KR100904226B1 - Adjuvant of Nanosome Form Comprising Pseudo Lecithin Emulsifier and Vaccine Composition Comprising same - Google Patents

Abstract

The present invention relates to a nanosome-type immunopotentiator containing a lecithin-like emulsifier and a composition for vaccine comprising the same. Specifically, the present invention relates to glyceryl cocoate / citrate / lactate or glyceryl citrate / lock as a lecithin-like emulsifier. It relates to an immunoadjuvant composition in the form of a nanosome containing any one of tate / linoleate / oleate, an oil and an aqueous component, and a composition for vaccines thereof.

The nanosomal immunopotentiator composition containing the lecithin-like emulsifier of the present invention induces a stronger immune response, sustains the release time of the drug, and greatly increases the safety when applied to animals, thereby increasing the shelf life of the product. It is very effective as a formulation of a vaccine such as increasing the unit cost and the service life.

Lecithin-like emulsifiers, nanosomes, immunopotentiators, vaccine compositions

Description

Adjuvant of Nanosome Form Comprising Pseudo Lecithin Emulsifier and Vaccine Composition Comprising same}

The present invention relates to a nanosome form of adjuvants using lecithin-like emulsifier and a vaccine composition comprising the same.

Bacteria, viruses, parasites and mycoplasma infections are widespread in veterinary animals such as cattle, pigs and familiar animals. Diseases caused by these infectious agents are often resistant to antimicrobial drug therapies, which defeats the means of treatment. As a result, vaccination approaches are increasingly used to suppress infectious diseases in veterinary animals. The entire infectious pathogen may be made suitable for use in vaccine formulation after chemical inactivation or suitable genetic engineering. Meanwhile, the protein subunits of the pathogen can be expressed in a recombinant expression system and purified for use in vaccine formulation.

An adjuvant generally refers to any substance that increases a humoral and / or cellular immune response to an antigen. Traditional vaccines consist of unprocessed preparations of dead pathogenic microorganisms, and impurities associated with the culture of pathogenic microorganisms can act as an adjuvant to enhance the immune response. However, when homogeneous preparations of pathogenic microorganisms or purified protein subunits are used as antigens for vaccination, the immunity triggered by such antigens is insufficient and therefore the addition of some foreign substance as an adjuvant is required. Moreover, synthetic and subunit vaccines are expensive to manufacture. Thus, smaller doses of antigen may be required to stimulate an immune response with the help of an adjuvant, thereby reducing the cost of vaccine production.

The use of traditional oil adjuvants is a combination of some antibodies, such as aluminum hydroxide, which has a high effect but involves side effects. The development of new oil adjuvant based on mineral oils or non-mineral oils can avoid these side effects. These adjuvants are classified according to their raw materials (minerals, bacteria, plants) and ingredients (emulsion suspensions). Commercially available immunopotentiators include aluminum hydroxide, carbopol mineral oil (mineral oil) or biodegradable oil.

Immunopotentiators are used in vaccines to amplify immune responses. Most attenuated venom (MLV) vaccines have high immunogenicity even if they do not contain excipients such as immunopotentiators, while subunit vaccines and genetic vaccines have weak immunogenicity and therefore excipients. Is urgently needed. In addition, the subunit vaccines and gene vaccines have fewer side effects, and are effective in infectious diseases of livestock such as bacteria, viruses, parasites and mycoplasma infections. Therefore, the development of excipients, ie, immunostimulants, is an important research task in this field. It is becoming.

The development of specific immunopotentiators for various vaccines is just as important as the purification of antibodies. The immunogenicity of the vaccine determines the direction and type of the induced immune system according to the type of excipient, and different excipients are required according to the type of pathogen in order to induce an accurate immune response. In addition, recent advances in the immune system have led to the development of immunopotentiators that produce specific immune responses to specific pathogens, but tests must be conducted to demonstrate that they are safe and do not cause side effects or local action or formation of pus. Should be. Therefore, the development of universal and universal immunostimulators for various vaccines is difficult to develop due to the variety of target species.

In fact, excipients are a major determinant of vaccine effectiveness and substantially affect the safety of the vaccine. Together with the vaccine, excipients are approved by the FDA. This approval is mainly based on the absence of injection site lesions at slaughter, while safety and efficacy with respect to side effects are considered in the overall approval of the vaccine.

A factor that distinguishes commercially available immunopotentiators is the nature of the formulation used. Vaccines containing carbopol or aluminum hydroxide are prepared as water-based suspensions, while vaccines containing oil adjuvant are produced in admixture with other emulsions.

Representative oil immunostimulants include oil-in-water (O / W), oil-in-water (W / O) and multiphase (W / O / W) emulsions.

Oil-in-water (oil in water, O / W) emulsion

Vaccines with oil-in-water (O / W) adjuvant have better immunostimulatory properties than vaccines with aluminum hydroxide adjuvant. However, the duration of immunostimulation is short due to the release of relatively fast antigens. Longer defenses require more powerful adjuvant oils. Mineral oil excipients in commercially produced vaccines are used in the form of oils to stimulate immunity. However, oil-in-water immunosuppressants are more aggressive and can cause local reactions such as inflammation and purulent reactions. The oil-sustaining form of the oil-in-water type is water, and mineral oil (mineral oil) exists as a form of diffusion in water.

Water-in-oil (water in oil, W / O) emulsion

Water-in-oil (W / O) adjuvants are prepared in the form of diffusion of very small droplets containing antigens in biodegradable oils. The oil containing the antigen is released very slowly after injection and thus smoothly stimulates the immune response for a long time. Water-in-water (W / O) antigens contain oils that continually elicit immune responses and tiny droplets scattered within them.

Focusing on single-dose vaccines with long-term effects, water-in-water (W / O) adjuvants, compared to mineral adjuvants containing mineral oil, release a gentle antigen that induces a progressive and sustained immune response. In addition, its biodegradability allows for a faster immune response, and the non-mineral oil-in-water type 2-oil adjuvant has the advantage of minimizing local reactions at the injection site.

Oil adjuvant is usually a water-in-oil (W / O) regimen that induces strong and long-lasting immune responses. It is based on mineral oils and is effective but sometimes produces antibody reactions at topical sites. Oils other than mineral oils are less effective against idiomatic but low immunogens. Therefore, a mixture of mineral oils and other oils can be a good alternative. These oils may vary depending on the type of target, the type of immune response, the route of inoculation, and the intention to achieve a good balance between stability and immunity.

Water-in-oil (W / O) adjuvants are strong and maintain immunity for a long time and are proven in the case of foot or mouth disease in animals. For cows, one dose will protect them for one year. Based on mineral oil this is very effective.

Occasionally, even if a local reaction occurs, water-in-oil emulsions are used because they have a better effect against certain diseases than other prescriptions.

Non-mineral oils are tolerant of the administered subject, but the immune response is less effective.

Multiphase (water in oil, W / O / W) emulsion

Multiphase emulsions can react with a variety of antibodies to elicit long-term immune responses, such as water-in-oil (O / W) adjuvant or as water-in-water (W / O) adjuvant. This is generally known to be more tolerant than water-in-oil prescriptions. External water phases give high fluidity and induce rapid antigen responses. The inner aqueous phase slows the release of antibodies into the oil-laden layer. Therefore, a long-term immune response can be maintained.

Nanosomes, a form of new immunopotentiator, are now called a new concept, "Immunosol." The role of this adjuvant is not yet clear and the mechanism is known to be different. In general, the principle of operation of the nanosome form of an adjuvant is understood as a reservoir of entrapped antibodies. The micro-diffusion of the particles through the immune system to increase the immune cells (eg B cells, T cells) or to promote the uptake of antibodies to the antigen presenting cells (APC). Other mechanisms include lymphocyte trapping or cytokine induction, and cell membrane disruption for induction and uptake of antibody formation.

The recent analogous documents registered as patents in the form of nanosomes prior to the present invention are US 2004 / 0258701A1, which is a US patent of US 2004 / 0258701A1, which is first emulsified using lecithin, and then added to an ultra-high pressure homogenizer together with an antigen to make nanosomes. Method and domestically this patent is registered as publication number 10-2006-0006909.

In the prior art described above, oil-based emulsion suspension enhancers are sometimes more effective than aluminum salts, but in the case of O / W enhancers, the initial immune response is strong but does not last long. In the case of enhancers, there are problems such as causing local side effects. In addition, the W / O / W adjuvant improves the disadvantages of the two adjuvants, but the immunoadjuvant containing lecithin has been developed in the form of nanosomes, but it is expensive and inferior in stability to general surfactants. have.

Accordingly, the present inventors have made intensive efforts to develop more effective and safe immunosuppressants, and as a result, W / O / W containing a lecithin-like emulsifier that overcomes the limitations of lecithin and has similar characteristics but has greatly improved oxidative stability and formulation stability. After confirming that the nanosomes in the form is useful as an adjuvant, the present invention was completed.

Accordingly, it is an object of the present invention to provide a nanosomal immunopotentiator composition using a lecithin-like emulsifier and a composition for vaccine thereof.

In order to achieve the above object, the present invention includes a nanosome containing a lecithin-like emulsifier glyceryl cocoate / citrate / lactate or glyceryl citrate / lactate / linoleate / oleate, oil and aqueous components It provides an adjuvant composition.

Another object of the present invention to provide a composition for vaccine comprising the adjuvant and antigenic material.

Another object of the present invention is to provide a method of increasing the antibody production rate against the antigen by injecting the vaccine composition into animals other than humans.

Hereinafter, the present invention will be described in more detail.

As used herein, the term “lecithin-like emulsifier” refers to a chemical having a chemical structure similar to that of lecithin, a natural surfactant contained in a large amount in egg yolk and soybean as a nonionic surfactant. In the lecithin-like emulsifier of the present invention, as shown in the following structure, two glycerols are ester-bonded to both sides of the ester bond of the citric acid and the lactic acid, and the coconut acid is ester-bonded to the two glycerin groups OH which are bonded again. R is composed of saturated fatty acids with 12 to 16 carbon atoms. The chemicals of the following structures, unlike lecithins bound to phosphatidylcholine, are linked by ester bonds of citric acid lactate glycerol coconut oil, and specific examples include glyceryl citrate / lactate / linoleate / oleate and glycerol. Reel cocoate / citrate / lactate, and the like. The lecithin-like emulsifier may be chemically synthesized, may be extracted from nature, or may be purchased and used commercially.

<Structure of Lecithin-like Emulsifier>

* R1 and R2 in the above structural formula are C12-C16 alkyl group.

The content of lecithin-like emulsifier in the adjuvant composition of the present invention is 0.01 to 20% by weight and preferably 0.1 to 5% by weight of the total weight of the adjuvant composition.

Polysorbate, sorbitan oleate, sorbitan sesquioleate may be used as a co-emulsifier of the lecithin-like emulsifier in the immunoadjuvant composition of the present invention, and the polysorbate may be, for example, twin-80, twin- 60, open-40 and tween-20, sorbitan oleate is Span 80 and sorbitan sesquioleate is Arlacel 83. The content of the co-emulsifier is 0.1 to 2% by weight, preferably 0.2 to 1% by weight based on the total weight of the adjuvant composition.

As used herein, "oil" refers to an inorganic oil, which is an oil that cannot be metabolized by the body of the animal and animal patient being administered.

As used herein, "animal" and "animal patient" refer to all non-human animals, such as cattle, sheep, horses, goats, and swine.

The above-mentioned "inorganic oil" refers to a mixture of liquid hydrocarbons obtained from petroleum through distillation techniques. The term encompasses "inorganic diesel" oils, ie oils which are similarly obtained by distillation of petroleum but with slightly lower specific gravity than liquid paraffin. Such oils include alkanes, alkenes, alkanes, and their corresponding acids and alcohols, ethers and esters thereof, and mixtures thereof. Preferably, the individual compounds of the oil are light hydrocarbon compounds, ie such components with 6 to 30 carbon atoms. The oil can be prepared synthetically or purified from petroleum products. The oil is a non-metabolized oil, which is a preferred nonpolar oil for use in the adjuvant and vaccine compositions of the present invention, for example mineral oil, squalene, hydrogenated polydecene, hydrogenated castol castrol), paraffin oil and cycloparaffin, and the like.

 The oil component in the adjuvant composition of the present invention is 0.1 to 50% by weight, preferably 1 to 20% by weight, and more preferably 1 to 10% by weight, based on the total weight of the adjuvant composition.

The aqueous component of the present invention may be water, buffered saline or any other suitable aqueous solution and is present in an amount of 1 to 90% by weight relative to the total volume of the adjuvant composition.

As used herein, the term "nanosome" refers to a nanolyzed liposome having the form of a conventional liposome and having an average droplet size of 50 to 500 nm, preferably 80 to 300 nm, and most preferably 100 to 200 nm. . The term "liposome" mentioned above is a colloidal particle aligned and aligned spontaneously with particles composed of amphiphilic molecules with water-soluble heads and insoluble tails, with a hydrophilic space inside and a closed double lipid membrane outside. It is a microvesicle (a type of pocket) which has a double membrane structure similar to a cell membrane. Nanosomes contain water-soluble molecules (including DNA) or drugs in the central hydrophilic space, and fat-soluble drugs can be attached to external lipid bilayers, or they can bind positively and negatively charged substances. The biggest characteristic of nanosomes is that they are easy to change their structure due to their diversity and elasticity, and by providing different structures and components, completely new physical properties, functions, and uses can be provided. Nanosomes have the advantages of excellent biocompatibility, degradability, and stability, and thus can be widely used as a delivery material for countless substances and drugs. In general, liposomes containing nanosomes are useful for drug and gene transfer, for four reasons: 1) Wrapping and protecting drugs or DNA to prevent destruction by enzymes in the blood; 2) the ability to attract substances, such as DNA, that are difficult to enter into the cell in its original state; 3) can act as a type of drug delivery system (DDS) that regulates drug release to be delayed; and 4) directs the therapeutic material to the target cell or prevents it from going to a specific site. As such, the nanosomes of the present invention have the advantage of minimizing side effects while maximizing the effect of the target drug.

Substances that can be included in the immunoadjuvant compositions of the present invention include anticancer agents (eg, adriamycin), antifungal agents (eg, amphotericin B), antibacterial agents, immunomodulators, antigens and antibodies. , Cytokines, imaging agents, hemoglobin, peptides such as growth factors, proteins, fats, and DNA, etc., and suitable and preferred additional ingredients such as preservatives, osmotic agents, biobinding molecules, and immunity. Stimulatory molecules. In addition, since the nanosome of the present invention is similar to a living cell membrane, it can be widely used as a model for biological membrane function research and drug penetration.

The immunopotentiator of the present invention is in the form of W / O / W, the external phase is water so it can penetrate gently into the blood vessel, longer duration than the W / O form, is safe as a biodegradable material, less stress on animals, W / O / W It is smaller than the immune enhancer and the outer membrane is firm to maximize the effect of the drug, such as to ensure the stability of the drug.

The “average droplet size” of the nanosomes of the present invention refers to the volume average diameter (hereinafter VMD) particle size within the volume distribution of the particle size. The VMD is calculated by multiplying and summing each particle diameter by the volume of all particles of that size. This is then divided by the total volume of all particles.

The present invention also provides a composition for vaccine containing the immunoadjuvant composition and antigenic substance.

In the present invention, the term "vaccine" is a biological preparation containing an antigen that immunizes a living body, and an immunogen that immunizes a living body by injection or oral administration to a human or an animal for the prevention of infection. Say In vivo immunization is largely divided into autoimmunity, in which immunity is automatically obtained after infection by pathogens, and passive immunity obtained by an externally injected vaccine. While autoimmunity is characterized by a long period of generation of antibodies related to immunity and continuous immunity, passive immunization with vaccines acts immediately to treat infectious diseases, but has a disadvantage of poor sustainability.

The antigenic substance of the present invention is any one selected from the group consisting of peptides, polypeptides, lactic acid bacteria, proteins expressing the polypeptide, lactic acid bacteria, oligonucleotides, polynucleotides, recombinant bacteria and recombinant viruses expressing the protein. can do. In specific embodiments, the antigenic material may be in the form of inactivated whole or partial cell preparations, or mycoplasma MYO, mycoplasma hypneumoniae in the form of antigen molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis. , Haemophilus somnus, Haemophilus parasuis, Bordetella bronchiceptica, Actinobacillus pleuroneumoniae, Pasteurella multocida, Manhemia haemolytica, Mycoplasma bovis, Myco Plasma Galanasium, Mycobacterium Vorbis, Mycobacterium Para Tuberculosis, Clostrial Spezes, Streptococcus Uberis, Streptococcus suis, Staphylococcus aureus, Erysefelotrix rusopatiia E, Campylobacter spezes, Fusobacterium necrophorum, Escherichia coli, Salonella enterica serovar , Pathogenic bacteria such as Leptospira Spanish sheath; Pathogenic fungi such as Candida; Protozoa such as Cryptosporidium parbium, neosporacanium, toxoplasma gondii, and Emeria spesses; There are antigens made from intestinal parasites such as asteria, couperia, hemoncus and pasiola. Additional antigens include canine parvovirus, bovine herpesvirus-1,3,6, bovine virus, in the form of inactivated whole or partial cell preparations, or antigenic molecules obtained by conventional protein purification, genetic engineering techniques or chemical synthesis. Sexual diarrhea virus (BVDV) types 1 and 2, bovine parainfluenza virus, bovine respiratory syncytial virus, bovine leukemia virus, Linderfest virus, foot and mouth virus, rabies, swine fever virus, African swine fever virus, swine parvovirus, Pathogenic viruses such as swine gastroenteritis virus, PRRS virus, swine circovirus, influenza virus, swine ripple virus, Techen fever virus, and pseudorabies virus.

The content of the antigen is 1 to 50% by weight, preferably 10 to 30% by weight, based on the total weight of the vaccine composition.

Compositions for vaccines according to the invention include stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylenes, bacterial cell walls, derivatives of bacterial cell walls , Bacterial vaccine, animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N--dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monoforce And at least one second adjuvant selected from the group consisting of foryl lipid A, dimethyldioctadecyl-ammonium bromide and mixtures thereof.

The vaccine composition according to the present invention is for the prophylaxis, colon cancer, lung cancer, breast cancer, ovarian cancer, head and neck cancer, vulvar cancer, bladder cancer, brain cancer and glioma, characterized in that for the prevention or treatment of any one or more diseases selected from the group selected can do.

The vaccine composition of the present invention may also comprise a veterinary acceptable carrier. "Vetically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorptive delay agents and the like. Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the vaccine composition of the present invention can be used in the form of oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions, respectively, according to conventional methods. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the lecithin-like emulsifier. (sucrose), lactose (lactose), gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, and the like may be used.In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used. May be included. Formulations for non-oral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.

The present invention also provides a method of increasing the antibody production rate against the antigen by injecting the vaccine composition into animals other than humans.

In the present invention, the "animal except human" may be characterized in that the mammal or bird, the "injection" is subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, It may be characterized in that it is carried out by any one method selected from the group consisting of transdermal administration and oral administration.

In the above, the "injection" or "administration" may vary depending on the age, sex, weight, etc. of the subject to be administered, and the dose of the vaccine also depends on the route of administration, degree of disease, sex, weight, age, and the like. Immunopotentiators can be used together with vaccine administration by parenteral, mucosal (oral and nasal, etc.) and transdermal routes.

The following Experimental Examples and Examples illustrate the content of the present invention, but the content of the present invention is not limited thereto.

Example 1. Nanosome  Preparation of Forms of Enhancer Formulation

To prepare a nanosome form of an adjuvant in the composition of Table 1 below.

Preparation of immunosome enhancer in the form of nanosomes (content: wt%) ingredient Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Experimental Example 6 Experimental Example 7 Experimental Example 8 Experimental Example 9 Experimental Example 10 Purified water to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 to 100 lecithin 0.1 One 4 10 20 - - - - - - - - - - A Glyceryl Citrate / Lactate / Linoleate / Olate - - - - - 0.1 One 4 10 20 - - - - Glyceryl Cocoate / Citrate / Lactate - - - - - - - - - - 0.1 One 4 10 20 B Mineral oil 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Twin 60 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 C Mycoplasma MYO 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20

The A and B phases were heated to 80 ° C., respectively, to completely dissolve. The B phase was mixed into the A phase, and the mixture was emulsified at 3500 rpm using a homomixer (TK-homomixer, Mark-2-F). The emulsified mixture was slowly cooled to 40 ° C., and then mixed with antigens of phase C at 40 ° C., followed by stirring the mixed solution of A, B, and C phases at 3500 rpm using the same homomixer, followed by a fine homogenizer. (Niro. Soavi, Italy) was used three times each at a pressure of 1000 Bar.

Glyceryl citrate / lactate / linoleate / oleate and glyceryl cocoate / citrate / lactate on A, the lecithin-like emulsifier, were purchased from Sasol, Germany, and used as an antigenic component, C phase. Mycoplasma MYO was used by dissolving Mycoplasma Hyopnemoniae OD410 = 0.1 in a buffer solution at 10% concentration.

Example 2. Particle Size Distribution of Nanosome Particles

In order to analyze the particle size distribution of the nanosome particles of the immunoadjuvant composition of the present invention, Comparative Examples 1 to 5 and Experimental Examples 1 to 10 of Example 1 were diluted in an appropriate concentration in 10% -Microplasma Hyopnemoniae OD410 = 0.1 buffer solution. After the particle size of the nanosomes was measured. The particle size of the nanosome particles was analyzed by the light scattering method using a Zetasizer Ns (Zetasizer Ns) equipment and the results are shown in the graphs of FIGS.

As can be seen in the graphs of Figures 1 and 2, the particle size distribution (Fig. 1) of the nanosome particles of the immunopotentiator of Experimental Example 8 of the present invention prepared with a lecithin-like emulsifier is nano of the composition of Comparative Example 3 containing lecithin Compared to the particle size distribution (Fig. 2), the uniformity was much better.

Example 3 Energy Filtration Transmission Electron Microscopy (EF-TEM) of Nanosome Particles

In order to determine whether a multi-layer formation of nanosome particles of the immunoadjuvant composition of the present invention comprising glyceryl cocoate / citrate / lactate, the interface of the nanosomes of the composition of Experimental Example 8 was subjected to an energy filtration electron microscope (EF). -TEM, Carl Zeiss) was taken, and the results are shown in FIG.

As can be seen from the results of Figure 3, it was confirmed that the interface of the nanosome particles of the immunostimulator composition of the present invention made of glyceryl cocoate / citrate / lactate as a lecithin-like emulsifier is composed of a double layer. It is judged that the shape is not circular because the strength of the particles is flexible.

Example 4 Stability Test of Immunostimulator Compositions

Stability tests of the compositions of Comparative Examples 1 to 5 and Experimental Examples 1 to 10 prepared in Example 1 were carried out. The stability evaluation was confirmed for 8 weeks under conditions of temperature cycling of 4 ° C., 25 ° C., 42 ° C., and 24 ° C. in cycles of 4 ° C., 25 ° C., 42 ° C., and a cycle of 24 hours. Shown in

Stability Test of Immunopotentiator Compositions stability Precipitation Deodorant color (Temperature) 25 ℃ 42 ℃ Temperature cycle 4 ℃ 42 ℃ 42 ℃ Comparative Example 1 stability stability Sedimentation Sedimentation Rancid Discoloration (yellow) Comparative Example 2 stability stability Sedimentation Sedimentation Rancid Discoloration (yellow) Comparative Example 3 stability stability Sedimentation Sedimentation Rancid Discoloration (yellow) Comparative Example 4 stability stability Sedimentation Sedimentation Rancid Discoloration (yellow) Experimental Example 1 stability stability stability stability stability Stable (White) Experimental Example 2 stability stability stability stability stability Stable (White) Experimental Example 3 stability stability stability stability stability Stable (White) Experimental Example 4 stability stability stability stability stability Stable (White) Experimental Example 5 stability stability stability stability stability Stable (White) Experimental Example 6 stability stability stability stability stability Stable (White) Experimental Example 7 stability stability stability stability stability Stable (White) Experimental Example 8 stability stability stability stability stability Stable (White) Experimental Example 9 stability stability stability stability stability Stable (White) Experimental Example 10 stability stability stability stability stability Stable (White)

As can be seen from the results in Table 2, in the case of nanosomes (Comparative Examples 1 to 4) made of lecithin, precipitation and coalescence were carried out at temperature cycling conditions circulating at 4 ° C., 42 ° C., and 24 ° C. at a cycle of 24 hours. The immunoadjuvant compositions of the present invention, including glyceryl cocoate / citrate / lactate, which occur but are lecithin-like emulsifiers, are stable without precipitation, discoloration or discoloration.

Example 5 Safety Test of Immunostimulator Compositions

In order to test the safety of the immunoadjuvant composition of the present invention, Experimental Example 8 of the present invention diluted in PBS was prepared for each content, and a safety test for mice, guinea pigs, rabbits, chickens, and piglets was performed as follows. The test results are shown in Table 3 below.

(1) Safety test for mice

Twenty mice weighing 15-20 g were disclosed and inoculated subcutaneously with 0.1 ml in 10 rats and 0.2 ml in the other 10 rats.

(2) safety testing for guinea pigs

15 guinea pigs weighing 300-400 g were published and inoculated with 5 ml of 1 ml in each muscle and subcutaneous observation for 7 days. Five mice were inoculated with 0.1 ml intradermally and observed for 7 days to confirm that they survived abnormally.

(3) safety testing for rabbits

Two rabbits of 2 kg body weight were inoculated with 2 ml muscle and observed for 7 days to confirm that they survived abnormally.

(4) safety testing for chickens

10 ml of 6 to 8 week old chickens were inoculated with 1 ml and observed for 14 days to ensure that they survived without any abnormalities.

(5) safety testing for piglets;

Two healthy pigs 1 to 2 months of age are inoculated twice as much as the vaccine dose into the muscle and there should be no hypersensitivity reaction within 1 to 2 hours after inoculation. Survival without side effects such as purulent and necrosis at the injection site for 10 days It was confirmed.

Safety Results of Adjuvant Compositions Composition content Inoculated Animal Inoculation number Inoculation amount Inoculation site Days observed Clinical observation Test result 10% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable 5 1 ml Subcutaneous 7 Normal 5/5 suitable 5 0.1 ml Intradermal 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 2/2 fit 20% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 2/2 fit 30% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable 5 1 ml Subcutaneous 7 Normal 5/5 suitable 5 0.1 ml Intradermal 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 22 suitable

Example 6 Safety Test and Efficacy Effect of Test Vaccine Containing Immunostimulator Composition

Test vaccines were prepared according to the content of the immunoadjuvant composition of the present invention, and the safety of the test vaccines and the antibody titer assay were performed. At this time, the test vaccine was prepared by the content of Experimental Example 8 as an immunostimulator composition, and prepared using Mycoplasma hyopnemoniae ( OD410 = 0.1) and PBS.

6-1. Safety test of test vaccine

Safety test of the test vaccine was carried out in the same manner as the safety test method for the animals performed in Example 5, the results are shown in Table 4.

Safety test by concentration of test vaccine Composition content Inoculated Animal Inoculation number Inoculation amount Inoculation site Days observed Clinical observation Test result One% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable 5 1 ml Subcutaneous 7 Normal 5/5 suitable 5 0.1 ml Intradermal 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 2/2 fit 10% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable 5 1 ml Subcutaneous 7 Normal 5/5 suitable 5 0.1 ml Intradermal 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 2/2 fit 20% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 2/2 fit 30% mouse 10 0.1 ml Subcutaneous 7 Normal 10/10 fit 10 0.2 ml Subcutaneous 7 Normal 10/10 fit Guinea pig 5 1 ml muscle 7 Normal 5/5 suitable 5 1 ml Subcutaneous 7 Normal 5/5 suitable 5 0.1 ml Intradermal 7 Normal 5/5 suitable rabbit 5 2 ml muscle 7 Normal 5/5 suitable chicken 10 1 ml muscle 14 Normal 10/10 fit pig 2 2 ml muscle 10 Normal 22 suitable

6-2. The antibody of the test vaccine is tested.

In order to determine the efficacy of the test vaccine comprising the immunoadjuvant composition of the present invention, the test was carried out as follows and the antibody titer was assayed.

Efficacy of the test vaccine was 30 mice, inoculated subcutaneously 1/10 (0.1ml) of the vaccine prescribed amount and 14 days after the blood was collected by measuring the mycoplasma hyon pneumoniae antibody titer by ELISA method and each test vaccine The antibody titers were compared. At this time, the antibody titer of the immunoadjuvant composition of the present invention was determined to have an efficacy effect when the control group was 10 times or less and the test group was 512 times or more.

Content of immunopotentiator composition of the present invention (Experimental Example 8) Average antibody (rounded to one decimal place) 0 (negative control: negative serum) 0 One% 757.9 10% 1515.7 20% 1777.5 30% 2746.7

As can be seen from the results of Table 5, the efficacy of the test vaccine was found to be excellent in all of the content of the immunostimulant composition of the present invention 1 to 30%, and the higher the content of the immunostimulant composition was observed to increase the efficacy of the vaccine. Could.

As described in detail above, the immunopotentiator composition of the present invention containing a lecithin-like emulsifier induces a stronger immune response, sustained release time of the drug, and excellent safety when applied to animals. More specifically, the adjuvant composition is easier to manufacture than the conventional adjuvant composition containing lecithin, uniform density, inhibits the precipitation of particles by preventing oxidation and rancidity, greatly increasing the stability of the formulation Therefore, it is very effective as a formulation of a vaccine, such as increasing the shelf life of the composition to increase the unit price and the period of use of the product.

Recently, as the oleochemical technology has evolved, various products are on the market, and while studying whether there is a compound that can replace the expensive lecithin, glyceryl cocoate / citrate / lock as in the present invention The present invention has been completed by discovering that nanosomes containing lecithin-like emulsifiers such as tate or glyceryl citrate / lactate / linoleate / oleate have a lower cost of living and a far superior effect than lecithin. The company developed nanosomes with excipients.

We are currently developing various animal vaccines by incorporating different antigens into nanosomes containing lecithin-like emulsifiers such as glyceryl cocoate / citrate / lactate or glyceryl citrate / lactate / linoleate / oleate. As a cosmetic, or agrochemical depositing agent, the development is underway.

Figure 1 is a graph showing the particle size distribution of the nanosome particles of an adjuvant prepared with lecithin.

Figure 2 is a graph showing the particle size distribution of nanosome particles of an adjuvant prepared with glyceryl cocoate / citrate / lactate.

3 is a transition electron microscope (TEM) photograph of nanosome particles of an adjuvant prepared with glyceryl cocoate / citrate / lactate.

Claims (9)

An immunopotentiator composition in the form of a nanosome containing a lecithin-like emulsifier, a non-metabolized nonpolar inorganic oil, and an aqueous component as defined by Structural Formula 1 below. <Structure 1>

In Formula 1, R1 and R2 are alkyl groups of C12-16. 2. The immunopotentiator composition of claim 1, wherein the lecithin-like emulsifier is glyceryl cocoate / citrate / lactate or glyceryl citrate / lactate / linoleate / oleate. 3. The immunoadjuvant composition according to claim 2, wherein the lecithin-like emulsifier content is 0.01 to 20% by weight based on the total weight of the composition. The method of claim 1, wherein the non-metabolized nonpolar inorganic oil is any one selected from the group consisting of mineral oil, squalene, hydrogenated polydecene, and hydrogenated castrol. Enhancer compositions. According to claim 1, wherein the adjuvant composition is in the form of a W / O / W multi-phase emulsion, wherein the average droplet size of the nanosomes is 50 to 500 nm. A vaccine composition comprising the immunoadjuvant composition of claim 1 and an antigenic substance. The method of claim 6, wherein the antigenic substance is any one selected from the group consisting of peptides, polypeptides, lactic acid bacteria expressing the polypeptide, antigen protein, lactic acid bacteria expressing the antigenic protein, oligonucleotides, polynucleotides, recombinant bacteria and recombinant viruses Vaccine composition, characterized in that one. According to claim 6, Prostate cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, head and neck cancer, vulvar cancer, bladder cancer, brain cancer and glioma, characterized in that the prevention or treatment of any one or more diseases selected from the group consisting of glioma Credit composition. A method of increasing the production rate of an antibody against an antigen by injecting the composition of claim 6 into an animal other than a human.

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