Abstract
The present invention relates to a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca, which are types of snakes, an extract thereof, or a fraction thereof; a quasi-drug composition; a food composition; a cosmetic composition; and a method of treating rheumatoid arthritis by using the composition. The snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca of the present invention can improve, alleviate, or treat symptoms of rheumatoid arthritis, which is a kind of autoimmune diseases, and thus will be able to be widely used in the development of therapeutic agents for various autoimmune diseases including rheumatoid arthritis.
Claims
1. A pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca, an extract thereof or a fraction thereof.
2. The pharmaceutical composition for preventing or treating rheumatoid arthritis according to claim 1, wherein the extract is obtained by extracting snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca with one or more solvents selected from the group consisting of water, alcohol with 1 to 4 carbon atoms and mixed solvents thereof.
3. The pharmaceutical composition for preventing or treating rheumatoid arthritis according to claim 1, wherein the fraction is obtained by fractionating the extract of snake venom with solvent selected from the group consisting of water, alcohol with 1 to 4 carbon atoms, hexane, ethyl acetate, chloroform, dichloromethane and mixed solvents thereof.
4. The pharmaceutical composition for preventing or treating rheumatoid arthritis according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, an excipient or a diluent.
5. A method of treating rheumatoid arthritis, comprising administering the pharmaceutical composition according to any one of claims 1 to 4 to a subject suspected of rheumatoid arthritis other than humans.
6. A quasi-drug composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca, an extract thereof or a fraction thereof.
7. A food composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca, an extract thereof or a fraction thereof.
8. A cosmetic composition for preventing or improving rheumatoid arthritis, comprising snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca, an extract thereof or a fraction thereof.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0066] FIG. 1 is a photograph showing the damaged joint area of arthritis model mice treated with snake venom sample derived from Agkistrodon piscivorus piscivorus, and show the change in symptoms after applying snake venom derived from Agkistrodon piscivorus piscivorus to the joint area with arthritis once/day for 2 weeks as external skin preparation, wherein it was confirmed that the group applied with PBS used as a control group did not improve but worsened.
[0067] FIG. 2 is a graph showing the change of recovered arthritic index score in arthritis model mice treated with snake venom sample derived from Agkistrodon piscivorus piscivorus and MTX (methotrexate).
[0068] FIG. 3 is a photograph showing the damaged joint area of arthritis model mice treated with snake venom sample derived from Naja melanoleuca, and show the change in symptoms after applying snake venom derived from Naja melanoleuca to the joint area with arthritis once/day for 2 weeks as external skin preparation, wherein it was confirmed that the group applied with PBS used as a control group did not improve but worsened.
[0069] FIG. 4 is a graph showing the change of recovered arthritic index score in arthritis model mice treated with snake venom sample derived from Naja melanoleuca and MTX (methotrexate).
[0070] FIG. 5 is a graph showing the results of flow cytometry analysis of CCR1 positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice orally administered with MTX.
[0071] FIG. 6 is a graph showing the results of flow cytometry analysis of CCR1 positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice treated with snake venom sample derived from Agkistrodon piscivorus piscivorus.
[0072] FIG. 7 is a graph showing the results of flow cytometry analysis of CCR1 positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice treated with snake venom sample derived from Naja melanoleuca.
[0073] FIG. 8a is a graph showing the results of measuring the change in CD4 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0074] FIG. 8b is a graph showing the results of measuring the change in CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0075] FIG. 8c is a graph showing the results of measuring the change in Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0076] FIG. 9a is a graph showing the results of measuring the changes in CD4 and CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0077] FIG. 9b is a graph showing the results of measuring the changes in CD25 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0078] FIG. 9c is a graph showing the results of measuring the changes in CD4 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0079] FIG. 9d is a graph showing the results of measuring the changes in CD4, CD25 and Foxp3 positive regulatory T cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0080] FIG. 10a is a graph showing the results of measuring the change in CD4 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0081] FIG. 10b is a graph showing the results of measuring the change in CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0082] FIG. 10c is a graph showing the results of measuring the change in Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0083] FIG. 11a is a graph showing the results of measuring the changes in CD4 and CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0084] FIG. 11b is a graph showing the results of measuring the changes in CD25 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0085] FIG. 11c is a graph showing the results of measuring the changes in CD4 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0086] FIG. 11d is a graph showing the results of measuring the changes in CD4, CD25 and Foxp3 positive regulatory T cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0087] FIG. 12a is a graph showing the results of comparing levels of CRP (C-reactive protein) in plasma in normal mice orally administered or skin-applied with snake venom derived from Agkistrodon piscivorus piscivorus.
[0088] FIG. 12b is a graph showing the results of levels of comparing AST (aspartate aminotransferase) in plasma in normal mice orally administered or skin-applied with snake venom derived from Agkistrodon piscivorus piscivorus.
[0089] FIG. 12c is a graph showing the results of comparing ALT (alanine transaminase) levels in plasma in normal mice orally administered or skin-applied with snake venom derived from Agkistrodon piscivorus piscivorus.
[0090] FIG. 13a is a graph showing the results of comparing levels of CRP (C-reactive protein) in plasma in normal mice orally administered or skin-applied with snake venom derived from Naja melanoleuca.
[0091] FIG. 13b is a graph showing the results of levels of comparing AST (aspartate aminotransferase) in plasma in normal mice orally administered or skin-applied with snake venom derived from Naja melanoleuca.
[0092] FIG. 13c is a graph showing the results of comparing ALT (alanine transaminase) levels in plasma in normal mice orally administered or skin-applied with snake venom derived from Naja melanoleuca.
[0093] FIG. 14 is a graph showing the results of comparing the level of IL-17 in plasma in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0094] FIG. 15 is a graph showing the results of comparing the level of IL-17 in plasma in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0095] FIG. 16 is a microphotograph showing the joint tissue of arthritis model mice orally administered with MTX or applied on skin with snake venom derived from Agkistrodon piscivorus piscivorus.
[0096] FIG. 17 is a microphotograph showing the joint tissue of arthritis model mice orally administered with MTX or applied on skin with snake venom derived from Naja melanoleuca.
BEST MODE FOR CARRYING OUT THE INVENTION
[0097] Hereinafter, the present invention will be described in more detail through working examples. However, these working examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these working examples.
Example 1: Production of Arthritis Model Mice
[0098] Type 2 collagen solution (10 mM), an adjuvant (Complete freund's adjuvant) and oil (mineral oil) were mixed at a ratio of 1:1:1 (v/v/v) to obtain a mixture, 50 μl of the mixture was injected into 1 cm site of the tail vein at the tail base of 8-week-old ICR mice, and 10 days later, was injected once more repeatly. After injection, mice showing arthritis symptoms at 10 to 15 days elapsed time point were selected and used as rheumatoid arthritis model mice.
[0099] On the other hand, in order to distinguish in detail the level of symptoms of the selected rheumatoid arthritis model mice, severity score is given as follows (Table 1), and when severity score was 2 or more, it was determined that rheumatoid arthritis has occurred.
TABLE-US-00001 TABLE 1 Severity score and symptom criteria of arthritis model mice Score Symptom 0 Normal condition with no symptom 1 Condition with minimal swelling or redness induced in any one of the toes, insteps ankles, etc. 2 Condition with significant edema and redness induced in at least two of the toes insteps, and ankles 3 Condition with inflammation and edema at three joint areas from the toes to the ankles 4 Condition in which significant edema and swelling are observed throughout the entire area from the toes to the ankles, or in which the feet cannot be used freely due to stiffness
[0100] Meanwhile, snake venom sample derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca used in the present invention was purchased from Sigma-Aldrich and used.
Example 2: Therapeutic Effect of Snake Venom on the Symptoms of Arthritis Model Mice
Example 2-1: Therapeutic Effect of Snake Venom Derived from Agkistrodon Piscivorus Piscivorus on the Symptoms of Arthritis Model Mice
[0101] On the skin with the damaged joint area of arthritis model mice produced in Example 1 above, snake venom sample derived from Agkistrodon piscivorus piscivorus was applied for 2 weeks, the therapeutic effect of snake venom above was visually verified (FIG. 1). At this time, as a control group, mice applied with PBS were used.
[0102] FIG. 1 is a photograph showing the damaged joint area of arthritis model mice treated with snake venom sample derived from Agkistrodon piscivorus piscivorus.
[0103] In addition, after snake venom sample derived from Agkistrodon piscivorus piscivorus was treated at the joint areas of the damaged 4 limbs (one pair of forelimbs and one pair of hind limbs) of arthritis model mice, each severity score was measured (FIG. 2). At this time, as a control group, mice applied with PBS were used, and as a comparative group, mice orally administered with MTX (methotrexate), known as rheumatoid arthritis therapeutic agents, were used.
[0104] FIG. 2 is a graph showing the change of recovered arthritic index score in arthritis model mice treated with snake venom sample derived from Agkistrodon piscivorus piscivorus and MTX (methotrexate), D1 represents the time point at which 1 day has elapsed after treatment, D11 represents the time point at which 11 days have elapsed after treatment, (◯) represents each arthritis model mouse, (-) is a link of the change between severity score measured at 1 day elapsed time point and severity score measured at 11 days elapsed time point in the same arthritis model mice, and a red mark represents the average value of severity score measured at 1 day and 11 days elapsed time points, respectively.
[0105] As shown in FIG. 2, in the case of a control group, severity score measured on days 1 and 11 did not show significant difference, but when MTX was orally administered (comparative group) or snake venom sample derived from Agkistrodon piscivorus piscivorus was treated, it was confirmed that the severity score measured on day 11 was significantly decreased compared to the severity score measured on day 1.
[0106] Therefore, snake venom sample derived from Agkistrodon piscivorus piscivorus was found to have the effect of improving the symptoms of rheumatoid arthritis.
Example 2-2: Therapeutic Effect of Snake Venom Derived from Naja melanoleuca on the Symptoms of Arthritis Model Mice
[0107] On the skin of the damaged joint area of arthritis model mice produced in Example 1 above, snake venom sample derived from Naja melanoleuca was applied for 2 weeks, the therapeutic effect of snake venom above was visually verified (FIG. 3). At this time, as a control group, mice applied with PBS were used.
[0108] FIG. 3 is a photograph showing the damaged joint area of arthritis model mice treated with snake venom sample derived from Naja melanoleuca.
[0109] In addition, after snake venom sample derived from Naja melanoleuca was treated at the joint areas of the damaged 4 limbs (one pair of forelimbs and one pair of hind limbs) of arthritis model mice, each severity score was measured (FIG. 4). At this time, as a control group, mice applied with PBS were used, and as a comparative group, mice orally administered with MTX (methotrexate), known as rheumatoid arthritis therapeutic agents, were used.
[0110] FIG. 4 is a graph showing the change of recovered arthritic index score in arthritis model mice treated with snake venom sample derived from Naja melanoleuca and MTX (methotrexate), D1 represents the time point at which 1 day has elapsed after treatment, D11 represents the time point at which 11 days have elapsed after treatment, (◯) represents each arthritis model mouse, (-) is a link of the change between severity score measured at 1 day elapsed time point and severity score measured at 11 days elapsed time point in the same arthritis model mice, and a red mark represents the average value of severity score measured at 1 day and 11 days elapsed time points, respectively.
[0111] As shown in FIG. 4, in the case of a control group, severity score measured on days 1 and 11 did not show significant difference, but when MTX was orally administered (comparative group) or snake venom sample derived from Naja melanoleuca was treated, it was confirmed that the severity score measured on day 11 was significantly decreased compared to the severity score measured on day 1.
[0112] Therefore, snake venom sample derived from Naja melanoleuca was found to have the effect of improving the symptoms of rheumatoid arthritis.
Example 3: Flow Cytometry Analysis in Arthritis Model Mice
Example 3-1: Change in CCR1 Positive Cells in Arthritis Model Mice Orally Administered with Methotrexate
[0113] Peripheral blood monocytes (PBMC) and lymph nodes (LN) were obtained from arthritis model mice orally administered with MTX, and then flow cytometry analysis was performed on these targets.
[0114] Roughly, an anti-mouse CCR1 antibody was added to cells isolated from peripheral blood monocytes and lymph nodes, and then reacted at 4° C. for 30 minutes. Then, in order to perform flow cytometry analysis, BD cantoII flow cytometer was used (FIG. 5). At this time, as a control group, mice administered with PBS were used.
[0115] FIG. 5 is a graph showing the results of flow cytometry analysis of CCR1 positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice orally administered with MTX (methotrexate).
[0116] As shown in FIG. 5, it was found that the level of CCR1 positive cells in the cell surface and cytoplasm of peripheral blood monocytes and lymph nodes was increased in arthritis model mice orally administered with MTX, known as rheumatoid arthritis therapeutic agents.
[0117] From the result of increasing the level of the CCR1 positive cells, it was found that the symptoms of inflammation were improved.
Example 3-2: Change in CCR1 Positive Cells in Arthritis Model Mice Treated on Skin with Snake Venom Derived from Agkistrodon piscivorus Piscivorus or Naja melanoleuca
[0118] On the skin of the damaged joint area of arthritis model mice produced in Example 1 above, snake venom sample derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca was applied on skin for 2 weeks, and peripheral blood monocytes (PBMC) and lymph nodes (LN) were obtained from the mice, and then flow cytometry analysis by the method of Example 3-1 above was performed on these targets (FIGS. 6 and 7). At this time, as a control group, mice applied with PBS were used.
[0119] FIG. 6 is a graph showing the results of flow cytometry analysis of CCR1 positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice treated with snake venom sample derived from Agkistrodon piscivorus piscivorus.
[0120] FIG. 7 is a graph showing the results of flow cytometry analysis of CCR1 positive cells on peripheral blood monocytes and lymph nodes of arthritis model mice treated with snake venom sample derived from Naja melanoleuca.
[0121] As shown in FIGS. 6 and 7, in the arthritis model mice treated with snake venom of the present invention, it was confirmed that the level of CCR1 positive cells was increased in peripheral blood monocytes and lymph nodes.
[0122] These results are similar to the results of oral administration of MTX, known as rheumatoid arthritis therapeutic agents, shown in FIG. 5, and compared with the results of FIG. 5 above, it was found that snake venom of the present invention shows therapeutic effect on rheumatoid arthritis.
Example 4: Flow Cytometry Analysis in Normal Mice
Example 4-1: Change in CD4, CD25 and Foxp3 Positive Cell in Normal Mice Orally Administered with Snake Venom Derived from Agkistrodon piscivorus Piscivorus
[0123] It is to confirm whether snake venom derived from Agkistrodon piscivorus piscivorus can increase the frequency of regulatory T cells.
[0124] For this, 0.1 μg or 1 μg of snake venom sample derived from Agkistrodon piscivorus piscivorus was orally administered to normal mice, and the levels of CD4 positive cells, CD25 positive cells and Foxp3 positive cells from these were confirmed through flow cytometry analysis (FIGS. 8a to 8c). At this time, as a control group, mice orally administered with PBS were used. In addition, CD4 positive cells and CD25 positive cells were confirmed by the method of Example 3-1. Finally, Foxp3 positive cells were confirmed through intranuclear staining, and roughly, the cells to be stained were washed twice with 1× permeabilization buffer and then fixed with Fix/perm buffer, and anti-mouse Foxp3 antibody was added and then reacted at 4° C. for 30 minutes.
[0125] FIG. 8a is a graph showing the results of measuring the change in CD4 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus, FIG. 8b is a graph showing the results of measuring the change in CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus, and FIG. 8c is a graph showing the results of measuring the change in Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0126] As shown in FIGS. 8a to 8c, among CD4 positive, CD25 positive and Foxp3 positive cells known as markers of regulatory T cells, it was confirmed that the frequency of CD25 positive and Foxp3 positive cells was increased by oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus.
[0127] Therefore, since oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus individually increased the frequency of CD25 positive and Foxp3 positive cells, which are the main characteristics of regulatory T cells, from this, it was analyzed that oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus would increase the level of regulatory T cells.
Example 4-2: Change in Regulatory T Cells in Normal Mice Orally Administered with Snake Venom Derived from Agkistrodon Piscivorus Piscivorus
[0128] Since the results of Example 4-1 above provided a clue that oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus would increase the level of regulatory T cells, in practice, it was to confirm whether oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus could increase the level of regulatory T cells.
[0129] For this, 0.1 μg or 1 μg of snake venom sample derived from Agkistrodon piscivorus piscivorus was orally administered to normal mice, and the levels of cells comprising CD4 positive, CD25 positive and Foxp3 positive in combination from these were confirmed through flow cytometry analysis (FIGS. 9a to 9d). At this time, as a control group, mice orally administered with PBS were used.
[0130] FIG. 9a is a graph showing the results of measuring the changes in CD4 and CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus, FIG. 9b is a graph showing the results of measuring the changes in CD25 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus, FIG. 9c is a graph showing the results of measuring the changes in CD4 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus, and FIG. 9d is a graph showing the results of measuring the changes in CD4, CD25 and Foxp3 positive regulatory T cells according to the dose in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0131] As shown in FIGS. 9a to 9d, it was confirmed that snake venom sample derived from Agkistrodon piscivorus piscivorus increased all of the level of each combination of CD4 positive, CD25 positive and Foxp3 positive, known as the main characteristics of regulatory T cells.
[0132] Therefore, it was found that oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus can increase the level of regulatory T cells and, due to the increase in the level of these regulatory T cells, can improve the inflammatory symptoms of rheumatoid arthritis.
Example 4-3: Changes in CD4, CD25 and Foxp3 Positive Cells in Normal Mice Orally Administered with Snake Venom Derived from Naja Melanoleuca
[0133] It is to confirm whether snake venom derived from Naja melanoleuca can increase the frequency of regulatory T cells.
[0134] For this, 0.1 μg or 1 μg of snake venom sample derived from Naja melanoleuca was orally administered to normal mice, and the levels of CD4 positive cells, CD25 positive cells and Foxp3 positive cells from these were confirmed through flow cytometry analysis (FIGS. 10a to 10c). At this time, as a control group, mice orally administered with PBS were used. In addition, CD4 positive cells and CD25 positive cells were confirmed by the method of Example 3-1. Finally, Foxp3 positive cells were confirmed through intranuclear staining, and roughly, the cells to be stained were washed twice with 1× permeabilization buffer and then fixed with Fix/perm buffer, and anti-mouse Foxp3 antibody was added and then reacted at 4° C. for 30 minutes.
[0135] FIG. 10a is a graph showing the results of measuring the change in CD4 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca, FIG. 10b is a graph showing the results of measuring the change in CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca, and FIG. 10c is a graph showing the results of measuring the change in Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0136] As shown in FIGS. 10a to 10c, among CD4 positive, CD25 positive and Foxp3 positive cells known as markers of regulatory T cells, it was confirmed that the frequency of CD25 positive and Foxp3 positive cells was increased by oral administration of snake venom sample derived from Naja melanoleuca.
[0137] Therefore, since oral administration of snake venom sample derived from Naja melanoleuca individually increased the frequency of CD25-positive and Foxp3-positive cells, which are the main characteristics of regulatory T cells, from this, it was analyzed that oral administration of snake venom sample derived from Naja melanoleuca would increase the level of regulatory T cells.
Example 4-4: Change in Regulatory T Cells in Normal Mice Orally Administered with Snake Venom Derived from Naja Melanoleuca
[0138] Since results of Example 4-3 above provided a clue that oral administration of snake venom sample derived from Naja melanoleuca would increase the level of regulatory T cells, in practice, it was to confirm whether oral administration of snake venom sample derived from Naja melanoleuca could increase the level of regulatory T cells.
[0139] For this, 0.1 μg or 1 μg of snake venom sample derived from Naja melanoleuca was orally administered to normal mice, and the levels of cells comprising CD4 positive, CD25 positive and Foxp3 positive in combination from these were confirmed through flow cytometry analysis (FIGS. 11a to 11d). At this time, as a control group, mice orally administered with PBS were used.
[0140] FIG. 11a is a graph showing the results of measuring the changes in CD4 and CD25 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca, FIG. 11b is a graph showing the results of measuring the changes in CD25 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca, FIG. 11c is a graph showing the results of measuring the changes in CD4 and Foxp3 positive cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca, and FIG. 11d is a graph showing the results of measuring the changes in CD4, CD25 and Foxp3 positive regulatory T cells according to the dose in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0141] As shown in FIGS. 11a to 11d, it was confirmed that snake venom sample derived from Naja melanoleuca increased all of the level of each combination of CD4 positive, CD25 positive and Foxp3 positive, known as the main characteristics of regulatory T cells.
[0142] Therefore, it was found that oral administration of snake venom sample derived from Naja melanoleuca can increase the level of regulatory T cells and, due to the increase in the level of these regulatory T cells, can improve the inflammatory symptoms of rheumatoid arthritis.
Example 5: Effects of Snake Venom Derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca on Metabolite Levels in Normal Mice
Example 5-1: Level of CRP (C-Reactive Protein) in Plasma
[0143] After oral administration or skin application of snake venom sample derived from Agkistrodon piscivorus piscivorus to normal mice, plasma from each mouse was collected, and then levels of CRP (C-reactive protein) in plasma, an inflammatory marker, were compared (FIG. 12a). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0144] FIG. 12a is a graph showing the results of comparing levels of CRP (C-reactive protein) in plasma in normal mice orally administered or skin-applied with snake venom derived from Agkistrodon piscivorus piscivorus.
[0145] As shown in FIG. 12a, when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered or applied on skin, all it was confirmed that the level of CRP in plasma were lowered.
[0146] As such, since it was confirmed that the level of CRP in plasma, an inflammatory marker, was decreased by snake venom derived from Agkistrodon piscivorus piscivorus, it was found that snake venom derived from Agkistrodon piscivorus piscivorus can improve the inflammatory symptoms of rheumatoid arthritis.
Example 5-2: Level of AST (Aspartate Aminotransferase) in Plasma
[0147] After oral administration or skin application of snake venom sample derived from Agkistrodon piscivorus piscivorus to normal mice, plasma from each mouse was collected, and then levels of AST (aspartate aminotransferase) in plasma were compared (FIG. 12b). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0148] FIG. 12b is a graph showing the results of comparing levels of AST (aspartate aminotransferase) in plasma in normal mice orally administered or skin-applied with snake venom derived from Agkistrodon piscivorus piscivorus.
[0149] As shown in FIG. 12b, when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered or applied on skin, all it was confirmed that the level of AST in plasma were lowered.
[0150] As shown in FIG. 12b, when the snake venom derived from Agkistrodon piscivorus piscivorus was orally administered, there was no significant difference from the control group, but when applied on skin, the level of AST in plasma tended to slightly decrease.
[0151] As such, since the level of AST in plasma did not change even when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered, it was analyzed that any particular toxicity in the body was not exhibited even when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered.
Example 5-3: Level of ALT (Alanine Transaminase) in Plasma
[0152] After oral administration or skin application of snake venom sample derived from Agkistrodon piscivorus piscivorus to normal mice, plasma from each mouse was collected, and then levels of ALT (alanine transaminase) in plasma were compared (FIG. 12c). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0153] FIG. 12c is a graph showing the results of comparing levels of ALT (alanine transaminase) in plasma in normal mice orally administered or skin-applied with snake venom derived from Agkistrodon piscivorus piscivorus.
[0154] As shown in FIG. 12c, when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered or applied on skin, all it was confirmed that the level of ALT in plasma was lowered compared to the control group. In particular, it was confirmed that the level of ALT in plasma exhibited significantly lower value in the case of oral administration than in the case of skin application.
[0155] As such, since levels of ALT in plasma tended to be rather decreased when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered, it was confirmed once again that any particular toxicity in the body was not exhibited even when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered.
Example 5-4: Level of CRP (C-Reactive Protein) in Plasma
[0156] After oral administration or skin application of snake venom sample derived from Naja melanoleuca to normal mice, plasma from each mouse was collected, and then levels of CRP (C-reactive protein) in plasma, an inflammatory marker, were compared (FIG. 13a). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0157] FIG. 13a is a graph showing the results of comparing levels of CRP (C-reactive protein) in plasma in normal mice orally administered or skin-applied with snake venom derived from Naja melanoleuca.
[0158] As shown in FIG. 13a, when snake venom derived from Agkistrodon piscivorus piscivorus was orally administered or applied on skin, all it was confirmed that the level of CRP in plasma were lowered.
[0159] As such, since it was confirmed that the level of CRP in plasma, an inflammatory marker, was decreased by snake venom derived from Naja melanoleuca, it was found that snake venom derived from Naja melanoleuca can improve the inflammatory symptoms of rheumatoid arthritis.
Example 5-5: Level of AST (Aspartate Aminotransferase) in Plasma
[0160] After oral administration or skin application of snake venom sample derived from Naja melanoleuca to normal mice, plasma from each mouse was collected, and then levels of AST (aspartate aminotransferase) in plasma were compared (FIG. 13b). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0161] FIG. 13b is a graph showing the results of levels of comparing AST (aspartate aminotransferase) in plasma in normal mice orally administered or skin-applied with snake venom derived from Naja melanoleuca.
[0162] As shown in FIG. 13b, when the snake venom derived from Naja melanoleuca was orally administered, there was no significant difference from the control group, but when applied on skin, the level of AST in plasma tended to slightly increase.
[0163] As such, since the level of AST in plasma did not change even when snake venom derived from Naja melanoleuca was orally administered, it was analyzed that any particular toxicity in the body was not exhibited even when snake venom derived from Naja melanoleuca was orally administered.
Example 5-6: Level of ALT (Alanine Transaminase) in Plasma
[0164] After oral administration or skin application of snake venom sample derived from Naja melanoleuca to normal mice, plasma from each mouse was collected, and then levels of ALT (alanine transaminase) in plasma were compared (FIG. 13c). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0165] FIG. 13c is a graph showing the results of comparing ALT (alanine transaminase) levels in plasma in normal mice orally administered or skin-applied with snake venom derived from Naja melanoleuca.
[0166] As shown in FIG. 13c, when snake venom derived from Naja melanoleuca was orally administered or applied on skin, all it was confirmed that the level of ALT in plasma was lowered compared to the control group. In particular, it was confirmed that the level of ALT in plasma exhibited relatively lower value in the case of skin administration than in the case of oral application.
[0167] As such, since levels of ALT in plasma tended to be rather decreased when snake venom derived from Naja melanoleuca was orally administered, it was confirmed once again that any particular toxicity in the body was not exhibited even when snake venom derived from Naja melanoleuca was orally administered.
Example 6: Cytokine Level Analysis
[0168] After oral administration of snake venom sample derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca to normal mice, plasma from mice was collected, and then levels of IL-17 in plasma were compared (FIGS. 14 and 15). At this time, as a control group, mice orally administered or skin-applied with PBS were used.
[0169] FIG. 14 is a graph showing the results of comparing the level of IL-17 in plasma in normal mice orally administered with snake venom derived from Agkistrodon piscivorus piscivorus.
[0170] FIG. 15 is a graph showing the results of comparing the level of IL-17 in plasma in normal mice orally administered with snake venom derived from Naja melanoleuca.
[0171] As shown in FIGS. 14 and 15, when snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca was orally administered, it was confirmed that the plasma IL-17 level was lowered compared to the control group.
[0172] Since said IL-17 is known as an inflammation-inducing cytokine, the result that snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca lowers the IL-17 level, that is, was analyzed to mean that snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca could improve the inflammatory symptoms of rheumatoid arthritis.
Example 7: Histological Analysis of Arthritis Model Mice
[0173] To the arthritis model mice produced in Example 1 above, MTX was orally administered or snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca was applied on skin, and after collecting the arthritis area tissue from mice, a tissue slice thereof was obtained, and was confirmed under a microscope to confirm whether inflammatory cells in the tissue are present (FIGS. 16 and 17). At this time, as a control group, PBS orally administered mice were used. In addition, the tissue slices were prepared by decalcifying the collected tissue in a decalcification solution, fixing it to make a paraffin block, cutting it to obtain a slice, and then performing H&E staining.
[0174] FIG. 16 is a microphotograph showing the joint tissue of arthritis model mice orally administered with MTX or applied on skin with snake venom derived from Agkistrodon piscivorus piscivorus.
[0175] FIG. 17 is a microphotograph showing the joint tissue of arthritis model mice orally administered with MTX or applied on skin with snake venom derived from Naja melanoleuca.
[0176] As shown in FIGS. 16 and 17, it was confirmed that inflammatory cells were present at the joint area in the tissue of the control group, but it was confirmed that no inflammatory cell was present at the joint area in the tissue of the arthritis model mice orally administered with MTX or applied on skin with snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca.
[0177] Therefore, it was found that snake venom derived from Agkistrodon piscivorus piscivorus or Naja melanoleuca could improve the inflammatory symptoms of rheumatoid arthritis.
[0178] From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed that, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents was included in the scope of the present invention.
