Composition for preventing radiation-induced inflammatory diseases, and use thereof

Abstract

The present invention relates to a composition capable of preventing inflammatory diseases caused by radiation therapy, or use thereof. Tranilast of the present invention can reduce rectal wall infiltration of mast cells and provide an anti-inflammatory effect against radiation-induced colitis, and thus radiation-induced inflammatory diseases can be effectively prevented when the tranilast of the present invention is pre-administered to patients in which radiation-induced inflammatory diseases have not yet occurred, such as patients receiving pelvic radiation therapy.

Description

Composition for preventing radiation-induced inflammatory disease and use thereof

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2023-0112617, filed on August 28, 2023, the entire disclosure of which is incorporated herein by reference.

[0002]

[0003] The present invention relates to a composition capable of preventing inflammatory diseases caused by radiation therapy and its use.

[0004]

[0005] The present invention was completed with the support of the Ministry of Science and ICT of the Republic of Korea under Project No. 2021R1A2C1093096 (1711181491).

[0006]

[0007] Radiation therapy (RT) is used for curative or palliative purposes in various types of malignancies, including gastrointestinal (GI), gynecological, and urological tumors. Radiation-induced colitis (RIC) is a common complication of GI diseases. Acute symptoms of RIC include bleeding, pain, urinary incontinence, bowel changes, and diarrhea. While acute RIC is self-limiting, chronic RIC can lead to thrombosis, submucosal fibrosis, endothelial degeneration, and ischemia, leading to proctitis.

[0008] Deep cystic colitis (CCP, Colitis cystica profunda) is a rare complication of chronic rectal inflammation (RIC), primarily occurring after radiotherapy for pelvic malignancies. It is an abnormal displacement of the normal mucosal crypts, sometimes containing mucus pools, within the submucosal and muscularis propria. Despite the absence of cytologic atypia, CCP can be mistaken for malignancy due to the ectopic localization of mucosal glands. It has been observed incidentally during endoscopic examinations in patients with isolated rectal syndrome, constipation, trauma, rectal prolapse, post-infectious enteritis, inflammatory bowel disease (IBD), malignancy, and post-radiation injury. Although the severity of acute RIC has been established to influence the later outcome of chronic RIC, the etiology remains unclear.

[0009] Mast cells play a key role in the pathogenesis of both acute and chronic RIC. Mast cells also secrete proteolytic enzymes, which play a crucial role in the invasion of malignant tumors into adjacent supporting tissue (stroma) during tumor progression. Mast cells are typically found at the so-called "invasive front."

[0010] Tranilast (N-(3',4'-dimethoxycinnamic)-anthranilic acid) is a membrane stabilizer widely used in the treatment of inflammatory diseases and can inhibit the release of histamine and other chemicals. However, the therapeutic role of tranilast in RIC has not been elucidated.

[0011]

[0012] [Prior Art Literature]

[0013] [Patent Document]

[0014] (Patent Document 1) KR 10-2020-0127547 (2020-09-29)

[0015]

[0016] The present inventors have made efforts to provide a preparation capable of preventing radiation-induced inflammatory diseases by pre-treating subjects who have not yet received radiation therapy, and as a result, have confirmed the correlation between the effect of tranilast treatment on mast cell infiltration and MMP-9 (matrix metalloproteinase-9) expression in radiation-induced colitis, and completed the present invention.

[0017]

[0018] Accordingly, the purpose of the present invention is to provide a composition or use thereof capable of preventing inflammatory diseases caused by radiation therapy.

[0019]

[0020] The present invention provides a pharmaceutical composition for preventing radiation-induced inflammatory diseases, comprising a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof:

[0021] [Chemical Formula 1]

[0022]

[0023] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1] is tranilast.

[0024] According to a preferred embodiment of the present invention, the radiation-induced means induced by pelvic radiation therapy.

[0025] According to a preferred embodiment of the present invention, the radiation-induced inflammatory disease is at least one selected from the group consisting of inflammation of mucosal tissues, such as radiation-induced colitis, proctitis, dermatitis, and cystitis.

[0026] According to a preferred embodiment of the present invention, the composition reduces mast cell infiltration and expression of MMP-9 (matrix metalloproteinase-9).

[0027] In addition, the present invention provides a health functional food composition for preventing radiation-induced inflammatory diseases, comprising a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof:

[0028] [Chemical Formula 1]

[0029]

[0030] In addition, the present invention provides a method for preventing radiation-induced inflammatory disease, comprising administering to a subject at risk of radiation-induced inflammatory disease a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof:

[0031] [Chemical Formula 1]

[0032]

[0033] In addition, the present invention provides the use of a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof for preparing a composition for preventing radiation-induced inflammatory diseases:

[0034] [Chemical Formula 1]

[0035]

[0036]

[0037] Hereinafter, the present invention will be described in more detail.

[0038]

[0039] Mast cells are the primary cells responsible for acute and chronic colitis caused by radiation-induced colitis (RIC). We investigated whether pretreatment with tranilast, a mast cell inhibitor, could alleviate RIC. A total of 23 Sprague Dawley (SD) rats were randomly divided into three groups: a control group (n=5, C), an irradiated group (n=9, RG), and a tranilast-pretreated irradiated group (n=9, TG). Tranilast (100 mg / kg) was administered intraperitoneally to the TG group for 10 days, followed by a single gamma irradiation (20 gray dose) into the pelvis of the RG and TG group. Ten weeks after irradiation, the rats were euthanized. Rectal tissue samples were stained, and the total inflammation score (TIS) and mast cell count were assessed. The expression of MUC2, MUC5AC, and matrix metalloproteinase (MMP)-9 was also evaluated immunohistochemically.

[0040] The present study revealed that the scores of TIS and some components of TIS were significantly lower in the TG than in the RG (Fig. 2, Table 1). Specifically, three of the seven components of the TIS of RIC (EA, VA, CCP) were higher in the RG than in the TG. In particular, the number of CCPs (8 / 9) was significantly higher in the RG than in the TG (p<0.05) (Fig. 1). These data confirmed the role of tranilast in preventing RIC in patients receiving pelvic radiotherapy.

[0041] The present invention suggests that tranilast may play a protective role and reduce inflammation associated with RIC. In the present study, the total number of infiltrating mast cells was higher in the RG than in the TG, but the difference was not significant. Interestingly, the number of infiltrating mast cells did not differ between the area near the CCP (23.67 vs. 18.91) and other areas of the RG.

[0042] The role of mast cells in the pathogenesis of colitis remains unclear. However, the present study suggests that mast cells may play a role in the development of RIC. Previous studies have shown that mast cells contribute to inflammation by releasing various proinflammatory mediators, such as histamine, cytokines, and chemokines, but mast cells are not essential for the development of colonic inflammation in a mouse model. In the present study, the total number of infiltrating MMP-9-positive cells was significantly higher in RG than in TG (Figure 3). Therefore, we conclude that RG exhibited more severe inflammation than TG. However, there was no significant correlation between TIS and the number of infiltrating mast cells (Table 3).

[0043] Assessing mast cell function in vivo is complicated by the fact that mast cells can release mediators with antagonistic effects and also play a role in wound healing and fibrosis. The involvement of mast cells in patients with ulcerative colitis remains unclear, as studies have reported variable results regarding mast cell counts, which can be increased, decreased, or stable in active IBD and control biopsies. In the present study, the overall and some component scores of the RIC were higher in the RG than in the TG.

[0044] Tranilast, a tryptophan derivative, has been used as an anti-allergic agent since 1982 and is approved for use in Japan and Korea as a treatment for bronchial asthma. However, the inhibitory activity of tranilast against radiation-induced colitis (RIC) has not been previously investigated. We propose that treatment with tranilast, a mast cell stabilizer, can prevent chronic RIC, including CCP formation, and could be used as a pretreatment drug for pelvic RT.

[0045] Colitis cystica profunda (CCP) is a rare intestinal disease associated with various clinical manifestations, including rectal prolapse syndrome, IBD, trauma, chronic radiation enteropathy, and malignant disease. The extent of chronic radiation damage, including CCP, can vary depending on the total dose, field size, and dose fraction of the initial radiation exposure. However, the contribution of mast cells to acute or chronic radiation enteropathy remains unclear. Mast cells infiltrating the intestinal wall play a protective or exacerbating role in acute radiation-induced enteropathy, but mast cells have been established to be associated with chronic enteropathy. In the present study, we confirmed that rectal CCP was induced by a single dose of 20 Gy, and this was positively correlated with the number of infiltrating mast cells. The number and histologic score of CCP were lower in TG than in RG. CCP was detected in most cases (8 / 9) after a single dose of 20 Gy, a dose appropriate for CCP induction.

[0046] To confirm the malignant potential, we performed mucin staining on the crypts of CCPs. Mucin expression in the gastrointestinal tract varies depending on the anatomical location and type of cancer. During the progression of colorectal cancer, mucin expression changes from MUC2 in the normal colon to MUC5AC in the stomach. Therefore, abnormal ectopic expression of mucin has been used as a marker of malignant transformation. Since the mucin expressed in CCPs was exclusively MUC2, whereas the mucin in the gastric mucosa was positive for MUC5Ac, our results indicate that CCPs have little malignant potential (Fig. 6).

[0047] It is unclear whether mast cells play a carcinogenic or preventive role during cancer progression. Mast cells are frequently found to induce invasion of adjacent non-tumorous tissues in various types of cancer, including colorectal cancer, suggesting a pro-oncogenic role for mast cells by secreting enzymes and cytokines, such as tryptase and MMPs. If CCPs were a histologic variant of normal glands that invades the mucosa, similar to malignant invasion, a greater number of mast cells should be observed in the rectal wall of RG, particularly around CCPs. However, we did not find a greater number of mast cells in lesions surrounding CCPs than in other stromal tissues. This finding suggests that, unlike cancer invasion, mast cells do not directly play a role in the pathogenesis of CCPs.

[0048]

[0049] Accordingly, the present invention can provide a pharmaceutical composition for preventing radiation-induced inflammatory diseases, comprising a compound represented by [chemical formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof:

[0050] [Chemical Formula 1]

[0051]

[0052] The term "derivative" of the present invention is interchangeable with each other and may refer to a natural or unnatural modification of at least one position of a given molecule. For example, a derivative of a given compound or molecule may be modified by any of the following: addition of a functional group or atom, removal of a functional group or atom, or change of a functional group or atom into another functional group or atom (including an isotope).

[0053] The 'pharmaceutically acceptable salt' of the present invention may be an acid addition salt formed by a pharmaceutically acceptable free acid. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanedioates, aromatic acids, and aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, Contains terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

[0054] The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound represented by [Chemical Formula 1] in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, the acid addition salt can be prepared by evaporating the solvent or excess acid from the mixture and then drying it, or by suction filtration of the precipitated salt.

[0055] Additionally, pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are pharmaceutically suitable. The corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0056] The term 'prevention' in the present invention means any action that suppresses or delays the onset of a radiation-induced inflammatory disease or related disease by using the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0057] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1] may be tranilast.

[0058] According to a preferred embodiment of the present invention, the radiation-induced may mean induced by pelvic radiation therapy.

[0059] According to a preferred embodiment of the present invention, the radiation-induced inflammatory disease may be at least one selected from the group consisting of radiation-induced colitis, proctitis, dermatitis, and cystitis. Preferably, the radiation-induced inflammatory disease may be radiation-induced colitis.

[0060] According to a preferred embodiment of the present invention, the composition may reduce mast cell infiltration and expression of MMP-9 (matrix metalloproteinase-9).

[0061] The pharmaceutical composition of the present invention may be administered orally or parenterally in various dosage forms. When formulating the composition, it may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0062] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0063] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants in some cases, and anticoagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments, and preservatives may be additionally included.

[0064] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0065] The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be formulated in the form of an external skin injection; an intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, epidural, or intracerebrovascular injection; or a transdermal injection; according to a method known in the art.

[0066] In the case of the above injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally include isotonic agents such as sugars or sodium chloride.

[0067] Transdermal administration includes forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, and aerosols. Transdermal administration, as described above, refers to topically administering a pharmaceutical composition to the skin, thereby delivering an effective amount of the active ingredient contained in the pharmaceutical composition into the skin.

[0068] The composition of the present invention can be administered individually or in combination with other drugs, sequentially or simultaneously with conventional drugs, and in single or multiple doses. That is, the total effective dose of the composition of the present invention can be administered to a patient as a single dose, or as multiple doses administered over a long period of time using a fractionated treatment protocol. Considering all of the above factors, it is important to administer the amount that achieves maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0069] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0070] The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0071] The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. When the pharmaceutical composition for preventing radiation-induced inflammatory diseases of the present invention is used as an external preparation for skin, it may additionally contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations for skin, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, or lipid vesicles. In addition, the above ingredients can be introduced in amounts commonly used in the field of dermatology.

[0072] When the pharmaceutical composition for preventing radiation-induced inflammatory diseases of the present invention is provided as an external skin preparation, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

[0073]

[0074] In addition, the present invention can provide a health functional food composition for preventing radiation-induced inflammatory diseases, comprising a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof:

[0075] [Chemical Formula 1]

[0076]

[0077] Since the above derivatives, pharmaceutically acceptable salts and prophylaxis are the same as the concepts used in the above pharmaceutical composition, the descriptions are replaced by their descriptions.

[0078] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1] may be tranilast.

[0079] According to a preferred embodiment of the present invention, the radiation-induced may mean induced by pelvic radiation therapy.

[0080] According to a preferred embodiment of the present invention, the radiation-induced inflammatory disease may be at least one selected from the group consisting of radiation-induced colitis, proctitis, dermatitis, and cystitis. Preferably, the radiation-induced inflammatory disease may be radiation-induced colitis.

[0081] According to a preferred embodiment of the present invention, the composition may reduce mast cell infiltration and expression of MMP-9 (matrix metalloproteinase-9).

[0082] The food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible plant oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), etc., which can be manufactured by adding the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof. In addition, as a nutritional supplement, it is not limited to this, but can be manufactured by adding the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof to capsules, tablets, pills, etc. In addition, as a health functional food, it is not limited to this, but for example, the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof itself can be manufactured in the form of tea, juice, and drinks, and can be consumed by liquefying, granulating, encapsulating, or powdering so that it can be consumed (health drinks). In addition, in order to use the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof in the form of a food additive, it can be manufactured and used in the form of a powder or concentrate. In addition, the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof can be manufactured in the form of a composition by mixing it with a known active ingredient known to have a radiation-induced inflammatory disease prevention effect.

[0083] When the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof is used as a health beverage, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener may be a natural sweetener such as thaumatin and stevia extract; or a synthetic sweetener such as saccharin and aspartame. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

[0084] In addition, the compound represented by [Chemical Formula 1] of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof may be contained as an active ingredient of a health functional food composition for preventing radiation-induced inflammatory diseases, and the amount thereof is not particularly limited to an amount effective for achieving the radiation-induced inflammatory disease prevention effect, but is preferably 0.01 to 100 wt% based on the total weight of the entire composition. The health functional food composition of the present invention may be prepared by mixing the compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof with another active ingredient known to be effective against radiation-induced inflammatory diseases.

[0085] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the health functional food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination.

[0086]

[0087] In addition, the present invention can provide a method for preventing radiation-induced inflammatory disease, comprising a step of administering a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof to a subject at risk of radiation-induced inflammatory disease:

[0088] [Chemical Formula 1]

[0089]

[0090] Since the above derivatives, pharmaceutically acceptable salts, prevention and administration are the same as the concepts used in the above pharmaceutical composition, the descriptions are replaced by those descriptions.

[0091] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1] may be tranilast.

[0092] According to a preferred embodiment of the present invention, the radiation-induced may mean induced by pelvic radiation therapy.

[0093] According to a preferred embodiment of the present invention, the radiation-induced inflammatory disease may be at least one selected from the group consisting of radiation-induced colitis, proctitis, dermatitis, and cystitis. Preferably, the radiation-induced inflammatory disease may be radiation-induced colitis.

[0094] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof may reduce mast cell infiltration and MMP-9 (matrix metalloproteinase-9) expression.

[0095]

[0096] In addition, the present invention can provide a use of a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof for preparing a composition for preventing radiation-induced inflammatory diseases:

[0097] [Chemical Formula 1]

[0098]

[0099] Since the above derivatives, pharmaceutically acceptable salts and prophylaxis are the same as the concepts used in the above pharmaceutical composition, the descriptions are replaced by their descriptions.

[0100] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1] may be tranilast.

[0101] According to a preferred embodiment of the present invention, the radiation-induced may mean induced by pelvic radiation therapy.

[0102] According to a preferred embodiment of the present invention, the radiation-induced inflammatory disease may be at least one selected from the group consisting of radiation-induced colitis, proctitis, dermatitis, and cystitis. Preferably, the radiation-induced inflammatory disease may be radiation-induced colitis.

[0103] According to a preferred embodiment of the present invention, the compound represented by the above [chemical formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof may reduce mast cell infiltration and MMP-9 (matrix metalloproteinase-9) expression.

[0104]

[0105] Since the tranilast of the present invention can reduce the infiltration of mast cells into the rectal wall and provide an anti-inflammatory effect on radiation-induced colitis, when the tranilast of the present invention is pretreated in patients who have not yet developed radiation-induced inflammatory diseases, such as patients receiving pelvic radiotherapy, radiation-induced inflammatory diseases can be effectively prevented.

[0106]

[0107] Figure 1 shows a comparison of the semiquantitative total inflammation score (TIS) between the colitis mouse models of radiation (RG) and tranilast-pretreated groups (TG). The TIS in the tranilast-pretreated group was significantly lower in the TG (n = 9) than in the RG (n = 9) (mean ± SD: 11.33 ± 3.00 and 6.89 ± 2.89, respectively, P = 0.02).

[0108] Figure 2 shows representative photomicrographs of marked CCP formation (A) and no CCP formation (B); and representative photomicrographs of high mast cell infiltration (MCI) (C) and low MCI (D). Representative photomicrographs of marked CCP formation (A, arrow) compared with no CCP formation (B) (no. 7 A for the radiation group, no. 3 RG and B, TG for the tranilast-pretreated group). Representative photomicrographs of high mast cell infiltration (C, arrow) compared with low mast cell infiltration (D, arrow) (no. 9 C for the RG, no. 3 D for the TG; A & B: H&E staining, C & D: toluidine blue staining, A, B: 400×, C, D: 100×; scale bar = 100 μm; inset: 400×; scale bar = 25 μm).

[0109] Figure 3 shows a comparison of the number of infiltrating mast cells in the radiation (RG) and tranilast-pretreated groups (TG). The number of infiltrating mast cells was significantly higher in the RG (n = 9) than in the TG (n = 9) (median and range: 20, 3-54 and 6, 3-25, respectively, P = 0.034).

[0110] Figure 4 shows representative photomicrographs of densely infiltrated matrix metalloproteinase-9 (MMP-9)-positive cells in colitis tissue and sparsely infiltrated MMP-9-positive cells in colonic tissue ((A) colitis tissue sample from radiation group (RG); (B) tranilast pretreatment, TG). Representative photomicrographs of two high-power fields (HPF, 400×) of MMP-9-immunostained slides of colitis tissue samples from RG and TG were selected for manual counting of MMP-9 expression. Using manual counting, 210 MMP-9-positive cells were observed in one HPF in slide RG No. 7 (A, arrow), whereas only 12 MMP-9-positive cells were observed in one HPF in slide TG No. 9 (B, arrow) (A, B; toluidine blue staining; 400×; scale bar = 50 μm).

[0111] Figure 5 shows the correlation between the total inflammation score (TIS) and the number of matrix metalloproteinase-9 (MMP-9)-positive cells. Semiquantitatively, the TIS and the number of MMP-9-positive cells showed a good correlation (correlation coefficient r=0.56, P=0.025): excellent (>0.80), good (0.60-0.79), fair (0.40-0.59), or poor (<0.40).

[0112] Figure 6 shows the mucin expression profile of CCP lesions. Immunohistochemically, CCP lesions were strongly positive for MUC2 (A), but negative for MUC5AC (B) (A, B: 100×; scale bar = 50 μm).

[0113]

[0114] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0115]

[0116] [Example 1]

[0117] animal testing

[0118] Twenty-three 5-week-old female Sprague Dawley (SD) rats were obtained from Orientbio (Seongnam, Korea). The rats were maintained at 23±3°C and 50%±10% relative humidity for 7 days (12-h light / dark cycle) and fed standard animal diet. They were randomly divided into three groups: control (C, n=5), radiation therapy (RG, n=9), and tranilast-pretreated radiation therapy (TG, n=9). Tranilast (100 mg / kg), donated by JW Pharmaceutical (Seoul, Korea), was administered intraperitoneally for 10 days starting on the day of pelvic irradiation. Except for the control group, rats in the other groups (RG and TG) received a single dose of 20 Gy (Gy) into the pelvis (1.5 cm from the anus). Gamma rays were administered at a dose rate of 300 cGy / min using a 6 MV linear accelerator (Mevatron MX-2, Siemens, USA) under general anesthesia via intraperitoneal injection of 30 mg / kg Zoletil (Virbac Korea, Seoul, South Korea). Ten weeks after irradiation, all mice were anesthetized with pentobarbital (40 mg / kg) and euthanized. Animals were cared for in accordance with the laboratory animal regulations of Uijeongbu St. Mary's Hospital.

[0119]

[0120] [Example 2]

[0121] Histopathological analysis

[0122] <2-1> TIS of histopathological RIC

[0123] The excised rectal tissue samples were fixed in 10% formalin and stained with hematoxylin-eosin (HE). Eighteen hours after colitis induction, the animals were euthanized, the abdominal cavity was opened, and a 5-cm segment of colon was removed from each animal. The colorectal tissue was sectioned and stored in a sample container. The samples were fixed in 10% formalin and stained with hematoxylin-eosin (HE). The stained samples were evaluated by a pathologist blinded to the samples. CCP was defined as an abnormal position of normal mucosal glands below the muscularis mucosa to the muscularis propria. The total inflammation score (TIS) of radiation-induced colitis (RIC) was measured by seven components: serosal thickening (TS, score 0, 1, 2, 3), mucosal ulceration (MU, score 0, 1, 2), epithelial atypia (EA, score 0, 1, 2, 3), vascular sclerosis (VS, score 0, 1, 2, 3), intestinal wall fibrosis (IWF, score 0, 1, 2, 3), lymphocytic congestion (LC, score 0, 1), and colitis cystic fibrosis (CCP) (score 0, 1, 2, 3). The CCP score was graded as follows: 1, submucosal inclusions; 2, submucosal cysts with polypoid elevation of the mucosa; and 3, large cysts extending into the muscularis propria. The TIS was calculated as the sum of the scores of these seven components.

[0124] As a result, histologically, the TIS of RIC was significantly higher in the RG (11.33±2.96) than in the TG (6.89±2.89) (p<0.01) (Fig. 1, Table 1). Among the seven components of the TIS of RIC, EA, VS, and CCP were significantly lower in the RG than in the TG: (EA: 1.67±0.50, 1.11±0.33; VS: 2.00±0.71, 1.00±0.71; CCP: 1.89±1.17, 0.33±1.00, p<0.05) (Table 1). In addition, the CCP score (8 / 9) was significantly higher in the RG than in the control group (0 / 5) and the TG (1 / 9) (p<0.05) (Table 1, Fig. 2).

[0125]

[0126] group a P-value RGTGInflammation ScoresMean ± SDMean ± SDThickening of serosa (TS)1.33±0.51.11±0.330.541Mucosal ulcerations (MU)1.11±0.600.67±0.50.223Epithelial atypia (EA)1.67±0.51.11±0.330.038*Vascular sclerosis (VS)2.00±0.711.00±0.710.025*Intestinal wall fibrosis (IWF)2.44±0.531.89±0.600.119Colitis cystica profunda (CCP)1.89±1.170.33±1.000.01*Total inflammation Score (TIS) 11.33±3.006.89±2.890.02* a P-value by Mann-WhitneyUtest with Bonferroni correction, *P<0.05

[0127]

[0128] group aP-value RGTGInflammation ScoresNo. of present / Total no.No. of present / Total no.Lymphocytic congestion (LC)8 / 97 / 91.00 a P-value by Fisher' exact test, *P = 1.00 > 0.05.

[0129]

[0130] <2-2> Quantitative assessment of mast cell expansion

[0131] Modified toluidine blue staining was performed to detect mast cells. Briefly, paraffin sections (3 μm) of rectal tissue samples were deparaffinized and rehydrated according to the procedure described above, immersed in toluidine blue for 3 minutes, and differentiated in 0.5% glacial acetic acid for 10–20 seconds at room temperature until mast cell nuclei were visible. The tissue sections were cleared with xylene, sealed with neutral gum, and photographed under a light microscope. A single image at medium magnification (×100) was randomly selected to count the number of mast cells.

[0132] For toluidine blue staining, rectal tissue samples were fixed with 4% paraformaldehyde at 4°C for 18 hours. Paraffin-embedded tissue samples were sectioned (thickness: 2.0 μm), deparaffinized in xylene, and dehydrated in graded ethanol solutions. Antigen retrieval was performed by heating the samples in citrate buffer (pH 6.0) (95°C in a rice cooker for 20 minutes), and endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes at room temperature.

[0133] As a result, the total number of infiltrating mast cells was higher in the RG (23.67±19.00) than in the TG (9.9±7.94), but the difference was not significant (p>0.05) (Fig. 3). The number of infiltrating mast cells did not differ between the area around the CCP (23.67±18.91) and the remaining area of ​​the RG (23.00±20.05) (p>0.05).

[0134]

[0135] [Example 3]

[0136] Quantitative assessment of MMP-9 expression in colon tissue samples

[0137] Immunostaining for MMP-9 was performed using the Polink-2 + HRP rabbit with DAB kit (GBI, Bothell, WA, USA) according to the manufacturer's instructions. Sections (4 μm thick) were microwaved in 0.1 M citrate buffer, treated with 3% H2O2 in methanol, blocked with DAKO Protein Block Serum-Free (Dako, Carpinteria, CA, USA), and incubated with rabbit polyclonal anti-MMP-9 antibody (1:250; cat. no. ab38898; Abcam, Cambridge, MA, USA). Sections were then incubated with a rabbit antibody-specific enhancer, followed by the addition of polymer-HRP for rabbit IgG, and visualized using DAB complex. Nuclear counterstaining was performed using hematoxylin according to the manufacturer's recommendations. Sections were developed with Impact DAB (Vector Laboratories).

[0138] All slides were washed with ddH2O, counterstained with hematoxylin, and mounted. All samples were examined blindly by two independent pathologists. Positive immunostaining was indicated by the presence of brown cytoplasmic granules. MMP-9-positive cells were manually counted in high-power fields (HPFs) of RG and TG slides.

[0139] As a result, the total number of MMP-9-positive cells was significantly higher in the RG (23.67±19.00) than in the TG (10.25±8.45) (p=0.024). The number of MMP-9-positive cells in one HPF of RG No. 7 was 210 (Fig. 4A), whereas in slide TG No. 9, the number was 12 (Fig. 4B).

[0140]

[0141] [Example 4]

[0142] Correlation between TIS and MMP-9 expression and mast cell infiltration in RIC

[0143] Total inflammation score (TIS) and MMP9 in radiation-induced colitis (RIC) showed a strong correlation (correlation coefficient r=0.6, p<0.05), whereas TIS and infiltrating mast cell count in RIC did not show a significant correlation (r=0.3, p>0.05) (Table 3, Fig. 5) ( a r= correlation coefficient: excellent, >0.80; good, 0.60-0.79; fair, 0.40-0.59; poor, <0.40).

[0144] a rP-value Total Inflammation Score (TIS) MMP90.6005<0.05 Mast cells0.3225>0.05

[0145]

[0146] [Example 5]

[0147] Expression of MUC2 and MUC5AC in CCP lesions

[0148] Immunohistochemistry (IHC) staining was performed with monoclonal antibodies, anti-MUC2 antibody (Leica Biosystems; clone, Ccp58; dilution, 1:100) and anti-MUC5AC antibody (Leica Biosystems; clone, CLH2; dilution, RTU, ready-to-use), and the Ventana ultraVIEW DAB detection kit (Roche Diagnostics, Basel, Switzerland).

[0149] All slides were washed with ddH2O, counterstained with hematoxylin, and mounted. All samples were examined by two independent pathologists. Positive immunostaining was indicated by the presence of brown cytoplasmic granules. Cytoplasmic reactivity was determined as positive for MUC2 and MUC5AC, and luminal membrane reactivity was determined as positive for CD10.

[0150] As a result, immunohistochemically, CCP lesions were strongly positive for MUC2 but negative for MUC5AC (Fig. 6).

[0151]

[0152] [Statistical Analysis]

[0153] All data are expressed as mean ± standard deviation (SD). MedCalc Statistical Software v. 18.2.1 (MedCalc Software, Ostend, Belgium) was used for statistical analysis. First, the Mann-Whitney U test with Bonferroni correction was performed to determine the differences in the seven components of the TIS between the RG and the TG. Then, the Mann-Whitney U test was performed to determine the differences in the number of infiltrating mast cells between the RG and the TG. Spearman's correlation analysis was performed to calculate the correlation coefficient between the TIS and the number of MMP-9-positive mast cells or between the TIS and the number of infiltrating mast cells, and the results were classified as excellent (>0.80), good (0.60–0.79), fair (0.40–0.59), or poor (<0.40).

[0154]

[0155] Since the tranilast of the present invention can reduce the infiltration of mast cells into the rectal wall and provide an anti-inflammatory effect on radiation-induced colitis, when the tranilast of the present invention is pretreated in patients who have not yet developed radiation-induced inflammatory diseases, such as patients receiving pelvic radiotherapy, radiation-induced inflammatory diseases can be effectively prevented, and thus, there is industrial applicability.

Claims

1. A pharmaceutical composition for preventing radiation-induced inflammatory diseases comprising a compound represented by [chemical formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 2. A composition according to claim 1, characterized in that the compound represented by [chemical formula 1] is tranilast.

3. A composition according to claim 1, characterized in that the radiation-induced radiation means induced by pelvic radiation therapy.

4. A composition according to claim 1, characterized in that the radiation-induced inflammatory disease is at least one selected from the group consisting of radiation-induced colitis, proctitis, dermatitis, and cystitis.

5. In the first paragraph, the composition is characterized in that it reduces the infiltration of mast cells and the expression of MMP-9 (matrix metalloproteinase-9).

6. A health functional food composition for preventing radiation-induced inflammatory diseases, comprising a compound represented by [chemical formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] 7. A method for preventing radiation-induced inflammatory disease, comprising administering a compound represented by [chemical formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof to a subject at risk of radiation-induced inflammatory disease: [Chemical Formula 1] 8. Use of a compound represented by [Chemical Formula 1], a derivative thereof, or a pharmaceutically acceptable salt thereof for preparing a composition for preventing radiation-induced inflammatory diseases: [Chemical Formula 1]

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