MXPA00008860A - Lipophilic microparticles containing a protein drug or antigen and formulation comprising same - Google Patents

Abstract

A lipophilic microparticle having an average particle size ranging from 0.1 to 200&mgr;m, comprising a lipophilic substance and an active ingredient selected from the group consisting of a protein or peptide drug and an antigen, retains the full activity of the active ingredient, and when formulated in the form of an oil dispersion or oil-in-water emulsion, it releases in an in vivo environment the active ingredient in a controlled manner over a long period.

Description

LIPOFILIC MICROPARTICLES THAT CONTAIN A PROTEIN OR ANTIGEN DROUGHT AND FORMULATION COMPRISING THE SAME FIELD OF THE INVENTION The present invention relates to microparticles coated with a lipophilic substance, comprising a protein drug or an antigen, and an effective release formulation for an effective in vivo delivery of the drug or antigen. BACKGROUND OF THE INVENTION It is well known that protein or antigen drugs have the problem of denaturation caused by heat, organic solvents and / or unfavorable pH (Weiqi Lu et al, PDA J. Pharm, Sci. Tech., 49, 13- 19 (1995)). They are usually administered by injection; however, due to their in vivo activities last only a short period of time after administration, they have to be administered repeatedly when long-term treatment is required. For example, to treat a child dwarfism with pituitary deficiency, human growth hormone (hGH) should be injected daily or every other day, for a period of 6 months or more. Therefore, there have been many efforts to develop sustained release formulations of effective drug proteins or antigens.

For example, extensive studies have been conducted to develop a sustained release microparticle formulation, prepared by coating a protein drug or an antigen with a synthetic biodegradable polymer, for example polylactide, polyglycolide, poly (lactid-co-glycolide), pol i -orto-ester or polyanhydride, which continuously releases the drug or the antigen as the polymer degrades in. the body (M. Chasin and R. Langer, ed., Biodegradable Polymers as Druq Delivery Systems (Biodegradable Polymers with Drug Release Systems), Marcel Dekker (1990), and Heller, J., Adv. Druq Del. Rev ., 10, 163 (1993)). Although these types of formulation have several advantages, it has a serious problem that the drug or antigen is subjected to denaturation upon contact with an organic solvent during its preparation process (Park, TG et al., J. Control, Reí., 33, 211-223 (1995)). The use of an organic solvent is unavoidable because a biodegradable polymer only dissolves in an organic solvent, for example methylene chloride, ethyl acetate, acetonitrile, chloroform or acetone. In order to avoid this undesirable contact of a drug or antigen with an organic solvent, Lee et al. Have prepared a type of microparticles by coating an antigen with a water-soluble polymer to obtain a primary particle; dispersing the primary particle in an organic solvent containing a biodegradable polymer; and drying the resulting dispersion to obtain a final microparticle (Lee, H. K. et al, J. Control, Re., 44, 283-294 (1997), and U.S. Patent No. 5,753,234). However, the process for preparing these microparticles is complicated and not economic. There have been many attempts to develop a obtained release formulation containing a natural polymer, for example gelatin, collagen, chitosan, carboxymethyl cellulose, alginate or hyaluronic acid. The natural polymer readily absorbs water to form a gel that has high viscosity that releases a drug or antigen slowly. For example, the patent of the U.S.A. No. 5,416,017 discloses an injection formulation with releases obtained from erythropoietin containing 0.01 to 3% hyaluronic acid gel; Japanese Patent Laid-Open No. 1-287041 (1989), an injection formulation with sustained insulin release containing a 1% hyaluronic acid gel; Japanese Patent Laid-Open No. 2-213 (1990), a formulation of releases obtained from calcitonin or human growth hormone containing 5% hyaluronic acid gel; and Meyer, J. et al., (J. Controlled Reí., 35, 67 (1995)), a sustained-release formulation of granulocyte colony stimulation factor (G-CSF) containing 0.5 to 4% gel. hyaluronic acid. These hyaluronic acid gel formulations have a sustained release effect because the protein drugs pass slowly through the gel matrix having high viscosity. Nevertheless, a gel having a concentration of hyaluronic acid of several units in% has high viscosity, for example in the order of 105 to 107 centipoises, which makes its injection difficult. In addition, since both the drug and hyaluronic acid dissolve in water, a hyaluronic formulation is easily diluted by the body fluid after injection, with a consequently rapid release of the drug, usually in one day. For example, Japanese Patent Laid-Open No. 1-287041 (1989) discloses that when a 1% hyaluronic acid gel formulation containing insulin is injected into a rabbit, the effect of reducing the level of glucose in the blood it was sustained for only 24 hours; and Meyer, J. and collaborators (see above) and the patent of. the U.S. No. 5,416,017, that when a 2% hyaluronic acid gel formulation containing G-CSF and a 1.5% hyaluronic acid gel formulation containing interferon-a together with whey protein are administered to an animal, the Blood levels of these protein drugs suddenly decrease or fall below 1/10 of the initial levels in 24 weeks. Benzyl hyaluronate (HYAFFMR, Fidia S.P.), a synthetic ester prepared by esterifying natural hyaluronic acid with benzyl alcohol, does not dissolve in water but in an organic solvent such as dimethyl sulfoxide (DMSO). A solid benzyl hyaluronate microparticle formulation containing a protein drug has been prepared by the solvent-emulsion extraction method (Nightlinger, NS et al., Proceed, Intern Symp, Control Relay Bioact. Mater., 22nd, Paper No., 3205 (1995); and Ilum, L. et al, J. Controlled Rei., 29, 133 (1994)), which is conducted by dissolving benzyl hyaluronate in DMSO; disperse the protein drug in the resulting solution; add the resulting dispersion to mineral oil to form an emulsion; and adding a solvent that is miscible with DMSO, for example ethyl acetate, to the emulsion to extract DMSO to obtain solid microparticles comprising the protein drug and benzyl hyaluronate. However, the benzyl hyaluronate formulation has the disadvantage that the protein drug can be easily denatured by the organic solvent used in the preparative step and also by the hydrophobic benzyl hyaluronate itself, An in vitro release test of a type of benzyl microparticles hyaluronate comprising the granulocyte macrophage colony stimulating factor (GM-CSF) showed that only 25% of GM-CSF was released in the initial 2 days and subsequently no more than GM-CSF (Nightlinger, NS et al., see above) , suggesting that the majority of the protein drug was denatured. Meanwhile, several attempts have been made to develop an oral formulation of a protein or antigen drug, using in particular the fact that a type of microparticles containing an antigen with a particle size of 5 μm or less can be phagocyte-fixed for a phagocyte. Therefore, it is expected that an antigen can be targeted to a specific location in the body, for example Payer patches of the small intestine. However, it is difficult for the M cells of the Payer patches to absorb an antigen due to their hydrophilicity and high molecular weight. To improve the absorption of an antigen by M cells, an absorption enhancing agent such as mycelium of fatty acid-bile salt in mixture, chelate, fatty acid, phospholipid, acrilcarnitine, surfactant, medium chain glycerides have been employed (Lee, VHL , Crit > Rev. Ther, Druq Carrier Systems, 5, 69-97 (1988), Yoshioka, S. et al, J. Pharm, Sci., 71, 593-597 (1982), Scott-Moncrieff, JC and collaborators, J. Pharm. Sci., 83, 1465-1469 (1994), Muranishi, S. et al., Chem. Pharm. Bull., 25, 1159-1163 (1977), Fix, JA et al., Am. J. Phvsiol., 251, G332-G340 (1986), Shao, Z. and collaborators, Pharm. Res., 10, 143-250 (1993), Constantinidas, PP et al., Proc. Int. Symp. Control. Bioactive Mater., 20, 184-185 (1993), and Bjork, E. et al., J. Druq Targeting, 2, 501-507 (1995)). Furthermore, it has been reported that when a type of microparticles prepared by encapsulating bovine serum albumin in liposome cholesterol lecithin is administered orally to an animal, an IgA response in the salivary glands is increased (Genco, R. et al., Ann. Acad Sci, .409, 650-667 (1983)). However, the only effective oral vaccine formulation that has been successfully developed is a polio vaccine for which there is a bowel receptor, and the liposome microparticles have problems of structural instability. COMPENDIUM OF THE INVENTION Accordingly, an object of the present invention is to provide a type of microparticles having improved stability and effective delivery of a protein drug or an antigen.

Another objective of the present invention is to provide a sustained release formulation comprising the microparticle. In accordance with one aspect of the present invention, lipophilic microparticles having an average particle size in the range of 0.1 to 200 μm, comprising a lipophilic substance and an active ingredient selected from the group consisting of a protein or drug are provided. peptide and an antigen. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing objects and features of the present invention will be apparent from the following description of preferred embodiments taken in conjunction with the accompanying drawings in which: Figure 1 shows the in vitro release profiles of Microparticles 11, 12 and 13; Figures 2A and 2B reproduce RP HPLC scans of the Microparticle extract solution 12 obtained in Test Example 3 and an aqueous solution hGH standard respectively; and Figures 3A and 3B illustrate SEC scans of the Microparticle extract solution 12 which is obtained in Test Example 3 and an aqueous solution hGH standard, respectively. DETAILED DESCRIPTION OF THE INVENTION The solid lipophilic microparticles of the present invention comprise a lipophilic substance and an active ingredient. The active ingredient that may be employed in the present invention is a protein drug, peptide drug or antigen. Representative peptide or protein drugs include a human growth hormone, bovine growth hormone, porcine growth hormone, growth hormone releasing hormone, growth hormone release peptide, granulocyte colony stimulating factor, colony stimulating factor. of granulocyte macrophage, macrophage colony stimulating factor, erythropoietin, morphogenic bone protein, interferon, insulin, atriopeptin-III, monoclonal antibody, tumor necrosis factor, macrophage activating factor, interleukin, tumor degenerative factor, growth factor type insulin, epidermal growth factor, tissue plasminogen activator and urokinase, but these do not limit the peptide drugs or proteins that can be employed in the present invention. Representative antigens include those obtained from: one or more pathogens selected from the group consisting of adenovirus type 4 and 7, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza A & amp; amp; B, Japanese encephalitis virus B, measles virus, epidemic parotitis virus, rubella virus, polio virus, hydrophobic virus, chickenpox virus, yellow fever virus and human immunodeficiency virus; one or more pathogens selected from the group consisting of Bordetella pertussis, Borrelia burgdorferi, enterotoxigenic Escherichia coli, Hemofilus in luenza type b, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria meningitidis A & C, Neisseria menin itidis B, Pseudomonas aeruginosa, Pseudomonas cepacia, Salmonella typhi, Shigella spp. , Streptococcus pneumoniae and Vibrio colerae; one or more pathogens selected from the group consisting of Coccidiodes immltis, Leishmania s. and Plasmodium sp.; one or more pathogens responsible for the disease selected from the group consisting of bovine symptomatic anthrax, bovine epidemic fever, bovine anthrax, bovine Akabane disease, bovine paw-and-mouth disease, mastitis (bovine mamilitis), infectious bovine nasotracheal inflammation, bovine viral diarrhea, infectious bovine gastroenteritis, swine cholera, porcine epidemic diarrhea, porcine atrophic gastritis, porcine disease caused by parvovirus, rotavirus porcine enteritis, chicken Newcastle disease, Marek's disease of chickens, chicken encephalomyelitis, rabies, dog distemper, dog enteritis caused by parvovirus and dog infectious hepatitis, the antigen is an attenuated, exterminated or recombinant antigen; or DNA, RNA, plasmid, CpG DNA or oligonucleotide extracted from the pathogen, but these do not limit the antigen that can be employed in the present invention. The lipophilic substance that may be employed in the present invention includes a lipid and its derivatives, a fatty acid and its derivatives, a wax and a mixture thereof. Representative lipids include lecithin, phosphatidylcholine, phosphatidylethanolamine, phosatidylserine and phosphatidylinositol. Representative lipid derivatives include arachididoyl phosphatidylcholine and stearoyl phosphatidylcholine. Representative fatty acids include myristic acid, palmitic acid, stearic acid, and their salts. Representative fatty acid derivatives include glyceryl stearate, sorbitan palmitate, sorbitan stearate, sorbitan monooleate and polysorbate. Representative waxes include an anionic emulsifying wax, carnauba wax and microcrystalline wax. Among those, lipophilic surfactant substances such as lecithin, phosphatidylcholine and phosphatidylethanolamine are preferred. The lipophilic microparticles of the present invention may comprise 0.001 to 99% by weight, preferably 0.1 to 10% by weight of an active ingredient and 1 to 99.999% by weight, preferably 5 to 50% by weight, of a lipophilic substance , based on the weight of the microparticle. In addition to the active ingredient, the microparticle of the present invention may further comprise hyaluronic acid or its inorganic salt. Representative inorganic salts of hyaluronic acid include sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zin hyaluronate and cobalt hyaluronate. Hyaluronic acid or its inorganic salt can be used in an amount in the range of 0.1 to 99% by weight, based on the weight of the microparticle. The microparticle of the present invention may further comprise a water-soluble excipient for the purpose of stabilizing the active ingredient. The water-soluble excipient that can be employed in the present invention includes a carbohydrate, such as hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, chitosan, alginate, glucose, xylose, galactose, fructose, maltose, sucrose, dextran and chondroitin sulfate.; a protein such as albumin and gelatin; an amino acid such as glycine, alanine, glutamic acid, arginine, lysine and its salt; a fatty acid such as stearic acid; an inorganic salt such as phosphate, a surfactant such as Tween ™ (ICI), poly (ethylene glycol) and its mixture. The water-soluble excipient may be employed in an amount in the range of 0.001 to 99% by weight preferably 0.1 to 50% by weight based on the amount of the microparticle. The lipophilic microparticles of the present invention can be prepared by coating a solid particle containing an active ingredient with a lipophilic substance according to any of the following methods. The lipophilic microparticles of the present invention can be prepared by dissolving an active ingredient in an aqueous solution containing other optional components such as hyaluronic acid and a water-soluble excipient; drying by dew or freezing the resulting solution, to obtain solid particles containing the active ingredient; dispersing the solid particles in an organic solvent containing a lipophilic substance; and then dry the dispersion to obtain solid, lipophilic microparticles. The organic solvent that may be employed in the above process includes ethanol, methylene chloride and isopropyl alcohol. Alternatively, the lipophilic microparticles of the present invention can be prepared by dissolving an active ingredient in an aqueous solution containing other optional components such as hyaluronic acid and a water-soluble excipient, adding a lipophilic surface-active substance such as lecithin, to allow the lipophilic surfactant substance is hydrated, and spray-drying the resulting solution. In the spray drying step, the lipophilic surfactant substance migrates to the surface of the droplets and coats the particles containing the active ingredient. The microparticle of the invention thus prepared has an average particle size in the range of 0.1 to 200 μm, preferably 1 to 50 μm, more preferably 1 to 10 μm. The microparticle of the invention that contains an active ingredient, has several advantages: (1) the active ingredient is not denatured and retains its full activity; (2) releases the active ingredient completely over a prolonged time; (3) it is easily dispersed in a lipophilic medium such as oil, and the dispersion thus obtained has low viscosity and retains all the activity of the active ingredient (4) is easily fused with the phospholipid membrane of the body for effective transport of the active ingredient to the body; (5) When administered orally, it can be translocated into M cells of Payer patches located in the small intestine. The microparticle of the present invention can be formulated in dispersion, emulsion and aerosol forms. Therefore, the present invention also provides a dispersion formulation prepared by dispersing the lipophilic microparticles of the invention in a lipophilic medium. The lipophilic medium which can be used in the present invention includes an edible oil, mineral oil, squalene, squalane, cod liver oil, mono-, di- or tri-glycerides and mixtures thereof. Representative edible oils include corn oil, olive oil, soybean oil, safflower oil, cottonseed oil. peanut oil, sesame oil, sunflower oil and their mixtures. The lipophilic medium may further comprise a dispersing agent or a preservative. The dispersion formulation can be used for injection or oral administration. The present invention also provides an oil-in-water emulsion formulation comprising an aqueous injection medium and the dispersant formulation. The aqueous injection medium includes distilled water and a buffered solution. In the emulsion formulation, the lipophilic microparticles are coated with an oil and remain in the oil phase while improving the formation and stabilization of the water-in-oil emulsion. The emulsion formulation can be used for injection. The emulsion formulation when its active ingredient is an antigen, can further comprise another antigen in the aqueous injection medium, thereby providing a mixed vaccine formulation. For example, a dispersion formulation comprising microparticles of hepatitis B surface antigen (HBsAg) dispersed in an edible oil can be mixed with a DTP vaccine containing the DTP antigen adsorbed on alum, in an aqueous solution, to obtain a formulation of oil-in-water emulsion formulation. The emulsion formulation thus obtained is composed of an aqueous phase which DTP antigen adsorbed on alum and an oil phase containing solid microparticles of HBsAg. In this emulsion formulation, the HBsAg antigen and DTP are present separately in the aqueous phase and the oil phase, respectively, in this way avoiding undesirable interactions between the HBsAg antigen and DTP. In contrast, a mixed vaccine formulation prepared by adsorbing several antigens in alum in an aqueous solution is known to suffer from a low vaccination effect due to undesirable interactions between the antigens. The present invention further provides an aerosol formulation comprising the microparticle of the invention. The aerosol formulation can be prepared according to a conventional method using a conventional excipient. The aerosol formulation can be administered by bronchial or nasal mucous membrane. The following examples are intended to further illustrate the present invention, without limiting its scope. In addition, percentages given below for mixing solid in liquid, liquid in liquid, and solid in liquid are on a weight / weight, volume / volume and weight / volume basis, respectively, unless specifically indicated otherwise. Example 1: Preparation of Lipophilic Microparticles Lecithin was added to a solution -damped with 10 mM phosphate (PBS) at a concentration of 2% (w / v) and hydrated completely. A recombinant hepatitis B surface antigen (HBsAg, LG Chemical Ltd.) is added at a concentration of 0.5 mg / ml and the resulting solution is supplied to a spray dryer (Buchi 191) at a flow rate of 0.55 ml / minute to obtain solid microparticles (Microparticles 1). At this stage, the inlet flow air temperature was 70 ° C and the outlet air temperature was 50 ° C. The particle size of the microparticles thus obtained was in the range of 0.1 to 5 μm.

Example 2: Preparation of Lipophilic Microparticles Recombinant HBsAg is dissolved in 10 mM PBS at a concentration of 0.5 mg / ml and carboxymethylcellulose is added at a concentration of 3% (w / v). The resulting solution is provided to a spray dryer (Buchi 191) at a flow rate of 0.55 ml / minute to obtain primary particles. In this stage, the inlet air temperature was 70 ° C and the outlet air temperature was 50 ° C. A 5% (w / v) lecithin solution in ethanol is prepared and the primary particles are dispersed at a concentration of 5% (w / v). The resulting dispersion is provided to a spray dryer (Buchi 191) at a flow rate of 1.0 ml / minute to obtain solid microparticles (Microparticles 2). In this stage, the inlet air temperature was 85 ° C and the outlet air temperature 50 ° C. The particle size of the microparticles thus obtained was in the range of 0.1 to 5 μm. Example 3: Preparation of Lipophilic Microparticles Carboxymethylcellulose was dissolved in 10 mM PBS at a concentration of 3% (w / v). The lecithin is added at a concentration of 2% (w / v) and hydrated completely. Recombinant HBsAg is then added at a concentration of 0.5 mg / ml. The resulting solution is provided to a spray dryer (Buchi 191) at a flow rate of 0.55 ml / minute to obtain solid microparticles (Microparticles 3). In this stage, the inlet flow air temperature was 70 ° C and the outlet flow air temperature 50 ° C. The particle size of the microparticles thus obtained was in the range of 0.1 to 5 μm. Examples 4 to 9: Preparation of Lipophilic Microparticles The procedure of Example 3 was repeated using various ingredients listed in Table I to obtain various solid microparticles (Microparticles 4 to 9).

Table I 11 Hepatitis B Antigen preS2 21 Staphylococcal Enterotoxin Cl mutant protein (LG Chemical Ltd.) (used in an amount that the concentration of the protein becomes 6% by weight based on the total weight of the ingredients) Example 10: Preparation of Lipophilic Microparticles Carboxymethylcellulose is dissolved in 10 mM PBS at a concentration of 3% (w / v), the resulting solution is filtered and sterilized. Lecithin is added at a concentration of 2% (w / v) and hydrates completely. A DNA extract of E. coli is dissolved in 10 mM PBS in an amount of 1 mg / ml and this solution is added to the above mixture at a concentration of 6% by weight, based on the total weight of the mixture, followed by mixing using a magnetic stirrer. The resulting suspension is provided to a spray dryer (Buchi 191) at a flow rate of 0.55 ml / minute to obtain solid microparticles (Microparticles 10). In this stage, the air pressure temperature was 70 ° C and the outlet air temperature 50 ° C. The size of the microparticles thus obtained was in the range of 0.2 to 3 μm. Example 11: Preparation of Lipophilic Microparticles Human growth hormone (hGH) is dissolved in 5 mM PBS at a concentration of 2 mg / ml and then Tween 80 is added in an amount of 0.01% by weight based on the weight of PBS. Sodium hyaluronate having a molecular weight of 1,000,000 is dissolved at a concentration of 0.2% (w / v). The resulting solution is provided to a spray dryer (Buchi 190) at a flow rate of 3 ml / minute to obtain primary particles. In this stage, the intake air temperature was 85 ° C. The average particle size of the primary particles thus obtained was 3 μm. Dissolve lecithin in ethanol at a concentration of 1% (w / v) and then the primary particles are suspended at a concentration of 1% (w / v). The resulting suspension is provided to a spray dryer (Buchi 190) to obtain microparticles (Microparticles eleven) . The average particle size of the microparticles obtained was 7 μm. Examples 12 to 24: Preparation of Microparticles Lipophilic The procedure of Example 11 was repeated using various ingredients listed in 1 to Table II to obtain various solid microparticles (Microparticles 12 to 24).

Table II a) bovine somatotropin b) porcine somatotropin c) granulocyte macrophage colony stimulating factor d) erythropoietin e) insulin-like growth factor- 1 - Example 25: Preparation of Oil Dispersion Formulations of Microparticle 3 Cotton Seed 3 Microparticles 3 prepared in Example 3 are added to cottonseed oil and dispersed using a magnetic stirrer to obtain five dispersions of cottonseed oil containing , 50, 100, 200 to 500 mg / ml of Microparticles 3, respectively. Dispersion Formulations 26: Preparation of Microparticle Edible Oil Dispersion Formulations 3 The procedure of Example 25 is repeated using soybean oil, corn oil and sesame oil, respectively, to obtain dispersions of soybean oil, soybean oil, and so on. corn and sesame oil each containing 100 mg / ml of Microparticles 3. Example 27: Preparation of Microparticle Edible Oil Dispersible Formulations 8 The procedure of Example 25 is repeated using Microparticles 8 to obtain dispersions of cottonseed oil , soybean oil, corn oil, and sesame oil, each containing 100 mg / m of Microparticles 8. Example 28: Preparation of Microparticle Edible Oil Dispersion Formulations 9 The procedure of Example 25 was repeated using Microparticles 9 to obtain dispersions of cottonseed oil, soybean oil, maize oil z and sesame oil, each containing 100 mg / ml of Microparticles 9. Example 29: Preparation of Microparticle Edible Oil Dispersion Formulations 10. The procedure of Example 25 was repeated using Microparticles 10 to obtain dispersions of cottonseed oil. , soybean oil, corn oil and sesame oil, each containing 100 mg / ml of Microparticles 10. Example 30: Preparation of Microparticle Cotton Seed Oil Dispersion Formulations 12 The procedure of example 25 was repeated using Microparticles 12 for obtaining five dispersions of cottonseed oil each containing 50,100, 200, 360 and 500 mg / ml of Microparticles 12, respectively. Example 31: Preparation of Microparticle Edible Oil Dispersion Formulations 12 The procedure of Example 25 is repeated using Microparticles 12 to obtain dispersions of soybean oil, corn oil and sesame oil each containing 100 mg / ml of Microparticles 12 Example 32: Preparation of Microparticle Edible Oil Dispersion Formulations 14 The procedure of Example 25 was repeated using Microparticles 14 to obtain dispersions of cottonseed oil, soybean oil, corn oil and sesame oil, each containing 3 £ 0 mg / ml of Microparticles 14. Example 33: Preparation of Microparticle Edible Oil Dispersion Formulations 15 The procedure of Example 25 is repeated using Microparticles 15 to obtain dispersions of cottonseed oil, soybean oil, corn oil and sesame oil each containing 360 mg / ml of Microparticle 15. Example 34: Preparation of Formulations of Dispersion of Microparticle Edible Oil 18 The procedure of Example 25 was repeated using Microparticles 18 to obtain dispersions of cottonseed oil, soybean oil, corn oil, and sesame oil each containing 360 mg / ml Microparticle 18 Example 35: Preparation of Microparticle Emulsion Formulations 3 Each of the dispersions of soybean oil, corn oil, and oil of sesame obtained in Example 26 are added to a volume 4 -fold of 0.9% NaCl solution to obtain a mixture of oil and water (1: 4) containing 20 mg / ml of Microparticle 3. The resulting mixture was mixed to obtain a homogenous opaque-white oil-in-water emulsion. Example 36: Preparation of Microparticle Emulsion Formulations 12 The procedure of Example 35 is repeated using the cottonseed dispersions obtained in Example 30 to obtain five homogeneous opaque-white oil-in-water emulsions, which respectively contains 5, 20, 50, 120 and 200 mg / ml of Microparticle 12, the oil to water ratios are 1: 9, 1: 4, 1: 3, 1: 2, and 2: 3, respectively. In these emulsion formulations, the solid microparticles were dispersed in the oil phase and the oil droplets containing the microparticles were stable due to the lipophilic surface of the microparticles. The emulsions were stable at room temperature for a period of more than 2 weeks. Example 37: Preparation of Microparticle Emulsion Formulations 12 The procedure of Example 35 is repeated using the soy oil, corn oil and sesame oil dispersions obtained in Example 31 to obtain oil-in-oil emulsion formulations. opaque-white water homogeneous. Example 38: Preparation of Microparticle Emulsion Formulations 22 The procedure of Example 25 is repeated using Microparticle 22 and soybean oil to obtain a soybean oil dispersion containing 100 mg / ml of Microparticle 22. Using the resulting dispersion, the procedure of Example 35 is repeated to obtain a homogenous white-opaque oil-in-water emulsion formulation. Example 39: Preparation of Microparticle Emulsion Formulation 23 The procedure of Example 25 was repeated using Microparticle 23 and soybean oil to obtain a soybean oil dispersion containing 100 mg / ml Microparticle 23. Using the resulting dispersion, the procedure of Example 35 was repeated to obtain a homogeneous white-opaque oil-in-water emulsion formulation. Example 40: Preparation of Microparticle Emulsion Formulations 24 The procedure of Example 25 was repeated using Microparticles 24 and soybean oil to obtain a soybean oil dispersion containing 100 mg / ml Microparticle 24. Using the resulting dispersion, the procedure of Example 35 is repeated to obtain a homogenous white-opaque oil-in-water emulsion formulation. Example 41: Preparation of Microparticle Emulsion Formulations 14 The procedure of Example 35 is repeated using the dispersions of cottonseed oil, soybean oil, corn seed oil and sesame oil obtained in Example 32 and a volume double 0.9% solution of NaCl, to obtain four homogenous white-opaque oil-in-water emulsion formulations. Each of the formulations thus obtained contains 120 mg / ml of Microparticle 14 in a mixture of oil and water (1: 2). Example 42: Preparation of Microparticle Emulsion Formulations The procedure of Example 35 is repeated using the cottonseed oil dispersions, soybean oil, corn seed oil and sesame oil, which is obtained in Example 33 and double volumes of 0.9% NaCl solution to obtain four homogenous white-opaque oil-in-water emulsion formulations. Each of the formulations thus obtained contains 120 mg / ml of Microparticle 15 in a mixture of oil and water (1: 2). Example 43: Preparation of Microparticle Emulsion Formulations 18 The procedure of Example 35 was repeated using the cottonseed oil dispersions, soybean oil, corn seed oil, and sesame oil obtained in Example 34 and double volumes of 0.9% NaCl solution to obtain homogenous white-opaque oil-in-water emulsion formulations of Microparticle 18. Each of the formulations thus obtained contains 120 mg / ml of Microparticle 18 in a mixture of oil and water (1: 2) Example 44: Preparation of Microparticle Emulsion Formulations 3 The procedure of Example 35 is repeated except that alum was used instead of 0.9% NaCl, to obtain white oil-in-water emulsion formulations-homogenous moles. Comparative Example 1: Preparation of Microparticle 1 Comparative The procedure of Example 2 is repeated without the use of lecithin to obtain comparative microparticles without coating with lipophilic substance (Comparative Microparticle 1). Comparative Example 2: Preparation of Comparative Microparticle Dispersion Formulations 1 The procedure of Example 25 was repeated using Comparative Microparticles 1 and soybean oil to obtain soybean oil dispersion containing 100 mg / m Comparative Microparticle 1 (Comparative Dispersion 1) , which contains non-homogeneously dispersed, aggregated microparticles. Comparative Example 3: Preparation of Comparative Microparticle Emulsion Formulation 1 The procedure of Example 35 is repeated using Comparative Dispersion 1 and physiological saline by injection, to obtain an emulsion formulation (Comparative Emulsion 1), which was not a homogeneous emulsion but showed a separation with aggregated microparticles. Comparative Example 4: Preparation of Comparative Microparticle 2 The procedure of Example 11 is repeated using 1 mg / ml of hGH, 0.1% (p.v.) of sodium hyaluronate having a molecular weight of 2,000,000 for Comparative Microparticle which does not have the lipophilic coating (Comparative Microparticle 2). Comparative Example 5: Preparation of Comparative Microparticle Dispersion Formulations 2 The procedure of Example 25 is repeated using Comparative Microparticle 2 and cottonseed oil to obtain a dispersion of cottonseed oil, containing 100 mg / ml of Comparative Microparticle 2 (Comparative Dispersion 2). Comparative Example 6: Preparation of Comparative Microparticle Emulsion Formulation 2 - The procedure of Example 35 was repeated using Comparative Dispersion 2. The resulting mixture (Comparative Emulsion 2) does not form a homogeneous emulsion but exhibits a phase separation, with the microparticles dispersed in both phases of oil and water.

Comparative Example 7: Preparation of Comparative Microparticle 3 The procedure of Example 11_ is repeated using 2 x 10 5 IU / ml interferon-α, 0.2 mg / ml of D-mannitol, 0.2 mg / ml serum albumin and 0.25% (w / v ) of sodium hyaluronate having a molecular weight of 2,000,000 at an inflow temperature of 105 ° C to obtain Comparative Microparticle which has no lipophilic coating (Comparative Microparticle 3) having an "average particle size"? of 3.5 μm. Comparative Example 8: Preparation of Comparative Microparticle Oil Dispersion Formulations 3 The procedure of Example 25 is repeated using Comparative Microparticle 3 and cottonseed oil to obtain a dispersion of cottonseed oil containing 100 mg / ml of Comparative Microparticle 3 (Comparative Dispersion 3).

Comparative Example 9: Preparation of Comparative Microparticle Emulsion Formulation 3 The procedure of Example 35 was repeated using Comparative Dispersion 3. The resulting mixture (Comparative Emulsion 3) does not form a homogeneous emulsion but exhibits a phase separation, with the microparticles dispersed in both phases of oil and water.

Example of Test 1: Microparticle Stability Test 1 and 3 To examine whether an antigen contained in one type of microparticle maintains its activity, each of Microparticles 1 and 3 dissolve in water and dilute with 1,000- to 100,000 -Times in volume of water and the antigenicity of HBsAb is examined using the AUZYME equipment (Abbott, USA). The results are illustrated in Table III. Table III As can be seen in Table III, the antigenicity of HBsAg contained in the microparticles of the invention was substantially unchanged before and after the preparation. Therefore, the antigen contained in the microparticle of the present invention preserves its antigenicity. Test Example 2: In Vitro Release Test of Microparticles 11, 12 and 13 Each of the Microparticles 11, 12 and 13 are dispersed in a buffered solution of (150 mM NaCl, 10 M phosphate, 0.05% sodium azide, pH 7.4) in such a way that the hGH concentration was 1.0 mg / m¿. An in vitro release test is conducted by stirring the resulting dispersion at 37 ° C and at a predetermined time, the dispersion is centrifuged at 800 g for 10 minutes and a supernatant mixture, which is taken in an amount of 1/10 volume of the dispersion is mixed with an equal volume of the previous buffer solution. hGH contained in the supernatant is quantified using the Lowry method with High Performance Liquid Chromatography (HPLC). Figure 1 shows the release profiles of Microparticles 11, 12 and 13 thus determined. As can be seen in the Figure. 1, as the molecular weight of the hyaluronic acid becomes higher, and as the hGH content decreases, the rate of hGH release becomes slower. Therefore, the release rate of drug protein can be controlled by adjusting the molecular weight of the hyaluronic acid and the protein drug content. In addition, the release test results in. vitro showed that an initial burst release of the drug protein does not occur and the rate of release is constant until it is released from 70% of the protein. Test Example 3: Microparticle Stability Test 12 To examine whether hGH contained in Microparticle 12 maintains its activity, the procedure of Test Example 1 is repeated and the amount of hGH released from Microparticle 12 in 48 hours is determined with both HPLC of reverse phase (RP) as size exclusion chromatography (SEC). RP HPLC is used to estimate the ratio of deamidative and oxidative denaturation of the protein; and SEC, the denaturation of the protein by aggregation. Figures 2A and 2B depict RP HPLC results for the Microparticle extract solution 12 and the aqueous hGH solution employed in the preparation of Microparticle 12, respectively. Figures 3A and 3B provide SEC results for the solution of Microparticle extract 12 and the aqueous hGH solution employed in the preparation of Microparticle 12, respectively. As can be seen in the Figures. 2 and 3, the amount of hGH released from the microparticle of the invention is identical to that of the original hGH aqueous solution according to the RP HPLC results; and the SEC results show that the monomeric hGH content is 95% or more. These results suggest that hGH is not denatured during the preparation of the microparticles of the invention. Test Example 4: In Vitro Microparticle Release Test 14 to 21 The procedure of Test Example 2 is repeated using each of Microparticles 14 through 21, and the cumulative amounts of the drug released in 10 and 72 hours as well as the Monomeric protein content of the 72-hour sample was determined. The results are illustrated in Table IV. Table IV As can be seen from Table IV, the micro particles of the present invention release the drug protein for a period of 3 days, without sign of denaturation of the drug protein. Test Example 5: Injection Test 1 To examine whether the microparticles of the present invention are dispersed homogeneously in an oil dispersion or an oil-in-water emulsion, injectability tests were performed. Injectability is defined as the force required to push a syringe filled with a test sample at a speed of 80 mm / minute. A 23 gauge injection needle was used. Samples used in these tests were the alum dispersion prepared dilute alum with PBS 20 times; soybean oil; the soybean oil dispersion of Example 26; the emulsion of Example 44, Comparative Dispersion 1 and Comparative Emulsion 1, and the results are illustrated in Table V. Table V As can be seen in Table V, the dispersion of Example 26 is easily injectable, showing that the Microparticles 3 of the present invention are well dispersed in oil as compared to Comparative Microparticles 1 without lecithin coating. Particularly, since the emulsion of the present invention has superior injectability, it can be used to prepare a mixed formulation. Test Example 6: Injection Test 2 The procedure of Test Example 5 is repeated for 0.9% aqueous NaCl solution, cottonseed oil dispersion, the cottonseed oil dispersion of Example 30, the emulsion of Example 36, Comparative Dispersion 2, and Comparative Emulsion 2, using a 26 gauge syringe needle. The results are illustrated in Table VI.

Table VI As can be seen in Table VI, the Microparticle 12 of the present invention has good dispersibility in cottonseed oil due to the lipophilic lecithin coating, and the seed oil dispersion of something, the present invention having an equivalent of injectability to that of cottonseed oil. In addition, the emulsion of Example 36 has lower injectability than cottonseed oil despite the high concentration of Microparticle 12. In contrast, Comparative Microparticle 2 has a lower dispersibility in cottonseed oil due to the absence of a lipophilic coating, causing the poor injectability of the Comparative Dispersion 2. Further, when the hydrophilic surface of the Comparative Microparticle 2 caused a phase separation in the Comparative Emulsion 2, with consequent leaching of the sodium hyaluronate component in the aqueous phase to raise the viscosity of the water layer. Therefore, Comparative Emulsion 2 has an even more poor injectability than Comparative Dispersion 2. Test Example 7: Injection Test 3 The procedure of Test Example 5 was repeated for the emulsions obtained in Examples 38 to 40, and Soybean oil as a control. The injectability of the soybean oil was 1.4 kgf, and those of the emulsions obtained in Examples 38 to 40 were in the range of 0.3 lgf similar to that of the aqueous solution at 0.9% NaCl. The lipophilic surface of the microparticles of the present invention is coated with soybean oil, resulting in a homogeneous oil-in-water emulsion having injectability equivalent to that of water.

Test Example 8: Animal Formulation Injection To examine the biological activity of the injection formulation of the present invention, Microparticle 3 is added to soybean oil to prepare four dispersions having charges of recombinant HBsAg of 0.5, 0.125, 0.03125 and 0.0078 μg protein / ml, respectively. As a comparative formulation, a hepatitis B vaccine (LG Chemical Ltd., Korea) containing alum as an immune adjuvant is diluted with PBS to prepare samples having HBsAg loading of 0.5., 0.125, 0.03125 and 0.0078 μg protein / ml, respectively. Each dispersion sample was injected intraperitoneally with 4-week-old male Balb / C (H-2d) mice (10 mice) and blood samples were taken 4 months after the injection. Sera were obtained from the blood samples and their antibody titers and antibody formation (%) were determined using the AUSAB EIA (Abott, USA) and ED50 (μg) microparticle was calculated using a statistical method (probit analysis). The results are illustrated in the Table VII.

Table VII As can be seen in Table VII, the microparticle of the present invention has a lower amount of ED50 than the comparative formulation and higher antibody titer than the comparative formulation with the same amount of antigen is injected. Therefore, the microparticle of the present invention functions as an adjuvant for superior antibody formation. Test Example 9: Oral Administration of Animal Formulation To examine the biological activity of the formulation of the present invention for oral administration, Microparticle 3 was added to soybean oil to prepare a dispersion having a recombinant HBsAg load of 5 μg. protein / it. As a comparative formulation, a hepatitis B vaccine (LG Chemical Ltd., Korea) is diluted with PBS to obtain the same loading of 5 μg protein / ml. Each dispersion sample was orally administered to 4-week-old male Balb / C (H-2d) mice (10 mice) and administered twice, 2 and 4 weeks later. Blood samples were taken from the mice after 8 weeks. Serums obtained from blood samples and antibody titers and antibody formation (%) were determined using the AUSAB EIA (Abbott, USA) equipment. The results are illustrated in Table VIII Table VIII As can be seen in Table VIII, the mice that were administered with the microparticle of the present invention had 80% antibody formation while the mice administered with the comparative formulation do not form antibodies. Therefore, the microparticle of the present invention has superior ability to form antibody in vivo, even when administered orally. This suggests that the microparticle of the present invention can be advantageously employed in a formulation for oral administration. Test Example 10: Cytotoxic Lymphocyte Test To examine whether the microparticle of the present invention induces a cell-mediated immune response, Microparticle 3 is added to soybean oil to obtain a dispersion having a loading of 10 μg protein / ml. As a comparative formulation, a hepatitis B vaccine (LG Chemical Ltd., Korea) is diluted with PBS at the same loading of 10 μg protein / ml. Each dispersion formulation is administered subcutaneously to 4-week-old male Balb / C (H-2d) mice (10 mice) and administered two or more times after 2 month intervals. Two weeks after the last administration, pre-cytotoxic lymphocyte cells (CTL) are obtained from the immunized mice and cultured to obtain effector cells (E). E cells were cultured together with target cells (T) varying the ratio of E cell to T cell, and then the number of cells of T lysed by E cells (specific lysis (%)) is determined as follows. ^ -Pm sample ~ cpm natural release Specific Lysis (%) = X 100 Cpm release maximum Cpm natural release A high specific lysis value means that the cell-mediated immune response is actively induced. The results are illustrated in Table IX. Table IX As can be seen in Table IX, the microparticle of the present invention induces the cell-mediated immune response while the comparative formulation does not. Test Example 11: Animal Test To examine the immune enhancing effect of the microparticle of the present invention, Microparticle 9 is added to soybean oil to obtain a dispersion having a SEC-SER load of 4.0 mg protein / ml. As a comparative formulation, SEC-SER antigen is mixed with a commercial ISA adjuvant according to the manufacturer's guide, and then diluted to the same 4.0 mg protein / ml load. As a control, microparticles prepared by repeating the procedure of Example 9 except that the antigen is omitted, are dispersed in soybean oil.

Each dispersion formulation is injected intramuscularly to cows in the 2nd or 3rd lactation period (5 cows) which has a large amount of somatic cell and are injected twice more, 2 and 4 weeks later. Blood samples are taken from the cows after 2, 6, 10 and 14 weeks and then the antibody titer is determined. Results expressed in the ELISA reader are illustrated in Table X.

Table X As can be seen in Table X, the formulation of the present invention induces the formation of a high level of antibody that lasts 14 weeks after the first injection. Therefore, the formulation of the present invention can be advantageously employed as a vaccine for animals. Test Example 12: Animal Test The administration effect of the inventive hGH-containing formulation is examined using 7-week-old female dwarf rats (body weight: approximately 100 g) having the inheritance of low growth hormone secretion.

The dispersion of Example 30, the emulsion of Example 36, Comparative Dispersion 2, Comparative Emulsion 2, and Utroprin ™ (LG Chemical Ltd., Korea), an aqueous formulation will be chosen for the test, and each was administered to a group of 10. dwarf rats at a hGH dose of 350 μg per rat and then a gain in weight was examined. As a control, rats that do not receive hGH were employed. The average cumulative net weight gains are illustrated in Table XI. Table XI (unit: g) As can be seen in Table XI, the average weight of the rats in Utropin ™ increased on the first day, but decreased on the second day. The weight was subsequently increased at a speed similar to that of the control group. The average weight of the rats in the group of Comparative Dispersion 2 was continuously increased while the rats in the Comparative Emulsion 2 group decreased on the third day. Because the microparticles in Comparative Emulsion 2 have a hydrophilic surface, the drug dissolves and therefore have a weight gain effect equal to that of the aqueous formulation Utropin ™. In contrast, the average weight of the rats administered with the dispersion of Example 30 and the emulsion of Example 36 was increased by 6 days at higher speeds than either Comparative Dispersion 2 or Comparative Emulsion 2. The microparticles of the present invention that have lecithin coating are covered with cottonseed oil even after injection and absorb water only slowly, thus releasing the drug at a constant speed. Test Example 13: Animal Test The dispersion of Example 32 and the emulsion of Example 41 were each administered to 7-week-old female dwarf rats (body weight: about 100 g) at a bST dose of 12.5 mg per rat. and then their weight gain was examined.As another control, rats not receiving bST were employed.The average cumulative net weight gain is illustrated in Table XII.Table XII (unit: g) As can be seen in Table XII, the average weight of the rats administered with any of the dispersions of Example 32 or the emulsion of Example 41 was continuously increased by 6 days and the rate of gain in daily weight was higher than the control group. . After 8 days, the gain in weight became insignificant, suggesting that the time of drug release is approximately 8 days in both cases. Test Example 14: Cytopathic Inhibition Test The emulsion of Example 43 Comparative Dispersion 3, aqueous formulation of interferon-a, each were administered to rabbits of 5 months of age (body weight: 2.5 kg) at a dose of interferon -a of 300 μg. The blood level of the drug is determined using a cytopathic inhibition test, which is conducted by treating cells with interferon-a, adding the viruses and then determining inhibition of cell pathology. Male calf kidney cells (MDBO CATCC CCF-22) and vesicular stomatitis virus (ATCC VR 158) were used in the test. Interferona- titers in the blood were measured for 5 days and the results are illustrated in Table XIII. Table XIII * not detected As can be seen in Table XIII, the interferon-a titers for the rats administered with the dispersion of Example 43 were elevated for the entire 5 day trial period., showing higher levels of interferon-a compared to the Comparative Dispersion group 3 from day 2. Therefore, the dispersion of the present invention have prolonged release characteristics due to the lipophilic surface of the microparticles. Comparative Test of ELj emplo 1 _ hGH is dissolved in 5mM PBS at a concentration of 2.3 mg / ml and then sodium hyaluronate having a molecular weight of 2,000,000 is dissolved at a concentration of 2% (w / v) to obtain gel formulation of hyaluronic acid. An in vitro release test is conducted using the gel formulation by repeating the procedure of Test Example 2. As a result, 100% hGH is released into the supernatant in 1 hour. Therefore, this gel formulation has a drug release time which is much shorter than the microparticles of the present invention. Comparative Test Example 2: Animal Test The method for preparing a hyaluronic acid gel formulation in the Test Example Comparison 1 is repeated using 1.5 mg / ml of hGH to obtain a gel formulation of hyaluronic acid that is not fluid. The gel formulation thus obtained is administered to dwarf rats at a dose of hGH of 150 μg per rat and then the average weight advance is examined for 6 days. As a comparative formulation, an aqueous solution formulation, Utropin ™ is administered to rats at the same dose of hGH. As a control, rats that do not receive hGH were employed. Results expressed in cumulative weight gains are illustrated in Table XIV. Table XIV As can be seen in Table XIV, the average weight gain of the rats administered with the gel formulation is similar to that of the Utropin ™ group. The weight gain rate after 2 days was not significantly different between the three groups, suggesting that the drug release from the gel formulation did not last more than 1 day. Example of Comparative Test 3 hGH is dissolved in PBS 5mM PBS at a concentration of 2 mg / ml and then Tween 80 is added at a concentration of 0.01% by weight. The resulting solution is provided to a spray dryer (Buchi 190) at a flow rate of 3 ml / minute to obtain microparticles. At this stage, the intake flow air temperature was 85 ° C. The average particle size of the microparticles thus prepared was 2.5 μm. Benzyl hyaluronate is dissolved in -dimethyl sulfoxide (DMSO) at a concentration of 6% and then the microparticles there are dispersed. The resulting dispersion is added to a mineral oil containing Aracel ARM (Atlas Chemical Ind.). The mixture is homogenized to obtain a microemulsion. The microemulsion is composed of the mineral oil as a continuous phase and the solution of benzyl hyaluronate / DMSO as a dispersed phase, Ethyl acetate is added to the microemulsion while a DMSO extract is stirred from the dispersed phase, giving microparticles of benzyl hyaluronate containing hGH. The average particle size of the microparticles was 5.5 μm and the hGH content was 45% by weight. An in vitro release test is conducted using the benzyl hyaluronate formulation thus obtained by repeating the procedure of Test Example 2 and the cumulative amounts of hGH released are illustrated in Table XV.

Table XV As can be seen in Table XV, the benzyl hyaluronate formulation released hGH only slightly after 5 hours and only 30% loaded hGH is released in 144 hours. Thus, the majority of hGH in the benzyl hyaluronate formulation binds to the benzyl hyaluronate matrix and does not release. The above benzyl hyaluronate formulation is dispersed in cottonseed oil and the resulting dispersion is administered to dwarf rats at a dose of hGH of 300 μg per rat. The average weight gain is determined and the results that are expressed in cumulative weight gain are illustrated in Table XVI. Table XVI As can be seen in Table XVI, the benzyl hyaluronate formulation does not show significant effects after 1 day. While the invention has been described with respect to the above specific embodiments, it will be recognized that various modifications and changes can be made to the invention by those skilled in the art that also fall within the scope of the invention as defined by the claims. annexes.

Claims (20)

1. Lipophilic microparticles having an average particle size in the range of 0.1 to 200 μm, comprising a lipophilic substance and an active ingredient selected from the group consisting of a peptide or protein drug and an antigen.

2. The lipophilic microparticles according to claim 1, characterized in that the average particle size is in the range of 1 to 50 μm.

3. The lipophilic microparticles according to claim 1, characterized in that the drug is selected from the group consisting of growth hormone, bovine growth hormone, porcine growth hormone, growth hormone releasing hormone, hormone releasing peptide Growth factor, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, erythropoietin, bone morphogenic protein, interferon, insulin, atri pep ina-III, monoclonal antibody, necrosis factor - tumor, macrophage activating factor, interleukin, tumor degenerating factor, "insulin growth factor, epidermal growth factor, tissue plasminogen activator and urokinase.

4. The lipophilic microparticles according to claim 1, characterized in that the antigen is obtained from: one or more pathogens selected from the group consisting of adenovirus type 4 and 7, hepatitis A virus, hepatitis B virus, hepatitis C virus, virus of influenza A and B viruses, Japanese encephalitis virus B, measles virus, epidermal parotitis virus, rubella virus, poliovirus, hydrophobic virus, chickenpox virus, yellow fever virus and human immunodeficiency virus; one or more pathogens selected from the group consisting of Bordetella Pertussis, Borrelia burqdorferi, enterotoxigenic Escherichia CQII, Haemophilus influenza type b, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria meningitidis A & C, Neisseria meningitidis B, Pseudomonas aeruginosa, Pseudomonas cepacia, Salmonella typhi, Shigella spp. , Streptococcus pneumoniae and Vibrio cholerae; one or more pathogens selected from the group consisting of Coccidiodes immitis, Leishmania sp. and Plasmodium sp.; oo more pathogens responsible for the disease selected from the group consisting of bovine symptomatic anthrax, bovine epidermal fever, anthrax bpvino, bovine Akabane disease, bovine paw-and-muzzle disease, bovine mastitis, infectious bovine nasotracheal inflammation, bovine viral diarrhea, infectious bovine gastroenteritis, swine cholera, porcine epidermal diarrhea, porcine atrophic gastritis, porcine disease caused by parvovirus, porcine enteritis caused by rotavirus, chicken Newcastle disease, chicken Marek's disease, chicken encephalomyelitis, rabies, dog distemper, enteritis of dog caused by parvovirus and infectious dog hepatitis, the antigen is an attenuated, exterminated or recombinant antigen; or DNA, RNA, plasmid, CpG DNA or oligonucleotide extracted from the pathogen.

5. The lipophilic microparticles according to claim 1, characterized in that the lipophilic substance is selected from the group consisting of a lipid, a lipid derivative, a fatty acid, a fatty acid derivative, a wax and mixtures thereof.

6. The lipophilic microparticles according to claim 5, characterized in that the lipid is lecithin, phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine and the lipid derivative arachididoyl phosphatidylcholine or stearoyl phosphatidylcholine.

7. The lipophilic microparticles according to claim 5, characterized in that the fatty acid is myristic acid, palmitic acid, or stearic acid, and the fatty acid derivative is glyceryl stearate, sorbitan palmitate, sorbitan stearate, sorbitan monooleate or polysorbate

8. The lipophilic microparticles according to claim 1, characterized in that it also comprises hyaluronic acid or an inorganic salt thereof.

9. The lipophilic microparticles according to claim 8, characterized in that the inorganic salt of hyaluronic acid is sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate or cobalt hyaluronate.

10. The lipophilic microparticles according to any of claims 1 and 8, characterized in that it also comprises a water-soluble excipient.

The lipophilic microparticles according to claim 10, characterized in that the water-soluble excipient is selected from the group consisting of a carbohydrate, a protein, an amino acid, a fatty acid, an inorganic salt, a surfactant, poly (ethylene), glycol) and mixtures thereof.

12. A dispersion formulations prepared by dispersing the lipophilic microparticles of any of claims 1 to 8 and 10, characterized in a lipophilic medium.

The dispersion formulations according to claim 12, characterized in that the lipophilic medium is an edible oil, mineral oil, squalene, squalane, cod liver oil, mono-, di- or triglycerides or a mixture thereof.

The dispersion formulations according to claim 13, characterized in that the edible oil is corn oil, olive oil, soybean oil, safflower oil, cottonseed oil, peanut oil, sesame oil, oil of sunflower or mixtures thereof.

15. The dispersion formulations according to claim 12, characterized in that the lipophilic medium further comprises a dispersing oil or a preservative.

16. The dispersion formulations according to any of claims 12 and 15, characterized in that it is used for injection or oral administration.

17. An oil-in-water emulsion formulation comprising an aqueous injection medium and the dispersant formulation of claim 12.

18. The oil-in-water emulsion formulation according to claim 17, characterized in that the aqueous injection medium is distilled water or a buffered solution. - -

19. The oil-in-water emulsion formulation according to claim 17, characterized in that the active ingredient is an antigen and the aqueous injection medium further comprises a second antigen.

20. An aerosol formulation comprising the lipophilic microparticles of any of claims 1 to 11.