KR101671376B1 - Sustained-Release Preparations for preventing or treating porcine Salmonella typhimurium infection and PRRS Virus and Method of Preparing the Same - Google Patents

Abstract

The present invention relates to a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus, and a method for producing the same, wherein the sustained-release preparation for preventing or treating a porcine salmonella infection and PRRS virus encapsulating bee venom according to the present invention in a biodegradable polymer After bee venom administration, the immune strengthening effect on Salmonella infection and PRRS virus is sustained for a long time.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sustained-release preparation for prevention and treatment of porcine salmonella infection and PRRS virus,

The present invention relates to a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus, and more particularly to a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus encapsulated in a biodegradable polymer, And a manufacturing method thereof.

Salmonella enterica serovar typhimurium ( S. typhimurium ) is the most common Salmonella serotype isolated from pigs. Zoonotic series contaminated pork of S. enterica (Brumme, S. et al , 2007. Impact of Salmonella typhimurium DT104 virulence factors invC and sseD on the onset, clinical course, colonization patterns and immune response of porcine salmonellosis, Vet Microbiol 124, 274-285.

Typical clinical symptoms of S. typhimurium- infected pigs include increased body temperature, enterocolitis (Volf, J. et al ., Salmonella enterica serovar typhimurium and enteritidis infection of pigs and cytokin signaling in palatinetonsils. Vet. Microbiol . 156 , 127-135 (2012)) and diarrhea. Although pigs of all ages are vulnerable to S. typhimurium , young pigs in the weaning and growing stages are most commonly affected and severe clinical symptoms (Boyen, F. et al ., Non-typhoidal Salmonella infections in pigs: a closer look at epidemiology, pathogenesis and control. Vet. Microbiol. 130, 1-19 (2008). After recovering from the disease, the pigs often stop or slow their growth and develop into pig farmers (Collado-Romero et al ., Quantitative analysis of the immune response against Salmonella typhimurium infection along the porcine intestinal gut. ), Which is a significant economic loss. In addition, infected, but the bacteria may remain in healthy pigs without any clinical symptoms. These animals are not easily detectable and are the source of contamination. Thus, pig salmonella infection has been associated with significant damage to the swine industry and has had a negative impact on its efficiency, leading to the production of pig production systems (Fosse, J. et al ., Prevalence and risk factors for bacterial food-borne zoonotic hazards in slaughter pigs: Public Health, 56, 429-454 (2009)).

On the other hand, the honeybee venom (HBV) of the bee ( Apis melifera ) has long been associated with pain and inflammation, rheumatoid arthritis, multiple sclerosis (Orslic, N., Bee venom in cancer therapy. (2012)), have been widely used as traditional alternative therapies to alleviate some immune-related diseases. Numerous studies have also shown that the components of total bee venom (HBV) can be used to enhance immunity (Son et al ., Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Pharmacol. Ther . , 246-270 (2007)), anticancer (Orslic, N., Bee venom in cancer therapy. Cancer. Metastasis. Rev. 31, 173-194 (2012)), radiation protection (Gajski et al ., Radioprotective effects of honeybee to have properties as a number of beneficial agents, such as in vitro study Int J. Toxicol 28, 88-98 (2009)) activities: venom (Apis melifera) against 915 -MHz microwave radiation-induced DNA damage in wistar rat lymphocytes... .

Bee venom (HBV) contains various biological activities such as enzymes, biogenic amines, and melittin (melittin, Orslic, N., Bee venom in cancer therapy. Cancer. Metastasis. Rev. 31, 173-194 Peptides, and melitin is known as an antimicrobial peptide that acts rapidly as a main component extracted from the water-soluble fraction of bee venom and has a wide activity range for bacteria, fungi, viruses and parasites (see, for example, ... Mataraci et al, In vitro activities of antibiotics and antimicrobial cationic peptides alone and in combination against methicillin-resistant Staphylococcus aureus biofilms Antimicrob Agents Chemother 56, 6366-6371 (2012);... Liu et al, Construction and expression of 487 sTRAIL-melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli. Appl. Microbiol. Biotechnol . 97, 2877-2884 (2013)). Melitin has also been shown to be useful in the treatment and prevention of immune and anticancer diseases (Son et al ., Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Pharmacol. Ther . 115, 246-270 )) And many other pharmacological effects.

Therefore, many commercial products have been produced with an emphasis on the melittin of the water soluble fraction as the only index compound of bee venom. However, the fat soluble fraction of bee venom has received little attention from bee venom manufacturers. Interestingly, flavonoids such as chrysin and pinocembrin, which are liposoluble compounds of bee venom, have anticancer, antioxidant and antimicrobial effects (Schnitzler, P. et al ., Antibiral activity and mode of action of propolis extracts and Rasul et al ., Pinocembrin: a novel natural compound with versatile pharmacological and biological activities, Biomed. Res. Int. 2013: 379850. Doi: 10.1155 / 2013/379850 (2013)). 11-eicosen-1-ol has also been reported to be abundant [Pickett, P. et al ., (X) -11-eicosen-1-ol and an infertant new pheromonal component from the sting of the honey bee, Apis melifera L. (Hymenoptera, Apodae.), J Chem Ecol 1982 Jan; 8 (1): 163-75].

The present inventors have found that a new bee venom-derived product containing melittin, chrysin, pinocembrin and 11-eicosen-1-ol has a T lymphocyte CD4 + / CD8 + ratio And the relative levels of interferon gamma (IFN-γ) and interleukin IL-12 mRNAs, the cytokines of Th1, and the virus removal from porcine reproductive and respiratory syndrome (PRRS) viral infected pigs However, this immunosuppressive effect of bee venom has a problem that it lasts for 7 days after bee venom administration.

Thus, the present inventors have made intensive efforts to produce a preparation having an immunosuppressive effect. As a result, it has been found that preparation of bee venom (HBV) nanoparticles (P-HBV) encapsulated with PLGA (poly-D, L-lactic acid-co-glycolic acid) , And delivering it to pigs via the work route, the protective effect against bacterial diseases, including pigs attacked with S. typhimurium, and the bacterial elimination effect in the pigs' stomach as an early stage of host cell immune response The present invention has been completed.

It is an object of the present invention to provide a sustained-release preparation for prevention and treatment of porcine salmonella infection and PRRS virus in which the immune strengthening effect against Salmonella infection and PRRS virus is sustained for a long time after bee venom administration, and a method for producing the same.

In order to achieve the above object, the present invention provides a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus, wherein the bee venom is encapsulated in a biodegradable polymer.

The present invention also provides a method for producing a biodegradable polymer, comprising the steps of: preparing a biodegradable polymer solution by adding an organic solvent to a biodegradable polymer; Preparing a W / O emulsion by adding a bee venom aqueous solution to the biodegradable polymer solution; And a step of dispersing the W / O emulsion in an emulsifier to prepare a W / O / W emulsion and drying the emulsion, and a method for producing a sustained release preparation for preventing or treating PRRS virus.

The present invention also provides a method for preventing or treating porcine salmonella infection and PRRS virus, which comprises administering an effective amount of a sustained-release preparation encapsulated in a biodegradable polymer through a porcine mucosal route.

When the bee venom according to the present invention is encapsulated in a biodegradable polymer, a porcine salmonella infection and a sustained-release preparation for the prevention or treatment of PRRS virus, when delivered through the rectal route, have a bacterial disease And the effect of removing bacteria is remarkably long lasting.

1 is a graph showing the results of high performance liquid chromatography (HPLC) and gas chromatography (GC) of a P-HBV sustained-release preparation obtained according to an embodiment of the present invention, and scanning electron microscopy , SEM) [(b) 5,000x magnification; (c) 10,000x magnification; (d) 30,000x magnification].
FIG. 2 is a graph showing FACS analysis of T lymphocyte composition ratio (a) and lymphocyte proliferation activity (b) in the peripheral blood of the P-HBV sustained-release preparation obtained according to the example of the present invention.
Figure 3 is a graph showing the effect of Th1 cytokine [(a) IFN-y (b) IL-12) and inflammatory response (c) TNF- alpha (d) IL-1 ?.
FIG. 4 is a graph showing the fecal state (a) and the S. typhimurium reduction rate (b) of the pig treated with the P-HBV sustained-release preparation obtained according to an embodiment of the present invention with time, , And a decrease rate (c) of Salmonella (S. typhimurium) in MLN tissue.
FIG. 5 is a graph showing the effect of a cytokine [(a) on a PBMC (Peripheral Blood Mononuclear Cell) infected with Salmonella (S. typhimurium) administered with the P-HBV sustained-release preparation obtained according to an embodiment of the present invention (B) IL-12, and (c) TNF-? (D) IL-? 1 mRNA.
6 is a graph showing the analysis of Th1, Th2 and inflammatory cytokines in tissues of pigs infected with S. typhimurium administered with the P-HBV sustained-release preparation obtained according to an embodiment of the present invention [(a ) Ileum, (b) cecum, (c) colon, and (d) mesenteric lymph nodes (MLN).

The present inventors have found that a new bee venom-derived product comprising imatinib (melittin), chrysin, pinocembrin and 11-eicosen-1-ol has a T lymphocyte CD4 + / CD8 + ratio And increased the relative levels of interferon gamma, interferon gamma (IFN-y and interleukin IL-12 mRNA, and cytotoxic Th1 cytokines, and virus removal from porcine reproductive and respiratory syndrome (PRRS) virus infected pigs .

However, the immune strengthening effect of bee venom persisted for 7 days after bee venom administration. Therefore, we have developed bee venom nanoparticles (P-HBV) encapsulated with PLGA (poly-D, L-lactic acid-co-glycolic acid) Because PLGA is a biocompatible, biodegradable, FDA-approved drug for sustained release of drugs due to its drug release characteristics, it protects proteins from protease-mediated degradation [Dwivedi V. et al ., PLGA nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus viral clearance in pigs. Vet. Microbiol. 166, 47-58 (2013)] and has been used in drug, protein and gene transfer applications due to its sustained release properties. PLGA containing hepatitis B, influenza, or PRRS virus, etc., has been effectively used to treat various viral diseases [Thomas C. et al ., Thomas, C., Rawat, A., Hope-Weeks, L., Ahsan, , 2011. Aerosolized PLA and PLGA nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis B vaccine. Mol. Pharm. 8, 405-415 (2011)] [Dwivedi V. et al ., PLGA nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccines in viral clearance in pigs. Vet. Microbiol. 166, 47-58 (2013).

Dube et al. [ Multimodal nanoparticles that provide immunomodulation and intracellular drug delivery for infectious diseases. Nanomedicine. pii: S1549-9634 (13) 00677-1. doi: 10.1016 / j.nano (2013) ] demonstrated that PLGA-coated rifampicin can carry four times more amount of macrophages than rifampicin solution. Thus, innovative nanotechnology using PLGA delivery systems based on the delivery system has been shown to be effective for anti-CD8 conjugated nanoparticles to target (Bicho A. et al ., Bicho, A., Peca, IN, Roque, ACA, Cardoso, mammalian cells expressing CD8. Int. J. Pharm. 399, 80-86 (2010)], Farokhzad OC et al ., Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc. Natl. Acad. Sci. 103, 6315-6320 (2006)], peptides [Geldenhuys, W. et al ., Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers. J. Drug. Target. 19, 837-845 (2011)] and prophylactic agents [Yang, S. et al . Novel intravaginal nanomedicine for the targeted delivery of saquinavir to CD4 + immune cells. Int. J. Nanomedicine. 8, 2847-2858 (2013)].

Nanomolecular-based delivery to mucosal sites can be achieved using specialized antigen presenting cells (APCs) [Inaba K. et al ., Dendritic cell progenitors, phagocytose particulates, including bacillus Calmette-Guerin organisms, and sensitizing mice to mycobacterial antigens in vivo. J. Exp. Med. 178, 479-488 (1993)].

The mucosal area (eye, nasal cavity, oral cavity, lung, vagina, and rectum) accounts for 80% of the body's immune cells as a whole [Holmgren and Czerkinsky, Mucosal immunity and vaccines. Nat. Med. 11, 45-53. Inaba, K., Inaba, M., Naito, M., Steinman, RM, 1993. Dendritic cell progenitors, phagocytose particulates, including bacillus Calmette-Guerin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178, 479-488 (2005)). Particulate antigens transmitted through the mucosal pathway are recognized by microfold cells (M cells) and strongly stimulate differentiation of T cells, and a specific adaptive immune response [ Renukaradhya, GJ et al ., Mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective. Anim. Health. Res. Rev. 13, 21-37 (2012)].

The present invention is based on the finding that a bee venom sealed with PLGA through nasal and rectal routes is transferred to pigs and compared with the immune enhancement efficacy of non-encapsulated bee venom within a given period, To evaluate the protective effect of PLGA bean curdled bee venom on bacterial diseases and the bacterial elimination effect of PLGA bean curd bean paste delivered to pigs as an early stage of host cell immune response using PLGA encapsulated bee venom.

Accordingly, in one aspect, the present invention relates to a porcine salmonella infection and a sustained release preparation for the prevention or treatment of PRRS virus, wherein bee venom is encapsulated in a biodegradable polymer.

The bee venom contains a water soluble component such as melittin and lipid soluble components such as chrysin, pinocembrin and 11-eicosen-1-ol. Apis melifera Bee venom, which contains water soluble components such as melitin and chrysin, pinosembrine and 11-eicosen-1-ol in the bees' bee venom, T1) cytokines (InF-γγ, iL-12, and iL-12, etc.) while enhancing the viral clearance and inducing an increase in CD4 + / CD8 + T lymphocyte ratio.

The biodegradable polymer contains bee venom while retaining the form of the preparation, and gradually dissolves the polymer, releasing the internal bee venom. Conventional polymers that can be used in the preparation can be used. Specific examples include poly-L-lactic acid, poly-D-lactic acid-co-glycolic acid, poly-L-lactic acid-co- glycolic acid, poly- -Valerolactone, poly-hydroxybutyrate, and poly-hydroxyvalerate, and preferably poly-D, L-lactic acid-co-glycolic acid can be used , But is not limited thereto.

The biodegradable polymer may have a weight average molecular weight of 60,000 or less.

L-lactic acid-co-glycolic acid (50:50) having a molecular weight of about 13,000, poly-D, L-lactic acid-co-glycolic acid (50:50) having a molecular weight of about 33,000, L-lactic acid-co-glycolic acid (75:25) having a molecular weight of about 20,000, poly-L-lactic acid-co- glycolic acid Lactic acid (100: 0) or the like can be used.

Such biodegradable polymers include, for example, Resomer L 206S, L 207S, L 209S, L 210, L 210S, LR 704S, LR 706S, LR 708, LR 927S, LG 824S, LG 855S, LG 857S, LC 703S, R 207S, RG 509S, X 206S, R 202S, R 202H, R 203H, R 205S, RG 502, RG 502H, RG 503, RG 503H, RG 504, RG 504H, RG 505 , RG 653H, RG 752H, RG 752S, RG 753S, RG 755S, RG 756S, RG 757S, RG 750S, RG 858S.

In the present invention, the bee venom in the sustained-release preparation containing bee venom can be appropriately selected according to the usage, the dose, and the characteristics of the bee venom protein. The bee venom content is 0.1 to 20% by weight, preferably 1 to 15 %, More preferably 2 to 12 wt%.

The diameter of the sustained-release preparation may be 1000 to 2000 nm, preferably 1500 to 1800 nm.

In the present invention, the sustained-release preparation may be selected from the group consisting of polyethylene glycol, chitooligosaccharide, chitosan, glycine, arginine, lysine, cyclodextrin, , Polyvinyl alcohol, hydropropyl methylcellulose and surfactants such as Tween®, Span®, Cremophor® or Poloxamer®, and sucrose. Or more species.

In the present invention, the sustained-release preparation may further comprise at least one selected from the group consisting of chitooligosaccharide, chitosan, glycine, arginine, lysine, cyclodextrin, sucrose, poly Polyvinyl pyrrolidone, polyvinyl alcohol, and the like. [0033] The term " a "

The sustained-release preparation may last for more than 7 days. Preferably more than 7 days, up to 21 days, more preferably more than 21 days.

Another aspect of the present invention is a method for producing a biodegradable polymer solution, comprising: preparing a biodegradable polymer solution by adding an organic solvent to the biodegradable polymer; Preparing a W / O emulsion by adding a bee venom aqueous solution to the biodegradable polymer solution; And dispersing the W / O emulsion in an emulsifying agent to prepare and drying the W / O / W emulsion; And a method for producing a sustained-release preparation for preventing or treating PRRS virus.

In the present invention, the organic solvent is not particularly limited as long as it is a volatile organic solvent which is excellent in solubility in a biodegradable polymer and easily removable by evaporation, but is preferably dichloromethane, ethyl acetate, chloroform, acetone, dimethylsulfoxide, One kind of solvent selected from dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethyl acetate, methyl ethyl ketone and acetonitrile or a mixed solvent of two or more kinds may be used, One kind of solvent selected from methane, ethyl acetate, chloroform and tetrahydrofuran, or a mixed solvent of two or more kinds may be used, and most preferably, one or two mixed solvents selected from dichloromethane and ethyl acetate are used . At this time, the organic solvent used in the present invention acts not only as a dissolving agent for dissolving the polymer but also as a dispersing agent for evenly dispersing the bee venom into the polymer solution.

In the present invention, the types of biodegradable polymers are as mentioned above.

In the present invention, the aqueous solution of bee venom is suitably adjusted depending on conditions, but is preferably 0.1 to 20% (w / v), more preferably 0.1 to 15% (w / v), most preferably 0.5 To 7% (w / v). The bee venom aqueous solution may contain an additive, and the kind of the additive is as mentioned above. The additive may be added in any concentration, but it is preferably 0.1 to 20% (w / v), more preferably 0.1 to 15% (w / v) and most preferably 1 to 7% (w / v). < / RTI > When the additive is added, bee venom can be dissolved in the aqueous solution of the additive.

The biodegradable polymer solution and bee venom aqueous solution can be appropriately adjusted according to the conditions, but are preferably mixed at a volume ratio of 1: 0.01 to 0.25, more preferably 1: 0.05 to 0.15, and most preferably 1: 0.05 to 0.13 .

The W / O emulsion and the emulsifier can be appropriately adjusted depending on the conditions, but are preferably mixed in a volume ratio of 1: 1 to 20, more preferably 1: 2 to 15, and most preferably 1: 3 to 12, .

The emulsifier used herein may be a lipophilic emulsifier dispersed in an organic solvent or a hydrophilic emulsifier dispersed in an aqueous solution. Examples of the hydrophilic emulsifier include chitooligosaccharide, chitosan, glycine, arginine, lysine, cyclodextrin, sucrose, polyvinyl pyrrolidone, And polyvinyl alcohol may be used as the emulsifying agent. At this time, it is most preferable to use the polyvinyl alcohol as the emulsifier. The polyvinyl alcohol preferably has a molecular weight of 10,000 to 200,000, more preferably 50,000 to 200,000, and most preferably 100,000 to 200,000.

W / O / W type double emulsion type emulsion can be formed by dispersing the water-in-oil type W / O emulsion which is the beesinized dispersion solution in an emulsifier aqueous solution.

The sustained-release preparation according to the present invention can be recovered through centrifugal separation and dried, and can be dried by any one of lyophilization, vacuum drying, spray drying and other conventional drying methods.

According to the present invention, bee venom is encapsulated in PLGA (P-HBV) so that bee venom is released slowly. The mucosal spray of P-HBV (beverage encapsulated with PLGA) increased the immunity of the pigs and proved to be effective in reducing Salmonella enterica serovar Typhimurium (pig diarrhea-causing hospital strain). After 2 weeks of P-HBV administration, lymphocyte proliferation in peripheral blood and increased expression of IFN-γ and IL-12 mRNA produced by Th1 lymphocytes were observed.

Therefore, the present invention relates to a method for preventing or treating porcine salmonella infection and PRRS virus, which comprises administering an effective amount of a sustained-release preparation encapsulating bee venom to a biodegradable polymer through a porcine mucosal route from another viewpoint.

The mucosal route may be eye, nasal, oral, lung, vaginal or rectal, and the like.

Salmonella enterica serovar After Typhimurium infection, the number of microorganisms in all tissue samples and excretion decreased in the P-HBV group, and the levels of Th1 cytokine and Th2 cytokine were significantly increased in tissue samples and peripheral blood. Therefore, P-HBV is expected to be a useful substance to prevent infection by improving immunity against Salmonella.

The present inventors focused on the response of T helper lymphocytes. (CD4 + / CD8 + lymphocyte ratio, proliferation of lymphocytes, cytokines associated with Th). In particular, the ratio of the two major T lymphocytes (CD4 + and CD8 +) is considered to be the most important variable for evaluation of the immune response, and the proportion of high CD + / CD8 + lymphocytes is generally observed in increased immune abilities or individuals. In addition, lymphocyte proliferation enhances immune activity and mitogenicity of T cells. The levels of CD4 + / CD8 + lymphocytes and IFN-γ and IL-12 (produced by Th1 lymphocytes) mRNA expression levels were significantly increased in the P-HBV group at day 7 and day 14. ConA-stimulated PBMCs also increased significantly in the P-HBV group. In addition, the P-HBV group showed a significant increase in CD4 + / CD8 + lymphocyte ratio and peripheral blood mononuclear cell (PBMC) proliferation on day 14 compared to the HBV group. These results are similar to the earlier results that HBV treatment increased the CD4 + / CD8 + T lymphocyte ratio and upregulated Th1 cytokines. However, the immune strengthening effect was maintained for only 7 days. In contrast, in the present invention, it was confirmed that the lymphocyte increase and cytokine production in the P-HBV group were increased until 14 days later. These results suggest that HBV wrapped in PLGA effectively enhances the Th1 lymphocyte-specific immune response and that this immune-strengthening effect lasts up to 14 days after administration of P-HBV. This is due to the fact that PLGA nanoparticles have been found to detect PRRS virus-infected pig vaccines. Dwivedi et al . [PLGA nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine. Vet. Microbiol. 166, 47-58 (2013).

The innate immune system consists of many mechanisms and factors, which rapidly remove pathogens into various groups of immune cells. Salmonella ( S. typhimurium ) responds to most of the host's immune defense period at the infectious stage, and there are a variety of strategies for destroying this immune response. Infection with S. typhimurium occurs as follows. (1) It is located in a person's intestines. (2) permeates epithelial cells. (3) spread through the lymphatic organs. Especially in the Mesenteric Lymph node (MLN) for the persistence of S. typhimurium infection. In this study, Salmonella ( S. typhimurium ) was identified in all organs and feces after infection and is reported in other studies. The administration of P-HBV showed a significant difference in the comparison of Salmonella ( S. typhimurium ) found in the ileum, intestines, colon, and mesentery lymph nodes. These results have been reported by Calveyra et al. [Effect of organic acids and mannanoligosaccharide on excretion of Salmonella typhimurium in experimentally infected growing pigs. Res. Vet. Sci. 93, 46-47 (2012)].

By forming colonies in the small intestine and mesenteric lymph nodes, S. typhimurium rapidly invades immune cells such as macrophages, dendritic cells, and white blood cells. Salmonella can survive in a variety of immune cells by inhibiting the antimicrobial response through several mechanisms (inhibiting the secretion of Th1-related cytokines such as fungicidal active oxygen, nitrogen species, TNF-α, and IFN-γ in the cell) . These cytokines are substances that are important for removing Salmonella in cells through priming of T lymphocytes. In the examples of the present invention, it was revealed through gene expression that P-HBV nanoformation is effective for producing TNF-a and Th1 cytokines. IFN-y, IL-2 and IL-12 also significantly increased in all parts of the P-HBV group. However, it was increased only in TNF-α, IL-1β, and P-HBV groups. Interestingly, IL-4 is significantly reduced in peripheral blood mononuclear cells (PBMCs) and in all tissues. IL-4 is considered to be the most important in the Th2 pathway (inhibiting Th1-related immune responses). Therefore, a decrease in IL-4 in the P-HBV group appears to be associated with upregulation of Th1 cytokines. These results are similar to the results of the 2013 study, in which IFN-γ and IL-18 were significantly increased in broiler chickens infected with S. Gallinarum.

In the present invention, the injection of P-HBV into the rectum was due to the absence of toxic symptoms, a longer period of immune response, and a greater immune enhancement effect than HBV. Mucosal immunity has several other advantages over conventional routes of administration. Optimal stimulation of mucosal immunity is highly effective in protecting against many infectious diseases because it can be easily done by untrained individuals and can promote both mucosal and systemic immunity. The intestinal tract includes a variety of lymphoid tissues that uptake pathogens entering the intestines and then induce mucosal immune responses such as lymphatic epithelial cell structure. Langranderie and colleagues have shown that enhancing rectal immunity in young mice is more effective in eliminating malignant Mycobacterium tuberculosis than subcutaneous vaccination. [Development of mixed Th1 / Th2 type immune response and protection against Mycobacterium tuberculosis after rectal or subcutaneous immunization of newborn and adult mice with Mycobacterium bovis BCG. Scand. J. Immunol. 55, 293-303 (2002)). In addition, Mayr et al. Fully protected rats at 50% lethal dose in rats vaccinated rectally with the bacteria generated from hemolytic Escherichia coli (EHEC) O157: H7. Immunized rats have an effect on humoral and cellular immune responses [Mayr, UB et al ., Rectal single dose immunization of mice with Escherichia coli O157: H7 induces efficient bacterial ghosts inducing humoral and cellular immune responses and protects against the lethal heterologous challenge . Microb. Biotechnol. 5, < / RTI > 502 283-294 (2012)). Therefore, the immune strengthening and sterilization effects of P-HBV are associated with rectal mucosal immunity boosting. In conclusion, the present invention demonstrates that P-HBV enhances the Th1-specific immune response rather than HVB in common pigs. During the infection with S. typhimurium, P-HBV entered the rectum and decreased bacterial counts, stimulated Th1 and inflamed the germs. However, the mechanism of immunity enhancement of HBV is unclear and further investigation is needed. The data presented in this study may lead to a new strategy for immunization enhancement and prevention of Salmonella infection in the pig industry, with additional confirmation of the protective effect of P-HBV on Salmonella infection.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example

Preparation Example 1: Preparation of HBV

2.5 g of CBV obtained by using a bee venom collector was extracted with 250 ml of distilled water and 250 ml of ethyl acetate at room temperature and filtered by using a 0.45 μm nylon filter. The two filters were mixed well and concentrated under reduced pressure at 40 < 0 > C. Melittin, Chrysin and Pinocembrin were analyzed by HPLC and HB-11-eicosen-1-ol was analyzed by GC. HPLC analysis was performed by passing 10 mg of HBV in 1 ml of distilled water through a 0.45 탆 filter (Millipore), and injecting with UPLC BEH C18 column (1.7 탆, 2.1 X 100 mm; Waters Corporation, Milford, MA, USA). Mobile phase A is 0.1% trifluoroacetic acid in distilled water and B is 0.1% trifluoroacetic acid in Acetonitrile. The moving phase conditions were 0 to 5 minutes B 0% (flow rate 0.2 mL / min), 10 to 20 minutes B 40% (flow rate 0.2 mL / min) and 20 to 25 minutes B 100% (flow rate 0.3 mL / min) . The detection wavelength was set to 220 nm. For GC analysis, 1 mg of HBV sample was dissolved in 1 mL of chloroform, passed through a 0.45 μm filter (Millipore), reacted with a derivatizing reagent, and subjected to GC analysis. The sample to be analyzed was a Gas Chromatograph Varian CP-2800, and the column was placed in DB-5 30 m x 0.25 mm (ID), 7 μm. The temperature of the injection part and the temperature of the detector were set at 280 ° C. and 300 ° C., respectively. The temperature of the oven was elevated by 10 ° C. per minute starting from 100 ° C., and then fixed at 300 ° C. for 5 minutes. The final HBV product consisted of 64% melittin, 3 ppm pinochemin and 20 ppm chromin. For use in the experiment, the fine HBV powder was dissolved in a solvent composed of 95.7 vol% of distilled water, 3.5 vol% ethanol and 0.8 vol% propylene glycol to adjust the final concentration to 2.1 mg / ml. The concentration was determined as the optimum concentration in the preliminary experiment. 11-eicosanol was dissolved in chloroform, and STD of each concentration and 200ul of 1mg / ml solution of analytical sample and 100ul of BSTFA were mixed in a reaction-specific vial and the derivatization reaction was carried out at 80 ° C for 1.5 hours. (Varian CP-2800, Column: DB-5, 30 m x 0.25 mm ID, 0.25 m, Injector Temp .: 280 ° C Detector Temp .: 300 ° C, Oven Temp .: 100 ° C ~ Carrier gas: N ₂ , 1 ml / min, Injection Vol .: 1 μl, Split ratio: 10: 1)]

Production Example 2: Synthesis of HBV-injected PLGA nanoparticles

The nanoparticles were prepared by slightly modified standard double emulsion solvent evaporation techniques. Briefly, 200 mg of PLGA is dissolved in 5 ml of DCM (dichloromethane) at room temperature. 15 mg of HBV and sucrose (5% w / v) were dissolved in 0.5 ml of distilled water at room temperature. Both solutions were emulsified (W / O) in a cold water bath at a speed of 20,000 rpm for 3 minutes with a homogenizer. The W / O emulsion was added to 30 ml of polyvinyl alcohol and the emulsion of W / O / W was made at a speed of 20,000 rpm for 5 minutes using a homogenizer. The DCM was allowed to evaporate with stirring overnight at room temperature. The PLGA nanoparticles were centrifuged at 4 ° C, 10,000 rpm for 30 minutes, rinsed once with 0.9% sodium chloride solution, and washed twice with distilled water. Encapsulated HBV by PLGA was lyophilized and stored at 4 ° C until use. PLGA without HBV as control was made in the same way.

Size distribution and zeta potential analysis of PLGA-encapsulated HBV were performed using a laser scattering analyzer (ELS-8000, Otasuka Electronics, Osaka, Japan). Samples were prepared by preparing lyophilized PLGA particles under reduced pressure using distilled water, adding a sample dispersion unit, and sonicating to minimize particle interactions. The obscuration range was maintained at 20% to 50%. The instrument was set up to measure 50 samples and the average volume mean diameter was obtained. The characteristics of PLGA encapsulated with HBV were observed by scanning electron microscope (SEM). The PLGA encapsulated with HBV was dropped onto the double-faced carbonates, vacuum coated with osmium for 50 seconds, and subjected to field-emission SEM (JEOL JSM7500; Thermo Scientific, Rockford, IL, USA) Respectively.

Pinocembrin, chrysin and melittin, which are components of HBV, were assayed by HPLC. As a result, pinocembrin, chrysin, The retention times of melittin were 37, 38 and 42.5 minutes, respectively. Eleven eicosenol (11-Eicosen-1-ol) was also assayed separately by GC.

Table 1 shows the particle size, zeta potential, EE%, and drug loading rate of PLGA and PLGA-HBV.

PLGA PLGA-HBV Size (nm) 1597 + 151.3 1780 ± 83.4 Zeta potential (mV) -23.2 ± 8.6 -20.8 ± 3.5 EE (%) * 96.48 ± 0.6 Drug loading rate (%) 4.86 ± 0.1

Data mean mean ± standard deviation.

* EE (%): encapsulation efficiency

The encapsulation efficiency of the PLGA-HBV particles was 96.48 ± 0.6%, the drug loading efficiency was 4.86 ± 0.1%, the particle size was approximately 1,780 nm, and the zeta-potential was -20.8 ± 3.5 mV. PLGA composed of glycolic acid and lactic acid showed negative reaction to zeta-potential. The appearance of the PLGA-HBV was observed by SEM. As a result, as shown in FIG. 1B, the PLGA-HBV was spherical and had a smooth surface. The particles are smooth and rounded, so they do not stimulate the tissues.

Example 1: Measuring the immunity-enhancing effect of PLGA in pigs

The experimental animals used healthy 4-week-old pigs that had never been infected with Salmonella ( S. t yphimurium). Pigs were randomly divided into four groups (5 per group) and HBV was injected through the rectum. Group 1: no treatment, Group 2: PLGA alone treatment, Group 3: HBV alone treatment, Group 4: PLGA coated HBV treatment. Blood samples were collected from all pigs at 7, 14 and 21 days after the start of the experiment. Body weight was continuously measured during the experiment. All animals were used in the experiment according to the prescribed guidelines.

Example 1-1: PBMC Isolation

Blood samples were collected in tubes coated with EDTA and diluted with the same volume of PBS. The diluted blood was placed in Lymphoprep and centrifuged at 800 xg for 30 minutes. RBCs were rinsed with RBC lysis buffer, washed twice with PBS, and then resuspended in RPMI-1640 medium (RPMI, 10% FBS, 2% Ab). Separated cells were used for lymphocyte proliferation test and T lymphocyte assay.

Example 1-2: Lymphocyte proliferation test

Lymphocyte proliferation assays were performed according to the method of Jung et al ., Immunoprophylactic effects of administering honeybee (Apis melifera) venom spray against Salmonella Gallinarum in broiler chicks. J. Vet. Med. Sci. 75, 1287-1295 Respectively. Live lymphocytes were stained with trypan blue and counted with a microscope. Cells were mixed at 3 × 10 ⁶ cells / ml in complete medium. The suspended cells were cultured in a 96-well plate with or without 5 ug / ml of ConA in the complete medium. After 48 hours of incubation, MTT solution was added and incubated again for 4 hours. The plate was centrifuged at 2000 xg for 10 min and the supernatant discarded. 200 ul of DMSO was added to each well and absorbance was measured at 540 nm.

Example 1-3: Measurement of T lymphocytes using FACS

The proportion of CD4 + CD8- T lymphocytes and CD4-CD8 + T lymphocytes in PBMC from blood was examined by FACS. Cells were washed with cold PBS and stained with FITC-conjugated mouse anti-pig CD4 and PE-conjugated mouse anti-pig CD8 antibody. The isotype control and the control that did not process anything were also measured and calibrated to derive exact values. After 30 min of shading, the cells were washed twice with PBS and analyzed by FACS.

As a result of FACS analysis of the blood after the HBV and P-HBV treatment, the ratio of CD4 + CD8- T lymphocytes and CD4 + / CD8 + lymphocytes was controlled by a group treated with HBV or P-HBV as shown in FIG. 2 Group. In particular, the ratio of CD4 + / CD8 + was significantly increased in HBV and P-HBV at 7 days and 14 days compared with control. At day 21, there was a slight increase in P-HBV group and no difference in other groups.

As shown in FIG. 2 (b), lymphocytes were proliferated by stimulation with ConA in the group treated with HBV and P-HBV, but the proliferation was decreased in the P-HBV group compared to the control group at 7 days. This trend was also observed at day 14 and the P-HBV group was significantly increased compared to the HBV and control groups.

Example 1-4: Measurement of cytokine mRNA expression level in lymphocytes

IL-2, IL-12, IL-4, TNF-α, and IL-1β mRNA expression in lymphocytes was measured in order to measure the immune potency of HBV and P-HBV.

Cytokines are important mediators of systemic immune and immune responses. HBV or P-HBV, and TNF-α and IL-1β, which are found in the inflammatory response of Th1 cytokines (IFN-γ, IL-12) As shown in FIG. 3, IFN-y and IL-12 mRNA seem to be increased in the group treated with P-HBV and HBV compared to the control group. In the P-HBV group, IFN-γ and IL-12 were significantly increased by day 14 compared to the control group. In the P-HBV group, IL-12 is still high on day 21. TNF-alpha, IL-1HBV and P-HBV groups.

Example 2: Salmonella ( S. typhimurium ), The bactericidal effect of HBV coated with PLGA in pigs infected with

Example 2-1: Salmonella ( S. typhimurium ) Infection experiment

Pigs were randomly divided into groups and placed PLGA, HBV, and P-HBV through the rectum. And 1 x 10 ⁹ CFU / ml of S. typhimurium bacteria were diluted in 10 ml and orally administered. After the experiment, all the pigs are sacrificed to collect the ileum, intestines, colon, mesenteric lymph nodes. All tissues were cleanly removed of bacteria and used for immunization experiments.

Example 2-1: Observation of medical symptoms

Infected pigs were monitored daily for signs of any symptoms (anorexia, lethargy, sneezing, vomiting, diarrhea). The body temperature and weight of each pig was measured twice a day. Infection rates of pigs were measured on days 1, 3, 5, 7 and 9 as 0 = normal feces, 1 = loose feces, 2 = 238 mild diarrhea and 3 = severe diarrhea.

All pigs are S. typhimurium. p and signs of infection were observed. Pigs showed signs of salmonella infection such as anorexia, vomiting, behavioral problems, and diarrhea. The temperature of the rectum increased rapidly to 39.5 ° C in the first to third days in all groups and there was no significant difference between groups during the experiment. Diarrhea (scores: 1-2) appeared in all infected pigs. Symptoms of diarrhea grew on the 3rd to 5th day and returned to normal on the 9th day.

As shown in FIG. 4 (a), during the entire experimental period, the P-HBV group received lower scores in feces than all other groups. However, no statistically significant difference was observed between the groups.

Example 2-2: Bacterial counts in rectal and tissue samples

Samples were prepared by adding PBS and diluting 10-fold. The diluted solution was plated on XLD agar medium containing 100 / / ml kanamycin and cultured at 37 캜 for 48 hours. About 30 ~ 300 colonies of black colonies were observed per plate and this was expressed as CFU / g.

The rate of bacterial reduction was measured in fecal, follicular, colon, mesenteric lymph nodes (MLN) of infected pigs. The fecal samples of the infected pigs were steadily increased after infection and peaked on the 3rd day, and gradually decreased over time. As shown in Fig. 4 (b), the P-HBV group significantly decreased on the third day. The bacterial mass was measured in the ileum, intestines, colon and MLN tissues. Pathogens were decreased in HBV and P-HBV groups in all tissues. As shown in FIG. 4 (c), it was confirmed that S. typhimurium was reduced in all tissues in the HBV and P-HBV groups.

Example 2-3: Analysis of cytokines of pigs infected with S. typhimurium

RNA from lymphocytes isolated from peripheral blood was extracted and cytokines were detected at the mRNA level.

The expression of cytokine mRNA was confirmed in lymphocytes and tissues. All cytokines showed a similar trend. As shown in FIG. 5, the inflammatory cytokines TNF-a, IL-1 beta and Th1 cytokines IFN-y and IL-12 were significantly increased in the P-HBV group compared to the control group. In the P-HBV group, Salmonella-infected ileum exhibited stronger responses than lymphocytes, and increased IFN-γ, IL-2, and IL-12 and increased inflammatory cytokines such as TNF-α and IL-1β .

In Fig. 6, the intestinal and colon tissues showed similar tendency to the ileum. However, the HBV group increased IFN-y only in both tissues.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (17)

L-lactic acid-co-glycolic acid, poly-D, L-lactic acid-co-glycolic acid, poly-L-lactic acid-co-glycolic acid, Characterized in that bee venom is encapsulated in at least one biodegradable polymer selected from the group consisting of caprolactone, poly-valerolactone, poly-hydroxybutyrate, and poly-hydroxyvalerate, and a porcine salmonella infection and PRRS virus prevention Or a sustained release formulation for treatment.
The method according to claim 1,
The bee venom is a sustained-release preparation for preventing or treating porcine salmonella infection and PRRS virus comprising a water-soluble component and a fat-soluble component.
3. The method of claim 2,
Wherein the water-soluble component is melittin, and the fat-soluble component is chrylsin, pinocembrin or eleven eicosanol. 9. A sustained-release preparation for prevention or treatment of porcine salmonella infection and PRRS virus,
delete The method according to claim 1,
Wherein the biodegradable polymer is poly-D, L-lactic acid-co-glycolic acid, and a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus.
The method according to claim 1,
Wherein the content of the bee venom is 0.1 to 20% by weight. The sustained-release preparation for prevention or treatment of porcine salmonella infection and PRRS virus.
The method according to claim 1,
Wherein the sustained-release preparation has a medicinal effect lasting more than 7 days, and a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus.
delete The method according to claim 1,
But are not limited to, polyethylene glycol, chitooligosaccharide, chitosan, glycine, arginine, lysine, cyclodextrin, polyvinyl alcohol, Wherein the composition further comprises at least one additive selected from the group consisting of hydropropyl methylcellulose, a surfactant, and sucrose. The sustained-release preparation for prevention or treatment of porcine salmonella infection and PRRS virus according to claim 1,
The method according to claim 1,
Chitin, chitosan, glycine, arginine, lysine, cyclodextrin, sucrose, polyvinyl pyrrolidone, and polyvinyl alcohol polyvinyl alcohol, and a sustained-release preparation for prevention or treatment of PRRS virus.
Poly-L-lactic acid-co-glycolic acid, poly-D-lactic acid-co-glycolic acid, poly-L-lactic acid-co- glycolic acid, poly- Preparing a biodegradable polymer solution by adding an organic solvent to at least one biodegradable polymer selected from the group consisting of lactone, poly-hydroxybutyrate, and poly-hydroxyvalerate;
Preparing a W / O emulsion by adding a bee venom aqueous solution to the biodegradable polymer solution; And
The method of claim 1, wherein the W / O emulsion is dispersed in an emulsifying agent to prepare a W / O / W emulsion and dried, and the method for producing a sustained-release preparation for preventing or treating a porcine salmonella infection and a PRRS virus according to claim 1.
delete 12. The method of claim 11,
Wherein the biodegradable polymer is poly-D, L-lactic acid-co-glycolic acid, and a method for producing a sustained-release preparation for preventing or treating PRRS virus.
12. The method of claim 11,
Wherein the organic solvent is selected from the group consisting of dichloromethane, ethyl acetate, chloroform, acetone, dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethyl acetate, methyl ethyl ketone and acetonitrile And a method for producing a sustained-release preparation for preventing or treating PRRS virus.
12. The method of claim 11,
The emulsifier may be selected from the group consisting of chitooligosaccharide, chitosan, glycine, arginine, lysine, cyclodextrin, sucrose, polyvinyl pyrrolidone, Wherein the virus is at least one member selected from the group consisting of polyvinyl alcohol, and a method for producing a sustained-release preparation for preventing or treating PRRS virus.
12. The method of claim 11,
The bee venom aqueous solution may contain at least one selected from the group consisting of polyethylene glycol, chitooligosaccharide, chitosan, glycine, arginine, lysine, cyclodextrin, polyvinyl alcohol, The present invention further provides a method for preventing or treating a porcine salmonellal infection and a PRRS virus, which method further comprises at least one additive selected from the group consisting of alcohol, hydropropyl methylcellulose, a surfactant, and sucrose. ≪ / RTI >
A method for preventing or treating porcine salmonella infection and PRRS virus, which comprises administering an effective amount of the sustained-release preparation of claim 1 encapsulated in a biodegradable polymer through a porcine mucosal route.

Images