Composition for preventing or treating inflammatory disease comprising the venom of <i>Agkistrodon piscivorus piscivorus </i>or <i>Naja melanoleuca </i>

Abstract

A pharmaceutical composition for preventing or treating inflammatory disease comprising snake venom is disclosed. More specifically, the pharmaceutical composition or quasi-drug includes venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca as an active ingredient. A method for preventing or treating inflammatory disease includes administering the venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca to a subject. The composition can increase the expression of C-C chemokine receptor type 1 (CCR1) in a mouse where skin ulcer is induced and thus has an excellent effect of treating skin ulcers, and thus can be effectively used for the treatment of skin ulcers, in particular Behcet's disease or Buerger's disease.

Claims

1. A method for treating a skin ulcer of a subject in need thereof, comprising administering an effective amount of a composition comprising venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca to the subject.

2. The method of claim 1, wherein the composition is a pharmaceutical composition or a quasi-drug.

3. The method of claim 2, wherein the composition is in a form of a liquid, an ointment, a cream, a lotion, a spray, a patch, a gel, or an aerosol. composition which is applied to an afflicted part of skin of the subject.

4. The method of claim 1, wherein the composition is an external application composition which is applied to an afflicted part of skin of the subject.

5. The method of claim 4, wherein the afflicted part has a skin ulcer.

6. The method of claim 1, wherein the skin ulcer is induced from Behcet's disease or Buerger's disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 shows images confirming the states of skin ulcer in mice with symptomatic Behcet's disease 5 days after administrating of the venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca of the present invention to the mice.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

(3) Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples

EXAMPLES

Example 1. Preparation of Mice

(4) Four-week-old ICR strain mice were used. Normal mice were subjected to hair cut before skin application, and then, a solution containing the venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca was applied to the mice using cotton swabs. In the case of oral administration, the same amount was orally administered. For the mice with Behcet's disease, the venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca was applied to the regions with skin symptoms (e.g. ulcers, erythema, etc.) using cotton swabs.

Example 2. Flow Cytometry

(5) After isolating the peripheral blood cells of each mouse, the red blood cells were removed and fluorescence-labeled anti-CCR1 antibody was reacted with cells and the results were analyzed by a flow cytometer and thereby the frequency of cells expressing CCR1 in each mouse was measured. Cells were isolated from the lymph node of each mouse and then reacted with the antibody in the same manner and analyzed.

Experimental Example

Experimental Example 1. Confirmation of Characteristics of CCR1 Expression in Mice with Behcet's Disease

(6) To confirm the characteristics of CCR1 expression in mice with Behcet's disease, PBMCs (peripheral blood mononuclear cells) and lymph nodes were isolated for each of normal mice (control group), mice with symptomatic Behcet's disease, and mice with asymptomatic Behcet's disease, and the CCR1 expression in each group was analyzed by flow cytometry. The results are shown in Table 1 below.

(7) TABLE-US-00001 TABLE 1 Normal Control BD BDN Surface Cytoplasm Surface Cytoplasm Surface Cytoplasm Lymph Lymph Lymph Lymph Lymph Lymph PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node 8.4 8.7 50.1 42.2 10.3 5.8 18.2 84.7 12.6 8.5 71 98.8 5.8 4.0 49.6 31.2 11.3 11.8 34.9 85.5 13.2 8.6 67.2 97.9 8.5 10.6 64.6 59.2 9 9.8 62.1 85.4 16.1 8.1 69.7 98.9 9.6 8.7 69 61.8 6 6.9 49.2 72.5 9.6 10.8 62.6 92.3 8.8 16.5 74.4 54.3 8.7 11.5 11.1 43.1 7.1 14.9 62 48.7 9.0 12.4 86.0 52.9 4 8.5 23.8 58 11.9 11.5 59.6 41.8 6.2 8.3 13.3 25.1 13 12.9 58.3 30.7 Avg 8.35 10.15 65.61 50.26 7.92 8.94 30.37 64.9 11.92 10.75 64.34 72.7 SD 1.32 4.18 14.15 11.52 2.73 2.43 19.47 17.62 3.09 2.59 4.60 26.88 p value 0.01 0.01 0.018 0.018 0.0007 0.0007 0.003 0.003

(8) As shown in Table 1 above, it was confirmed that the CCR1 expression was lower in the mice with symptomatic Behcet's disease (PBMC: surface 7.92±2.73%, cytoplasm 30.37±19.47%, and Lymph Node: surface 8.94±2.43%, cytoplasm 64.90±17.62%), compared to those in the mice of the control group (PBMC: surface 8.35±1.32%, cytoplasm 65.61±14.15%, and Lymph Node: surface 10.15±4.18%, cytoplasm 50.26±11.52%) and the mice with asymptomatic Behcet's disease (PBMC: surface 11.92±3.09%, cytoplasm 64.34±4.60%, and Lymph Node: surface 10.75±2.59%, cytoplasm 72.7±26.88%). Accordingly, contrary to those in the mice where Behcet's disease has not occurred and the mice where asymptomatic Behcet's disease has occurred, the CCR1 expression was shown to be significantly lower in the PBMCs and lymph nodes of the mice with symptomatic Behcet's disease.

Experimental Example 2. Confirmation of Characteristics of CCR1 Expression in Normal Mice by the Venom of Naja melanoleuca

(9) To confirm the effects of the venom of Naja melanoleuca (NM) on CCR1 expression in normal mice, the venom of Naja melanoleuca was applied to each of the skin of normal mice (local administration) and oral cavity (oral administration), and the PBMCs and lymph nodes were isolated and the CCR1 expression therein was analyzed by flow cytometry. The results are shown in Table 2 below.

(10) TABLE-US-00002 TABLE 2 Normal Control NM Skin NM Oral Surface Cytoplasm Surface Cytoplasm Surface Cytoplasm Lymph Lymph Lymph Lymph Lymph Lymph PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node 8.4 8.7 50.1 42.2 6.2 12.0 72.7 44 12.3 10.0 54.9 33.3 5.8 4.0 49.6 31.2 7.8 11.2 59.6 48.7 1.2 7.4 47.8 87.4 8.5 10.6 64.6 59.2 9.1 10.7 90.5 66.3 10.8 8.5 72.9 47.0 9.6 8.7 69 61.8 7.9 10.4 75.6 58.2 11.2 8.4 75.3 59.6 8.8 16.5 74.4 54.3 12.6 13.4 64.5 58.0 20 13.4 49.9 61.6 9.0 12.4 86.0 52.9 5.4 13.5 62.7 64.8 14.9 16.3 70.1 74.2 Avg 8.35 10.15 65.61 50.26 8.16 11.86 70.96 56.66 11.73 10.66 61.81 60.5 SD 1.32 4.18 14.15 11.52 2.53 1.34 11.36 8.79 6.17 3.46 12.3 19.14

(11) As shown in Table 2 above, it was confirmed that the CCR1 expression was slightly increased in the mice where the venom of Naja melanoleuca was applied to the skin of normal mice (PBMC: surface 8.16±2.53%, cytoplasm 70.93±11.36%, and Lymph Node: surface 11.86±1.34%, cytoplasm 56.66±8.79%) and in the mice where the venom of Naja melanoleuca was administered to oral cavity of normal mice (PBMC: surface 11.73±6.17%, cytoplasm 61.81±12.3%, and Lymph Node: surface 10.66±3.46%, cytoplasm 60.5±19.14%), compared to the CCR1 expression in normal mice without any treatment (PBMC: surface 8.35±1.32%, cytoplasm 65.61±14.15%, and Lymph Node: surface 10.15±4.18%, cytoplasm 50.26±11.52%).

Experimental Example 3. Confirmation of Characteristics of CCR1 Expression in Normal Mice by the Venom of Agkistrodon piscivorus piscivorus

(12) To confirm the effects of the venom of Agkistrodon piscivorus piscivorus on CCR1 expression in normal mice, the venom of Agkistrodon piscivorus piscivorus was applied to each of the skin of normal mice (local administration) and oral cavity (oral administration), and the PBMCs and lymph nodes were isolated and the CCR1 expression therein was analyzed by flow cytometry. The results are shown in Table 3 below.

(13) TABLE-US-00003 TABLE 3 Normal Control AP Skin AP Oral Surface Cytoplasm Surface Cytoplasm Surface Cytoplasm Lymph Lymph Lymph Lymph Lymph Lymph PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node 8.4 8.7 50.1 42.2 6.1 10.7 38.2 30 17.5 12.5 31.2 38.6 5.8 4.0 49.6 31.2 7 16.7 42.2 30.4 11.1 12.3 36.2 52.6 8.5 10.6 64.6 59.2 7 18.6 66.7 57.1 6.7 15.3 66.5 79.7 9.6 8.7 69 61.8 6 9.3 65.4 56.8 7.8 22.2 62.5 67 8.8 16.5 74.4 54.3 14.4 16.6 71.5 67.6 7.5 20.1 54.3 72.9 9.0 12.4 86.0 52.9 12.8 16.6 65.4 73.7 10.6 22.7 44.4 73.1 Avg 8.35 10.15 65.61 50.26 8.88 14.75 58.23 52.6 10.2 17.51 49.18 63.98 SD 1.32 4.18 14.15 11.52 3.71 3.78 14.20 18.50 3.98 4.74 14.27 15.44 p value 0.07 0.07 0.017 0.017

(14) As shown in Table 3 above, it was confirmed that the CCR1 expression was slightly increased in the mice where the venom of Agkistrodon piscivorus piscivorus was applied to the skin of normal mice (PBMC: surface 8.88±3.71%, cytoplasm 58.23±14.20%, and Lymph Node: surface 14.75±3.67%, cytoplasm 50.6±18.50%) and in the mice where the venom of Agkistrodon piscivorus piscivorus was administered to oral cavity of normal mice (PBMC: surface 10.2±3.98%, cytoplasm 49.18±14.27%, and Lymph Node: surface 17.51±4.74%, cytoplasm 63.98±15.44%), compared to the CCR1 expression in normal mice without any treatment (PBMC: surface 8.35±1.43%, cytoplasm 65.61±14.15%, and Lymph Node: surface 10.15±4.18%, cytoplasm 50.26±11.52%).

Experimental Example 4. Confirmation of Effects of the Venom of Agkistrodon piscivorus piscivorus (AP) or Naja melanoleuca (NM) on CCR1 Expression in Mice with Symptomatic Behcet's Disease

(15) To confirm the effects of the venom of Agkistrodon piscivorus piscivorus (AP) or Naja melanoleuca (NM) on CCR1 expression in mice with symptomatic Behcet's disease, the venom of Agkistrodon piscivorus piscivorus was applied to the skin of mice with symptomatic Behcet's disease (local administration), and the PBMCs and lymph nodes were isolated and the CCR1 expression therein was analyzed by flow cytometry. The results are shown in Table 4 below. Additionally, the venom of Agkistrodon piscivorus piscivorus was administered to mice with symptomatic Behcet's disease, and 5 days thereafter, the resulting changes in skin ulcer of the mice are shown in FIG. 1.

(16) TABLE-US-00004 TABLE 4 BD BD + AP BD + NM Surface Cytoplasm Surface Cytoplasm Surface Cytoplasm Lymph Lymph Lymph Lymph Lymph Lymph PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node PBMC Node 10.3 5.8 18.2 84.7 14.2 18.7 54.9 20.4 9.3 16.5 67.8 47.9 11.3 11.8 34.9 85.5 13.2 17.6 42.9 15.2 16.7 13.5 79.7 46.1 9 9.8 62.1 85.4 4.7 23.1 71.6 33.4 17.8 15.3 79.8 39.7 6 6.9 49.2 72.5 19.9 21.4 61.1 37 18.9 27.6 89.8 70.4 8.7 11.5 11.1 43.1 25.1 26.7 48.5 69.3 18.3 28.8 85.4 62.9 4 8.5 23.8 58 24.1 34.4 59.9 51.1 23.6 31.2 67.7 53.4 6.2 8.3 13.3 25.1 23.8 32.6 65.4 52.7 27.6 23.3 79.5 50.7 27.2 29.5 63.8 28.9 Avg 7.92 8.94 30.37 64.9 19.02 25.5 58.51 38.5 18.88 22.31 78.52 53.01 SD 2.73 2.43 19.47 17.62 7.70 6.30 9.35 18.12 5.72 7.19 8.27 10.46 p value 0.003 0.003 0.0006 0.0006 0.00002 0.00002 0.0005 0.0005 0.003 0.003 0.00006 0.00006 0.0008 0.0008 0.03 0.03

(17) As shown in Table 4 above, it was confirmed that the CCR1 expression was significantly increased in the group of mice with symptomatic Behcet's disease where the venom of Agkistrodon piscivorus piscivorus was administered (PBMC: surface 19.02±7.70%, cytoplasm 58.51±9.35%, and Lymph Node: surface 25.5±6.30%, cytoplasm 38.5±18.12%) or in the group of mice with symptomatic Behcet's disease where the venom of Naja melanoleuca was administered (PBMC: surface 18.88±5.72%, cytoplasm 78.52±8.27%, and Lymph Node: surface 22.31±7.19%, cytoplasm 53.01±10.46%), respectively, compared to the group where none of the venom was administered (PBMC: surface 7.92±2.73%, cytoplasm 30.37±19.47%, and Lymph Node: surface 8.94±2.43%, cytoplasm 64.9±17.62%).

(18) The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that the present invention may be embodied in various other forms without departing from the essential characteristics thereof. Therefore, the disclosed embodiments should be considered from an illustrative aspect rather than a restrictive aspect. The scope of the present invention is represented in the accompanying claims rather than the foregoing description and all of the differences within the scope of equivalents thereof should be construed as being included in the present invention.

INDUSTRIAL APPLICABILITY

(19) As described above, the composition for preventing or treating inflammatory disease comprising the snake venom has an excellent effect for treating skin ulcers. Therefore, the composition can be effectively used by applying to pharmaceutical drugs, etc. including skin external agents for the treatment of skin ulcers, in particular for the treatment of Behcet's disease or Buerger's disease.

ADVANTAGEOUS EFFECTS

(20) The venom of Agkistrodon piscivorus piscivorus or Naja melanoleuca according to the present invention can increase the expression of C-C chemokine receptor type 1 (CCR1) in a mouse where skin ulcer is induced and thus has an excellent effect of treating skin ulcers.