COMPOSITION FOR PREVENTING OR TREATING INFLAMMATORY DISEASES OF DIGESTIVE SYSTEM OR COLITIS, COMPRISING REOVIRUS AS ACTIVE INGREDIENT

Abstract

The present disclosure relates to a composition for preventing, alleviating, or treating an inflammatory disease of the digestive system, particularly, colitis, comprising a reovirus or a variant thereof as an active ingredient.

Claims

1. A method for preventing, alleviating, or treating colitis, the method comprising administering a composition comprising a wild-type reovirus or a variant thereof as an active ingredient to a subject having or suffering from colitis.

2. The method of claim 1, wherein the wild-type reovirus is Type 1 (Lang), Type 2 (Jones) or Type 3 (Dearing and Abney) wild-type reovirus or a variant thereof.

3. The method of claim 2, wherein the variant is RP116.

4. The method of claim 1, wherein the reovirus variant is a heat-inactivated variant of the wild-type reovirus.

5. The method of claim 1, wherein the colitis is selected from the group consisting of acute colitis, bacterial colitis, viral colitis, pseudomembranous colitis, inflammatory bowel disease, chronic colitis, ulcerative colitis, Crohn's disease, ischemic colitis, Behcet's colitis, drug-induced colitis, collagenous colitis, lymphocytic colitis, and radiation colitis.

6. The method of claim 1, wherein the composition is formulated for oral administration.

7. The method of claim 1, wherein an effective dose of the active ingredient 10.sup.7 to 10.sup.9 pfu.

8. The method of claim 1, wherein the composition provides the effects of a decrease in disease activity index (DAI), an increase in body weight or an increase in colon length.

9. (canceled)

10. The method of claim 1, wherein the composition is a pharmaceutical composition or a health functional food composition.

Description

DESCRIPTION OF DRAWINGS

[0073] FIG. 1 illustrates a series of processes of orally administering a reovirus-containing composition to a DSS-induced animal model.

[0074] FIG. 2 illustrates the results of measuring cell viability after heat-inactivation of a wild-type reovirus (RC402).

[0075] FIG. 3 shows a disease activity index (DAI) reduction effect exhibited by the composition of the present disclosure including a reovirus.

[0076] FIG. 4 shows the effect of suppressing the body weight loss in a subject exhibited by the composition of the present disclosure including a reovirus.

[0077] FIG. 5 shows the effect of suppressing the decrease in colon length in a subject exhibited by the composition of the present disclosure including a reovirus.

MODES OF THE INVENTION

[0078] Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for describing the present disclosure, and it will be obvious to those skilled in the art that the scope of the present disclosure is not interpreted to be limited by these examples.

Example 1. Heat-Inactivation of Reovirus

[0079] To prepare a reovirus for use in the present disclosure, the reovirus was obtained by heat-inactivating a wild-type reovirus.

[0080] Specifically, wild-type reovirus human type 3 (Dearing) was purchased from the American Type Culture Collection (ATCC, VR-824). The reovirus was first isolated from the stool of a child with diarrhea around 1955 and was donated by Dr. Albert Sabin. A BHK21 cell line, HEK293 or L929 cells were inoculated with virus at a multiplicity of infection (MOI) of about 1 to 10 and cultured at 37° C. for 48 to 72 hours to propagate a wild-type reovirus. Thereafter, the cells were freeze-thawed, crushed, and then centrifuged to remove cell debris, and the titer of the liberated reovirus was confirmed using the plaque assay method using a L929 monolayer and the cells were used while being stored at −80° C.

[0081] After the obtained wild-type reovirus was heat-inactivated by being stiffed in a water bath at about 60° C. for about 20 minutes, the ability of the virus to kill L929 cells was confirmed using a wst1 assay (Roche kit), and the inactivation efficiency was confirmed through this.

[0082] As a result, a heat-inactivated reovirus (referred to as ‘heat-inactivated RC402’) with a titer of 10.sup.8 PFU/100 μl PBS was obtained.

Example 2. Construction of DSS-Induced Colitis Animal Model

[0083] The animals used in this experiment were 7- to 8-week-old female BALB/C mice and purchased from Orient Bio Co., Ltd., Seoul, Korea. The mice were tested after an adaptation period of 7 days in an animal laboratory of the ViroCure Institute and the supply of water and feed to the mice was not limited during the adaptation period. The experimental animals were provided with a standardized environment, day and night were maintained at 12-hour intervals, and room temperature (23±2° C.) and humidity (50 to 55%) were maintained at appropriate levels.

[0084] As illustrated in FIG. 1, colitis was induced by changing the normal drinking water of the mice to 2.5% (w/v) dextran sulfate sodium salt (DSS) (MP Biomedicals, Cat #. 9011-18-1) and supplying it for 10 days (1 to 10 days), and thereafter, a recovery period of about 12 days (11 to 12 days) was given to the mice by changing the 2.5% (w/v) DSS to normal drinking water (filtered & autoclaved normal water) and colitis was again induced by re-supplying DSS (2.5%) from approximately day 29.

Example 3. Oral Administration of Composition Including Reovirus

[0085] Experiments were performed by dividing the mice used in this experiment into a normal control, a DSS-induced colitis group, and a reovirus-administered group.

[0086] The method of administering a reovirus to the colitis animal model was performed using an oral administration method.

[0087] A heat-inactivated reovirus (heat inactivated RC402) was administered orally at about 10.sup.8 PFU/100 μl PBS at 2-day intervals from approximately day 23 and then administered daily from approximately day 29.

Example 4. Analysis of Therapeutic Effect of Composition Including Reovirus Using DSS-Induced Colitis Animal Model

[0088] From approximately day 29, experiments were performed while observing the presence or absence of colitis and the degree of symptoms by measuring the body weight and observing the stool and anal conditions. On approximately day 36, changes in mouse colon length were measured.

[0089] 4-1. Visual Evaluation by Measurement of Disease Activity Index (DAI)

[0090] In order to measure the colitis intensity of colitis animal model mice treated with oral administration of the composition including the reovirus of Example 3, a disease activity index (DAI) was measured by confirming the changes in body weight, stool consistency, and the presence or absence of bloody stool, which is observed with the naked eye in the stool or anus daily for 7 days according to the disease activity index (DAI) grades in the following Table 1.

TABLE-US-00001 TABLE 1 Presence or absence of Grade Reduction in body weight Stool consistency bloody stool 0 No change in body weight Normal Normal 1 1 to 5% — — 2 5 to 10% Loose stool Occult blood 3 10 to 20% — — 4 >20% Diarrhea Bloody stool observed with naked eye

[0091] In the DSS-administered mice, loose stool and visually observed bloody stool began to be seen from day 4, and diarrhea and bloody stool could be observed in all mice on day 7.

[0092] In the reovirus administration group using the oral administration method, diarrhea was relatively reduced and bloody stool was ameliorated, so it was confirmed that the disease activity index was decreased during the administration period compared to the DSS colitis group (FIG. 3).

[0093] From this result, it can be seen that not only a wild-type reovirus, but also a reovirus variant, such as a heat-inactivated reovirus, can provide a significant effect in treating and ameliorating colitis and a bowel disease accompanied by inflammation through oral administration, particularly without inducing observable side effects over a long period of time.

[0094] 4-2. Measurement of Changes in Body Weight and Colon Length

[0095] The colitis animal models exhibits symptoms of a decrease in body weight and a decrease in colon length compared to the normal control. As a result of oral administration of a composition including the reovirus of the present disclosure to the colitis animal model, it was confirmed that body weight loss was suppressed and a decrease in colon length was significantly suppressed (FIGS. 4 to 5).

[0096] Through this, it can be seen that not only a wild-type reovirus, but also a reovirus variant, such as a heat-inactivated reovirus, can provide a significant effect in treating and ameliorating a disease of the digestive system accompanied by inflammation, including colitis, upon oral administration by suppressing the body weight loss and suppressing the decrease in colon length, particularly without inducing observable side effects over a long period of time.

Example 5. Statistical Analysis

[0097] Statistical analysis was performed using GraphPad Prism 6. Among the One-way ANOVA tests, the Dunnett's multiple comparison test was used to compare differences between the normal control, the DSS-induced colitis group, and the reovirus-administered group. Differences with P-values less than 0.05 were considered statistically significant. Data is presented as mean and SEM.

[0098] From the foregoing description, it will be understood by those skilled in the art to which the present disclosure pertains that the present disclosure can be implemented in other concrete forms without modifying the technical spirit or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are only exemplary in all aspects and are not restrictive. The scope of the present disclosure is represented by the claims to be described below rather than the detailed description, and it should be interpreted that the meaning and scope of the claims and all the changes or modified forms derived from the equivalent concepts thereto fall within the scope of the present disclosure.

INDUSTRIAL APPLICABILITY

[0099] A composition including the reovirus according to the present disclosure or a variant thereof is expected to be usefully utilized in treating and ameliorating a disease of the digestive system accompanied by inflammation, including colitis, particularly without inducing observable side effects over a long period of time.