Pharmaceutical composition for preventing or treating inflammatory bowel disease

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease in which a peptide exhibits an anti-inflammatory effect through the regulation of inflammation-related factor expression, the inhibition of nitric oxide production, and the like without exhibiting cytotoxicity in intestinal epithelial cells and macrophages, and which can thus be effectively used as a composition for preventing, treating, or alleviating inflammatory bowel disease.

Description

Pharmaceutical composition for preventing or treating inflammatory bowel disease

[0001] The present invention relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease.

[0002] Inflammatory bowel disease (IBD) is a chronic, unexplained inflammation of the intestines. It can be divided into ulcerative colitis and Crohn's disease. Ulcerative colitis is a disease in which erosions or ulcers continuously form on the mucosa of the colon, causing bloody or bloody stool, diarrhea, and abdominal pain. In severe cases, it can also cause systemic symptoms such as fever, weight loss, and anemia. Crohn's disease is a disease in which ulcers and other lesions develop discontinuously anywhere in the digestive tract from the mouth to the anus. In addition to abdominal pain, diarrhea, and bloody stool, severe cases can also cause fever, weight loss, malaise, and anemia. The cause and pathophysiology of IBD are not yet clearly known, but it is believed that a combination of genetic factors, environmental factors such as gut bacteria and diet, and immunological factors are involved in its pathogenesis.

[0003] The incidence of ulcerative colitis and Crohn's disease was traditionally known to be higher in Westerners. However, due to recent changes in lifestyle habits, including dietary habits, the number of patients has been rapidly increasing in East Asian countries, including Korea. Despite this, due to unclear causes and other factors, a fundamental treatment method has not yet been established. Therefore, rather than aiming for a complete cure, medications are used to delay and alleviate the progression of symptoms and maintain this condition for as long as possible. Medications primarily used for this popular treatment include aminosalicylates, corticosteroids, immunosuppressants, and TNF-α monoclonal antibodies. However, various side effects have been reported. For example, sulfasalazine, a frequently used aminosalicylates, has been reported to cause nausea, vomiting, anorexia, rash, headache, leukopenia, abnormal red blood cells, proteinuria, and diarrhea. In addition, prednisolone, an adrenocortical steroid, is used orally, as an enema, as a suppository, or as an intravenous injection, but has strong side effects such as gastric ulcers and femoral head necrosis with long-term use. Infliximab, a TNF-α monoclonal antibody, was approved by the US FDA in 1998 as a treatment for Crohn's disease and has been used to treat patients with Crohn's disease, but side effects such as pancytopenia, drug-induced lupus, and hepatitis B / tuberculosis reactivation have appeared. In addition, the US FDA warns doctors that the use of infliximab and other tumor necrosis factor (TNF) inhibitors may increase the risk of lymphoma and other cancers.

[0004] To address these issues, there is a need to develop safer and more effective treatments for inflammatory bowel disease that can replace the currently used treatments.

[0005] The purpose of the present invention is to provide a composition for preventing, treating or improving inflammatory bowel disease.

[0006] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a peptide having an amino acid sequence represented by sequence number 1 as an active ingredient.

[0007] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory bowel disease, which contains the peptide as an active ingredient.

[0008] According to the present invention, a peptide that does not exhibit cytotoxicity in intestinal epithelial cells and macrophages and exhibits anti-inflammatory effects through regulating the expression of inflammation-related factors and inhibiting nitric oxide production, etc., can be usefully utilized as a composition for preventing, treating, or improving inflammatory bowel disease.

[0009] Figure 1 is a general schematic diagram of the production of an animal model of ulcerative colitis.

[0010] Figure 2 shows the results of analyzing the cytotoxicity of the peptide (hereinafter referred to as TPS-39) synthesized in Experimental Example 1 below in intestinal epithelial cells and macrophages. *p<0.05 vs. normal group (peptide-untreated group).

[0011] Figure 3 shows the results of analyzing the effect of TPS-39 on the expression of inflammation-related factors.

[0012] Figure 4 shows the results of analyzing the effect of TPS-39 on NF-κB activity.

[0013] Figure 5 shows the results of analyzing the effects on nitric oxide (NO) production. ###p<0.001 vs. normal group; and ***p<0.001 vs. control group.

[0014] Figure 6 shows the results of analyzing the effect of TPS-39 on iNOS (Inducible nitric oxide synthase) expression.

[0015] Figure 7 shows the results of analyzing the effect of TPS-39 on body weight.

[0016] Figure 8 shows the results of analyzing the effect of TPS-39 on colon length. ###p<0.001 vs. normal group; and *p<0.05, ***p<0.001 vs. control group.

[0017] Figure 9 shows the results of analyzing the effect of TPS-39 on colon tissue recovery.

[0018] Hereinafter, the present invention will be described in more detail.

[0019]

[0020] The present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a peptide having an amino acid sequence represented by sequence number 1 as an active ingredient.

[0021] The amino acid sequence represented by the above sequence number 1 is as follows.

[0022] [Sequence number 1]

[0023] TFKK (T; Threonine, F; Phenylalanine; and K; Lysine)

[0024] The above peptide can inhibit the activity or expression of one or more selected from the group consisting of, but not limited to, interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor kappa B (NF-κB), and nitric oxide (NO).

[0025] iNOS expression is increased by various factors, including bacterial toxins, inflammation, immune responses, and increased oxidative stress, and induces NO production from L-arginine. In particular, NO production resulting from increased iNOS expression has been reported to play a significant role in the pathophysiology of various diseases.

[0026] NF-κB is a representative transcription factor that regulates the expression of inflammatory mediators and has been reported to be associated with various pathologies, including inflammation and cancer. A recent study reported that NF-κB activity is increased in colon tissues of patients with colitis.

[0027] Nitric oxide (NO) is a highly reactive biomolecule and an active oxygen species induced by oxidation processes. Excessive NO damages biomolecules such as fats and proteins in the body, promoting inflammatory responses such as blood permeability and edema. It has been reported to exacerbate inflammatory responses, particularly those that cause tissue damage.

[0028] Additionally, the peptide can promote IL-10 (interleukin-10) expression.

[0029] The above inflammatory bowel disease may be at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, and ischemic colitis, but is not limited thereto.

[0030] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0031] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0032] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0033] The above pharmaceutical composition may be formulated in the form of one or more external skin preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas, but is not limited thereto.

[0034] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0035] The pharmaceutical composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) and can be formulated as an injection solution, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.

[0036] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition, weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0037]

[0038] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, which comprises a peptide having an amino acid sequence represented by sequence number 1 as an active ingredient.

[0039] The present invention can be generally used as a commonly used food.

[0040] The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with functional properties useful to the human body as defined in the Health Functional Food Act. "Functionality" refers to ingestion for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects.

[0041] The above health functional food composition may contain conventional food additives, and its suitability as the above “food additive” is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0042] Items listed in the above “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0043] The food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. For example, among health functional foods in capsule form, hard capsules can be manufactured by mixing and filling a composition according to the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be manufactured by mixing a composition according to the present invention with additives such as excipients and filling a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0044] The definitions of terms for the above excipients, binders, disintegrants, lubricants, flavoring agents, etc. are described in literature known in the art and include those with identical or similar functions. There are no specific restrictions on the type of food, and all health functional foods in the conventional sense are included.

[0045] In the present invention, the term “prevention” refers to any act of suppressing or delaying the disease by administering a composition according to the present invention.

[0046] In the present invention, the term “treatment” refers to any act of improving or beneficially changing the symptoms of the disease by administering a composition according to the present invention.

[0047] In the present invention, the term “improvement” means any act of improving the bad condition of the disease by administering or ingesting the composition of the present invention to a subject.

[0048] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0049]

[0050] [Experimental Example 1] Peptide Synthesis

[0051] To synthesize novel peptides, Merrifield's liquid-solid method using an Fmoc amino group protection vessel was used. The solid phase method uses Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group for the Nα-amino group of amino acids. Rink Amide MBHA-Resin was used as a starting material for peptides with a carboxyl terminal in the -NH2 form, and Fmoc-amino acid-Wang Resin was used as a starting material for peptides with a carboxyl terminal in the -OH form. Peptide chain elongation by coupling of Fmoc-amino acid was performed by the DCC (N-hydroxybenzotriazole(HOBt)-dicyclo-hexylcarbodiimide) method. Fmoc-amino acid was coupled to the amino terminus of each peptide, and the Fmoc group was removed with a 20% piperidine / N-methylpyrrolidone (hereinafter referred to as NMP) solution, washed several times with NMP and dichloromethane (DCM), and dried with nitrogen gas. Then, a trifluoroacetic acid (TFA)-phenol-thioanisole-H2O-triisop-ropylsilane (85:5:5:2.5:2.5, vol. / vol.) solution was added and reacted for 3 hours to remove the protecting group, and the peptide was separated from the resin. Through the above manufacturing process, TPS-39, a peptide (amino acid sequence: TFKK) having four amino acids, Threonine (T), Phenylalanine (F), Lysine (K), and Lysine (K), was manufactured.

[0052]

[0053] [Experimental Example 2] Preparation of a cell model

[0054] 2-1. Intestinal epithelial cells

[0055] HT-29 cells, a human intestinal epithelial cell line, were purchased from the Korea Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum) at 37°C in an incubator controlled with 5% CO2 and 95% humid air.

[0056]

[0057] 2-2. Macrophages

[0058] RAW264.7 cells, a mouse macrophage cell line, were purchased from the Korea Cell Line Bank (Seoul, Korea). The cells were cultured in DMEM medium supplemented with 10% FBS at 37°C in an incubator controlled with 5% CO2 and 95% humid air.

[0059]

[0060] [Experimental Example 3] Preparation of an animal model

[0061] An animal model of acute ulcerative colitis was constructed as follows. Seven-week-old C57BL / 6J mice were purchased from Orient Bio (Seongnam, Korea) and acclimated for 7 days in a housing environment with constant humidity, temperature, and a 12-hour light / dark cycle, with free access to solid food and water. After the acclimation period, the mice were divided into five experimental groups, as follows:

[0062] 1) Control group: Normal water intake + PBS

[0063] 2) Control group (DSS only): Drinking water containing 3% (w / v) DSS + PBS

[0064] 3) Peptide treatment group 1 (DSS+TPS-39 5mg / kg): drinking water containing 3% (w / v) DSS + oral administration of 5mg / kg TPS-39

[0065] 4) Peptide treatment group 2 (DSS + TPS-39 10 mg / kg): drinking water containing 3% (w / v) DSS + oral administration of 10 mg / kg TPS-39

[0066] 5) Positive control group (DSS+5-ASA 50 mg / kg): Drinking water containing 3% (w / v) DSS + oral administration of 5-ASA (5-acetylsalicylic acid) 50 mg / kg

[0067] As shown in Fig. 1, the normal group was allowed to freely consume normal drinking water during the experimental period (days 0 to 11 of the experiment), and the remaining experimental groups (control group, peptide treatment group, and positive control group) excluding the normal group were allowed to freely consume drinking water supplemented with 3% (w / v) DSS (Dextran Sulfate Sodium, MP bio, USA) for 5 days from day 0 of the experiment to induce acute ulcerative colitis in the mice. After that, the mice were allowed to freely consume normal drinking water for 6 days from day 5 of the experiment. After that, the normal and control groups were orally administered PBS once a day for 5 days from day 5 of the experiment, and the other experimental groups were administered TPS-39 (peptide treatment group) or 5-ASA (positive control group) dissolved in PBS. The single PBS dose per mouse in all experimental groups was set to the same value of 200 μl, the single TPS-39 dose per mouse in the peptide-treated group was set to 5 mg / kg or 10 mg / kg, and the single 5-ASA dose per mouse in the positive control group was set to 50 mg / kg. After the experiment, the mice were sacrificed, the large intestine from the cecum to the rectum was removed, and the feces were drained by injecting physiological saline solution and then dried. During the experimental period, the mice were checked daily for weight loss, stool condition, and bleeding.

[0068]

[0069] [Example 1] Cytotoxicity analysis

[0070] 1-1. Intestinal epithelial cells

[0071] To determine the cytotoxicity of TPS-39 in intestinal epithelial cells, 5 × 10 HT-29 cells, a human intestinal epithelial cell line, were seeded in 96-well plates. 3 Cells were dispensed per well and treated with TPS-39 diluted at various concentrations. Cell viability was measured using the WST assay 24 hours after treatment with TPS-39.

[0072] As a result, as shown in Fig. 2, TPS-39 did not exhibit cytotoxicity in intestinal epithelial cells up to a concentration of 100 μg / mL.

[0073]

[0074] 1-2. Macrophages

[0075] To confirm the cytotoxicity of TPS-39 in macrophages, cell viability was measured using mouse macrophage RAW264.7 cells using the same method as in Example 1-1.

[0076] As a result, as shown in Fig. 2, TPS-39 did not exhibit cytotoxicity in mouse macrophages up to a concentration of 100 μg / mL.

[0077]

[0078] [Example 2] Analysis of anti-inflammatory effects (in vitro)

[0079] 2-1. Analysis of expression of inflammation-related factors

[0080] To determine the effect of TPS-39 on the expression of inflammatory factors, qRT-PCR analysis was performed. HT-29 cells were pretreated with various concentrations of TPS-39 and treated with TNF-α (20 ng / mL) to induce an inflammatory response. Then, gene expression of inflammatory factors [IL-6 (NCBI: NM_000600), IL-10 (NCBI: NM_000572), ICAM-1 (NCBI: NM_000201), and COX-2 (NCBI: NM_000963)] was measured using qRT-PCR.

[0081] As a result, as shown in Fig. 3, the expression of IL-6, ICAM-1, and COX-2 significantly increased in the control group (TNF-α only treatment group) compared to the normal group (TPS-39 and TNF-α untreated group), whereas the expression of IL-6, ICAM-1, and COX-2 significantly decreased in a concentration-dependent manner in the peptide treatment group compared to the control group, and the expression of IL-10 significantly increased.

[0082]

[0083] 2-2. NF-κB activity analysis

[0084] To determine the effect of TPS-39 on NF-κB activity, Western blot analysis was performed. HT-29 cells were pretreated with TPS-39 (10 μg / mL) and treated with TNF-α (20 ng / mL) to induce an inflammatory response, and changes in NF-κB activity were measured.

[0085] As a result, as shown in Fig. 4, NF-κB activity increased in the control group (TNF-α only treatment group) compared to the normal group (TPS-39 and TNF-α untreated group), whereas NF-κB activity decreased in the peptide treatment group compared to the control group.

[0086]

[0087] 2-3. NO production analysis

[0088] To determine the effect of TPS-39 on nitric oxide (NO) production, RAW264.7 cells were pretreated with various concentrations of TPS-39 and then treated with LPS (Lipopolysaccharide, 1 μg / mL) for 24 h. The medium was then recovered, and the amount of NO produced in the cells was measured using the Griess test.

[0089] As a result, as shown in Fig. 5, NO production significantly increased in the control group (LPS only treatment group) compared to the normal group (TPS-39 and LPS untreated group), whereas NO production significantly decreased in the peptide treatment group compared to the control group.

[0090]

[0091] 2-4. iNOS expression analysis

[0092] To determine the effect of TPS-39 on the expression of iNOS (Inducible Nitric Oxide Synthase), an inflammatory mediator, Western blot analysis was performed. RAW264.7 cells were pretreated with various concentrations of TPS-39 and then treated with LPS (Lipopolysaccharide, 1 μg / mL) for 24 hours. iNOS expression was then measured by Western blot.

[0093] As a result, as shown in Fig. 6, iNOS expression significantly increased in the control group (LPS only treatment group) compared to the normal group (TPS-39 and LPS untreated group), whereas iNOS expression significantly decreased in the peptide treatment group compared to the control group.

[0094]

[0095] [Example 3] Analysis of anti-inflammatory effects (in vivo)

[0096] 3-1. Weight analysis

[0097] To confirm the effect of TPS-39 on body weight, the body weight of the animal model of Experimental Example 2 was measured daily from day 0 of the experiment and the change in body weight was analyzed.

[0098] As a result, as shown in Figure 7, body weight was significantly reduced in the control group compared to the normal group, whereas body weight loss was significantly suppressed in the peptide treatment group and positive control group compared to the control group, and the weight loss suppression effect was more significant in the peptide treatment group than in the positive control group.

[0099]

[0100] 3-2. Colon length analysis

[0101] To determine the effect of TPS-39 on colon length, the length of the colon extracted from the animal model of Experimental Example 2 was measured.

[0102] As a result, as shown in Fig. 8, the colon length was significantly reduced in the control group (4.8±0.33cm) compared to the normal group (7.82±0.34cm), whereas the colon length reduction was significantly suppressed in the peptide treatment group 1 (6.8±0.9cm), peptide treatment group 2 (7.33±0.41cm), and positive control group (6.5±0.83cm) compared to the control group, and the effect of suppressing colon length reduction was more significant in the peptide treatment group than in the positive control group.

[0103]

[0104] 3-3. Colon tissue recovery analysis

[0105] To determine the effect of TPS-39 on colonic tissue recovery, histological changes were observed after staining colonic tissue with H&E (Hematoxylin & Eosin). The colon extracted from the animal model of Experimental Example 2 was divided into three parts (proximal, middle, and distal), and the rectal part was fixed in a 10% neutral formalin solution for pathological analysis. The fixed rectal tissue was sectioned into paraffin tissue specimens at 5 μm thickness, stained with H&E for histological examination, and observed using an optical microscope.

[0106] As a result, as shown in Fig. 9, the number of goblet cells decreased in the control group compared to the normal group, damage to the surface epithelium and the entire tissue was observed, and it was confirmed that a large number of immune cells had infiltrated the colon tissue. In the positive control group, the structure of the mucosa in the colon tissue was partially maintained, but goblet cells and surface epithelium were lost, and a large number of immune cells had infiltrated the colon tissue. On the other hand, the structure of the mucosa in the colon tissue was maintained in the peptide-treated group, and a colon tissue structure similar to the normal group was observed.

[0107]

[0108] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a peptide having an amino acid sequence represented by sequence number 1 as an active ingredient.

2. A composition according to claim 1, characterized in that the peptide inhibits the activity or expression of at least one selected from the group consisting of IL-6 (interleukin-6), ICAM-1 (intercellular adhesion molecule-1), COX-2 (cyclooxygenase-2), iNOS (inducible nitric oxide synthase), NF-κB (Nuclear factor kappa B), and nitric oxide (NO).

3. A composition according to claim 1, characterized in that the peptide promotes IL-10 (interleukin-10) expression.

4. A composition according to claim 1, characterized in that the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, irritable bowel syndrome, intestinal Behcet's disease, and ischemic colitis.

5. A health functional food composition for preventing or improving inflammatory bowel disease, comprising a peptide having an amino acid sequence represented by sequence number 1 as an active ingredient.

Images