EXOSOME AND VARIOUS USES THEREOF

Abstract

The present invention relates to a novel exosome comprising glucosamine, glucosamine derivatives, or salts thereof. The exosome provided by the present invention has an excellent anti-inflammatory effect, and thus can effectively prevent, ameliorate, or treat various inflammatory diseases. The exosome also has an excellent effect of treating intestinal diseases, improving the skin, treating wounds, or treating hair loss.

Claims

1. An exosome comprising glucosamine, a glucosamine derivative or a salt thereof, wherein the exosome is isolated from prokaryotic cells or eukaryotic cells.

2. The exosome according to claim 1, wherein the exosome is isolated from culture medium of prokaryotic cells or eukaryotic cells.

3. The exosome according to claim 1, wherein the exosome is isolated from one or more microorganisms selected from the group consisting of Lactobacillus genus, Leuconostoc genus, Pediococcus genus, Lactococcus genus, Streptococcus genus, Aerococcus genus, Carnobacterium genus, Enterococcus genus, Oenococcus genus, Bifidobacterium genus, Sporolactobacillus genus, Tetragenococcus genus, Vagococcus genus, Weisella, Propionibacterium genus, Pediococcus genus, Staphylococcus genus, Peptostrepococcus genus, Bacillus genus, Micrococcus genus, Listeria genus, Escherichia genus, Debaromyces genus, Candida genus, Pichia genus, Torulopsis genus, Aspergillus genus, Rhizopus genus, Mucor genus, Penicillium genus, Bacteroides genus, Clostridium genus, Fusobacterium genus and Melissococcus genus.

4. The exosome according to claim 1, wherein the glucosamine derivative is characterized in that hydrogen of at least one hydroxyl group in glucosamine is substituted with an acyl or alkyl group.

5. The exosome according to claim 1, wherein the glucosamine, glucosamine derivative or salt thereof is included in an amount of more than 0 and 20% by weight (“wt. %”) or less.

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. A pharmaceutical composition for anti-inflammation, comprising the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1.

11. The pharmaceutical composition for anti-inflammation according to claim 10, wherein the composition is used to prevent or treat inflammatory diseases.

12. The pharmaceutical composition for anti-inflammation according to claim 11, wherein the inflammatory diseases are any one of atopy, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis media, pharyngitis, tonsillitis, cystitis, nephritis, pelvicitis, inflammatory bowel disease, ankylosing spondylitis, systemic lupus erythematosus (SLE), atherosclerosis, asthma, arteriosclerosis, edema, rheumatoid arthritis, delayed allergy (type IV allergy), transplant rejection, graft versus host disease, autoimmune encephalomyelitis, multiple sclerosis, cystic fibrosis, diabetic retinopathy, rhinitis, ischemic-reperfusion injury, vascular restenosis, glomerulonephritis and gastrointestinal allergy.

13. A pharmaceutical composition for prevention or treatment of intestinal diseases, comprising the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient.

14. The pharmaceutical composition according to claim 13, wherein the intestinal diseases are any one of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), traveler's diarrhea, constipation, acute diarrhea, enteritis, gastroenteritis, abdominal pain or abdominal distension.

15. The pharmaceutical composition according to claim 14, wherein the inflammatory bowel disease is Crohn's disease, intestinal lesions involved in Behcet's disease, ulcerative colitis, hemorrhagic rectal ulcer and ileal cystitis.

16. A food composition for improving intestinal function or promoting bowel movement, comprising the exosome which includes glucosamine, a glucosamine derivate or a salt thereof according to claim 1, as an active ingredient.

17. A pharmaceutical composition for prevention or treatment of hair loss, comprising the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient.

18. A cosmetic composition for skin improvement, comprising the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient.

19. The cosmetic composition according to claim 18, wherein the skin improvement includes skin whitening, wrinkle improvement, elasticity enhancement, skin regeneration, skin moisturizing, anti-aging, alleviation of skin irritation, acne prevention or improvement, or atopic prevention or improvement effects.

20. A pharmaceutical composition for prevention or treatment of wounds, comprising the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient.

21. (canceled)

22. (canceled)

23. A method for prevention or treatment of inflammatory diseases, comprising administering the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient to a subject suffering from inflammatory disease.

24. (canceled)

25. A method for prevention or treatment of intestinal diseases, comprising administering the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient to a subject suffering from the intestinal disease.

26. (canceled)

27. A method for prevention or treatment of hair loss, comprising administering the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient to a subject having hair loss.

28. (canceled)

29. A method for prevention or treatment of wounds, comprising administering the exosome which includes glucosamine, a glucosamine derivative or a salt thereof according to claim 1, as an active ingredient to a wounded subject.

30. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0121] FIG. 1 is a graph illustrating results of measuring a change in the expression level of inflammatory cytokine (IL-2) as compared to the control, after treatment of Jurkat cells in Experimental Example 1 with the exosome containing glucosamine isolated from each microorganism strain.

[0122] FIG. 2 is a graph illustrating results of measuring a change in the expression level of inflammatory cytokine (IL-2), as compared to the control, after treatment of Jurkat cells in Experimental Example 2 with a Lactobacillus kefirgranum conditioned medium, the exosome containing glucosamine separated from the conditioned medium or the conditioned medium from which the exosome was removed, respectively.

[0123] FIG. 3 is a graph illustrating results of measuring a change in the expression level of inflammatory cytokines (IL-2) as compared to the control, after treatment of Jurkat cells in Experimental Example 3 with the exosome containing glucosamine, liposome containing glucosamine or glucosamine alone, respectively.

[0124] FIGS. 4A to 4D illustrate cross-sectional photographs following H & E staining of colon tissues of mice after treatment of a colitis mouse model in Experimental Example 4 with the exosome containing glucosamine.

[0125] FIG. 5 is a graph illustrating scores evaluated by observing a length and appearance of the large intestine, after treatment of the colitis mouse model in Experimental Example 4 with the exosome containing glucosamine.

[0126] FIG. 6 is a graph illustrating results of evaluating homeostasis of feces, after treatment of the colitis mouse model in Experimental Example 4 with the exosome containing glucosamine.

[0127] FIG. 7 is a graph illustrating results of evaluating a degree of bleeding in feces, after treatment of the colitis mouse model in Experimental Example 4 with the exosome containing glucosamine.

[0128] FIG. 8 is a graph illustrating results of measuring a change in the body weight of mice, after treatment of the colitis mouse model in Experimental Example 4 with the exosome containing glucosamine.

[0129] FIG. 9 is a graph illustrating results of measuring a change in the cell proliferation rate, after treatment of the keratinocytes (HaCaT) in Experimental Example 5 with the exosome containing glucosamine by concentration.

[0130] FIG. 10 is a graph illustrating results of measuring a change in the cell proliferation rate, after treatment of skin fibroblasts (HDF) in Experimental Example 5 with the exosome containing glucosamine by concentration.

[0131] FIG. 11 is a graph illustrating results of measuring a change in the cell proliferation rate, after treatment of the adipose-derived stem cells (ASC) in Experimental Example 6 with the exosome containing glucosamine.

MODE FOR CARRYING OUT INVENTION

[0132] The present invention relates to an exosome including glucosamine, a glucosamine derivative or a salt thereof.

[0133] Hereinafter, the present invention will be described in more detail by means of the following examples. These examples are proposed only for describing the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

EXAMPLES

[Example 1] Confirmation of Glucosamine in Exosome

[0134] 1. Isolation of Exosome

[0135] Lactobacillus kefirgranum, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Weissella koreensis, Tetragenococcus halophilus and Bifidobacterium animalis strains were inoculated in MRS medium (including 10 g of proteose peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of D-glucose, 1 ml of Tween 80, 2 g of K2HPO4, 5 g of sodium acetate, 2 g of diammonium hydrogencitrate, 0.2 g of MgSO4.7H2O, 0.2 g of MnSO4.H2O and 1 L of distilled water, pH 6.2-6.5), followed by stationary incubation at 30° C. and 0 rpm. Further, Staphylococcus aureus, Listeria monocytogens and Escherichia coli were inoculated in LB medium (including 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl and 1 L of distilled water, pH 6.8-7.2), followed by stationary incubation at 30° C. and 0 rpm.

[0136] The culture medium thus obtained for each strain was centrifuged at 300 g and 4° C. for 10 minutes, the supernatant was centrifuged at 1,200 g and 4° C. for 20 minutes, and then the supernatant was taken again at 10,000 g and 4° C. for 30 minutes. After centrifugation, the supernatant was taken and centrifuged at 110,000 g and 4° C. for 1 hour and 10 minutes through an ultracentrifuge, and then the supernatant was removed to suspend the precipitate with PBS to obtain exosome.

[0137] 2. Measurement of Glucosamine Content

[0138] As described above, a content of glucosamine in the exosome separated from each strain was measured by hexosamine assay. Specifically, the content of hexamine was measured by a 3-methyl-2-benzothia-zolinone hydrazone hydrochloride (MBTH) method (Frederik et al., 2000). That is, 100 μl of 1M HCl solution was added to a cap tube containing 100 μl of the sample, heated in a Reacti-thermometer at 110° C. for 2 hours, then cooled, and 400 μl of 2.5% sodium nitrate was added thereto, followed by leaving the same at room temperature for 15 minutes. Then, 200 μl of 4M ammonium sulfamate was added to the mixed solution, left at room temperature for 5 minutes, and 200 μl of 0.25% MBTH was further added and incubated in a water bath at 37° C. for 30 minutes. Thereafter, 200 μl of 1.0% ferric chloride was added and incubated in a water bath at 37° C. for 5 minutes, followed by cooling the same to measure absorbance at 650 nm. A content of hexamine was determined according to the standard curve prepared with D-glucosamine, and results of the content of glucosamine in each exosome are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Content of glucosamine Item (wt. %) P1 Control — P2 Lactobacillus kefirgranum 2.61 P3 Lactobacillus kefiranofaciens 2.50 P4 Lactobacillus kefiri 2.76 P5 Staphylococcus aureus 1.85 P6 Listeria monocytogens 0.75 P7 Escherichia coli Substantially not detected P8 Weissella koreensis Substantially not detected P9 Tetragenococcus halophilus Substantially not detected P10 Bifidobacterium animalis 2.74 P11 Cyclosporin A —

[0139] As shown in Table 1, it was determined that glucosamine was present in the exosomes of P2 to P6 and P10, respectively, but glucosamine was not substantially detected in the exosomes of P7 to P9.

[Experimental Example 1] Confirmation of Anti-Inflammatory Effect of Exosome Containing Glucosamine (1)

[0140] After inoculating Jurkat cells with 4×104 cells/well in each well of a 96-well plate, each exosome prepared in Example 1 was used in an amount of 100 μl/well to treat the cells, and cultured for 24 hours. Thereafter, an expression level of cytokine IL-2 related to proliferation and differentiation of T cells acting as a cause of inflammatory bowel disease was measured by ELISA, and a change in the expression level of IL-2 compared to the untreated control was determined. Results thereof are shown in Table 2 below and FIG. 1. However, as a positive control (P11), cyclosporin A, which is used as a therapeutic agent for inflammatory bowel disease, was used in the same amount as the exosome to treat the cells.

TABLE-US-00002 TABLE 2 Item IL-2 (%) P1 100 P2 48 P3 61 P4 57 P5 50 P6 67 P7 106 P8 106 P9 98 P10 48 P11 71

[0141] As shown in Table 2 above and FIG. 1, it was confirmed that IL-2 expression level was significantly reduced when treated with the exosomes (P2 to P6 and P10) containing glucosamine according to the present invention, and it was confirmed that the exosome of the present invention has superior results over cyclosporin A used as a typical therapeutic agent for inflammatory bowel disease.

[0142] As such, it can be seen that the exosome containing glucosamine according to the present invention may be used as an anti-inflammatory agent.

[Experimental Example 2] Confirmation of Anti-Inflammatory Effect of Exosome Containing Glucosamine (2)

[0143] In order to demonstrate whether the anti-inflammatory effect of Experimental Example 1 is based on effects of the exosome containing glucosamine only, the culture medium (P2-W) of Lactobacillus kefirgranum in Example 1, the exosome (P2-V) containing glucosamine separated from the culture medium, and the culture medium (P2-R) from which the exosome was removed, respectively, were used to treat Jurkat cells in the same manner as in Experimental Example 1, followed by measuring the expression level of IL-2. Results thereof are shown in Table 3 below and FIG. 2.

TABLE-US-00003 TABLE 3 Item IL-2 (%) P1 Control 100 P2-W Lactobacillus kefirgranum conditioned medium 62 P2-V Exosome containing Lactobacillus kefirgranum- 53 derived glucosamine P2-R Lactobacillus kefirgranum conditioned medium 88 with exosome removed

[0144] As shown in Table 3 above and FIG. 2, the IL expression level was significantly reduced when the exosome containing glucosamine (P2-V) or the culture medium (P2-W) including the same was used for treatment according to the present invention. In particular, for the exosome containing glucosamine (P2-V), it was found that a degree of reduction in the IL-2 expression level was superior over that of the culture medium. However, it can be confirmed that IL-2 expression reduction effects are insignificant when treated with the culture medium (P2-R) from which the exosome was removed.

[0145] As such, it can be seen that the exosome containing glucosamine according to the present invention may be used as an anti-inflammatory agent.

[Experimental Example 3] Confirmation of Anti-Inflammatory Effect of Exosomes Containing Glucosamine (3)

[0146] In order to demonstrate whether the anti-inflammatory effects of Experimental Examples 1 and 2 are not due to glucosamine alone but to the effect of the exosome containing glucosamine, the exosome containing glucosamine, which was separated from the culture medium of Lactobacillus kefirgranum in Example 1, liposome containing glucosamine (Lipofectamine 2000) and glucosamine itself, respectively, were used at a dose of 4 μg/ml to treat Jurkat cells as in Experimental Example 1 above, followed by measuring the IL-2 expression level. Results thereof are shown in Table 4 below and FIG. 3.

TABLE-US-00004 TABLE 4 Item IL-2 (%) P1 Control 100 P2 Exosome containing Lactobacillus kefirgranum- 62 derived glucosamine L1 Liposome containing glucosamine 89 G1 Glucosamine 94

[0147] As shown in Table 4 above and FIG. 3, when the exosome (P2) containing glucosamine was used for treatment according to the present invention, the IL-2 expression level was significantly reduced. However, when the liposome (L1) containing glucosamine or glucosamine itself (G1) was directly used for treatment, it could be confirmed that there was almost no effect of reducing IL-2 expression.

[0148] As such, it can be seen that the exosome containing glucosamine according to the present invention may be used as an anti-inflammatory agent.

[Experimental Example 4] Confirmation of Therapeutic Effects of Glucosamine-Containing Exosome Against Intestinal Disease

[0149] In BALB/c mice, colitis was induced with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Specifically, after anesthetizing each mouse with ether, a solution in which 2.5 g of 2,4,6-trinitrobenzene sulfonic acid (TNBS) is mixed with 50% ethanol was administered into the large intestine through the anus using a syringe of 1 ml dose having a round end by 0.1 ml, followed by holding the mouse vertically for 30 seconds to cause inflammation. On the other hand, 0.1 ml of physiological saline was orally administered to the normal group. Thereafter, the exosome (P2) containing glucosamine obtained in Example 1 was administered orally at a dose of 600 μg/mouse (30 mg/kg) once a day for 10 days from the next day. After the mouse was subjected to euthanasia with suffocation using carbon dioxide the day after sample administration was completed, the large intestine was cut between the cecum and the site immediately before the anus. On the other hand, as a positive control, prednisolone (P12) as a therapeutic agent for colitis was administered orally in an amount of 2 mg/kg.

[0150] (1) Tissue Appearance Assessment

[0151] Cross-sectional photographs, which are taken after H & E staining of the extracted large intestine tissue, are shown in FIGS. 4A to 4D. In addition, length and appearance of the extracted bowel tissue were observed to score according to the criteria stated in Table 5 below (Hollenbach et al., 2005 criteria for colitis). Results thereof are shown in FIG. 5.

TABLE-US-00005 TABLE 5 Macroscopic score Standard 0 No ulcers and inflammation found 1 Redness without bleeding found 2 Congestive ulcers found 3 Ulcer and inflammation found in only one site 4 Ulcers and inflammation found in two or more sites 5 Ulcer enlarged to 2 cm or more

[0152] As shown in FIG. 4B, severe epithelial necrosis, bleeding, inflammatory cell infiltration, edema and ulcers were observed in the large intestine of the colitis mouse model. However, in the case of administering the exosome (FIG. 4C, P2) containing glucosamine according to the present invention, it could be confirmed that symptoms of colitis were significantly alleviated at a level equal to or higher than the case of administering the prednisolone (FIG. 4D, P12).

[0153] Further, as shown in FIG. 5, when the exosome (P2) containing glucosamine according to the present invention was administered to the colitis mouse model, bleeding in the bowel was reduced and it was confirmed that such therapeutic effects are substantially equal to the case of administering the positive control, that is, the prednisolone in a large amount of 2 mg/kg.

[0154] (2) DAI Evaluation

[0155] DAI (disease activity index) is a method for scoring symptoms due to TNBS causing acute colitis, which begins at a time of providing the exosome of the present invention, in order to measure a concentration and check color of the stool at the same time every day during scoring. Stool consistency, stool bleeding and the weight of the mouse were measured, and the measured results are shown in FIGS. 6 to 8, respectively. In this case, the evaluation criteria of DAI are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Stool consistency Stool bleeding 0 Formed 0 Normal color 2 Loose stool 2 Fecal occult blood test positive 4 Diarrhea 4 Gross bleeding

[0156] As shown in FIGS. 6 to 8, when the exosome (P2) containing glucosamine according to the present invention was administered to the colitis mouse model, it could be confirmed that the DAI value was lowered to a level equal to or higher than that of the prednisolone (P12) as a positive control, thereby improving the bowel movement, while not exhibiting a significant change in the body weight of the mouse.

[0157] As such, it can be seen that the exosome containing glucosamine according to the present invention may be used as a therapeutic agent for intestinal diseases.

[Experimental Example 5] Effect of Promoting Proliferation of Skin Cells by Exosomes Containing Glucosamine

[0158] After inoculating 3,000 keratinocytes (human keratinocyte cell line, HaCaT) and 3,000 human fibroblasts (HDF) into DMEM medium containing 1% FBS, as shown in Table 7 below, the cell line was treated with the exosome of the present invention, EGF or FBS. After 48 hours, a change in cell viability was measured, and results thereof are shown in Tables 8 and 9 below and FIGS. 9 and 10.

TABLE-US-00007 TABLE 7 Item Treatment group Concentration P1 Negative control — P2(L) P2 exosome 1 × 10{circumflex over ( )}8/ml P2(M) P2 exosome 1 × 10{circumflex over ( )}9/ml P2(H) P2 exosome  1 × 10{circumflex over ( )}10/ml E1 EGF 500 ng/ml

TABLE-US-00008 TABLE 8 Item HaCaT proliferation rate (%) P1 93.84 P2(L) 107.25 P2(M) 114.20 P2(H) 116.68 E1 160.38

TABLE-US-00009 TABLE 9 Item HDF proliferation rate (%) P1 97.18 P2(L) 101.39 P2(M) 120.95 P2(H) 134.62 E1 143.04

[0159] As shown in Tables 8 and 9 above and FIGS. 9 and 10, when the exosome according to the present invention was used to treat keratinocytes (HaCaT) and skin fibroblasts (HDF), it could be seen that the cell proliferation rate was significantly increased depending on the concentration.

[Experimental Example 6] Effect of Promoting Proliferation of Stem Cells by Exosome Containing Glucosamine

[0160] Adipocytes derived stem cells (ADCs) were cultured in DMEM medium (Invitrogen) containing 10% fetal calf serum and 1% penicillin/streptomycin at 37° C. and 5% CO2 conditions, and the medium was changed every 3 days. After culturing for 24 hours, the cells were treated under the conditions shown in Table 10 below, and further cultured for 24 hours. After removing the treated material from each cell and dispensing the CCK-8 solution thereto, the cells were incubated at 37° C. in a CO2 incubator for 2 hours, and absorbance was measured at 450 nm. Results thereof are shown in Table 11 below and FIG. 11.

TABLE-US-00010 TABLE 10 Item Treatment group Concentration CONT Negative control — P2(L) P2 exosome 1 × 10{circumflex over ( )}(n − 1)/ml P2(M) P2 exosome 1 × 10{circumflex over ( )}(n)/ml  .sup.  P2(H) P2 exosome 1 × 10{circumflex over ( )}(n + 1)/ml F1 FBS 10 wt. % in medium

TABLE-US-00011 TABLE 11 Item ASC proliferation rate (%) P1 99.61 P2(L) 136.54 P2(M) 140.91 P2(H) 174.44 E1 186.10

[0161] As shown in Table 11 above and FIG. 11, when adipose-derived stem cells (ASC) were treated with the exosome according to the present invention, it could be confirmed that the cell proliferation rate was significantly increased depending on the concentration, and the degree of increase was substantially equal to the level when treated with fetal bovine serum (FBS).

[Experimental Example 7] Effect of Promoting Proliferation of Dermal Papilla Cells by Exosome Containing Glucosamine

[0162] After dispensing a serum medium with 10,000 cells/well of papillary cells (Dermal papilla cells, DPC) in a 96-well microplate, the cells were incubated overnight at 37° C. in a CO2 incubator. After incubation, the papillary cells were treated as shown in Table 12 below, and further cultured for 24 hours. After removing the treated material from each cell and dispensing the CCK-8 solution thereto, the cells were incubated in the CO2 incubator at 37° C. for 2 hours, and absorbance was measured at 450 nm. The measured results are shown in Table 13 below.

TABLE-US-00012 TABLE 12 Item Treatment group Concentration P1 Negative control — P2(M) P2 exosome 1 × 10{circumflex over ( )}(n)/ml  .sup.  P2(H) P2 exosome 1 × 10{circumflex over ( )}(n + 1)/ml M1 Minoxidil 10 μM

TABLE-US-00013 TABLE 13 Item DPC proliferation rate (%) P1 97.98 P2(M) 108.86 P2(H) 126.75 M1 118.97

[0163] As shown in Table 13 above, when the exosome according to the present invention was used to treat dermal papilla cells (DPC), it could be confirmed that the cell proliferation rate was significantly increased depending on the concentration, and the degree of increase was higher than the case of treatment using minoxidil, which is commonly used as a therapeutic agent for hair growth.

[0164] As such, it can be seen that the exosome containing glucosamine according to the present invention may be used as a therapeutic agent for hair loss.

INDUSTRIAL APPLICABILITY

[0165] The present invention provides novel exosomes and various uses thereof.