Method for delivering biologically active compound onto mucosa-associated lymphoreticular animal tissue, method for obtaining composition and composition for delivering biologically active compound

FIELD: veterinary science.

SUBSTANCE: the suggested methods and compositions provide transfer of biologically active compound, antigen predominantly, in animal body. Efficient quantity of biologically active compound should be put into microcapsules made of biocompatible material the size of which do not exceed 10 mcm, then one should introduce efficient quantity of these microcapsules, perorally, preferably, for animals under immunization. As material for microcapsules one usually applies a biologically active polymer or copolymer being of capacity to pass through gastro-intestinal tract or being localized at mucosal surface not being affected by biodegradation. This provides the transfer of biologically active compound onto Peyer's patches or other mucosa-associated lymphatic tissues, that provides inducing systemic immunity and activization of mucosal immunity system.

EFFECT: higher efficiency.

22 cl, 12 ex, 19 tbl

 

The state of the problem

The invention relates to a method of oral administration and preparation of biologically active compounds, a prisoner in an indifferent environment from one or more biocompatible polymeric materials forming microcapsules with active substance, which due to their size and physico-chemical properties provide delivery of this substance and its inclusion in the aggregated lymphatic follicles (Peyer's patches) of the digestive organs of animals without loss of activity during the passage through the gastro-intestinal tract. Similar units exist in the respiratory organs, the urinary system, the colon and other organs, with the mucous membrane. In the further exposition these formations are referred to as associated with mucosal lymphatic tissue.

Microcapsules are widely used for protection of biologically active compounds from decomposition. Usually such compounds involve one or more layers of protective material, usually polymeric nature. The active compound may be surrounded by a layer of polymeric material in the form of a shell (microcapsules) or evenly distributed in it (microspheres). Further, the term "microcapsules", we will refer to both forms of the drug. The amount of active substance in the microcapsules can is about to vary depending on the drug, from very small quantities up to 95% or more of the total composition of the microcapsules. The size can vary from less than 1 μm to 3 ml and more.

Peyer's patches are aggregated lymphatic nodules, localized in the walls of the thin and thick gut and blind process, and are an important component of the protective system of the body against infectious organisms and other foreign material. Antigens are substances that induce the formation of antibodies and/or cellular immune response due to the presence in their composition of foreign proteins or tissues.

Reactions induced by the interaction of antigens with the immune system, can be positive or negative in the sense of the body's ability to produce antibodies or to answer mediated through cells of the immune responses to repeated exposure to the same antigen. The concept of mediated through cells of the immune system include the ability to cause destruction of foreign cells or tissues, "mediated through cell cytotoxicity and allergic reactions of the delayed type. Antibodies belong to a class of proteins called immunoglobulins (Ig), which are formed under the influence of antigens and form with them a specific complexes. Such systems enable the removal of the antigen from the body, their formation is accompanied by the death of the people live antigens (for example, pathogenic agents, foreign or cancer cells) and neutralization activity of toxins or enzymes. A major class of immunoglobulins in the secret surrounding mucous, are immunoglobulin A (sIgA). Secreting their antibodies prevent the adhesion of pathogens to the mucosal or their penetration into her. The same applies to other antigens.

Numerous antigens enter the body through mucous membranes, but the most common methods of immunization, for example, intramuscular or subcutaneous injection of vaccines or antigens, rarely cause sIgA in the secretions of mucous. Their products are the most intensively induced by direct immunization associated with mucosal lymphatic tissues, most of which is present in the body in the form of Meyerovich plaques of the gastrointestinal tract.

Peyer's plaques are composed of precursor cells, so-called cells, which are localized in the lamina propria of the gastrointestinal tract and upper respiratory system and differentiate into Mature cells that synthesize IgA. Research heremans kept carrying out renovation and Bazin on the formation of the ability to synthesize IgA in mice after oral administration of antigen showed that there is a sequence of formation of antigen-specificclick, producing IgA: they first appear in the mesenteric lymph nodes, then in the spleen and, finally, in the lamina propria of the gastrointestinal tract (Bazin, N., Levi G. & Doria G. Predominant contribution of IgA antibody-forming cells to an immune response detected in extraintestinal lymphoid tissues of germ-free mice exposed to antigen via the oral route / the Leading role of cells producing IgA antibodies in immune responses detected in extra-intestinal lymphoid tissues amicrobic mice after oral administration of antigen /, I. Immunol. 105: 1049; 1970 and Crabbe P.A., D.R. Nash, Bazin H., Eyssen H. & J.F. heremans kept carrying out renovation Antibodies of the IgA type in intestinal plasma cells of germ-free mice after oral or parenteral immunization with ferritin / IgA - antibodies in the cells of the intestinal plasma amicrobic mice after oral or parenteral immunization with ferritin /, J. Exp. Med.130: 723; 1969). Subsequent studies showed that oral administration of antigens leads to the formation of sIgA antibodies in the digestive organs, as well as their appearance in secretions such remote from the digestive tract organs, as the bronchi, mammary gland (milk and colostrum), salivary gland, and in tears (Mestecky j, McGhee J.R., R.R. Arnold, S.M. Michalek, Prince S.J. & Babb J.L. Selective induction of an immune response in human external secretions by ingestion of bacterial antigen / Selective induction of immune responses in external secretions after oral administration of bacterial antigen /, J. Clin Invest. 61: 731; 1978, Montgomery R.S., Rosner B.R. & Cohen J. The secretory antibody response. Anti-DNP antibodies induced by dinitrophenylated Type II pneumococcus / Secretory response of antibodies. Antibodies to DNP induced dinitrofluorobenzene the pneumococcus type III /, Immunol. Commun. 3: 143; 1974; & Hanson L.A., Ahistedt S., Carlsson C., Kaijser Century, Larsson P., Mattsby Baltzer, A., Sohl Akerlund, A., Svanborg Eden C. & Dvennerhoint A.M. Secretory IgA antibodies to enterobacterial virulence antigens: their induction & possible relevance / Secretion of IgA antibodies to viral antigens enterobacteria: Their induction and possible role / Adv. Exp. Med. Biol. 1007; 165; 1978). The data of these authors suggest that Peyer's plaques are rich sources of progenitor cells into IgA-producing cells, which after sensitization with antigen enter the circulation and cause the expression of antibodies not only in the place of the source activity and antigen, but away from areas of mucosa. This transport system In cells in different parts of the mucosa after sensitization provides a reaction of the immune system by penetrating into the gastrointestinal tract of environmental antigens and potential pathogens.

Of particular importance from the point of view of the present invention has the ability of antigens to induce the formation of protective antibodies after oral administration. It is known that the intake of antigens in the body of animals with food leads to the formation of specific antibodies that can be detected in bronchial aspirates or nasal cavity. If IP is the studies on volunteers also shows introduction Antigrippin vaccine (oral) induces the secretion of the corresponding antibodies in the nasal cavity.

Numerous studies have shown that oral immunization to induce the activity of the immune system of the mucosa. At the same time it was shown, however, that this requires very high doses of antigen. Therefore, with rare exceptions, this method may not have practical value. It is obvious that any drug for oral administration or how the latter should provide protection of the active ingredient from destruction in the gastrointestinal tract during transport to aerowyn plaques. Failure to do so will lead to the fact that if this ingredient and reaches plaques, the number or activity will not be sufficient for the manifestation of the desired effect.

In this regard, a clear need in the way of oral immunization, which would ensure effective stimulation of the immune system of the mucosa and helped solve the problem of protecting the antigen from destruction while passing through the gastrointestinal tract to aerowyn plaques. Even more important task is to develop a method that provides selective transport of antigens in Peyer's patches and his release in these formations. Remains unsolved and problems the immunization using mucosal other bodies, that would avoid the destruction of the antigen and provided it to arrive just in associated with mucosal lymphatic tissue. In addition, to solve the issues of protection of the antigen from destruction after its receipt by the mucous membrane, provide and/or further optimization of the transport of antigen from associated with mucosal lymphatic tissues and release of the biologically active material of the pharmacological drug after the introduction of the latter into the body.

Summary of invention

The invention relates to a method of introduction and preparation for the directional migration and subsequent release of biologically active compounds in the animal body after contact with mucous membranes, especially to the way oral and intra-lungs injection. The biologically active compound is enclosed in a microcapsule of a biologically compatible polymer or copolymer, which can pass through the gastrointestinal tract and stored on the mucosal surface without being destroyed or subjected him to a minor extent, which ensures the supply of biologically active compounds in Peyer's plaques or other associated with mucosal lymphatic tissues and entry to them at the original effective amounts. The term biologically comparable the first polymeric material is indicated by the polymer, non-toxicity, carcinogenic or inflammatory action in the body. Preferably, indifferent polymeric material of the microcapsules were subjected to biodegradation, i.e. destroyed during physiological processes to products that do not accumulate in tissues and flushed out of the body. The microcapsules should have such dimensions and physico-chemical properties, which would ensure their effective selective input in Peyer's plaques. The invention solved the problem of directional transfer of biologically active compounds in Peyer's patches and other associated with mucosal tissue, and include.

The purpose of the invention is to develop a method of oral administration of antigen to animals, in which he reaches of Meyerovich plaques and is included in them, thus stimulating the immune system of the mucosa, without loss of immunogenic activity in the transport process along the gastro-intestinal tract.

Another object of the invention is to develop a method of oral administration to animals of the antigen when it reaches of Meyerovich plaques and is included in them without loss of activity in the transport process along the gastro-intestinal tract, thus stimulating a systemic immune system.

Another objective of the invention is to develop a way of introduction of antigen animals, in which on the reaches associated with mucosal lymphatic tissues thus stimulating the immune system of the mucosa, without loss of activity due to the destruction on the surface of the latter.

Another aim of the invention is to develop a way of introduction of antigen animals, in which he reaches associated with mucosal lymphatic tissues and is included in them, thus stimulating a systemic immune system, without loss of activity due to degradation on the surface of the mucous membrane.

The purpose of the invention consists in developing a method of oral administration of biologically active compounds to animals, providing its transport and incorporation into Peyer's plaques, to create a local or systemic concentration of the drug.

The purpose of the invention consists in developing a method of introducing animals biologically active compounds, providing for its receipt and the inclusion associated with mucosal lymphatic tissue to create a local or systemic concentration of the drug.

The purpose of the invention is the development of pharmaceutical forms containing biologically active ingredient and containing the polymer or copolymer indifferent material, preferably amenable to biodegradation, which is suitable for transfer to the mucous membranes of the above described methods.

Another objective of the invention to provide an improved system which we vaccinate, to avoid the use of stimulants immunogenesis.

Another aim of the invention is the creation of an improved system of vaccination for the induction of immunity in the process of pulsating release of antigen after a single injection in microcapsules.

The purpose of the invention is to create an improved system for vaccination, which avoids the use of stimulants immunogenesis and allows to induce an immune response through the pulsating release of antigen exclusively of microcapsules after a single injection of the drug.

The purpose of the invention is to develop compositions for achieving the above objectives.

Detailed description of the invention

Below are examples of implementation of the invention. They illustrate how directed and programmed transfer of antigens (trinitrophenyl hemocyanin marine saucer and toxoide vaccine staphylococcal enterotoxin) and medication (etretinate) in associated with mucosal lymphatic tissue of mice in the composition of the microcapsules of the polymer of DL-lactide-glycolide (50:50).

It should be noted that for this purpose it is possible to use other polymers such as polyglycolide, poly(DL-lactide-coglycolide), cooliosity, polikar the lactone, poly(lactide-coproduction), poly(esteramide), polyarteritis and poly-8-oximosilane acid, and polyanhydrides and other polymeric compounds.

In addition, you can use other, in addition to the above, the biologically active ingredients. These include antigens of antiviral vaccines, vaccines against bacterial protozoal and fungal diseases (influenza, parainfluenza, other respiratory diseases, (Hemophilus influenzae, Bordetella pertussis, Neisseria gonorrhoeae, Streptococcus pneumoniae & Plasmodium falciparum), as well as vaccines against other diseases caused by pathogens; diseases caused by pathogenic microelemental; and anti-allergic vaccines. In addition, biologically active ingredients used immunomodulators, nutrients, drugs, peptides, lymphokines, cytokines and other compounds.

I. Microencapsulation

A. Manufacture of microcapsules with dyes

In order to trace the penetration of microcapsules in Peyer's plaques, made luminous capsule from resistant to biodegradation of the polymer polystyrene containing water-soluble fluorescence dye coumarin. The procedure of manufacture is as follows:

First prepare the polymer solution, diluting of 4.95 g of polystyrene (Type 685D, Dow Chemical Company, Midland, MI) 29.5 g of methylene chlorine is Yes (Reagent Grade, Eastman Kodak Rochester, NY). Then in the polymer solution add approximately 0.05 g of coumarin (Polysciences Inc., Warrington, PA) and placed the mixture on a magnetic stirrer.

In a separate container prepare a 10%aqueous solution of polyvinyl alcohol (PVA)is used as a working environment. For this purpose, 40 g of PVA (Vinol 2050, Air Products & Chemical, Allentown) is dissolved in 360 g of deionized water.

The finished solution is saturated 6 g of methylene chloride. It is then poured into the mixer from a synthetic resin (ACE Glass, Inc., Vineland, NY) turbine impeller (2.5 inches) and a motor Fischer and stirred at a rotation speed of 380 rpm/min Capacity mixer 1 liter

Thus obtained mixture of coumarin with polystyrene transferred into a container with a solution of PVA, using a funnel with a diameter of 7 mm with a long stem. This forms a stable water-oil emulsion, which is additionally stirred for 30 min at atmospheric pressure to obtain oil droplets of the desired size. Then the container is closed and gradually lower the pressure to 520 mm Hg, using a water pump connected to a pressure gauge, and an exhaust valve. The contents of the tank is stirred under reduced pressure for 24 hours until complete evaporation of the methylene chloride. After centrifugation, collect the hardened microcapsules and dried them for 72 hours in a vacuum is the Amer at room temperature.

B. Fabrication of microcapsules with antigen

Soluble antigen TNF - SMC enclosed in a capsule of poly(DL-lactide-coglycolide) - biocompatible biodegradable biodegradation of polyester. Use the following procedure.

First preparing a polymer solution by dissolving 0.5 g of poly(DL-lactide-coglycolide) in the ratio of 50:50 in 4.0 g of methylene chloride. Then 300 μl of antigen TNF-SMC (46 mg antigen/ml after dialysis) are distributed uniformly in a solution of poly(DL-lactide-cullenalice), using the mixer Vortex-Genie 2 (Scientific Industries Inc., Bohemia, NY).

In a separate container prepare a 8%aqueous solution of PVA, diluting 4.8 g PVA in 55,2 g deinitializes water. After dissolution of the PVA solution was poured into the mixer from a synthetic resin with a turbine impeller is made of Teflon with a diameter of 1.5 inches on the rotating shaft (Kontes Glass Inc., Vineland, NY). Then through a funnel with a length of 7 mm with a long stem and add the polymer solution, while stirring the solution of PVA at rotation speed of about 650 rpm/min. thus Obtained water-in-oil emulsion is stirred for another 10 min, the contents of the tank is transferred into 3.5 l of deionized water in a vessel 4 l and stirred at about 800 revs/min using a stainless steel impeller (2 inches). The resulting microcapsules for 30 min, washed with deionized in the DOI, collected by zentrifugenbau, washed twice with deionized water to remove residual PVA and again gather in the conditions of freeze drying. Thus obtained microcapsules are spherical particles with a diameter from 1 to 10 μm. In a similar way we obtain other microcapsules, for example, to include staphylococcal enterotoxin Century

Antigen content of TNF-HMC in microcapsules, or rather their core, determined by weighing 10 mg containing capsules in a centrifugal tube 12 ml of the test tube pour 3.0 ml of methylene chloride and stirred to dissolve the poly(DL-lactide-coglycolide). Then in a test tube add 3.0 ml of deionized water and vigorously stirred for another 1 min the resulting mixture was centrifuged to separate the aqueous and organic phases. The aqueous layer was transferred into a volumetric flask of 10 ml, Repeat the extraction, combining the aqueous phase in a volumetric flask and refilling it with deionized water to final volume. Method of protein samples conduct quantitative determination of antigen TNF-HMC in a volumetric flask and calculate its concentration in the microcapsules. The latter must contain the antigen in the amount of 0,2% of the total weight. Similarly define the content of staphylococcal enterotoxin In enclosing microcapsules.

II. Proniknout the e microcapsules with dye in Peyer's patches after oral administration

Peyer's plaques form the bulk of the tissue, which is induced by the formation of IgA antibodies. These isolated Islands lymphoreticular tissue located along the entire length of the small intestine and blind process. Directed admission of intact antigen, accompanied by its local accumulation, according to the modern view is the most effective way of induction of diffuse mucosal immune response. Biodegradable capsules are the best media for this targeted vaccination.

Example 1. Microcapsules made of polystyrene

The incorporation of microcapsules in lymphoreticular tissue of the digestive system and its limitation by the size of the microcapsules was studied in experiments on oral administration to mice polystyrene capsules containing fluorescence dye coumarin. Mice of BALB/C on an empty stomach and without anesthesia was administered 0.5 ml of a suspension containing 100 mg/ml fluorescent microcapsules of different sizes (from less than 5 to 8-50 μm in diameter). The suspension was prepared in tap water and introduced into the stomach by means of the probe. At different times after injection (0.5; 1 and 2 hours) mice were scored and took the small intestine. Segments of intestine length of 1 cm with isolated peyrovani plaques were washed from the content, was turned inside out and quickly amraiwadi. Frozen sections were examined under fluorescent microscope, determining the number, location and size of the microcapsules included in the Peyer's patches of the gut lumen.

Separate microcapsules after washing remained between the fibers, however, their inclusion in other tissues, except of Meyerovich plaques were absent. After 30 min after oral administration of microcapsules was found in Meyerovich plaques proximal, but not distal small intestine. Over time they migrated through peristaltic contractions in other parts of digestive system and after 2 hours they were found throughout the gastrointestinal tract, including Peyer's patches of the ileum. Subjected to endocytosis microcapsules were characterized by predominantly peripheral localization, remote from the apical part of the plots plaques, and therefore it seems that the physical capture of the microcapsules between the body plaques and adjacent fibers when the motility promotes their inclusion. Comparison of the effect of the inclusion of microcapsules with a size less than 5 microns compared with larger capsules (8-50 μm) suggests that Peyer's plaques quickly and selectively include microcapsules with a size of 1-10 μm, whereas the incorporation of microcapsules with a diameter of more than 10 µm is missing. This allows us to consider microcapsules made from undergoing biodegradation of the material, effective directional transfer of antigens in lymphoreticular fabric to induction of immune responses at the mucosal surface.

Example 2. Microcapsules of poly(DL-lactide-coglycolide) in the ratio of 85:15

1. The inclusion of biocompatible and biodegradable microcapsules in Peyer's plaques

Different groups of mice through the gastric tube was introduced suspended in water microcapsules of biodegradable material containing fluorescence dye coumarin-6. The material of the microcapsules served as poly(DL-lactide-coglycolide) in the ratio of 85:15, with the ability to resist biodegradation for 6 weeks. Later 1-35 days after administration of the selected three representative Peyer's plaques, tissue sample large mesenteric lymph nodes and spleen, and after special processing was preparing frozen sections.

In the study under fluorescent microscope using the appropriate filters and excitation sources localization dark green fluorescing dye was determined by the distribution of microcapsules with a diameter of much less than 1 micron. In order to identify the total number of microcapsules in individual organs or tissues microscopical who were subjected to all of the available slices. Using ocular calibration of the micrometer was determined by the size of each of the microcapsules and registered its localization in the tissue or organ.

The inclusion of microcapsules, regardless of size, was observed even after 24 hours after administration; they remained in Meyerovich plaques in all terms of a 35-day observation period (table 1). There was no single case of penetration of the microcapsules in the intestinal tissue outside of Meyerovich plaques. The total number of microcapsules in these formations has increased during the first 4 days after injection and decreased over the next 31 days up to 15% of the maximum value.

This observation is consistent with the data on that free microcapsules are present on the surface of the intestinal villi in 1, 2 and 4-th day after injection. Interestingly, about 10 hours after oral administration of microcapsules in the form of a suspension containing coumarin material was detected in the faeces. These observations were carried out using a source of ultraviolet radiation, and in a day the main part fluorescing material was derived from the intestine. Thus, the continuous incorporation of microcapsules in Peyer's plaques within 2-4 days after injection can be explained by the capture of a small number of the mucosa of the intestinal wall in between the villi. In addition, online is newest inclusion captured microcapsules, apparently, several orders of magnitude higher than that of the microcapsules present in the lumen of the intestine above the surface of the mucosa. This observation is important from a medical point of view, since the mass of lymphatic tissue of Meyerovich plaques in humans compared with mice immeasurably more, and duration of passage of the particles through a thin human intestine longer, which can significantly improve the effectiveness of their inclusion in the Peyer's plaques.

During all periods of observation, as shown in table 1, in Meyerovich plaques were present microcapsules of any size. 1, 2 and 4 days after their introduction, the relative share of microcapsules with a diameter less than 2 microns (45-47%), from 2 to 5 μm (31-35%) and greater than 5 microns (18-23%) remained fairly constant. After 7 days, and especially in the later stages the distribution of microcapsules of different sizes were changed, and the largest of them (more than 5 microns in diameter) began to prevail over a number of smaller (less than 2 μm and from 2 to 5 μm). This shift has coincided with a decrease in the total number of microcapsules in Meyerovich plaques, starting with 7 days. These observations indicate selective migration of microcapsules small and medium size of Meyerovich plaques and the delay predominantly large microcapsules.

These data are corroborated by the observations of the locale is the situation of the microcapsules in the morphological structures of Meyerovich plaques. They usually were found in the epithelium directly at the point of penetration into plaques (within 200 μm from them) or in the depths of lymphoid tissue at a distance of 200 μm or more from the nearest layer of the epithelium (table 1). Microcapsules, characterized the last type of localization were usually represented by small - and medium-sized forms. On the first day after the introduction of 92% of the microcapsules was localized in the epithelium near the domed surface of the plaque. The relative number of particles penetrating to a greater depth, increased to 24% of their total number on the 4th day, then gradually decreased to 2% on the 14th and the following days. Thus, small and medium-sized microcapsules migrate through Peyer's patches and are derived from them, while large, with a diameter greater than 5 microns, for a long time remain within the dome.

2. Migration of microcapsules in the mesenteric lymph nodes and spleen

A small amount of microcapsules was found in the lymph nodes of the mesentery on the day of injection, after which their number was progressively increased up to 7 days (table 2). Then it declined, but part of microcapsules remained at these sites for at least 35 days. The analysis of the distribution of microcapsules size revealed a relatively high content of m is lcih microcapsules (less than 2 microns in diameter) compared with the microcapsules of medium size (2-5 μm) in the lymph nodes in the early stages after the introduction and the absence of large microcapsules with a diameter greater than 5 microns, indicating a more intensive migration in them small particles. In addition, for the first time after the introduction of the majority of the microcapsules was localized directly beneath the shell of the site or its podkapsulnaya sinus. Later microcapsules moved into the deeper structure of the node and to a 14-day, 90% of the microcapsules was localized within cortical medullary substance. Initial localization of microcapsules in subcapsular sinus or near it suggests that they penetrate into the nodes through the lymphatic vessels draining the Peyer's plaques. A progressive increase in the number of small microcapsules in the depth of the lymph nodes to 4 days after their oral administration and the gradual reduction of their total number over the next 14 days and at a later date indicates that the microcapsules migrate through host tissue and out through the efferent lymph.

Similar studies in the spleen revealed the absence of microcapsules up to 4 days after injection. The maximum number of them were registered not earlier than 14 days. As in the case of mesenteric lymph nodes, the spleen was absent microcapsules with a size greater than 5 microns. During all periods of observation microcapsules was localized in the depth of cortical tissue. It should be noted, is the maximum number of microcapsules in the spleen was found during when the content in the mesenteric lymph nodes was decreased and remained in them microcapsules was localized in the deepest parts of the sites. This observation is consistent with the known sequence of the lymph from Meyerovich plaques in the mesenteric lymph nodes, and from there into the bloodstream through the thoracic duct. Thus, before you enter the spleen microcapsules pass through Peyer's patches and lymph nodes of the mesentery, and then carried by the blood.

In additional experiments using histochemical and immunohistochemical methods were investigated sections of Meyerovich patches, mesenteric lymph nodes and spleen, containing absorbed microcapsules from polylactide-coglycolide in the ratio 85:15. In addition, these studies found that absorbed by peyrovani plaques microcapsules are located inside macrophagecolony cells, which use color-Schiff reagent with period acid (PCS) has revealed carbohydrates, mainly glycogen, and antigens class II major histocompatibility complex (MHC). Also present in the mesenteric lymph nodes and spleen microcapsules typically transferred there in these PCS and MHC positive cells. Thus, the microcapsules containing the antigen include antigen-is transforming adventitious cells (ATPC) of Meyerovich plaques and spread them to other lymphatic tissue.

The data show that the nature of immune responses induced by oral administration enclosed in microcapsules vaccine can be adjusted by selection of the particles of a certain size. Microcapsules with a size less than 5 μm is output from Meyerovich plaques in the composition ATPC and release the antigen in the lymph tissue that serves as a place for the induction of systemic immune responses. In contrast, microcapsules with a diameter of 5-10 μm are trapped in Meyerovich plaques and ATPC for a long time and release the antigen directly into the plaques inducyruya products sIgA antibodies.

Example 3. The mapping included in Peyer's plaques microcapsules of 10 different materials

In order to identify indifferent materials, the most suitable for controlled delivery of biologically active compounds to the target tissues, conducted experiments with microcapsules of different materials, simultaneously estimating physico-chemical properties of the latter, providing directed absorption of microcapsules associated with mucosal lymphatic tissue. As for the last task, previous studies showed more intense fisicos hydrophobic particles by cells of the reticuloendothelial system compared to hydrophilic. In connection with timbila studied the absorption peyrovani plaques microcapsules ranging in size from 1 to 10 μm, made from 10 different polymeric materials that have varying degrees of hydrophobicity. Among them were: polystyrene, poly(L-lactide), poly(DL-lactide), 50:50 poly(DL-lactide-coglycolide), 85:15 poly(DL-lactide-coglycolide), poly(hydroxybutyric acid), poly(methyl-methacrylate), ethylcellulose, cellulose acetate - phthalate hydrogen, triacetylcellulose. Set the absorbance of the microcapsules 7 of the materials used and their preferential localization in the body of Meyerovich plaques within 48 hours after administration in the form of a suspension containing 20 mg of microcapsules, oral (table 3). Cases of penetration of the microcapsules in the intestinal wall outside of Meyerovich plaques are not registered. With one exception (ethylcellulose), absorption efficiency was correlated with the relative hydrophobicity indifferent material of the capsules. With the introduction of the most hydrophobic materials: polystyrene, polymethyl methacrylate and policymaking acid, in three representative of Meyerovich plaques mice had up to 1500 microcapsules. When using capsules of the less hydrophobic materials: poly(L-lactide), poly(DL-lactide), 85:15 poly(DL-lactide-coglycolide) or 50:50 poly(DL-lactide-coglycolide), their number in the same samples was 200-1000 pieces. Cellulose microcapsules were not absorbed.

It is established that the dependence and the election of absorption of the particles from the physico-chemical properties of their constituent material is mediated by surface processes at the interface between peyrovani plaques and the cavity of the intestine. Therefore, the change in the surface characteristics of the microcapsules by chemical modification of the polymer or properties of the shell is fashionable to use as a means of regulation of the efficiency of capture of microcapsules and delivery of biologically active compounds associated with mucosal lymphatic tissues or ATPC. As non-limiting examples of materials for such layers can result in chemicals, polymers, antibodies, bioadhesive compounds, proteins, peptides, carbohydrates, pectins and similar materials, both natural and artificial origin.

III. The production of antibodies induced by a vaccine composition of the microcapsules

Materials and methods:

Mouse: mouse strain BALB/C mice aged 8 to 12 weeks (in all experiments).

Trinitrophenyl hemocyanin: Hemocyanin derived from marine saucer (Megathura crenulate), the production company Calbiochem (San Diego, USA), was manuginobili with nitrovanillin the hapten (TNF-GCMS) using 2,4,6-trinitrobenzene-sulfonic acid, as described Rittenburg and Amkraut (Immunogenecity of trinitrophenyl-hemocyanin: Production of primary and secondary anti-hapten precipitins / Immunogenic properties trinitrophenyl-hemocyanin: Obtain primary and secondary antiJapanese of precipitins / J. Inununol., 97, 421, 1966). The degree of substitution according to the spectrophotometric determination was equal to FBL861- SMC (when CoE is viciente molar extinction 15400 and a wavelength of 350 nm to 30%absorption correction GCMS under these conditions).

Vaccine with staphylococcal enterotoxin In:

Formalin vaccine staphylococcal enterotoxin In (CMEA) was prepared by the method of Warren J.R., Spero L., & Metzger J.F. (Anfcigenicity of formalin-inactivated gets enterotoxin In / Antigenic properties formalin inactivated staphylococcal enterotoxin / J. Immunol., 111, 885, 1973). Briefly, 1 g of enterotoxin were diluted in 0.1 m sodium phosphate buffer pH 7.5 to a final concentration of 2 mg/ml the resulting solution was added formalin to obtain a molar ratio of formaldehyde:enterotoxin 4300:1. The solution was placed in an incubator at 37°C and slow shaking, maintaining the pH at 7.5+0,1 daily. After 30 days, the toxin was concentrated, washed borate buffer (BB) in the filtration chamber (Amicon) under pressure and sterilized by filtration method. Turning enterotoxin in enterotoxin was confirmed by the absence of weight reduction rabbits weighing 3.0 to 3.5 kg after intramuscular injection of 1 mg toxoiding material.

Immunization: the Antigen in the microcapsules and without them suspended at a certain concentration in a solution consisting of 8 parts sterilized by ultrafiltration water and 2 parts of a 7.5%sodium bicarbonate solution. 0.5 ml of suspension using a gastric probe was injected into mice - recipients on an empty stomach, as described Babb J.L., Kiyono H., S.M. Michalek, & Me Ghee JR. (LPS regulation of the immune responce: Suppression of immune response to orally-administered T-dependent antigen/LDF Regulation of immune response: Suppression of immune response to oral administration of T-dependent antigen /, J. Immunol 127, 1052, 1981).

Obtaining biological fluids:

1. Blood plasma: Blood was collected calibrated capillary pipettes after puncture of the retro-orbital plexus. After the formation of a clot, the serum was centrifuged for the separation of erythrocytes and platelets were subjected to thermal inactivation and stored until analysis at -70°C.

2. Intestinal fluid: Mice 4x was injected at 0.5 ml wash liquid (25 mM NaCl, 40 TM PA2SO410 TM KCl & 20 TM Panso3and 48,5 mm of polyethylene glycol by osmotic 530 mOsm) with 15-minute intervals (see Elson S.O., Ealding, W. & Lefkowitz J. A lavage technique allowing repeated measurement of IgA antibody on mouse intestinal secretions /Method of washing, providing a re-determination of IgA antibodies in the intestinal fluid of mice/, J. Immunol. Meth. 67: 101; 1984). After 15 min after the last washing mice were anestesiology and after another 15 min intraperitoneally injected them with 0.1 mg of pilocarpine. Over the next 10-20 min stimulated defecation. Highlight was collected in a Petri dish containing 3 ml of 0.1 kg/ml soybean trypsin inhibitor (Sigma, St. Louis, MO) in 50 mm EDTA, vigorously stirred and centrifuged to remove suspend the fragmented material. The supernatant was transferred into a centrifuge tube made of polycarbonate with a rounded bottom and was added 30 μl of a 20 mm solution of fluoric phenylmethylsulfonyl (FFMS, Sigma). The resulting mixture was osvetleni by centrifugation at 27000 × g for 20 min at 4°C. thereafter was added 20 µl of FFMS and 1% of sodium azide and the resulting solution was diluted FCS to a final concentration of 10% to obtain an alternative substrate for the remaining proteases.

3. Saliva: Simultaneously with the bowel movement was intense salivation. The Pasteur pipette was selected 0.25 ml of saliva was added to 20 μl of trypsin inhibitor, FFMS, of sodium azide and FCS, after which he osvetleni.

4. The swabs from the bronchi and alveoli: the Wash liquid from the bronchi and alveoli received after irrigation lungs 1.0 ml of phosphate buffer solution. To introduce the latter into the trachea inserted the probe, which was fixed with suture material. Then washed five times the trachea and other respiratory tract buffer solution, the resulting washings were added to 20 μl of trypsin inhibitor, FFMS, of sodium azide and FCS and the resulting mixture was osvetleni using centrifugation.

5. Reagents for immunohistochemistry studies:

Specific for mouse IgM, IgG and IgA goat antibodies (polyclonal)adsorbed on the solid phase and purified by the method of affin the th chromatography was obtained from a commercial source (Southern Biotechnology Associates, Birmingham). Their specificity when used for radioimmunoassay determinations was evaluated by binding with the corresponding purified monoclonal antibodies and proteins myeloma.

6. Radioimmunological determination on solid phase:

Purified antibodies were labeled free from carrier iodine-125 (Amersham) using a T-chloraminated method (Hunter W.W. Radioimmunoassay / Radioimmunological method /in: Handbook of Experimental Immunology, M. Weir (editor), Blackwell Scientific Publishing, Oxford p.14.1, 1978). Test strips (Immulon Removawell, Dynatech) were dokriani conjugate FBL with bovine serum albumin (BSA) or staphylococcal enterotoxin In a concentration of 1 μg/ml BB with 4°C. Control strips were left uncovered. All strips within 2 hours was kept in 1%solution of BSA and BB at room temperature. BB was used as solvent for the preparation of all samples and labeled with iodine-125 reagents. Samples of biological fluids appropriately diluted, was placed in washed cells (in triplicate) and incubated for 6 hours at room temperature. After washing in each cell was added in 1000000 pulse/min labeled with I125specific immunoglobulin and incubated over night at 4°C. Unbound labeled I125antibodies were washed, and the remaining cells of the radioactive material was estimated at 5500-gamma spectrometer (Beckman Instruments, San Ramon, CA). In the determination of TNF-specific antibodies calibration was made by serial twofold dilutions of a standard serum (Miles Scientific, Naperviile, IL)containing a known amount of immunoglobulins, cells, covered with a labeled specific antibodies (1 μg/cell). To construct the calibration curve and the interpolation of unknown parameters used data from a computerized statistical analysis by type “Logit-Log” or “Four Parameter Logistic (Basic Technology Center, Vanderbilt Medical Center, Nashville, TN). In the case of specific antibodies against staphylococcal enterotoxin b, the results were expressed through the degree of dilution of serum at which the signal level was more than 3 times higher than in the corresponding dilution control sera from comparable groups of animals (on the final test results).

A. Injection of microcapsules vaccine

1. Increasing the immunogenic activity of the microcapsules

Example 1. Immunogenic activity of the vaccine in its intraperitoneal administration in microcapsules

Conducted in our laboratory studies have shown that antibodies or vaccine composition of the microcapsules significantly enhances an immune response in a variety of experimental systems. As an example, the results of the direct comparison of titles and izotopicheskogo RA the distribution of antibodies to staphylococcal enterotoxin b - the causative agent of staphylococcal food poisoning after immunization enterotoxemia in the form of a solution or in the composition of the microcapsules. Mice are divided into several groups were administered different doses toxoide vaccine enclosed in microcapsules from polylactic-coglycolide (in the ratio 50:50), or in the form of a solution. In both cases, the introduction was produced by intraperitoneal injection. After 10 and 20 days after immunization received samples of blood plasma and the results of titration-specific isotype Immunoradiometric method defined titles anticarcinogen antibodies (table 4). The optimal dose dissolved toxoid (25 μg) caused a significantly weaker immune response, which only manifested itself in the production of IgM antibodies. In contrast, the introduction of 25 μg of toxoid in the composition of the microcapsules induced the formation of not only IgM, but IgG antibodies that can be detected at dilutions of plasma to 1/2560 on the 20th day after immunization. In addition, the microcapsules were allowed to increase the input dose of toxoid, without reducing the intensity of the immune response, as was observed with increasing doses of dissolved material to 50 mcg. By measuring the release of active ingredient from the microcapsules, their application gave the opportunity for 4-5 fold increase of the input dose without loss of sensitivity the spine of tissue in the zone of action of the toxin, which greatly increases the degree of immunization. Immunopathology activity microcapsules are even stronger after secondary and tertiary immunization (table 6).

Titles anticarcinogen IgG antibodies on the 20th day after immunization were 512 times higher with the introduction of 50 μg of toxoid in the composition of the microcapsules than the introduction of optimal immunizing dose of the dissolved material. Moreover, to achieve the same antibody titers, as after a single immunization with 100 μg enterotoxaemia in the composition of the microcapsules was required three times the optimal dose of toxoid in the form of a solution. Similar potentiate the action was discovered and immunization such widely used in laboratory practice protein antigens, as hemocyanin marine saucer or influenza vaccine.

Example 2. Immunogenic activity of the vaccine in its subcutaneous injection in the microcapsules

The proposed system immunization is also effective in intramuscular and subcutaneous administration of the vaccine. Comparative study of intraperitoneal and subcutaneous routes of administration of microcapsules mice to assess the intensity of the immune response and its dynamics. The results are presented in table 7.

Subcutaneous injection of 100 μg enterotoxaemia in the composition of the microspheres at 4 points on the back of mice resulted in the formation of Ig anticarcinogen antibodies in the same title, after intraperitoneal administration, albeit in a slightly later date. Nevertheless, these results indicate the possibility of the introduction of microcapsules with vaccines not only intraperitoneally, but in other ways. The results of the secondary immunization intraperitoneal and subcutaneous injection also did not differ significantly in regard to the maximum antibody titers, although the response to subcutaneous administration developed a little later than intraperitoneal (table 8).

2. The mechanism immunopotentiating steps of microcapsules

Example 1. Increase immunogenic microcapsule is not associated with own potentiate the activity of polymeric material

There are three possible mechanism by which the microcapsules of the lactide-coglycolide size of 1-10 μm patentiert immune response to those antigens. The first one is to increase the duration of release of antigen compared to those observed with the direct introduction of the latter into the body. Secondly, it is experimentally shown that the microspheres of the specified size quickly undergo phagocytosis by cells that digest the antigen. Therefore, the directional migration of relatively large quantities of intact antigen directly on the cells, causing the immune response dependent on the T cell antigens, is the second reason potentiate the action of microcapsules. The third reason could be to own immunogenic activity of microcapsules and their ability to activate immune cells via the same mechanism, which is similar to the activation is carried out such adjuvantly as bacterial lipopolysaccharides or muramyl-dipeptide. The last mechanism is characterized by the fact that his manifestation is possible with the simultaneous introduction of antigen adjuvant.

With the aim of ensuring that the microcapsules own immunogenic activity, mediated through their ability to induce nonspecific activation of the immune system, we compared the actions enterotoxaemia contained in capsules at a dose of 100 μg, and an equal number enterotoxaemia mixed with does not contain the antigen of the microcapsules (placebo). Different forms of the antigens were injected intraperitoneally to mice of BALB/c, are divided into groups of 10 animals, specific IgM and IgG antibodies to enterotoxin was determined by the results of the final titration with radioimmunological determination (table 9).

The immune response to the introduction optimal dose enterotoxaemia in the form of a solution (25 µg) was significantly weaker. The maximum antibody titers of IgM and IgG, respectively, at 10 and 20 days after injection of antigen was $ 1800. With the introduction of equal doses of antigen in the microcapsules was observed a significant increase in antibody titers of either type, which continued to increase until at least 30 days.

The simultaneous introduction of a solution enterotoxaemia and microcapsules placebo, comparable in weight, size, composition and doses with capsules containing the antigen, was not accompanied by the formation of antibodies in the credits, in excess of antibody titers, registered after the introduction of only dissolved antigen. Similar results were obtained with the introduction of the antigen for 1 day before or 1, 2 or 5 days after placebo. Thus, these data indicate that immunomoduliruushim effect in cases of introduction of the antigen in the microcapsules of polylactic-glycolide size of 1-10 μm is not a consequence they have the ability to activate the immune system. The obtained data rather suggest depositing effect, directed the transfer of antigen ATPC or a combination of both mechanisms.

Example 2. The slow release of antigen from the microcapsules with a size of 1-10 μm enhances the formation of antibodies and delays the manifestation of the maximum immune response.

Were compared immune response after intraperitoneal administration of 4 types of microcapsules, characterized by the rate of release of the antigen. The latter depended on two f the Ktorov: rate of diffusion through the pores of the material of the walls and the intensity of hydrolysis (bioerosion) microcapsules. Party 605-026-1 and 514-140-1 was characterized by different initial rate of release of the antigen through the pores and the presence of the second phase of release due to degradation of the capsule wall during hydrolysis. On the contrary, the parties 697-143-2 and 298-060-00 microcapsules were made of a dense homogeneous material, impeding the release of the antigen through the pores, and therefore it was primarily a function of hydrolysis of the walls of the microcapsules. In the last two batches of microcapsules were characterized by the ratio of lactide and glycolide. Resistance to hydrolysis when the value of this ratio of 85:15 was the reason for the reduced speed release enterotoxaemia.

Maximum immune response after injection of microcapsules from the party 605-026-1 (60% of the antigen was released 48 hours) was recorded 20 days after injection, when the titers of IgG antibodies reached 1:6400 (table 10). At the same time the maximum response was developed with the use of microcapsules from the party 514-140-1, ensuring the release of 30% of the antigen within 48 hours. However, in this case a high concentration of IgG antibodies remained on the 30th day.

Immunization with antigen in the microcapsules of the party 697-143-2 responsible for release of 10% of the active ingredient within 48 hours, accompanied by a maximum increase in the titres of IgG antibodies to 1:102400 30 and 45 days after vaccination, which is much higher than the increase in the use of variants of the microcapsules provided earlier release of antigen. Further decrease in the rate of release in the microcapsules of polylactic-glycolide with a mixing ratio of 85:15 (party 928-060-00, the release of the antigen is not earlier than 48 hours after vaccination) were detained peak production of antibodies to 45-60 days, but further enhance the immune response when this occurred.

The results show that the slow or delayed release of antigen to enhance the immune response. At the same time, some features of this reaction using different types of microcapsules show that depositing the effect is not the only mechanism immunopotentiating actions. The higher initial rate of release of antigen, the lower the maximum antibody titers. This is consistent with the model, according to which the antigen is released through the pores within the first 48 hours, the effectiveness of actions on the immune system does not exceed the antigen is introduced in the form of a solution. Significantly delayed the beginning of the release is used by macrophages to phagocytosis of microcapsules, efficient conversion of antigen and development of the response, and the degree of the latter is determined by the amount of antigen entering sensitive kletno delayed release later than a certain date, when the entire quantity of the antigen must be received in corresponding cells is not accompanied by further activation of the immune reaction, and causes only delay the onset of peak antibody.

2. Pulsating release of the vaccine of microcapsules for programmable immunization after a single injection

When immunization with any vaccine to obtain good results, it requires the introduction of two, three or more replications. Usually the first injection is made with the purpose of obtaining the primary reaction, the second for the induction of secondary reactions, and the third to obtain tertiary reactions. The need for multiple injections due to the fact that to stimulate a powerful response from the immune system it is necessary to re-interaction of the antigen with the corresponding cells. So after the first vaccination, the patient several times to go to the doctor to get a second, third and subsequent injection of the vaccine and provide robust immune protection of an organism. In practice, patients often do not visit the doctor for re-vaccination.

Intended for injection vaccines typically include antigen and adjuvant. The antigen, for example, can be associated with alum. The combination of antigen with adjuvant at first vaccination has important significance from the point of view that the last Wuxi which supports the immune reaction. In the second and third vaccination antigen itself stimulates the immune system, so when re-immunization introduction adjuvant not so necessary, as at the first.

Corporation Also proposed a method of vaccination, providing a continuous release of antigen and immunopotentiator funds (adjuvant) to stimulate the immune response (U.S. patent No. 4455142). The present invention differs from the patented, at least two important aspects. First, it does not require the use immunopotentiating means to stimulate the immune response and, secondly, does not provide for continuous release of antigen from the system of delivery to target tissues.

The invention relates to a dosage form of the vaccine (antigen) in the form of microcapsules or microspheres, which is a structure amenable to biodegradation of polymeric material (for example, polylactic-coglycolide) concluded with the active substance. In a more particular case, receive different microcapsules and mix them so that by a single injection of the vaccine to provide an improved primary reaction, and then the pulsating release of antigen to obtain secondary, tertiary and subsequent reactions.

The mixture of microcapsules consists of particles larger and smaller sizes. Small microcapsules is, diameter less than 10 microns, preferably less than 5 microns, and optimally from 1 to 5 μm, stimulate a primary immune response (without the use of adjuvant), due to the fact that they are easily recognized and included by macrophages. After absorbing these cells microcapsules release the antigen, which undergoes transformation during intracellular processes and flows on the surface of macrophages, providing a manifestation of the primary reaction. Microcapsules larger (greater than 5 microns, preferably 10 microns in diameter), but not so large so as not to succumb to the introduction by injection (preferably not more than 250 μm in diameter, are made of different polymeric materials in such a way that they release the antigen in the form of individual portions in the process of biodegradation at different speeds.

According to this invention the composition of the microcapsules containing the antigen for the induction of primary immune response, is not significantly different from the composition of the microcapsules to stimulate secondary, tertiary and subsequent reactions. In all cases, to make them use the same class amenable to biodegradation of polymers. The maximum response to an antigen comprising such microcapsules is achieved by selecting the size and pulsed nature of the release of antigen.

It is preferable to use polymer materials, the rate of biodegradation can be controlled by simply changing the ratio of monomer components, such as polylactic-coglycolide, so that the microcapsules used for the induction of secondary reactions, faster subjected to biological degradation than microcapsules used for the induction of subsequent reactions. This provides a pulsating release of antigen.

Thus, changing the size of the microcapsules is practically the same composition, it is possible to optimize the effect of antigen on the immune system. An equally important aspect of the invention is the use of small particles (less than 10 microns in diameter, preferably less than 5 μm and especially from 1 to 5 μm). The use of the delivery system for antigen potentiate the immune system, in the form of microcapsules small size is especially important if you need to induce an immune response in a relatively weak immunogenic agents, such as killed vaccines, vaccines containing low molecular weight proteins or their subagency, etc. connections.

Example 1. The combined introduction of free vaccine and a vaccine comprising microcapsules

Researched the vaccine against Japanese encephalitis virus (Biken). The virus was obtained from the Research Foundation for Microbial Disease (Osaka University, With the ITA, Osaka, Japan). According to the recommendations of the manufacturers immunization should be threefold: first two doses with an interval of 1-2 weeks, and then (after a month) third dose. We have compared the results of immunization of mice according to the recommended Protocol and by a single injection of this animal vaccine for Japanese encephalitis virus, one part of which consisted of standard material, and the other two represented the antigen in the composition of the particles larger than 10 microns. Results immunization by both methods were compared by determining the titers of serum antibodies against Japanese encephalitis detected immunofermentative analysis. The last method allowed to identify present in the serum specific antibodies to specific components of a vaccine against Japanese encephalitis, but did not provide a definition in the same samples of virus - neutralizing antibodies. The title of the latter was determined using the method of inhibition of the cytopathic effect of the virus (CAV) and methods for inhibiting belascoaran. Below are the results of these tests.

The experiment was carried out in 4 groups of mice: (1) control animals not receiving the vaccine, (2) mice, treated on day 0 were injected normal (without capsules) vaccine for Japanese encephalitis at a dose of 3.0 mg, (3) mouse, which is the tier 0 was introduced encephalitis vaccine, and then repeated injections at 14 and 42 days (standard Protocol) and (4) mice, treated on day 0 were injected 3.0 mg routine vaccines and the same amount of the drug in the form of microcapsules. Control animals were used to determine basal levels of antiviral neutralizing antibodies, which compared the titers of antibodies in experimental mice. In turn, the latter group treated with 3.0 mg routine vaccines at day 0, was used for comparison with the group of animals treated with combined injection of normal or microcapsules vaccine. The results show that the introduction of the vaccine composition of microcapsules significantly enhances the immune system's response to free vaccine, administered once at a dose of 3.0 mg Animals treated with three injections of free vaccine, served as a control for comparison with mice treated with microcapsules, to ascertain the comparability of the immunogenic activity of the combined vaccination of free and microencapsulated vaccine after a single dose and activity of the antiviral vaccines in its threefold introduction according to the standard Protocol.

The 10 animals of each experimental group 21, 49 and 77 days, took blood samples and tested the serum on the inhibition of the cytopathic effect of a standard dose (100 TCID50) virus Japanese is about encephalitis. Table 11 presents the results of the evaluation of inhibitory activity, expressed in terms of the maximum dilution of serum at which they are at 50% inhibited CAV. As can be seen from the table, the control animals showed a significant virus - neutralizing activity of sera, regardless of the timing of (group 1). 1 out of 10 animals treated with a single injection of 3.0 mg antiviral vaccine on day 0 (group 2), neutralizing antibodies were also absent. The maximum titer of antibody in the remaining 9 animals reached 1:254 49 days post-vaccination. The average geometric titers of antiviral antibodies in this experimental group was highest on day 49. In 8 of 10 animals treated with antiviral vaccine in accordance with the standard Protocol (group 3), noted a decrease in the immune response between 49 and 77 days. Geometric average antibody titers in animals of this group were reduced by more than 50% between 40 and 77 days. Antibodies to Japanese encephalitis virus were detected in all 10 mice group 4, treated with the vaccine composition of the microcapsules. Geometric average antibody titers in this group was increased from 21 to 77 days after vaccination. The average titer of antibody in these animals on day 49 was significantly lower than in mice treated with three injections of the vaccine (group 3) (p=0.006). However, unlike the animals of the latter group, antibody titers in mice of group 4 continued to grow from 49 to 77 days of observation. On day 77, the average antibody titers in animals of both groups did not significantly differ (p=0.75). This shows that when introducing the vaccine in the form of microcapsules titers of antiviral antibodies reached a comparable level control to 77 days after vaccination. In contrast, animals treated with three injections of the vaccine (group 3), the animals of group 4, treated with microcapsules, the serum titers of antiviral antibodies continuously increased throughout the observation period. Another difference between mice of the two groups was that in the latter case (group 4) the increase in average antibody titers between 49 and 77 days reached double value. The average titers of antiviral antibodies on day 21 the animals receiving the vaccine composition of the microcapsules, and in mice treated with a single injection of free vaccine at day 0, were approximately the same (p=0,12). However, between 49 and 77 days of the differences in antibody titers in animals of these groups were highly significant (p=0.03 and P=0.03 respectively). These results show that the titers of antibodies to Japanese encephalitis virus, are celebrating after immunization according to the standard Protocol, can be achieved with a single administration of the vaccine in the composition of the microcapsules. Although used in the present study, the years of indifferent material of the vaccine did not provide such a rapid increase in titers of virus-neutralizing antibodies, which have been reported with the use of standard vaccines, their ultimate value was comparable with the value of credits, achieved after standard triple vaccination.

To confirm the results of these studies were conducted analysis of the combined serum samples from each of the 4 groups of animals. The analysis was performed in another laboratory, which were transferred to the appropriate samples. Assessed the ability of sera to inhibit the formation of plaques in the standard provocative test with Japanese encephalitis virus. The test results presented in table 12 and confirm the above data. Although titers of antiviral antibodies in animals that received the vaccine in the microcapsules, reached the maximum value is not as fast as after immunization conventional vaccine, their final levels in animals of both groups were quite comparable. Moreover, after the introduction of the microcapsules antibody titers remained elevated for a longer period than after the introduction of the standard vaccine. These observations serve as additional evidence that a single injection of the vaccine composition of microcapsules gives almost the same results as three times the introduction of standard vaccines in accordance with normal Protocol.

Example 2. Combined vaccine introduction in the microcapsules resorable 10 and less than 10 micrometers

One of the advantages of the use of polymeric microcapsules for injection of vaccines is the ability to control the time and/or rate of release of the antigen. The mode of release can be adjusted to cause maximum production of antibodies in response to a single immunization. One of the modes that enhance the immune response to vaccination, is the pulsating release of antigen, providing the same effect as in conventional immunization with live vaccine.

The possibility of using such a regime was studied following subcutaneous injection of 100 μg enterotoxaemia mice several groups of microcapsules with a size of 1-10 μm (the ratio of the components in polylactid-coglycolide 50:50 containing enterotoxaemia 1,51 weight percent), microcapsules size 20-125 μm (the ratio of the components in polylactid-coglycolide 50:50, the content enterotoxaemia of 0.64 wt%) or a mixture of microcapsules with a size of 1-10 and 20-125 μm with the same content enterotoxaemia in each of their varieties. The blood of the experimental mice were made at intervals of 10 days and by the end of the titration of IgG antibodies in the plasma at specific isotypes radioimmunometric analysis using sorbed on the solid phase enterotoxin evaluated the induced immune response. IgG antibodies poyavlyal the ü in the blood on day 10 after injection enterotoxaemia comprising microcapsules with a diameter of 1-10 μm. Their titres were increased to the maximum value of 1:102400 for 30 or 40 days and decreased to 1:25600 60 day. In contrast, the response to the microcapsule size 20-125 μm developed no earlier than 30 days after injection. To 50-60 days antibody titers were increased to 1:51200. Combined with the introduction of equal parts enterotoxaemia comprising microcapsules with a diameter of 1-10 or 20-125 μm immune response of mice within the first 30 days was generally the same as with the introduction of some small microcapsules. However, since 40 days the reaction of the mice treated with enterotoxic simultaneously in the composition of the capsules of size 1-10 and 20-125 μm, progressively increased, and the titers of virus-neutralizing antibodies to the 60th day after the injection was increased to 1:819200, which is significantly higher than the total response for the separate introduction of microcapsules.

The immune response observed when combined with the introduction contains enterotoxin capsules size 1-10 and 20-125 μm, indicates a two-phase nature of the release of antigen (burst release). The first phase is the result of rapid absorption and rapid decomposition of small microcapsules in the process of tissue histocytosis, accompanied by enhanced primary reaction due to the localization of large quantities of antigen on accessory cells and, apparently, their activation. The second phase of release of the antigen on the due to the biodegradation of the microcapsules in the size 20-125 μm, value which prevents their digestion by phagocytes. In this phase, the antigen is released in a pre-stimulated body and induces an anamnestic immune response. Thus, it is possible create a system for a single vaccination on the basis of microcapsules from polylactic-coglycolide with a mixing ratio of 50:50, which potentional immune response due to the presence of microcapsules with a size of 1-10 μm and provides active secondary immunization with antigen in the microcapsules on 20-125 μm, controlled in terms of timing and duration. In addition, by changing the ratio of polymer components, you can obtain drugs, which provide even a later release of the antigen and, therefore, tertiary and Quaternary immunization in the absence of additional injection.

Summarizing, we can conclude that there are a large number of possible approaches to vaccination using injectively microcapsules according to the present invention. They include, in particular, multiple injections of small microcapsules, preferably 1-5 microns in diameter, which are absorbed by macrophages and avoids the use immunopotentiator funds. Another approach is to use a mixed injection of free antigen for the induction of primary immune reaction is AI and antigen in the composition of microcapsules with a diameter of 10 μm or more to ensure the pulsating release of antigen to enhance secondary and tertiary reactions and achievements of immunization with a single administration of the vaccine. You can also use small microcapsules for the induction of primary immune response to those antigens in combination with the larger microcapsules to enhance secondary and subsequent reactions that eliminates the use of immunopotentiators funds and multiple injection.

B. Oral administration of microcapsules with vaccines

Example 1. Microcapsules for oral administration, containing TNF-SMC antigen, induce simultaneous immune response in the form of production of serum antibodies and antibodies mucosa

Microcapsules for heptenophos protein antigen in the form of horseradish trinitrophenyl hemocyanin sea plate (TNF-SMC antigen) produced from polylactide-coglycolide in the ratio of 50:50. Microcapsules were sorted by size, for further work were collected microcapsules with a size of 1-5 microns, containing the 0.2 wt% of the antigen. The suitability of these microcapsules as a system of delivery of antigen to the target tissues after oral administration was evaluated by feeding experimental animals in the form of a suspension (10 mg/ml) in the amount of 0.5 ml (10 μg antigen). The suspension was prepared in water (sterilized) bicarbonate buffer solution and was administered using a gastric probe for 4 consecutive days. For comparison, the additional group of mice were immunized free antigen TNF-HMC in the form of a solution, containing 20 μg/ml of antigen in a dose of 0.5 ml of the Control mice orally was administered one solvent.

At 14 and 28 days after the last injection of antigen in 5 mice of each group on an empty stomach made the blood, saliva and gastrointestinal secretions. The obtained samples were analyzed using an isotype-specific radioimmunoassay method for determining specific antigen TNF-HMC and total antibodies of classes IgM, IgG and IgA (table 13). In saliva and intestinal secretion was present almost exclusively IgA antibodies, in accordance with the results of previous studies. This served as confirmation of the absence of contamination of these fluids material blood sampling. In none of the experimental groups not registered significant changes in the overall level of immunoglobulins in the investigated biological fluids. In the sera of control mice were found specific to the antigen of the antibody of natural origin, related to the isotypes IgM and IgG. In addition, in the serum and intestinal fluid were identified IgA antibodies. In all cases, the antibody titers were low. At the same time, the introduction of equal doses of TNF-SMC antigen in the composition of the microcapsules (30 µg) for three consecutive days caused the emergence of a large number of antibodies in the intestinal secretion and antic the l of all isotypes in the serum on day 14 after immunization (see the last column of table 13). The titers of these antibodies was increased to 28 days. In contrast, oral administration of the same amount of antigen in the form of a solution did not induce specific antibodies, regardless of their class or subjected to analysis of biological fluids.

The results deserve attention from several points of view. First, the introduction of microcapsules induces the formation of large quantities of specific antibodies to the antigen in the serum and intestinal fluid (IgA) at that time, as by the usual method of systemic immunization this reaction is absent or expressed very weakly. In this regard, it can be expected that the proposed method of immunization will significantly enhance the processes of immunogenesis in the mucous membrane, which serves as a gateway infection or place of development of pathological process in case of infection with a variety of bacterial and viral pathogens. Secondly, microencapsulated drugs antigen has proved to be a powerful immunogenic means when administered orally, while the available antigens do not have this property. Thus, the use of antigen in the composition of the microcapsules significantly increases its efficiency by directed injection directly into the target tissue and increased inclusion in Peyer's plaques. In-t is etlich, phase induction of the immune response is characterized by a greater duration. Systemic immunization with protein antigens in the absence of adjuvant maximum antibody titers are reached after 7 to 14 days after injection, whereas in the microcapsules of the same antigens cause a similar reaction after 28 days after oral administration, when the antibody titers are higher than on day 14. This suggests that the biological destruction of the material of the capsules and release of antigen occur over a fairly long period of inducyruya reaction more than the introduction of free antigen, duration.

Example 2. Oral administration of microcapsules containing toxoid CMEA, induce simultaneous production of serum antibodies and mucosal antibodies to the toxin CMEA

The above results show that (a) with the introduction of microencapsulated antigen high adjuvant activity of the drug and (b) microcapsules with a diameter of 5 μm penetrate into the mesenteric lymph nodes and spleen after inclusion in Peyronie plaques. These data show that systemic immunization by oral administration containing the antigen of microcapsules amenable to biodegradation of the material with the selection of their size depending on the purpose of the drug. So the option was shown in experiments, in which separate groups of mice were immunized by injection of 100 μg of staphylococcal enterotoxaemia In the form of a solution or composition of microcapsules from polylactic-glycolide (mixing ratio 50:50) as the indifferent material. In these experiments, solutions and microcapsules with toxoid was administered to mice using a gastric probe three times, at intervals of 30 days, after which, after 10 and 20 days after each immunization, produced blood sampling for plasma. Table 14 presents the results of the final titration of antibodies IgM and IgG antibodies to toxin 20 days after primary, secondary and tertiary oral immunization of animals.

In mice treated with the vaccine composition of the microcapsules was observed a sustained increase in the titers of specific antibodies after each subsequent immunization, whereas a similar effect of dissolved antigen was absent. In the described experiments used the same batch of microcapsules and the same evaluation methods as in the experiments, the results of which are reflected in the previously presented tables 4, 5 and 6. Thus, these data are a direct confirmation of the increased effectiveness of immunization with microcapsules containing staphylococcal enterotoxin In, for oral administration, compared with optimal doses dissolved entero is oxoid for parenteral injection.

Mice of the same group studied the reaction of IgA antibodies using both methods of immunization. This proceeded from the assumption that the microcapsules have the same range in size from less than 1 to 10 μm, containing enterotoxin, will be accompanied by the release of antigen some of them after inclusion in Peyer's plaques. After 10 and 20 days after tertiary oral immunization received samples of saliva and intestinal secretion and analyzed them for the presence of specific IgA antibodies (table 15). In contrast, dissolved toxoid, not possessing the ability to induce an immune response after oral administration, the feeding of the mice of equal amounts of toxoide vaccine composition of microcapsules resulted in a significant increase in the number of sIgA antibodies in saliva and gastrointestinal fluids. It should be noted that when collecting the last of her was diluted to a final volume of 5 ml Although the degree of dilution can not be accurately assessed, it is safe to assume that the concentration of sIgA antibodies in the mucous membrane of the bowel, at least 10 times higher than in the studied samples, which, however, was not taken into account when measurements are carried out in the described experiments.

The data show that microcapsules with enterotoxemia are effective induction coord is anticarcinogen sIgA antibodies in the mucous membrane of the intestines and other organs after oral administration. Moreover, using a mixture of microcapsules of different diameters (from less than 1 to 10 microns), can cause the formation of large quantities of these antibodies in the mucous and their simultaneous appearance in the blood. This suggests the possibility of preparation of various vaccines with high efficiency and convenient for practical use on the basis of microcapsules technology.

Century Microcapsules with a vaccine for introduction into the trachea

Example 1. The introduction of the microcapsules with the CMEA toxodon in the trachea causes simultaneous production of serum antibodies and mucosal antibodies to the toxin CMEA

Aggregated lymphatic follicles, such aerowyn the plaques of the gastrointestinal tract, are also associated with mucosal lymphatic tissues other anatomical localization, for example in the respiratory system. The function of these formations is not different from the function of Meyerovich plaques: they can absorb different materials from the lumen of the respiratory tract and serve as places of induced immune response, which is characterized by increased production of antibodies. Explored the possibility of immunization using functions associated with mucosal lymphatic tissues of the bronchi. Different groups of mice were administered 50 μl of phosphate buffer solution containing 50 μg of the CMEA of toxoid, either in the form of mi is recaps, either in the form of dissolved material. The introduction was made directly into the trachea. After 10, 20, 30 and 40 days after the introduction of the produced samples of blood, saliva, swabs from the intestines and broncho-alveolar fluid.

The results of the analysis of blood plasma for the presence of antibodies after injection of dissolved CMEA of toxoid indicate the absence of its inducing effect on the production of antibodies of all isotypes (table 16). In contrast, the introduction into the trachea of an equal amount of a vaccine of the CMEA in the composition of the microcapsules induced the formation of antibodies of all classes. The vaccine was maximal after 30 days after injection and was maintained during the subsequent 10-day period in respect of antibodies IgM, IgG and IgA titres which were reached, respectively, 1:400, 1:51300 and 1:400.

Similarly, the introduction of microcapsules was accompanied by the formation of specific anticarcinogen antibodies in the mucous membrane of the respiratory tract, while the free vaccine did not cause activation of the immune system (table 17). The study of the kinetics of IgG anticarcinogen antibodies showed that their appearance in bronchoalveolar fluid is delayed relative to blood plasma, and 20 days after the vaccine it had only IgG antibody titers were low in comparison with final values in more than the later stages. 30 day celebrated peak titres of IgG and IgA antibodies (respectively 1:1280 and 1:320). They remained at least 40 days. IgM antibodies in swabs from the bronchi and alveoli in all periods after immunization with microencapsulated vaccine is not detected, which confirms the absence of producing their cells in the lung and the inability of large molecules antibodies to penetrate from the blood through the walls of the alveolar capillaries that serve as a filter material with a molecular weight greater than 200,000.

The results show that the introduction of microcapsules allows to induce an immune response to an antigen CMEA of toxoid directly in the respiratory tract, whereas similar effects of the vaccine in the form of a solution is missing. Antibodies to this antigen in this method of immunization was found in the secret surrounding mucosa of the respiratory tract and in the blood. It should be noted that the proposed method provides the induction of IgA antibodies, which are assumed to be formed and are secreted locally in the upper respiratory system, i.e. the area which is not protected IgG antibodies, which are received in the lower part of the respiratory system from the blood. Thus immunization with antigens in the composition of the microcapsules injected directly into the trachea through inhalation aerosol preparations, can be an effective means of inducing antic is l, protects against infections of the upper respiratory system.

He Microcapsules with vaccines for immunization using different routes of administration

When tested on humans and animals, it was found that systemic immunization in combination with the introduction of the antigen in the mucous membranes to a greater extent induces an immune response last than any other combination methods of vaccine administration (N.F. Pierce and J.L. Gowans Cellular kinetics of the intestinal immune response to cholera toxoid in rats / Cellular kinetics of the immune response of the intestine to Vibrio toxoid rats/, J. Exp. Med., 142, 1550, 1975). The mice were divided into 3 groups, were injected intraperitoneally with 100 µg of microcapsules containing CMEA enterotoxin, and after 30 days, 100 μg of the same drug using intraperitoneal, oral or nutritherapy ways of introduction. The purpose of the study was to obtain direct confirmation of the benefits of using microcapsules with antigen for the induction of antibodies.

20 days after immunization with microencapsulated drug received samples of blood plasma, swabs from the gastrointestinal tract to the broncho-alveolar cavity and by titration with radioimmunological analysis determined the character of the distribution in these liquids of different isotypes of antibodies to toxin CMEA (table 18). Repeated immunization of mice the same way as per the ranks immunization (intraperitoneally), caused the appearance in the blood plasma and the secrets of the gastrointestinal and respiratory systems of IgG antibodies in high titers, whereas IgA antibodies in all studied samples was absent. In contrast, secondary immunization by injection containing toxoid CMEA microcapsules orally or directly into the trachea was accompanied by the appearance in plasma as a specific anticarcinogen IgG antibody titers before re-immunization reached 1:51200, and sIgA antibodies, which aspirates the gastrointestinal tract and bronchial cavities were also present in large quantities. Oral administration of microcapsules mice pre-immunized by intraperitoneal injection induced the secretion of sIgA antibody isotype in gastrointestinal fluid in quantities comparable with the number of them after three oral dosage forms (compare tables 15 and 18). With the introduction of the microcapsules into the trachea of mice pre-immunized by intraperitoneal injection, the greatest effect was noted in relation to the induction of diffuse mucosal immune response to the simultaneous occurrence of large quantities of antibodies IgG and sIgA in swabs from the gastrointestinal tract and broncho-alveolar cavity.

These observations are of especially great interest in what manisali against a variety of infectious agents, pathophysiological action which is performed by an acute infection of the respiratory tract. Present in the respiratory antibodies originate from two different sources. Antibodies of the IgA class predominate in the mucosa and mucus surrounding the nasopharynx and bronchial tree (Soutar S.A. Distribution of plasma cells and other cells containing immunoglobulin in the respiratory tract of normal man and class of immunoglobulin contained therein / Distribution of plasma cells and other cell types containing immunoglobulins, in the respiratory system of healthy people, and the types present in immunoglobulins/, Thorax 31: 58; 1976 and Kaltreider H.B. and Chan M.K.L. The classspecified immunoglobulin composition of fluids obtained from various levels of canine respiratory tract. / The specific composition of immunoglobulins of different classes in liquids received from different departments of the respiratory system of dogs /, J. Immunol, 116, 423, 1976). These antibodies are formed in the local plasma cells present in the lamina propria of the upper respiratory system. Unlike the nasopharynx and bronchial tree in the bronchial tubes and alveoli contain predominantly IgG antibodies that passively receives them from the blood in the extravasation process (Reynolds H.Y. and Newdfll, H.H. Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage / Analysis of proteins and cells of the human respiratory system, obtained from the lungs and bronchial aspirates/, J. Lab. Clin. Med., 84, 559, 1974). Thus, to effectively protect the lungs need the AK circulating IgG antibodies and antibody isotype sIgA in the mucous membrane.

The data show that a mixed method immunization using microcapsules provides the greatest efficiency induction simultaneously circulating antibodies and mucosal antibodies. The experiments consisted of primary immunization and subsequent activation of the immune system, which required the introduction to each of these stages of new portions of microcapsules with prisoners in them the antigen. However, the flexibility of the proposed method to provide a controlled pulsating release of antigen after a single injection of microcapsules for maximum stimulation simultaneous systemic and secretory immune response. As an example we can mention the introduction of the antigen simultaneously by mouth and by injection during a single visit to a patient of a medical institution. By varying the ratio of lactide and glycolide polymeric material of the microcapsules, enter both ways, you can achieve the situation in which systemic injection of antigen induces a few days later activation of the immune system and oral introduced the antigen is released from Meyerovich plaques at a later date, potenziare mucosal immune response.

IV. Absorption of medicines

P is evidenee following examples show that microcapsules with a size up to 5 μm, preferably from 1 to 5 μm, can improve the absorption of drugs, including vaccine antigens in the body. From polylactic-glycolide (in the ratio 50:50) produced microcapsules containing etretinate (ethyl ester E-9) 4-methoxy-2-3,6-trimethyl)phenyl-3,7-dimethyl-2,4,8-nonatetraenovoy acid. Used microcapsules with a diameter of 0.5 to 4.0 μm, containing 37,2 weight percent of the preparation. Microcapsules, as well as free etretinate by means of a gastric probe was administered to mice in a 1%solution of tween-80 in water. The introduction was made once, at a dose of 50 mg etretinate /kg body weight. In certain periods produced the collection of blood samples and conducted quantitative analysis of etretinate / or products of its transformation in serum using high-performance liquid chromatography (table 19). The results show that the introduction of etretinate mice in the composition of the microcapsules is accompanied by a much more pronounced increase of its concentration in the blood compared with mice treated with Neopalimovsky the drug. As in the case of microcapsules with vaccines size less than 5 microns, microcapsules containing etretinate, ensure its receipt in the blood of Meyerovich plaques (lymphatic tissue of the gastrointestinal tract). A similar approach can be used clausilia absorption of other medicines, that can be found particularly wide application in systems for targeted delivery of such biologically active compounds such as peptides, proteins, nucleic acids and other materials.

Table 11.
The results of the test of the inhibiting effect of sera after vaccination
Dilution of serum causing 50% inhibition of the cytopathic effect of the virus
Animals21 days49 day77 day
Group I: without vaccination
renewamerica.<101111
the average titre<101111
max<10 16<20
min.<10<10<10
Group 2: 3 mg Neopalimovsky vaccine/br on day 10
renewamerica.447350
the average titre559571
max127254160
min.<1013<10
Group 3: 3 mg Neopalimovsky vaccine/br at 0, 14 and 42 days
renewamerica.5073,8801,576
the average titre9345,3632,951
max4,064>10,240>10,240
min.160806254
Group 4: 3 m Neopalimovsky vaccine and 3 mg vaccine in the microcapsules in/barrel per day 0
renewamerica.777181,341
the average titre8031,2302,468
max3205,12010,240
min.13160254



Table 12.
The test results belascoaran with blood sera after vaccination
Dilution of sera
GroupFormDay50%80%
IaControl0<10<10
14<10<10
21<10<10
42<10<10
49<10<10
84<10<10
2bWithout capsules 0<10<10
1416020
21NDcND
4232080
4932040
84640160
   
3gWithout capsules0<10<10
1416040
212,560640
421,280640
495,1202,560
842,5601,280
4dCapsules0<10<10
1416020
2132080
425,120640
495,120640
8410,0002,560
andunvaccinated control
banimals were treated with 3.0 mg Neopalimovsky vaccine is the tier 0 (br)
withthe definition was not conducted because of the small sample size
ganimals were treated with 3.0 mg Neopalimovsky vaccine at 0, 14 and 42 th days (br)
danimals were treated with 3.0 mg Neopalimovsky vaccines and 3.0 mg of the vaccine in the microcapsules at day 0 (br)

1. The method of delivery of biologically active compounds to associate with the mucous membranes lymphoreticular tissue of the animal, characterized in that exercise

a) encapsulating effective amounts specified biologically active compounds in biologically compatible excipient to form microcapsules with a size of 1-10 μm, and

b) introducing an effective amount of microcapsules animal so that required for therapeutic action, the number of microcapsules reached and joined in the specified associated with the mucous membranes lymphoreticular fabric.

2. The method according to claim 1, characterized in that the introduction is carried out orally.

3. The method according to claim 1, otlichalis the same time, the introduction is carried out through the nose.

4. The method according to claim 1, characterized in that the introduction is carried out rectally.

5. The method according to claim 1, characterized in that the introduction is carried out through the eyes.

6. The method according to claim 1, characterized in that the introduction is carried out by oral inhalation.

7. The method according to claim 1, characterized in that the said compound is selected from the group consisting of drugs, nutrients, immunomodulator, lymphokine, monokine, cytokine, antigen or allergen.

8. The method according to claim 1, characterized in that the microcapsules have a size of 5-10 μm, which ensures their retention in the specified associated with mucous membrane lymphoreticular tissue.

9. The method according to claim 8, characterized in that said biologically active compound is selected from the group consisting of an antigen and an allergen, to ensure immunity of the mucous membrane of the animal.

10. The method according to claim 1, characterized in that the microcapsules have a size of about 1-5 microns, which ensures penetration associated with the mucous membranes lymphoreticular fabric.

11. The method according to claim 10, characterized in that said biologically active compound is selected from the group consisting of an antigen and an allergen, to ensure systemic immunity of the animal.

12. The method according to claim 1, characterized those who, the microcapsules include a microcapsule type I size of 1-5 μm and a microcapsule type II size of about 5-10 μm, and the introduction provides the delivery of a mixture of microcapsules type I and II to ensure that systemic immunity and immunity of the mucous membrane.

13. A method of obtaining a composition for administration of biologically active compounds in associated with the mucous membranes lymphoreticular tissue of the animal, characterized in that an effective amount specified biologically active compounds conclude in a biocompatible excipient to form microcapsules with a size of 1-10 microns.

14. The method according to item 13, wherein the specified vehicle selected from the group consisting of drugs, nutrients, immunomodulator, lymphokine, monokine, cytokine, antigen or allergen.

15. The method according to item 13, characterized in that the microcapsules have a size of 5-10 μm, which ensures their retention in the specified associated with mucous membrane lymphoreticular tissue.

16. The method according to item 13, characterized in that the microcapsules have a size of 1-5 microns, which ensures penetration associated with the mucous membranes lymphoreticular fabric.

17. The method according to item 13, characterized in that the microcapsules include microcapsules I t is and the size of 1-5 μm and a microcapsule type II size of 5-10 microns, moreover, the introduction provides the delivery of a mixture of microcapsules type I and II to ensure that systemic immunity and immunity of the mucous membrane.

18. Composition for delivery of biologically active compounds in associated with the mucous membranes lymphoreticular tissue of the animal, characterized in that it contains an effective amount of the compounds encased in a biocompatible excipient to form microcapsules with a size of 1-10 microns.

19. The composition according to p, characterized in that said biologically active compound is selected from the group consisting of drugs, nutrients, immunomodulator, lymphokine, monokine, cytokine, antigen or allergen.

20. The composition according to p, characterized in that the microcapsules have a size of 5-10 μm, which ensures their retention in the specified associated with mucous membrane lymphoreticular tissue.

21. The composition according to p, characterized in that the microcapsules have a size of 1-5 microns, which ensures penetration associated with the mucous membranes lymphoreticular fabric.

22. The composition according to p, characterized in that the microcapsules include a mixture of microcapsules type I size of 1-5 μm and microcapsules type II size of 5-10 μm, and their introduction induces both systems the initial immunity, and the immunity of the mucous membrane.



 

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