination of organic solvent and/or cleaning steps and stages of the stabilization of the nanoparticles obtained using cross-linking agents. This method may not necessarily provide additional the stage of lyophilization.

Brief description of drawings

The figure 1 presents a graph showing the dependence of the release of ovalbumin (%) of compositions NP-I NP-II, NP III NP-IV time (days).

The figure 2 presents a graph showing the dependence of the levels of specific antibodies against ovalbumin (IgG1, IgG2aafter transdermal immunization of Balb/c mice with a solution of ovalbumin (OVA) in a free form, ovalbumin adsorbed at alhydrogel (OVA-Alum), and blank nanoparticles (NP), NP-I NP-II, NP III NP-IV in time.

The figure 3 presents a graph showing the dependence of the levels of specific antibodies against ovalbumin (IgG1, IgG2a) after oral immunization of Balb/c mice with a solution of ovalbumin (OVA) in free form and blank nanoparticles (NP), NP-I NP-II, NP III NP-IV in time.

The figure 4 presents a graph showing the dependence of the levels of specific antibodies against ovalbumin (IgG1, IgG2a) after oral immunization of Balb/c mice with different doses of encapsulated ovalbumin (NP-III25 and NP-III50) and blank nanoparticles (NP) in time.

The figure 5 presents a graph showing the dependence of the levels of specific antibodies against ovalbumin (IgG1after transdermal immunization of Balb/c mice with a solution of ovalbumin-free (OVA), ovalbumin absorbed on alhydrogel (OA-Alum), and blank nanoparticles (NP), NP-I NP-II, NP III NP-V NP-VI and NP-VII in time.

The figure 6 presents a graph showing the dependence of the levels of specific antibodies against ovalbumin (IgG2aafter transdermal immunization of Balb/c mice with a solution of ovalbumin-free (OVA), ovalbumin absorbed on alhydrogel (OVA-Alum), and blank nanoparticles (NP), NP-I NP-II, NP III NP-V NP-VI and NP-VII in time.

The figure 7 presents a graph showing the dependence of the concentration of serum IL-10 after transdermal immunization of Balb/c mice with a solution of ovalbumin-free (OVA), ovalbumin absorbed on alhydrogel (OVA-Alum), and blank nanoparticles (NP), NP-I NP-II, NP III NP-V NP-VI and NP-VII in time.

The figure 8 presents a graph showing the dependence of the levels of specific antibodies against ovalbumin (IgG1, IgG2aafter transdermal immunization of Balb/c mice with a solution of ovalbumin, ovalbumin absorbed on alhydrogel (OVA-Alum) OVASAL and solution of ovalbumin (OVA) in time.

The figure 9 presents the result of the separation of the extract is NOT by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) and Coomassie staining of protein (10 μg per well).

The figure 10 shows the result of Western blot turns extract NOT before (a) and after (B) encapsulation using a mixture of sera deg. of infection is the R chickens.

The figure 11 presents a graph showing the results of the experiment by intraperitoneal protection in Balb/c mice with a lethal dose of 102colony forming units (CFU)S. enteritidis3934.

The figure 12 presents a bar chart of the release of IFN-γ (A) and IL-4 (B) spleen cells of Balb/c mice, restimulating extract NOT.

Figure 13 presents a bar chart showing the results of indirect ELISA in the sera of Balb/c mice in comparison with the extract using antibodies IgG1and IgG2a.

Detailed description of the invention

Unexpectedly it was found that the nanoparticles-based copolymer metilfenidato ether maleic anhydride, optionally containing an allergen or antigen and/or an immunostimulating agent that can stimulate or enhance the immune response when administered to a patient, which allows their use in immunotherapy and vaccines.

The term "patient"as used in this description, includes any animal having an immune system, preferably a mammal, more preferably human.

One aspect of the present invention is a composition for stimulating an immune response, indicated in the present description as the composition of the invention containing nanoparticles-based copolymer of metilfenidato what about the ether maleic anhydride. These nanoparticles may optionally contain an allergen or antigen and/or an immunostimulating agent that can be contained within these nanoparticles and/or at least partially cover the surface of these nanoparticles. If desirable, these nanoparticles can also contain cross-linking agent.

Under used in the present description, the term "nanoparticles" understand colloidal system of solid particles with a size of less than 1.0 micrometer, preferably in the range from 10 to 900 nanometers (nm), and these include the matrix of the nanospheres and vesicular nanocapsules. In a specific embodiment of the present invention, the average size of these nanoparticles is less than 400 nm.

The nanoparticles present in the compositions of the present invention include a copolymer metilfenidato ether maleic anhydride or PVM/MA. The specified copolymer (PVM/MA is a well known product that can be obtained by suitable methods, for example by polymerization of acetylene with maleic anhydride, or can be purchased on the market. In this sense, International Specialty Products (ISP) makes the copolymer (PVM/MA with different molecular mass, presents under the trademark Gantrez® AN. As a rule, to implement the present invention in practice, the mod is ekulama weight of the specified copolymer (PVM/MA can vary in a very wide range from 100 to 2400 kDa, more preferably, from 200 to 2000 kDa. In one embodiment of the present invention, the preferred molecular weight of the copolymer (PVM/MA is the molecular weight of 180 to 250 kDa.

Application of the above copolymer (PVM/MA has special advantages, allowing you to use it widely in the pharmaceutical industry due to its low toxicity (LD50=8-9 g/kg oral) and excellent biocompatibility. In addition, it is easy to obtain and can react with other hydrophilic substances, due to its functional groups, without the use of commonly used organic reagents (derivatives of glutaraldehyde and carbodiimide), with considerable toxicity [Arbs et al., J. Controlled Rel., 83 (2002) 321-330]. The copolymer (PVM/MA insoluble in water, but found it anhydrite group is hydrolyzed, forming a carboxyl group. The solution is weak and depends on the conditions in which it is received. Thanks to the availability of functional groups in PVM/MA during incubation in aqueous medium is covalent binding of molecules with nucleophilic groups such as hydroxyl or amino groups. These nanoparticles PVM/MA also have bioadhesive properties [Arbs et al., Int J Pharm, (2002) 129-136]thus, when administered orally they interact with peyrovani plaques, which contain 20% of all l is Mazitov body and launch an augmented immune response to those antigens and/or allergens, which were introduced together with the water solution.

In a specific embodiment of the present invention the composition according to the present invention includes nanoparticles based on PVM/MA, does not contain the antigen or allergen and immunostimulating agent. These nanoparticles in the present description are called "empty" nanoparticles and can be easily obtained by the method, for example, described in patent application WO/069938, the full contents of which are described in this description as a reference. To illustrate these empty nanoparticles easily get desolately water-alcohol solution phase copolymer (PVM/MA in acetone. The resulting nanoparticles can be brought into stable aqueous suspensions or lyophilized. Optional can be entered cross-linking agent. Can be used virtually any cross-linking agent containing one or more functional group which can interact with anhydrite groups of the copolymer (PVM/MA, mainly polyamine or a carbohydrate, such as amino acid, protein-oz or sid and the like, for example, lysine, arginine, histidine, soluble proteins, poly-L-lysine, poly-L-arginine, and the like, preferably, 1,3-diaminopropan.

These empty nanoparticles can act as an adjuvant in vaccination or in them is urotherapy, when combined with the introduction of vaccines or compositions for immunotherapy (immunotherapy compositions)containing the antigen or allergen, respectively, causing the effect of the stimulation of the immune response after vaccination or immunotherapy compositions and empty nanoparticles. The figure 12 shows that the introduction of empty nanoparticles induces the secretion of significant amounts of IFN-γ. Joint administration of the vaccine or immunotherapy composition and nanoparticles can be simultaneous or sequential in different times, in any order, for example, the first can be entered vaccine or immunotherapy composition, and then the nanoparticles, or Vice versa. Alternatively, this vaccine or immunotherapy composition and these nanoparticles can be introduced simultaneously. Vaccine or immunotherapy composition and nanoparticles can also be entered in the same composition or different compositions. The dose of injected blank nanoparticles may vary within wide limits, for example, from about 0.01 to about 10 mg/kg body weight, preferably from 0.1 to 2 mg/kg of body weight.

In another specific embodiment of the present invention the composition according to the present invention includes nanoparticles based on PVM/MA, filled with allergen or ant the genome and/or immunostimulating agent.

In one of the embodiments of the present invention the composition according to the present invention includes nanoparticles based on PVM/MA, filled with allergen or antigen, where these nanoparticles based on PVM/MA include the allergen or antigen.

Used in the present description, the term "allergen" refers to a substance to which the patient is sensitive and which is the cause immune reactions, for example the allergen is pollen, allergen insects, food allergen or allergens food components present in the saliva, ticks and biting insects that cause a hypersensitive response in a patient, compounds present in plants, inducing the reaction sensitivity of the patient, etc. Therefore, for example, can also be used protein in pollen of plants, such as pollen (Lolium perenne, Poa pretense, Cynodon dactylon, Festuca pratensis, Dactylis glomerata, Secale cereale, Hordeum vulgare, Avena sativa, Triticum sativa) pollen from other plants (such as,Artemisia vulgaris, Chenopodium album, Plantago lanceolata, Traxacum vulgare, Parietaria judaica, Salsola kali, Urtica dioica), or pollen of trees (such as,Olea europea, Plantus sp., Cupressus sp.) etc. can Also be used in protein insects such as dust mites (such as,Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acarus siro, Blomia tropicalis, Euroglyphus maynei, Glyciphagus domesticus, Lepidoglyphus destructor, Tyrophagus putrescentiaeand so Another allergen can be obtained skribkov and epithelium of the animal ( Alternaria alternata, Cladosporium herbarumthe epithelium of dogs, the epithelium cats, the epithelium of the horse, a mixture of feathers and down of different species of waterfowl,Penicillium notatumetc), as well as components of food, etc. in fact, the compositions of the present invention can be used any allergen to fill the nanoparticles; however, in the specific embodiment of the present invention the specified allergen is an ovalbumin (OVA) protein, which is widely used as experimental models of Allergy.

Used in the present description, the term "antigen" refers to natural or recombinant immunogenic product derived from a higher organism or microorganism, such as bacteria, virus, parasite, protozoa, fungus, etc. containing one or more epitopes, such as structural components of the specified body; toxins, such as exotoxins, etc. in fact, the compositions of the present invention can be used any antigen to fill the nanoparticles; however, in the specific embodiment of the present invention the specified antigen extract is NOTSalmonella enteritidis.

As you know,Salmonella enteritidisin food products is a common agent in the gastrointestinal tract of a person, most often detected in food poisoning (60% Sluch is s, which was allocated to this agent). Poultry and offal of the poultry is recognized as the main source ofSalmonellaand the most important source of human infections caused bySalmonella enteritidis. Infection is a zoonotic disease, transmitted by digestion of contaminated food or water, and is currently a pandemic. For the control of salmonellosis world Health Organization (who) and the European Union has published guidelines on monitoring and destroy infectionSalmonella enteritidisin birds because of the obvious effectiveness of the human population. For controlSalmonellabirds use antibiotics, competitive exclusion, genetic selection of birds and vaccines, improved hygienic conditions on farms for breeding birds. Of these measures the most common and most practical should consider vaccination as the most simple and cost efficient method. Despite widespread use of attenuated live vaccines (killed bacteria) vaccine both of them are quite ineffective on poultry farms (chicken and other bird species) [Zhang-Barder et al., Vaccine, 17 (1999) 2538-2545]. In addition to its low efficiency, a great disadvantage of live vaccines is their virulence potential in animals with depressed immunity, as well as their ability to modify invasive status. In addition to the, the vaccine should be injected parenterally and they interact with conventional serological diagnostic tests. Inactivated (killed) bacteria do not have residual virulence, but they do not induce a cellular immune response. Require the introduction of a strong adjuvant and there is a need multiple redaktirovaniya. An alternative would be the use of subcellular vaccines that can stimulate an appropriate immune response against infections caused bySalmonella enteritidis. Despite the high degree of protection described for these vaccines at the stage of the experiment, as in the case of killed bacterial vaccines require multiple booster doses to obtain an acceptable degree of protection [Powell, Pharm Res, 13 (1996) 1777-1785]. On the other hand, when oral administration is denaturation and breakdown in the gastrointestinal tract [Langer et al., Adv Drug Deliv. Rev, 28 (1997) 97-119], and thus, this type of vaccine must be administered parenterally, that is related with certain logistical and economic difficulties. AntigensSalmonella enteritidiscan be encapsulated in nanoparticles PVM/MA, described in the present description above, with the aim of solving these problems.

The allergen or antigen, is present in this embodiment of the composition according to the present invention may at least an hour is ichno to cover the surface of these nanoparticles and/or to fill in these nanoparticles. In a specific embodiment of the present invention the specified allergen or antigen covers all or part of the surface of these nanoparticles. This variant embodiment of the invention is used for selective stimulation of the Th2 response in the patient. In another specific embodiment of the present invention the specified allergen or antigen encapsulated within a specified PVM/MA nanoparticles. This variant embodiment of the invention is used to stimulate a balanced Th1 and Th2 response or for the predominance of Th1 response.

PVM/MA nanoparticles filled with allergen or antigen, can be easily obtained by the method similar to that described in patent application WO02/069938. To illustrate these nanoparticles filled with allergen or antigen, can be easily obtained by desolately in the liquid phase, for example water-alcohol phase, such as liquid phase formed by ethanol and water, a solution of the copolymer (PVM/MA in an organic solvent such as a polar organic solvent, for example acetone. The resulting nanoparticles can be brought into a state of suspension in water or they can be dried. Depending on the moment, which was added to the allergen or antigen, will be received different formulations with different ratios of allergen or antigen (see compositions, so is e as, NP I, NP II NP III NP IV, and NP is NOT 3934, described in examples 1 and 6). As an illustration, if necessary, coating the allergen or antigen all or part of the surface of the nanoparticles indicated the allergen or the specified antigen is incubated after evaporation of organic solvents. Similarly, if you want to encapsulate the allergen or antigen nanoparticles inside the specified allergen or specified antigen is incubated, the allergen or antigen when dispersed in a solvent, preferably in a solvent, which is a solution of the copolymer (PVM/MA, or can be mixed with the specified solvent, such as, preferably, a polar organic solvent or solvent, able to be mixed with the solvent of the polymer solution, such as acetone. Before the introduction of the specified liquid phase are desolately solution of copolymer (PVM/MA. If necessary, optional can be entered cross-linking agent, which is mentioned above in the context of empty nanoparticles.

Specifically, in another aspect, the present invention relates to a method for producing the composition according to the present invention containing nanoparticles copolymer (PVM/MA, filled with allergen or antigen, where these nanoparticles copolymer (PVM/MA include the allergen or antigen, the method involves the following stages:

a) is asolutely organic solution of a copolymer (PVM/MA, dissolved in an organic solvent with a water-alcohol solution;

b) removing the organic solvent to obtain nanoparticles; and

(C) the introduction of specified allergens or specific antigen in the specified organic solution of a copolymer (PVM/MA before desolately specified organic solution of a copolymer (PVM/MA, or, alternatively, incubation of the specified allergen or a specified antigen with these nanoparticles, obtained in stage b).

Organic solvent in which is dissolved copolymer (PVM/MA, can be any solvent in which to dissolve the specified copolymer, typically a polar solvent such as a ketone, for example acetone. The liquid phase used for the purpose of such desolvatation may be any water-alcohol solution containing alcohol and water, such as ethanol and water, such as alcohol and water, pharmaceutical grade (according to the application purified water or water for injection). Mainly the ratio of the copolymer solution: water-alcohol solution is from 1:1 to 1:10, preferably 1:4. Then the organic solvents are removed in any suitable way, for example by evaporation under reduced pressure, and immediately formed nanoparticles with stable water turbid suspension.

The introduction of the allergen or anti is on in the organic solution of the copolymer (PVM/MA before desolately specified organic solution of a copolymer (PVM/MA allows to obtain nanoparticles, inside of which contains the allergen or antigen. Mainly, the specified allergen or antigen introduced, dissolved or dispersed in the same organic solvent and the organic solution of the copolymer (PVM/MA, or, which is able to mix with the specified solvent such as a polar organic solvent, for example acetone. Alternatively, the incubation of the specified allergen or a specified antigen with nanoparticle obtained in stage b), allows to obtain nanoparticles, in which the allergen or antigen covers all or part of the external surface of the nanoparticles. In a specific embodiment of the present invention the incubation of allergen or antigen with nanoparticle is carried out in the aquatic environment.

If necessary, optional can be entered cross-linking agent to improve the stability of the nanoparticles, as described above, in the context of empty nanoparticles.

The obtained nanoparticles filled with allergen or antigen can be purified in a suitable way, for example by centrifugation, ultracentrifugation, tangential filtration or evaporation, including the use of vacuum.

And finally, if necessary, the nanoparticles filled with allergen or antigen, can be dried with the purpose of long term storage and preservation the purpose. Can be used with standard cryoprotectants, preferably in a concentration factor of 0.1 to 10% by weight of the total composition to facilitate lyophilization.

Nanoparticles filled with allergen or antigen, can act as adjuvant in vaccination or immunotherapy to stimulate the immune response after their introduction to the patient, as shown in examples 3-6.

Doses for the introduction of nanoparticles filled with allergen or antigen, vary over a wide range, for example, from about 0.01 to 10 mg/kg body weight, preferably from 0.1 to 2 mg/kg of body weight.

In another embodiment of the present invention the composition according to the present invention contains nanoparticles based on PVM/MA, filled immunostimulating agent, where these nanoparticles based on PVM/MA contain immune-stimulating agent.

Used in the present description, the term "immune-stimulating agent or immunomodulator" refers to a product that can enhance specific or nonspecific immune response, for example, to proteins or peptides, acting as natural adjuvants that stimulate the immune system response to an allergen or antigen, bacterial lipopolysaccharides, components of the cell walls of gram-positive bacteria (muramyl dipeptide (MDP)), CpG sequence DN is, secreted by the plant, mainly saponin, secreted by the plant, etc. in fact any immunostimulating agent can be used to produce nanoparticles filled immunostimulating agent composition of the present invention, however, in a specific embodiment, the present invention specified immunostimulating agent lipopolysaccharide is a majorBrucella ovis.

Specified immunostimulating agent may at least partially cover the surface of these nanoparticles and/or contained in these nanoparticles. In a specific embodiment, the present invention specified immunostimulating agent covers all or part of the surface of these nanoparticles PVM/MA, while in another specific embodiment, the present invention specified immunostimulating agent encapsulated in these nanoparticles PVM/MA.

Nanoparticles PVM/MA, filled immunostimulating agent, can easily be obtained in a manner analogous to that described above for nanoparticles PVM/MA, filled with allergen or antigen, but with substitution of the specified allergen or antigen immunostimulating agent. Thus, depending on the moment, which was introduced immunostimulating agent, will be received by various structures with different aspect] the agreements (see example 1 or 4). As an illustration, when you want an immunostimulating agent to cover the whole or part of the surface of the nanoparticles indicated immunostimulating agent is incubated after evaporation of organic solvents. Similarly, if necessary, an immunostimulating agent encapsulate inside the nanoparticles. For this specified immunostimulating agent is incubated. The agent is dispersed or dissolved in a solvent, mostly the same, which is a solution of the copolymer (PVM/MA, or in a solvent, able to mix with the specified solvent of the copolymer. Preferably it is a polar organic solvent or solvent, able to be mixed with the solvent of the polymer solution, such as acetone. Before the introduction of the specified liquid phase are desolately solution of copolymer (PVM/MA. If necessary, optional can be entered cross-linking agent, which is mentioned above in connection with empty nanoparticles to improve the stability of the nanoparticles.

Nanoparticles filled immunostimulating agent, can be cleaned by suitable means, as mentioned above in connection with nanoparticles filled with allergen or antigen.

If you nanoparticles filled immunostimulating agent, can also be sublimated the standard of cryoprotectants, preferably, the concentration factor of 0.1 to 10% by weight of the total composition.

Nanoparticles filled immunostimulating agent, can act as adjuvant in vaccination or immunotherapy to stimulate the immune response after their introduction into the patient.

Doses for the introduction of nanoparticles filled immunostimulating agent, vary over a wide range, for example, from about 0.01 to about 10 mg/kg body weight, preferably from 0.1 to 2 mg/kg of body weight.

In another embodiment of the present invention the composition according to the present invention contains nanoparticles of PVM/MA, filled with allergen or antigen with an immunostimulating agent, where these nanoparticles PVM/MA contain the allergen or antigen and immune-stimulating agent.

The allergen or antigen, as well as a stimulating agent that is present in this embodiment of the composition according to the present invention may at least partially cover the surface of these nanoparticles and/or to fill in these nanoparticles.

In a specific embodiment of the present invention the specified allergen or antigen covers all or part of the surface of these nanoparticles, although immunostimulating agent contained within these nanoparticles, we discovered that this particular implementation of the image is etenia allows you to selectively stimulate a Th2 response in a patient.

In another specific embodiment of the present invention the specified allergen or antigen encapsulated in nanoparticles PVM/MA, as indicated stimulating agent that at least partially covers the surface of these nanoparticles.

This variant embodiment of the invention is used to stimulate a balanced Th1 and Th2 response or response with a predominance of Th1 response.

In another specific embodiment of the present invention as an allergen or antigen, and immunostimulating agent encapsulated in these nanoparticles PVM/MA.

Nanoparticles PVM/MA, contains the allergen or antigen and immune-stimulating agent, can easily be obtained in a manner analogous to the method described above. Depending on the time at which the allergen or antigen and immune-stimulating agent has been introduced, will be received different formulations with different ratios of these products. As an illustration, similarly, if necessary, an immunostimulating agent covers all or part of the surface of the nanoparticles, the specified immunostimulating agent is incubated after evaporation of organic solvents. Similarly, if necessary, an immunostimulating agent encapsulate inside these nanoparticles. For this specified immunostimulating agent incubi the comfort. Immunostimulating agent is dispersed or dissolved in a solvent mainly containing the solution of copolymer (PVM/MA, or in a solvent, able to mix with the specified solvent of the copolymer. Preferably it is a polar organic solvent or solvent, able to be mixed with the solvent of the polymer solution, such as acetone. Before the introduction of the specified liquid phase are desolately solution of copolymer (PVM/MA. Similarly, if you want the allergen or antigen coated all or part of the surface of the nanoparticles, the allergen or antigen is incubated after evaporation of organic solvents. Similarly, if necessary, the allergen or antigen encapsulate inside these nanoparticles. For that particular allergen or antigen is incubated. The allergen or antigen is dispersed or dissolved in a solvent mainly containing the solution of copolymer (PVM/MA, or in a solvent, able to mix with the specified solvent of the copolymer. Preferably it is a polar organic solvent or solvent, able to be mixed with the solvent of the polymer solution, such as acetone. Before the introduction of the specified liquid phase are desolately solution of copolymer (PVM/MA. Similarly, when you want allerg is h or antigen and immune-stimulating agent was encapsulated inside these nanoparticles, the allergen or antigen is incubated, optionally mixed with the specified immunostimulating agent, which is dispersed or dissolved in a solvent mainly containing the solution of copolymer (PVM/MA, or can be mixed with the specified solvent, such as preferably a polar organic solvent or solvent, able to be mixed with the solvent of the polymer solution, such as acetone. Before the introduction of the specified liquid phase are desolately solution of copolymer (PVM/MA. In any case, if necessary, may not necessarily be introduced cross-linking agent to the obtained nanoparticles to improve their stability, as described above in connection with empty nanoparticles.

Namely, in another aspect, the present invention relates to a method for producing the composition according to the present invention containing nanoparticles copolymer (PVM/MA, filled with allergen or antigen and the immunopotentiating agent, where these nanoparticles copolymer (PVM/MA include the allergen or antigen and immune-stimulating agent. This method involves the following stages:

and desolvatation organic solution of a copolymer (PVM/MA, dissolved in an organic solvent with a water-alcohol solution;

b) removing the organic solvent to obtain nanoparticles; and

(C) the introduction of specified allergens or specific antigen and/or specific immunostimulating agent specified in the organic solution of the copolymer (PVM/MA before desolately specified organic solution of a copolymer PVM/MA or alternatively, the incubation of the specified allergen or a specified antigen or specific immunostimulating agent with the specified nanoparticles obtained in stage b).

Stages a) and b) are conducted in a manner analogous to that described above in the context of the method of obtaining the composition of the present invention containing nanoparticles copolymer (PVM/MA, filled with allergen or antigen. Stage C) is carried out in a manner analogous to the method described above in connection with the specified way, while making appropriate modifications for the introduction of the immunostimulating agent, or for a specified organic solution of a copolymer (PVM/MA before desolately specified organic solution of a copolymer (PVM/MA, or for the optional introduction of the allergen or antigen, or alternatively incubated specified immunostimulating agent with the specified nanoparticles obtained in stage b), optionally partially covered by the specified allergen or antigen, as described above.

If necessary, optional can be entered cross-linking agent to improve the stability of the obtained nanoparticles. The obtained nanoparticles filled with allergen or antigen and immunostimulatory agent can be purified in a suitable way, for example centrifuging what W, ultracentrifugation, tangential filtration or evaporation, including the use of vacuum.

And finally, if necessary, the nanoparticles filled with allergen or antigen, can be lyophilized for long term storage and preservation. Can be used with standard cryoprotectants, preferably in a concentration factor of 0.1 to 10% by weight of the total composition to facilitate lyophilization.

Nanoparticles filled with allergen or antigen and the immunopotentiating agent, can act as adjuvant in vaccination or immunotherapy and stimulate the immune response after their introduction to the patient, as shown in examples 3-6.

Dose nanoparticles, filled with allergen or antigen and the immunopotentiating agent, vary over a wide range, for example, from about 0.01 to 10 mg/kg body weight, preferably from 0.1 to 2 mg/kg of body weight.

If necessary, the composition according to the present invention can be in lyophilized form or in a form suitable for oral administration or parenteral administration. The lyophilization is conducted in a suitable way, not necessarily in the presence of suitable cryoprotectants such as sucrose, mannitol, trehalose, glycerol, lactose, sorbitol, polyvinylpyrrolidone and the like, preferably, the concentration is s is 0, 1 to 10% by weight of the total composition. To obtain various forms suitable for oral or parenteral administration, can be used suitable fillers and carriers that are suitable for obtaining the necessary pharmaceutical forms for administration. Information about these media, and fillers, as well as information about these forms suitable for oral or parenteral compositions of the present invention, may be found in pharmaceutical directories.

As mentioned previously, the composition according to the present invention stimulates or enhances the immune response after its introduction to the patient and, thus, can be used as an adjuvant in vaccines or immunotherapy. Indeed, the composition of the present invention has the ability to selectively stimulate one of the two pathways of the immune response (Th1 or Th2 dysbalance) or both ways simultaneously sbalansirovanno, and thus, it can be used in the vaccine or immunotherapy compositions according stimulated response. As an illustration, typically, the vaccine composition is required depending on the mechanisms of pathogenicity of the organism from which the antigen (intracellular or extracellular, exensively, egotistically etc.), stimulation of Th1 response (vnutri mocny, as in the case ofBrucella, Salmonellaand the like) or Th2 dysbalance response (intracellular, as in the case ofStaphylococcus,Escherichia coli, enterotoxigenic bacteria and the like). Similarly, as an illustration, you want tolerance induction for immunotherapy composition due to the presence of two types of response, such as a balanced induction of Th1 and Th2 dysbalance answers.As a rule, to stimulate selective Th2 dysbalance answer compositions will contain nanoparticles PVM/MA, fully or partially covered by the allergen or antigen, while for a balanced stimulation of Th1 and Th2 dysbalance responses or with a predominance of Th1 response, the compositions will contain nanoparticles PVM/MA, in which the antigen or allergen is encapsulated, and these nanoparticles are mainly contain cross-linking agent. In any of the previously mentioned cases, if necessary, the nanoparticles may contain an immunostimulating agent.

In addition, another aspect of the present invention relates to a vaccine or immunotherapy composition, comprising a therapeutically effective amount of a composition for stimulating an immune response (the composition of the present invention together with a pharmaceutically acceptable carrier or excipient. This vaccine or immunotherapy composition may be in any pharmaceutical form, entered the nd in any way, for example, oral, parenteral, rectal, etc. In a specific embodiment, the present invention this vaccine or immunotherapy composition is a pharmaceutical form for oral administration, although in another specific embodiment, the present invention this vaccine or immunotherapy composition is a pharmaceutical form for parenteral administration, such as intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.), intraperitoneal (I.P. Pavlova.), intradermal (i.d.) etc. Doses for various forms of administration of drugs in General and methods for their preparation can be taken in "Tratado de Farmacia Galénica", .Faulí I Trillo, 1stEdition, 1993, Luzán 5, S.A. de Ediciones.

The composition of the present invention, while in the specified immunotherapy vaccine or composition obtained according to the present invention, includes nanoparticles PVM/MA. These nanoparticles may also contain an allergen or antigen and/or an immunostimulating agent that can be contained within these nanoparticles and/or at least partially cover the surface of these nanoparticles. Similarly, these particles may not necessarily contain a cross-linking agent.

In a specific embodiment of the present invention, the composition p of the present invention, while in the specified immunotherapy vaccine or composition obtained according to the present invention includes a blank nanoparticles PVM/MA, necessarily introduces a cross-linking agent. In this case, the composition of the present invention is administered in conjunction with a vaccine or immunotherapy compositions containing an antigen or allergen, respectively stimulating the immune response after injection of the indicated vaccine or immunotherapy compositions and empty nanoparticles. Joint introduction of specified vaccines or immunotherapy compositions and empty nanoparticles can be carried out simultaneously or sequentially at different times, in any order, for example, the first can be entered vaccine or immunotherapy composition and then empty nanoparticles, or Vice versa. Alternatively, the vaccine or immunotherapy composition and these empty nanoparticles can be introduced simultaneously. In this case, the vaccine or immunotherapy composition and empty nanoparticles can be entered in the same composition or different compositions.

In another specific embodiment of the present invention the composition of the present invention, while in the specified immunotherapy vaccine or composition obtained by the present the mu to the invention, includes nanoparticles PVM/MA, where these nanoparticles PVM/MA, in addition, contain the allergen or antigen, and, if necessary, a cross-linking agent.

In another specific embodiment of the present invention the composition of the present invention, while in the specified immunotherapy vaccine or composition obtained according to the present invention, includes nanoparticles PVM/MA, where these nanoparticles PVM/MA, in addition, contain an immunostimulating agent and, if necessary, a cross-linking agent.

In another specific embodiment of the present invention the composition of the present invention, while in the specified immunotherapy vaccine or composition obtained according to the present invention includes nanoparticles PVM/MA, where these nanoparticles PVM/MA, in addition, contain the allergen or antigen and immune-stimulating agent, and if necessary cross-linking agent.

Immunotherapy vaccine or composition obtained according to the present invention contains a composition of the present invention in a therapeutically effective dose, for example, in a quantity which is suitable for enhancing or stimulating an immune response. In addition, the number of compositions of the present invention, in the vaccine or immunotherapy whom is osili, obtained according to the present invention may vary within a wide range depending on other factors, including the type of nanoparticles (empty or filled with allergens or antigens and/or immunostimulatory agent), etc. in Addition, in a specific embodiment of the present invention, the invention relates to a vaccine or immunotherapy composition containing:

Component% of the total mass
Nanoparticles PVM/MA84-99,998%
Cross-linking agentof 0.001-1%
The allergen or antigenof 0.001-15%

Specified immunotherapy vaccine or composition optionally may contain cryoprotector in a quantity sufficient to protect the nanoparticles during the process liofilizirovanny.

In a specific embodiment of the present invention the specified allergen contains allergens extracted from pollen, allergens extracted from insects, or allergens extracted from food.

In a specific embodiment, the present invention specified antigen soda is separated by immunogen, extracted from an organism, for example, the extracted membraneSalmonella spp.

As mentioned previously, the composition according to the present invention can stimulate or enhance the immune response after administration to the patient, thus, it can be used as an adjuvant in vaccines or immunotherapy and, in particular, is capable of selectively stimulate one of the two pathways of the immune response (Th1 or Th2), or even both simultaneously and in a balanced manner.

In addition, in another aspect, the present invention relates to the use of a composition according to the present invention in the production of vaccines or immunotherapy composition.

In another aspect, the present invention relates to the use of a composition according to the present invention in the manufacture of pharmaceutical compositions for the selective stimulation or stimulation with domination or enhancing Th1 immune response.

In another aspect, the present invention relates to the use of a composition according to the present invention in the manufacture of pharmaceutical compositions for the selective stimulation or stimulation with domination or enhance Th2 immune response.

In another aspect, the present invention relates to the use of a composition according to the present invention in the manufacture of pharmaceutical compositions for sbalansirovany the th stimulation or enhancement of Th1 and Th2 immune responses.

The use of a composition according to the present invention for vaccination or immunotherapy has certain advantages, which include:

- the use of pharmaceutical forms that are biodegradable under normal conditions, the body and which are derived from polymers and materials, acceptable in pharmacological and medical practice;

- conducting immunotherapy using nanoparticles and the introduction of the vaccine preparation based on nanoparticles, which protects the encapsulated allergen or antigen from premature inactivation;

- receiving immunotherapy drug and vaccine based on nanoparticles that exhibit slow release over time; the period of time during which it will be controlled release, will depend on conditions microarrays environment at the injection site;

- introduction of nanoparticle adjuvant based on nanoparticles, who pass on their characteristics (safety, biodegradation, the avirulence), it can be used in humans and animals for immunotherapy purposes and vaccination through different types of introduction; and

- introduction of nanoparticle adjuvant based on nanoparticles, which transmit their bioadhesive potential in the gastrointestinal tract, making oral administration possibly the first form of the introduction.

The following examples illustrate the invention but do not restrict it.

Examples

The following examples describe various types of nanoparticles based on PVM/MA, which does not necessarily contain allergens or antigens and/or immunostimulating agent, and demonstrate the ability of these nanoparticles to act as adjuvants in immunotherapy or vaccination.

The main method of obtaining nanoparticles

The main way to obtain nanoparticles of PVM/MA includes desolately specified copolymer in acetone with subsequent introduction of ethanol. In the resulting solution entered the water, which immediately forms a particle in the middle in the form of a turbid suspension. Then the organic solvents (ethanol and acetone) are removed by evaporation at low pressure, the particles remain in a stable aqueous suspension [Arbos et al., J Control Rel, 83 (2002) 321-330]. Depending on the time at which were introduced allergens or antigens, and where possible applied immunostimulating agent, you will receive various compositions with different ratios (see examples 1, 5 and 6). As an example:

And

To obtain compositions containing the allergen or antigen, encapsulated in nanoparticles (compositions NP I and NP (VI): incubation with the allergen or antigen for encapsulating is performed on the Le evaporation of organic solvents.

In

To obtain compositions containing the allergen or antigen, fully or partially covering the outer surface of the nanoparticles (compositions NP II NP III NP IV, NP, V, NP, VII, OVASAL and NP HE): before the introduction of ethanol and water incubated allergen or antigen, dispersed in acetone.

The next step is the introduction of a cross-linking agent, which in this case is 1,3-diaminopropanol, NP III NP V NP VII, OVASAL, NP HE (a total of 5 μg/mg of polymer) and NP (IV (10 μg/mg of polymer).

The compositions NP V NP VI and NP VII contain an immunostimulating agent, consisting of a large lipopolysaccharide (R-LPS)Brucellaovis. Briefly, for its introduction in the case of the composition of the NP V incubation with the specified immunostimulating agent is carried out after evaporation of organic solvents, although in the case of compositions NP VI and NP VII R-LPS incubated after the introduction of ethanol and water (see examples 1.4.2, 1.4.3 and 1.4.4).

Purification of the nanoparticles is carried out by ultracentrifugation. Finally, the purified nanoparticles lyophilizer to increase retention and preservation.

Characterization of nanoparticles

The size and Zeta-potential of the nanoparticles were determined in Zetamaster (Malvern Instruments/Optilas, Spain).

The content of the encapsulated protein (active ingredient) was determined by analysis with micromechanical acid (Micro BCA, Pierce, USA).

Large lipopolysaccharide Brucella ovis(R-LPS), adsorbed or encapsulated in nanoparticles was determined indirectly through one of its exclusive markers DO (3-deoxy-D-manno-Octo-2-oleanolova acid) method using thiobarbituric acid [Warren, J Biol Chem, 243 (1959) 1971-1975].

Determination of antibodies against OVA, cytokine and IL-10

Serum antibodies against OVA was investigated by ELISA with conjugates of anti-IgG1and anti-IgG2A(Sigma-Aldrich Chemi, Germany). Briefly, for the purposes of this study were used 96-well plates (EB, Thermo Labsystems, Vantaa, Finland), each well contained 1 μg of ovalbumin, which was fixed at 4°C for 15 hours; were carried out consistently 5 washes, each for 1 minute in the washing device ELISA (Thermo Labsystems, Vantaa, Finland), sera were introduced in serial solutions, ranging from 1:40, they were kept at 37°C for 4 hours, and were conducted sequentially 5 washes, each for 1 minute; then the conjugate was incubated for 2 hours with peroxidase were also carried out consistently 5 washes, each for 1 minute, the substrate [ABTS (2,2'-Azino-bis(3-ethylbenzo-thiazoline-6-sultanekova acid) (Sigma-Aldrich Chemi, Germany)] was introduced and the reading was carried out at 405 nm in the reader (iEMS Reader MF, Labsystems, Vantaa, Finland) after 30 minutes incubative room temperature.

Cytokines (IFN-γ, IL-4) were investigated using ELISA kits (Biosource, USA) supernatant of restimulation of spleen cells pre-immunized mice.

IL-10 was studied in the blood serum of the pre-centrifuged at 800 x g for 10 minutes. IL-10 was investigated by ELISA kit (Biosource, USA).

Method elecrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE)

The profile of protein samples was determined using SDS-PAGE [Laemmli, Nature, 227 (1970) 680-685] at 15% concentration of acrylamide and bisacrylamide (Biorad), taken in the ratio of 37.5:1, respectively, in 125 mm Tris-HCl (pH of 6.8) and 0.1% SDS in the gel. Used electrode buffer contained 30 mm Tris-HCl (pH 8,3), 192 mm glycine and 0.1% SDS. The sample was processed at 100°C for 10 minutes in 62.5 mm Tris-HCl (pH 6,8), 10% glycerol, 2% SDS, 5% β-mercaptoethanol and 0.002% of Bromphenol blue. Electrophoresis was carried out in 8 x 7 cm gel with a constant density of 15 mA/gel. The gel was stained Coomassie blue [King, Anal Biochem, 71 (1976) 223-230]. For this purpose, it was incubated in 3% aqueous solution of trichloroacetic acid for 1 hour to secure. Staining was performed with 0.25% solution of Coomassie blue incubation in 50% methanol and 10% acetic acid for 1 hour, and the samples were discolored 50% methanol and 20% acetic acid until yet become visible. The approximate molecular weight what s the components of the sample were identified by comparing their electrophoretic mobility with a token with standard molecular weight (Rainbow, Amersham Pharmacia Biotech, Uppsala, Sweden)containing myosin (220 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (of 20.1 kDa) and lysozyme (of 14.3 kDa).

The Western blot turns

The Western blot turns was performed according to previously described Protocol [Towbin & Staehelin, Proc Natl Acad Sci USA 76 (1979) 4350-4354]. Briefly, after treatment of the samples SDS-PAGE gel was passed through a nitrocellulose membrane with a pore size of 0.45 µm (Schleicher &Schuell, Dassel, Germany). The pass was performed using the semi-dry method by Trans-Blot® SD, Semy-Dry transfer Cell system (Bio-Rad, Richmond, USA) at 200 mA, 5 V for 30 minutes in the transfer buffer 25 mm Tris-HCl (pH 8,3), 192 mm glycine and 10% methanol.

Example 1

Immunotherapy

Fabrication and characterization of nanoparticles based copolymer metilfenidato ether maleic anhydride with ovalbumin

Selected protein was albumin (OVA), because at the moment it is widely used as experimental models of Allergy.

About 50% of the protein contained in white eggs, is ovalbumin. He is Monomeric phosphoglycoprotein containing 385 amino acids with a molecular mass of from 43 to 45 kDa [Johnsen and Elsayed, Mol Immunol, 27 (1990) 821]. Ovalbumin is the egg white with the highest level of allergenicity, immediately inducing hypercast is of type I, mediated by IgE.

The following method is suitable for dosage forms with nanoparticles colloid type, which can be used for immunotherapy.

1.1 Getting empty nanoparticles (NP)

100 mg of copolymer metilfenidato ether maleic anhydride (PVM/MA) [Gantrez® AN-119] were dissolved in 5 ml of acetone. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. The resulting mixture was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was brought by water to 10 ml

The solution was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

Received empty nanoparticles (NP) had an average size of less than 200 nm and the total surface charge -45,1 mV (see Table 1).

1.2 Obtaining nanoparticles, coated with ovalbumin (NPI)

100 mg of copolymer metilfenidato ether maleic anhydride (PVM/MA) [Gantrez® AN-119] would and dissolved in 5 ml of acetone. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. The resulting mixture was subjected to homogenization for 5 minutissima nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was reduced with water to 5 ml of This solution was incubated for 1 hour at room temperature with 5 ml of an aqueous solution of ovalbumin containing 10 mg of protein.

The mixture was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

The obtained nanoparticles (NPI) had an average size of less than 300 nm and the total surface charge -61,3 mV and content of ovalbumin reached 54.7 ág/mg polymer (see Table 1).

1.3 Obtaining nanoparticles with encapsulated ovalbumin (NPII, NPIII and NPIV)

100 mg of copolymer metilfenidato ether maleic anhydride (PVM/MA) [Gantrez® AN-119] were dissolved in 4 ml of acetone, in addition, while 5 mg of ovalbumin were dispersed in 1 ml of acetone by ultrasonic treatment Microson TMor ultrasonic bath for 1 min with cooling. The variance of ovalbumin was introduced in the polymer suspension and stirred for 30 minutes at room temperature. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. The resulting mixture was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was brought by water to 10 ml of the Obtained nanoparticles were encapsulated OVA (NPII). In the case of compositions NPIII and NPIV then the same could be introduced cross-linking agent: 0.05 ml and 0.1 ml of 1% solution of 1,3-diaminopropane respectively.

The mixture was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

The resulting nanoparticles had an average size of less than 300 nm and the total surface charge -41,4 mV in the case of NPII, -50,8 mV in the case NPIII, -57,5 mV in the case of NPIV, and the content of ovalbumin was close to 30 µg/mg polymer (see Table 1).

1.4 Received the e nanoparticles with ovalbumin and large lipopolysaccharide Brucella ovis(NPV, NPVI, NPVII)

1.4.1 Selection and characterization of complex large-scale lipopolysaccharideBrucella ovis

Used the extract is a major polysaccharideBrucella ovis(R-LPS). R-LPS was obtained in the way described earlier (Galanos et al., Eur. J. Biochem. (1969), 245-249). After culturing bacteriaBrucella ovisthey were resuspendable in absolute ethanol with stirring at a temperature of 4°C overnight in a closed container. Then cells were centrifuged (6000 x g, 20 min, 4°C) and resuspendable in acetone with stirring for 12 hours at 4°C in a closed container. Then cells were collected by centrifugation (6000 x g, 20 min, 4°C), they were resuspendable in ethyl ether and were kept at room temperature for 3 to 4 hours with magnetic stirring. Then they were again centrifuged (6000 x g, 20 min, 4°C) and were subjected to evaporation to dry. For carrying out the extraction cells were mixed before homogenization with petroleum ether, simply ether of phenol and chloroform in the ratio 8:5:2 respectively. The mixture was centrifuged (8000 x g, 15 minutes, room temperature) and collected the supernatant. The precipitate in the test tube was extrahieren under the same conditions twice. The entire supernatant was combined and petroleum ether and ether chloroform were in party in a rotary evaporator. Phenolic residue was precipitated with distilled water and centrifuged (8000 x g, 30 minutes, room temperature). Then it was washed twice with phenol and twice with ethyl ether. After the last centrifugation the remaining ethyl ether was removed under vacuum, the residue in the tube was resuspendable in deionized water and was subjected to dialysis. The dialysis bag was centrifuged (100000 x g, 6 hours, 4°C), the precipitate in the test tube was resuspendable in deionized water and subjected to lyophilization.

The number of R-LPS was determined indirectly through one of its exclusive markers KDO using method using thiobarbituric acid.

1.4.2Nanoparticles with encapsulated ovalbumin and R-LPSBrucella ovison the surface (NP V).

100 mg of copolymer metilfenidato ether maleic anhydride [Gantrez® AN-119] were dissolved in 4 ml of acetone, in addition, while 5 mg of ovalbumin were dispersed in 1 ml of acetone by ultrasonic treatment (MicrosonTMor ultrasonic bath for 1 min with cooling. The variance of ovalbumin was introduced into the copolymer suspension and stirred for 30 minutes at room temperature. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. Resulting from mesh was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was brought by water to 9 ml of This solution was maintained for 1 hour at room temperature with 1 ml of water dispersion (previously was held sonication for 1 minute), containing 1 mg of R-LPSBrucella ovis.

The mixture was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

The obtained nanoparticles (NP V) had an average size of less than 300 nm and the total surface charge -46,1 mV, the content of ovalbumin was 64.1 ág/mg of polymer and 15.2 ág of R-LPS /mg of polymer (see Table 1).

1.4.3Nanoparticles encapsulated with R-LPSBrucella ovisand ovalbumin on the surface (NP (VI).

100 mg of copolymer metilfenidato ether maleic anhydride [Gantrez® AN-119] were dissolved in 4 ml of acetone, in addition, at the same time, 1 mg of R-LPSBrucella oviswas dispersed in 1 ml of acetone by ultrasonic treatment (MicrosonTMor ultrasonic bath for 1 min with cooling. The variance of R-LPS was the introduced in the copolymer suspension and stirred for 30 minutes at room temperature. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. The resulting mixture was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was reduced with water to 5 ml of This solution was incubated for 1 hour at room temperature with 5 ml of an aqueous solution containing 5 mg of ovalbumin.

The mixture was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

The obtained nanoparticles (NP (VI) had an average size of less than 300 nm and the total surface charge -56,9 mV, the content of ovalbumin was 68.5 ág/mg of polymer and 12.1 ág of R-LPS /mg of polymer (see Table 1).

1.4.4Nanoparticles with encapsulated ovalbumin and R-LPSBrucella ovison the surface (NP (VII)

100 mg of copolymer metilfenidato ether maleic anhydride [Gantrez® AN-119] were dissolved in 3 ml of acetone, in addition, at the same time, 5 mg of ovalbumin were dispersed in 1 ml of acetone pose the STV ultrasonic treatment (Microson TMor ultrasonic bath for 1 minute, cooling, and 1 mg of R-LPSBrucella oviswas dispersed in 1 ml of acetone, was held ultrasonic treatment with cooling. The variance of ovalbumin was introduced in the variance of R-LPS and this mixture was introduced into the copolymer suspension and stirred for 30 minutes at room temperature. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. The resulting mixture was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was brought by water to 10 ml

The mixture was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

The obtained nanoparticles (NP (VII) had an average size of less than 300 nm and the total surface charge -46,1 mV, the content of ovalbumin reached 54.7 μg/mg of polymer and 13.8 µg of R-LPS /mg of polymer (see Table 1).

1.5 Characterization of nanoparticles

Physico-chemical features and advantages of the ka of various compositions shown in Table 1.

According to the results, it was found that when the nanoparticles were coated with ovalbumin (NP I and NP (VI), continued to increase in size and Zeta potential became more negative, while if the ovalbumin was inside the nanoparticles (NP II NP III NP IV, NP V NP (VII), the size as well as the Zeta-potential was not changed compared with empty nanoparticles (NP).

At the same time it was found that the size of the nanoparticles decreased as reduced the number of cross-linking agent and slightly reduced the amount of ovalbumin.

The presence of R-LPS on the surface of the nanoparticles causes the number of encapsulated ovalbumin increase (NP V in comparison with NP II), while if R-LPS is mainly inside the nanoparticles, the amount of adsorbed ovalbumin does not change (NP VI in comparison with NP (I).

Table 1
Physico-chemical characteristics of different compositions (with the data as mean±standard deviation, n=10)
Size (nm)Zeta-potential (mV)Encapsu-
isolation
OVA (ág/mg)
The effectiveness of encapsu-
population
(%)
R-LPS (µg/mg)
NP158±3-45,1±0,5---------
NP I300±4-61,3±3,867,8±20,247,50±2,12---
NP II205±1-41,4±2,536,1±3,850,48±5,32---
NP III239±4-50,8±2,930,1±4,542,14±6,30---
NP IV270±2-57,5±3,131,4±4,244,08±5,88---
NP V152-46,164,1±2,989,74±4,0615,2±0,5
NP VI287-56,968,5±2,4 47,95±1,6812,1±0,6
NP VII135-43,754,7±1,976,58±2,6613,8±3,0

The output of the nanoparticles was approximately 70%.

Example 2

Testin vitrorelease of ovalbumin nanoparticles

To assess the allocation of ovalbuminin vitrothe nanoparticles were incubated in 1 ml PBS (phosphate buffer saline pH 7.4) in test tubes "Eppendorf" with a concentration of approximately 8 mg/ml These test tubes were incubated in a thermostat at 37°C with rotation, and the samples were subjected to centrifugation at pre-defined intervals when 26500 x g for 20 minutes, collecting the supernatant for further research. Released ovalbumin was measured in the supernatant using the method using bicinchoninic acid.

The obtained curve selection ovalbumin shown in figure 1; it was found that the composition of NP I ovalbumin released much faster than in the compositions of NP II NP III NP IV, which showed a similar profile release. This is because in the song NP I ovalbumin adsorbed on the outer surface of the nanoparticles, so you initiated the order his (explosion) is much sharper, than other compositions, in which a portion of ovalbumin encapsulated (NP II, III and IV). At the same time, the release profiles NP II NP III NP IV can be observed that as the number of cross-linking agent increases, the percentage of free ovalbumin slightly reduced.

Example 3

Quantifying the production of antibodies against OVA after immunization with ovalbumin in mice BALB/c

Were immunized 65 BALB/c mice, then divided into 13 groups according to the scheme of administration.

For control were used: a solution that is free from ovalbumin (OVA) (10 μg intradermally and 25 mg orally), empty nanoparticles (NP) (intradermally and oral) and ovalbumin adsorbed on alhydrogel (OVA-Alum) (10 μg intradermally), as a positive control to induce a Th2 response, characterized by high titers of IgG1[Faquim-Mauro et al., Int Immunol, 12 (2000) 1733-1740].

The remaining groups were inoculated intradermally (10 μg OVA) or oral (25 μg OVA), differences in treatment was:

A) a solution of ovalbumin (OVA) intradermally

B) a solution of ovalbumin (OVA) oral

(C) ovalbumin adsorbed on alhydrogel (OVA-Alum), intradermally

D) blank nanoparticles (NP) intradermally

E) blank nanoparticles (NP) oral

(F) nanoparticles coated with ovalbumin (NP I), vnutri is tenderly

G) nanoparticles coated with ovalbumin (NP I)oral

H) nanoparticles with inkapsulirovanne by ovalbumin (NP II) intradermally

I) nanoparticles with inkapsulirovanne by ovalbumin (NP II) oral

J) nanoparticles with a cross-linking agent is 1,3-diaminopropanol with inkapsulirovanne by ovalbumin (NP III NP (IV) intradermally

K) nanoparticles with a cross-linking agent is 1,3-diaminopropanol with inkapsulirovanne by ovalbumin (NP III NP (IV) oral.

Sequential extraction of blood were conducted at 7, 14, 28 and 35 days after immunization. The sera of each group were collected and frozen at a temperature of -80°C for further studies.

The levels of antibodies against OVA (IgG1and IgG2a) were identified in various sera by ELISA, and the data obtained are shown in figure 2 and 3. It was found that the nanoparticles containing ovalbumin (NP I, NP II NP III NP (IV), each of which is covered nanoparticles or was encapsulated in them, strengthened the immune response to ovalbumin in solution. On day 14 after administration of these compositions with nanoparticles increased the titer IgG1sera of mice at 3 or 6 logarithmic units depending on type. At the same time, in the serum of mice, which was introduced OVA-Alum, antibody IgG2Awas absent during the whole experiment, while in mice, which was introduced NP III 35 day after immunization, they reached titers in 5120.

This shows that these NP composition (NP I, NP II NP III NP (IV) is able to increase the titers of antibodies and Th1 and Th2, but, in particular, the composition NP III is the most immunogenic.

At the same time, if the titers of mice in the group treated NP III, compared with the titers of the mice group treated NP IV, you can see that there is an optimal cross-linking (Example 2, last paragraph), because, although they have similar curves IgG1titres of IgG2Afor NP III slightly longer than in the case of NP IV.

Comparing the results obtained in mice when administered orally, it was found that only NP III was the music that induced measurable levels of antibody IgG1and IgG2A(figure 3).

After confirmation of this in the same experimental conditions, the composition of the NP III was the most effective, a similar study was conducted by modifying the input dose NP III. In this case, the following compositions were introduced (oral) animal:

A) blank nanoparticles (NP)

In) 25 mcg inkapsulirovanie of ovalbumin nanoparticles, cross-linked with 1,3-diaminopropane with encapsulated ovalbumin (NP III - 25)

(C) 50 μg inkapsulirovanie of ovalbumin nanoparticles, cross-linked with 1,3-Daminova is with an encapsulated ovalbumin (NP III-50).

In this case, the results obtained (figure 4) show that the levels of IgG2Aagainst OVA in serum were significantly higher when the dose was 50 mg.

Example 4.

Quantifying the production of antibodies against OVA and interleukin 10 (IL-10) after the introduction of the ovalbumin nanoparticles with lipopolysaccharideBrucella ovis

Were immunized 40 BALB/c mice, divided into 8 groups according to the shape of the introduction. In all groups was performed intradermal injection (10 μg of ovalbumin). This time the differences in treatment were:

A) a solution of ovalbumin (OVA)

C) ovalbumin absorbed on alhydrogel (OVA-Alum)

With empty nanoparticles (NP)

(D) nanoparticles coated with ovalbumin (NP I)

(E) nanoparticles cross-linked with 1,3-diaminopropane with inkapsulirovanne by ovalbumin (NP (III)

(F) nanoparticles cross-linked with 1,3-diaminopropane with inkapsulirovanne by ovalbumin and covered withBrucella ovisR-LPS (NP V)

G) nanoparticles encapsulated withBrucella ovisR-LPS and coated with ovalbumin (NP (IV)

H) nanoparticles cross-linked with 1,3-diaminopropane with encapsulated ovalbumin andBrucella ovisR-LPS (NP (VII).

The group, which was introduced ovalbumin adsorbed on alhydrogel (OVA-Alum), was taken as a positive control of the experiment.

Sequential extraction of blood were PR is maintained at 7, 14, 28, 35, 42 and 49 days after immunization. The samples were processed (see example 3) and the results obtained are shown in figures 5 and 6.

The IgG response1(Figure 5): NP III NP V NP VII induced high levels of IgG1more quickly and more intensely than the control composition (OVA-Alum). Only on the 49th day after immunization compositions based on nanoparticles and control (OVA-Alum) showed similar levels. Finally, R-LPS on the surface of the nanoparticles had no inducing effect on the levels of IgG1.

At the same time NP I also shows the great potential of inducing production of antibodies IgG1. In addition, as in the previous case, this phenomenon was faster and more intense than the control composition. When R-LPS ingested nanoparticles coated with OVA, it was found a significant reduction in serum titers of IgG1in comparison with NP I and the control compositions.

The IgG response2Aon the figure 6 shows the titers of IgG2Ain the serum of mice. In this case, none of the control compositions (OVA, NP, OVA-Alum) not induced antibody titers. However, compositions with encapsulated OVA were able to provide high titers of IgG2Amainly when R-LPS was adsorbed on the outer surface of nanoparticles (composition NP (V). At the same time NP I proved to be much more effective than NP IV to induce a Th1 response, measured by the CSOs in the titer of serum IgG 2A.

IL-10: figure 7 shows the levels of IL-10 serum, which were determined by an ELISA kit (Biosource, Camarillo, USA). In this case, none of the control compositions were not able to induce the secretion of IL-10 serum. In addition, all compositions with nanoparticles to a greater or lesser extent induced secretion of the cytokine. Composition with ovalbumin adsorbed on the surface of the NP (NP I), allows to induce the maximum value of IL-10 is much faster than the rest of the composition. In addition, the introduction of LPS (NP (VI) significantly slows down (14 days) secretion of the cytokine. At the same time, NP III showed that is the most effective composition for inducing IL-10 serum, resulting in levels that were two times higher than that of NP I, although the delay time was one week. The introduction of R-LPS in encapsulated particles OVA (NP V NP (VII) allows to induce more discrete levels of IL--10, but with a maximum value within two weeks after injection. This potential compositions to induce significant levels of IL-10 suggests their use as a possible treatment for diseases characterized by the imbalance in the balance of Th1/Th2 through the regulation of this cytokine [Zuany-Amorim et al., J Clin Invest, 95 (1995) 2644-2651; Stampfli et al., Am J Respir Cell Mol Biol, 21 (1999) 586-596; all et al, Vaccine, 21 (2003) 549-561].

Example 5

Obtaining, characterization, and the maintenance of nanoparticles formulated with ChE Salmonella enteritidis

5.1 Selection of the extract ChESalmonella enteritidis

ChE (atrophy extract) bacterial extract was obtained by following the Protocol described previously Altman et al. (Altman et al., Biochem J (1982) 505-513). Inoculum (inoculate)Salmonella enteritidisin the stable phase was incubated in flasks containing 400 ml of medium BHI (broth with cardio-cerebral extract Brain Heart Infusion, Difco Lab., Detroit, USA) at 37°C for 48 hours, without stirring. Cells were sequentially centrifuged (7000 x g, 30 minutes) and washed with PBS (phosphate-buffered saline; 10 mmol; pH 7,4). The bacterial extract was obtained after treatment of the cell precipitate in the test tube 3M KSCN/PBS with magnetic stirring (1 hour, room temperature), then centrifuged (35,000 x g, 30 minutes). The supernatant, which contained the extract of ChE, was collected and subjected to dialysis for the first time from PBS and then with deionized water. And finally, it was dried and stored at 4°C until further use.

5.2 Obtaining nanoparticles with ovalbumin and ChESalmonella enteritidis(OVASAL)

100 mg of copolymer metilfenidato ether maleic anhydride [Gantrez® AN-119] were dissolved in 3 ml of acetone, at the same time, 5 mg of ovalbumin and 5 mg of the extract ChE were dispersed in 2 ml of acetone by ultrasonic treatment (MicrosonTMin those who tell 1 min with cooling. The variance of ovalbumin and variance ChE were introduced into the polymer suspension and stirred for 30 minutes at room temperature. Consistently in this phase were introduced in 10 ml of ethanol and 10 ml of deionized water under magnetic stirring. The resulting mixture was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was brought by water to 10 ml and Then this solution was incubated with 0.1 ml of 1% solution of 1,3-diaminopropane.

The mixture was subjected to purification by ultracentrifugation (20 minutes at 27000 x g). Supernatant were removed and the residue was resuspendable in water or in 5% aqueous sucrose solution. Finally, the resulting suspension of nanoparticles was freeze-dried.

5.3Quantifying the production of antibodies against OVA after immunization with OVASAL in mice BALB/c

Intradermally 10 μg of ovalbumin were immunized with 15 mice divided into 3 groups, according to the received processing:

A) a solution of ovalbumin (OVA)

C) ovalbumin adsorbed on alhydrogel (OVA-Alum)

(C) ovalbumin nanoparticles and ChESalmonella enteritidis(OVASAL)

After the introduction of various compositions at 7, 14, 28, 35, 42 and 49 days was taken as the blood of retroorbital the th plexus and the samples were processed as described in example 3. The obtained results are shown in figure 8. Suddenly OVASAL significantly increased the titer IgG1although the titer of IgG2Aremained at very low levels. This shows that this composition was only able to enhance Th2 response, which was also much more rapid and intense than that of the control composition (OVA-Alum). These results allow us to think about the possible application of the inventive compositions as an adjuvant in vaccines with high levels of antibodies or against toxins produced by microorganisms. It would also be possible use in certain autoimmune diseases characterized by excessive Th1 response in the acute phase.

Example 6

Fabrication and characterization of biodegradable nanoparticles formulated with NOTSalmonella enteritidis(NP NOT 3934)

6.1 Selection and characterization is NOT antigenic complex 3934Salmonella enteritidis

Used the antigen is an extract of surface proteinsS. enteritidisnamed NOT (hot salt exractor Hot Saline Extract) due to the fact that it allocates antigenic complex in saline medium and heating. This does NOT contain phospholipids, surface proteins and pure lipopolysaccharide (S-LPS). The extract was obtained by the method described previously [Gamazo et al., Infect. Immun. (1989) 1419-1426]. After cultivation the project for bacteria Salmonella enteritidis3934 in tubes containing tripleksovanii broth (TSB), the bacteria were centrifuged at 7000 x g for 30 minutes and were twice washed with saline. Live cells were resuspendable in saline solution (10 g of wet cells in 100 ml) and heated in a steam flow at 100°C for 15 minutes. After centrifugation (12000 x g, 10 min) the supernatant was subjected to dialysis for 5 days to remove the deionized water. Subjected to dialysis material was ultracentrifugation for 4 hours at 60,000 x g, and the precipitate in the test tube (NOT) was resuspendable in deionized water, lyophilized and stored at room temperature.

Characterization includes the determination of the percentages of protein and lipopolysaccharide. Determining the amount of protein was carried out using the method of Lowry. Extract antigenSalmonella enteritidis3934 contains about 31% protein. The number of LPS was determined directly by one of its exclusive markers KDO with the method of using thiobarbituric acid. Through this method it was found 0,86% KDO, corresponding to 65% S-LPS.

6.2 Production and physico-chemical characteristics of nanoparticles formulated with NOTSalmonella enteritidis3934 (NP HE 3934)

First, 100 mg of copolymer metilfenidato ester of maleic anhydride were attoreny in 5 ml of acetone; antigenic extract (4 mg), as well resuspending in 1 ml of acetone, was introduced into this solution and subjected to mixing with magnetic stirring. This solution was incubated for 15 minutes at room temperature. Consistently in this phase were introduced in 10 ml of ethanol and the same volume of water. The resulting mixture was subjected to homogenization for 5 minutes. The suspension of nanoparticles was then subjected to evaporation at low pressure as long as both of an organic solvent has not been removed, and the final volume was brought by water to 10 ml

The suspension was subjected to purification by ultracentrifugation (10 minutes at 35000 x g). Supernatant were removed and the residue was resuspendable a 5% aqueous solution of sucrose m/o (weight/volume). Finally, the resulting suspension of nanoparticles was freeze-dried, which allowed us to retain all its original qualities.

The final composition of the particle suspension prior to lyophilization:

The copolymer metilfenidato
ether maleic
anhydride [Gantrez® AN-119]
1,0% w/v (m/o)
Extract antigen0,4%
Sucrose5,0%
Water for injectionto 10 ml

The size and surface charge of the nanoparticles was determined before lyophilization. The average size of the obtained nanoparticles was less than 200 nm and a negative total surface charge (-20,1±3,2 mV). The final product yield was determined after lyophilization process. The original weight of the copolymer was 100 mg, at the end of the process it was determined the number of converts to the nanoparticles, and it was expressed as a percentage of the original weight of the copolymer.

The quantity of the extract was determined using the method of using bicinchoninic acid. With this in mind, take an aliquot (1 ml) suspension of nanoparticles to cross-linking agent. Before checking these data, this mixture was investigated by colorimetric method using bicinchoninic acid in the protein. The nanoparticles were digested by addition of 0,1 n NaOH and the mixture was analyzed in relation to protein colorimetric method with bicinchoninic acid. In this sample was introduced a solution of bicinchoninic acid with 5% copper sulfate in a ratio of 100:2; the mixture was stirred for 1.5 hours at 37°C and then measured by a spectrophotometer with a wavelength of 562 nm.

The occupancy of the extract was expressed as the amount of extract in µg per mg nanoparticles, and the effectiveness of the Inca is salazie was determined by comparing the total amount of the encapsulated extract to the original number.

Table 2 shows the occupancy extract nanoparticles (µg extract/mg nanoparticles) and encapsulation efficiency (%).

Table 2
Physico-chemical characteristics of nanoparticles Gantrez® AN-119, filled extract antigen
TrackSize (nm)Encapsulated
extract (µg/mg)
The effectiveness of
Encapsulationand(%)
NP171,4±3,9------
NP-HE
S. enteritidis
178±55,418,1±6,645,2±5,4
and: the encapsulation efficiency is the percentage of encapsulated and NOT he, was calculated as the number of encapsulated extract antigen, multiplied by 100 and divided by the original number.

The figure 9 shows the standard protein HES. enteritidisin which the various components can be divided into: porins (approximately 36 kDa), OmpA (34 kDa) and fibrin SEF 14 (14 kDa) and SEF21 (21 kDa). By electrophoresis with dodecyl sodium sulfate on high the nom gel (SDS-PAGE) and Western blot turns was confirmed, what capsulotomy extract retained structural integrity and antigenic properties. The figure 10 shows the antigenic profile of the extract before and after encapsulation.

Example 7

Study of protection in mice, provided by the introduction of vaccines administered intraperitoneally NP NOT 3934

We used group devyatisilnyi BALB/c mice, each group consisted of 10 animals. Vaccination was carried out by a vaccine preparation with nanoparticles (NP NOT 3934), described in Example 6, in the ratio of 30 μg of extract per animal, including control animals: (i) the same number of corresponding blank nanoparticles; (ii) 30 μg/animal extract in a free form, unencapsulated public; (iii) 200 μl of a commercial vaccine Salenvac® (Intervet UK Limited, Walton, England); (iv) salt solution.

Ten days after vaccination, they were administered intraperitoneally inoculated with a lethal dose of 102SOME strains ofS. enteritidis3934 and after this inoculation was determined the number of animals who died from salmonellas. The results of the experiment for the protection show that the preparation of a vaccine based on nanoparticles (NP NOT 3934) when introduced subcutaneously protects fromS. enteritidislike the protection caused by commercial vaccine Salenvac®, as well as extract antigen entered in free form (NOT Se 3934).

The results show that encapsulation of extracts NOT on what chastity not increase the levels of protection in mice. At the same time, immunization of mice with empty nanoparticles induces nonspecific immune response that provides protection for three weeks. In addition, it should be noted that the method of introduction is NOT antigen in free and encapsulated form, was held intraperitoneal. In earlier studies it was assumed that oral or intranasal introduction unencapsulated public antigens could be the cause of their degradation before they reach the places of interaction with antigen-presenting cells.

In addition, 10 days after vaccination studies have been conducted immunity provided by the nanoparticles entered at the time of the experimental infection was determined by the number of IFN-γ and levels of production of IL-4 (typical of Th1 and Th2 types of immune response, respectively), produced by spleen cells of mice (figure 12). It was also determined the production of antibody IgG2aand IgG1in the blood (Th1 and Th2, respectively) (figure 13).

To study the immune balance of Th1/Th2 induced by free and encapsulated extracts in mice involved in the study were identified IFN-γ and levels of production of IL-4 produced by spleen cells of immunized animals (figure 12). Panel a shows the induced levels of IFN-γ, demonstrating the higher is their specified production in mice immunized nanoparticles (NP HE 3934). In contrast to the Panel shown as slightly increasing the level of production of IL-4 in mice immunized extract NOT (NOT 3934) in free form and in mice immunized with nanoparticles NP HE 3934. Taking into account the obtained results, it can be confirmed that the encapsulation of the extracts are NOT in nanoparticles promotes Th1-type immune response, confirmed by increased production of IFN-γ compared with Th2.

Was investigated by direct ELISA the production of specific antibodies against extracts are NOTS. enteritidis3934 from immunized mice (figure 13). In non-immunized mice or mice immunized with empty nanoparticles (empty NP), did not producirovanie antibody IgG2aor IgG1against extract NOTS. enteritidis3934. The levels of IgG2adefined by ELISA, were significantly higher than the levels of IgG1all sera obtained from mice. In addition, there was no increase in serological responses in mice immunized with extracts NP NOT (NOT 3934), in comparison with the reaction shown by mice immunized with extracts NOT (NOT 3934) in free form.

IgG2ais the dominant isotype antibodies in Th1 immune responses, although IgG1has the same function for Th2 immune responses, which could p is tverdal results obtained in the study of the release of IFN-γ and IL-4, showing that intraperitoneal nanoparticles with extracts NOTS. enteritidisinduces the predominance of Th1-type immune response. This introduction provides the same degree of protection against salmonellosis, as with the introduction of antigen in a free form. In addition, as described earlier, the use of these non-encapsulated antigens through the mucous membrane could not give these levels of protection observed in this experiment, because of the acid and the destruction of the enzymes that they could experience, passing through the gastrointestinal tract of the animal.

1. The use of nanoparticle-based copolymer metilfenidato ether and maleic anhydride (PVM/MA) as an adjuvant to obtain a vaccine containing the antigen, or to receive immunotherapy compositions containing the allergen.

2. The use according to claim 1, where these nanoparticles based on PVM/MA contain the specified allergen or the specified antigen.

3. The use according to claim 2, where the specified allergen or antigen is at least partially covers the surface of these nanoparticles and/or is within the above-mentioned nanoparticles.

4. The use according to claim 1 or 2, where these nanoparticles based on PVM/MA additionally contain an immunostimulating agent.

5. The use according to claim 4, where the specified immunostimulatory the second agent at least partially covers the surface of these nanoparticles and/or is within the above-mentioned nanoparticles.

6. The use according to claim 4, where the specified allergen or antigen and immune-stimulating agent at least partially covers the surface of these nanoparticles and/or is within the above-mentioned nanoparticles.

7. The use according to claim 1, where these nanoparticles also include cross-linking agent.

8. The use according to claim 1, where the average size of these nanoparticles is from 10 to 900 nm, preferably equal to 400 nm or less.

9. The use according to claim 1, where the molecular weight of the specified copolymer (PVM/MA is from 100 to 2400 kDa, preferably from 200 to 2000 kDa, more preferably from 180 to 250 kDa.

10. The use according to claim 1, where the specified vaccine or immunotherapy composition is in lyophilized form.

11. The use according to claim 1, where the specified vaccine or immunotherapy composition is in a form suitable for oral or parenteral administration.

12. The use according to claim 1, where the specified allergen is an allergen extracted from pollen, allergen extracted from insects, or allergen extracted from food.

13. The use according to claim 1, where the specified antigen is immunogenic extract of the organism.

14. Use item 13, where the specified organism is Salmonella spp., preferably, S.enteritidis.

15. Use item 13, where indicated the p antigen extract is NOT S.enteritidis or extract ChE S.enteritidis.

16. The use of nanoparticle-based copolymer metilfenidato ether and maleic anhydride (PVM/MA) to obtain a composition for enhancing an immune response to an antigen or allergen in their joint introduction.

17. The use of nanoparticle-based copolymer metilfenidato ether and maleic anhydride (PVM/MA) together with the antigen or allergen to obtain a pharmaceutical composition for the selective stimulation of the Th1 immune response or to obtain a pharmaceutical composition for the selective stimulation of the Th2 immune response, or to obtain a pharmaceutical composition for the balanced stimulation of Th1 and Th2 immune responses.

18. The application of item 16 or 17, where the specified allergen is an allergen extracted from pollen, allergen extracted from insects, or allergen extracted from food.

19. The application of item 16 or 17, where the specified antigen is immunogenic extract of the organism.

20. The application of claim 19, where the specified organism is Salmonella spp., preferably, S.enteritidis.

21. The application of claim 19, where the specified antigen extract is NOT S.enteritidis or extract ChE S.enteritidis.

22. The product containing separately (i) a composition comprising an allergen or antigen, and (ii) a composition comprising nanoparticles-based copolymer metilfenidato e the Ira and maleic anhydride (PVM/MA) as the composition, amplifying the immune response to the specified allergen or antigen for joint simultaneous or sequential introduction of one patient to enhance the immune response to the specified allergen or antigen in the specified patient.

23. Composition for stimulating the immune response containing nanoparticles-based copolymer metilfenidato ether and maleic anhydride (PVM/MA) and the antigen or allergen, where the specified immunogenic antigen contains an extract of the microorganism, and the specified allergen contains the allergen extracted from pollen, allergen extracted from insects, allergen extracted from food allergen extracted from mushrooms or allergen extracted from the epithelium of animals.

24. The composition according to item 23, where the specified organism is Salmonella spp.

25. The composition according to item 23, where the specified antigen extract is NOT S.enteritidis or extract ChE S.enteritidis.

26. The composition according to item 23, where the specified allergen or antigen is at least partially covers the surface of these nanoparticles and/or is within the above-mentioned nanoparticles.

27. The composition according to item 23, where these nanoparticles based on PVM/MA additionally contain an immunostimulating agent.

28. The composition according to item 27, where the specified immunostimulating agent at least partially covers the surface of the nanoparticles shown and/or is within the above-mentioned nanoparticles.

29. The composition according to item 23, where these nanoparticles also include cross-linking agent.

30. The composition according to item 23, where this composition is in lyophilized form.

31. The composition according to paragraph 24, where this composition is in a form suitable for oral or parenteral administration.

32. Vaccine containing a therapeutically effective amount of a composition for stimulating an immune response, PP-31 and a pharmaceutically acceptable carrier or excipient.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular to pharmaceutics, and concerns pharmaceutical compositions, which contain as active substance therapeutically effective quantity of ladasten, and as target additives - starch, stearic acid and/or its salt or ludipress and stearic acid and/or its salt with definite ratio of said components. Composition is made in form of pills, contains optimal quantity of target additives, which allows to obtain easy swallowed pills. Pills meet all requirements of State Pharmacopoeia XI edition.

EFFECT: medication form easily releases active substance, which provides its high bioaccessibility.

13 cl, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to virology, immunology and medicine. The composition contains antigen bound or fused with virus-like particle (VLP), completed with immunostimulating nucleic acid containing at least one unmathylated CpG-sequence and ligand of toll-like receptor (TLR). There are also disclosed vaccine composition and method for potentiating immune response in an animal.

EFFECT: invention can be used for inducing strong antibody and T-cell response and, particularly it is effective in treatment of allergy, tumours and virus chronic diseases, and also other chronic diseases.

25 cl, 12 dwg, 1 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to medical products and concerns an ellagic acid composition for immune system enhancement, differing that additionally is contains chitosan beta-1.3/1.6-glucans or oligosaccharides.

EFFECT: offered composition possesses enhanced immunostimulating effect.

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to pharmacology and can be used in veterinary science and medicine for chemotherapy. There is disclosed immunostimulating and antioxidant lithium composition containing lithium ascorbate and lithium aspartate in percentage ratio 50:50.

EFFECT: expansion of the list of low-toxicity immunomodulators applicable for manufacture of drugs.

1 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly therapy, and can be applied in chronic disease treatment. Method involves causative agent extraction or determination of antibodies to causative agents or extraction of genetic components from blood, body fluid or smear. Treatment is performed by vaccine drugs specific to one or several identified causative agents, additionally immunomodulators are administered.

EFFECT: activated repair processes, arrested development of disease state of organs and systems due to elimination of identified pathogen from organism and to immune correction.

3 cl, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly granulocytopoiesis stimulants, radioprotectors and immune stimulants. Under cytostatics effect Cyclophilin A amplifies granulocyte precursor removal from marrowbone and stimulates granulocytopoiesis.

EFFECT: in case of sublethal radiation, cyclophilin A stimulates migration of marrowbone stem elements and participates in recovery of blood cells and immune system, and shows redioprotection properties.

4 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: medicinal agent contains alpha and/or beta and/or gamma human recombinant interferon, tocopherol acetate or other tocopherol derivatives, ascorbic acid and/or its salts, pantothenic acid or calcium pantothenate, or dexapanthenol, riboflavin or levocarnitine, and orotic acid and/or ornithine or its derivative - citrulline malate, or Glutoxim, antibacterial, antimycotic agents, additives: emulsifiers, stabilisers, preservatives, antioxidants and base. The formulation is suppositories in certain component ratio.

EFFECT: efficiency for severe infectious diseases and mixed infections.

9 cl, 18 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, be more specific to oncology, and concerns the substances stimulating maturing of dendritic cells (DC). Application of fucoidan from Fucus evanescens or polysaccharide composition from Fucus evanescens consisting of fucoidan in amount of 60-80% and poly-mannuronic acid in amount of 20-40%, as an agent possessing ability to induce maturing of dendritic cells is offered. The declared fucoidan preparations have a standardised composition and, hence, possess direct biological effect. They keep the properties for long time (3 years).

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8 dwg

FIELD: medicine, veterinary science.

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EFFECT: rising of efficiency of the received preparation at treatment and prevention of infectious diseases of animals.

2 tbl

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria. The immunogene compositions containing a transferrin-binding fiber and Hsf, and a way of their reception are offered. It is shown, that the combination of these two antigens synergically influences on production of antibodies with high activity in the analysis of bactericidal Serum. The composition can be used in vaccines against Gram-negative bacteria, including Neisseria meningitides, Neisseria gonorrhoeae.

EFFECT: creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria.

56 cl, 10 ex, 1 dwg

FIELD: medicine.

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EFFECT: retention of functional properties of protein during its encapsulation.

6 cl, 2 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: invention concerns oncological area of medicine (along with genetic engineering and biochemistry) and methods of obtaining magnetosensitive liposome systems of medication delivery with controlled release. Method of obtaining magnetosensitive liposomes carrying medication involves dissolution of phospholipids in chloroform, addition of magnetic effect carrier and ultrasonic processing. Badger's fat is used as phospholipid source, ferromagnetic metal nanopowder with 2-5 nm particle size, obtained by gas phase method, is used as magnetic effect carrier and coated with carbon shell, and liposome system is sterilised in betatron chamber.

EFFECT: novel method of magnetosensitive liposome obtainment.

2 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: invention relates to topical formulation with: spironolactone nanoparticles, containing medium-diameter nanoparticles measured by photonic correlation spectroscopy technique, in the range approximately 300 nm to 900 nm, and dispersion of solid polar lipids crystals in polar liquid, with additional stabiliser. The nanoparticles are included in crystal structure formed by solid polar lipids crystals; the lipids mentioned being directed their hydrophilic ends outside, and hydrophobic ends inside, relative to spironolactone nanoparticles. Items of the patent application are also as follows: method of producing the formulation, the crystal lattice arrangement of solid polar lipids crystals with spironolactone nanoparticles included, and use of the nanosuspension with structure mentioned for medication producing, intended for therapy in anti-androgen responding conditions.

EFFECT: achieving of high stability formulation and increased bioavailability of spironolactone.

17 cl, 4 ex, 8 tbl, 11 dwg

FIELD: medicine.

SUBSTANCE: means has micro-particles of practically pure active substance immersed into biologically compatible pharmacologically permissible polymer. The polymer is ester of polyol and copolymer of polylactide and glycolide. The polyol has at least three hydroxide groups and molecular weight not greater than 20000.

EFFECT: enhanced effectiveness in introducing drug as depot; supporting therapeutically effective dose of active substance.

9 cl, 1 tbl

FIELD: chemistry of polymers, medicine.

SUBSTANCE: invention describes a micelle-formed composition including hydrophobic core covered by hydrophilic envelope and a therapeutic agent as a component of micelle said wherein hydrophobic core is chosen from group consisting of poly-(ortho-ester), polyanhydride, pseudopoly-(amino acid) prepared from tyrosine, polyphosphazene or poly-(β-benzyl-L-aspartate) and their combinations, or polyester chosen from group consisting of poly-(glycolic acid), poly-(lactic acid), poly-(D-lactic acid), poly-(D,L)-lactic acid), copolymers of lactide/glycolide, polycaprolactone and their derivatives and wherein hydrophilic envelope represents poly-(N-vinyl-2-pyrrolidone). Indicated composition can be easily dispersed or dissolved repeatedly after addition of water or aqueous solution to a form obtained after sublimation drying the indicated micelle-formed composition. Using hydrophilic envelope allows avoiding aggregation of micelles. Also, invention provides simplifying stages in preparing micelles.

EFFECT: improved and simplified preparing method.

10 cl, 4 tbl, 1 ex

FIELD: medicine, biochemistry, pharmacy, biotechnology.

SUBSTANCE: invention relates to a method for preparing polyelectrolyte microparticles containing the end substance and showing sensitivity to alteration of the environment composition. Method involves preparing oppositely charged polyelectrolytes on microaggregates containing an encapsulated substance. These polyelectrolyte microparticles can be used both in medicine as systems used in delivery drugs and providing pH-sensitive release of encapsulated substance and in biotechnology as biocatalysts stabilized with respect to unfavorable conditions. Invention provides preparing polyelectrolyte microparticles characterizing by the high content of active substance - up to 90% of microparticles mass. Proposed method is sample and involves lesser amounts of steps.

EFFECT: improved preparing method.

9 cl, 3 tbl, 2 dwg, 61 ex

The invention relates to a pharmacy and concerns microspheres

The invention relates to the field of pharmaceutical industry and relates to a pharmaceutical composition in the form of a colloidal suspension of nanocapsules, which contains an oil phase formed mainly oil containing in solution a surfactant and a suspension of nanocapsules having a diameter below 500 nm, comprising the aqueous phase is formed mainly by solution or suspension of a therapeutically active substance, surface-active substances and in some cases ethanol, as well as the method of obtaining this composition

FIELD: medicine.

SUBSTANCE: invention can be used in manufacturing of vaccines for Streptococcus pyogenes - streptococci of group A (SGA) and Streptococcus agalactiae - streptococci of group B (SGB). Substance of the invention involves development of recombinant DNA pB1 derived from PCR with using chromosomal DNA of strain 090R Ia of serotype SGB, primers Pb1 and Pb2 and following cloning with using expression plasmid pQE-30 in E coli M15. Recombinant DNA pB1 codes recombinant protein PB1 expressing protective properties in relation to specified streptococci which has no enzymatic activity and causes synthesis of anti-Pb1 antibodies expressing protective properties in relation to Streptococcus pyogenes and Streptococcus agalactiae. In the invention there is developed recombinant plasmid DNA pQE-pB1 representing plasmid DNA pQE-30 that bears recombinant DNA pB1, and strain-producer E. coli M15-PB1 enabling to express recombinant protein PB1.

EFFECT: no enzymatic activity of produced recombinant protein allows application as an ingredient of the vaccine for Streptococcus pyogenes and Streptococcus agalactiae.

7 cl, 7 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: according to the invention, the method provides combined cultivation of nonadherent fraction of mononuclear cells (MNCs) recovered from peripheral blood of the patients with pulmonary tuberculosis, with dendritic cells (DCs) processed with the antigen Mycobacterium tuberculosis and prepared of monocytes of adherent MNC fraction with using mature DCs being prepared by adding a maturing inducer representing lipopolysaccharide E coli to antigen-activated immature DCs. The combined cultivation of specified cells is carried out with recombinant human interleukine-18 (IL-18) added.

EFFECT: intensified proliferative potential of specific cytotoxic cells of mononuclear origins, stimulation of IFN -γ production level, formation of cytotoxic cells in response to the specific antigen M tuberculosis.

3 tbl

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