Agents that suppress the decline in intestinal epithelial barrier function in non-infectious inflammatory bowel disease

Abstract

To provide a composition that can suppress the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease. [Solution] An agent containing human milk oligosaccharides that suppresses the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease.

Description

[Technical Field]

[0001] This invention relates to an agent for suppressing the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a disease characterized by inflammation and ulcers in the intestinal tract, causing symptoms such as diarrhea, abdominal pain, weight loss, and fever. Among IBDs, non-infectious IBDs (such as Crohn's disease and ulcerative colitis) have seen an increase in the number of patients in recent years (Non-Patent Literature 1), but there is no established treatment that leads to a complete cure for non-infectious IBDs (Non-Patent Literature 2). Non-infectious IBDs are associated with a decrease in the barrier function of the epithelium of the intestinal tract, including the small and large intestines. This decrease in the barrier function of the intestinal epithelium is caused by inflammatory cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). In non-infectious IBDs, this decrease in the barrier function of the intestinal epithelium leads to further excessive activation of immune and inflammatory responses, causing (or exacerbating) various symptoms of the disease (Non-Patent Literature 3). Therefore, it is expected that if the decline in the barrier function of the intestinal epithelium caused by inflammatory cytokines can be suppressed, non-infectious inflammatory bowel disease can be treated, prevented, or improved.

[0003] Symptomatic treatment for non-infectious inflammatory bowel disease includes long-term treatment with anti-TNF-α antibodies and treatment with anti-inflammatory drugs for acute inflammation (Non-Patent Literature 4). On the other hand, it is known that there are human patients for whom treatment with anti-TNF-α antibodies is ineffective (primary failure), or human patients for whom the effect of anti-TNF-α antibodies is lost due to long-term administration (secondary failure), and there is a need for symptom improvement methods through TNF-α-independent mechanisms (Non-Patent Literature 4). Furthermore, as mentioned above, although anti-inflammatory drugs for inflammation in non-infectious inflammatory bowel disease are known, the development of bioactive substances that can treat, prevent, or improve impaired intestinal barrier function, leaky gut, etc. is insufficient.

[0004] Human milk oligosaccharides (hereinafter also referred to as "HMOs") are a general term for oligosaccharides found in breast milk, and are the third most abundant component among the solid components of breast milk, after lactose and lipids. Among the most representative HMOs are 2'-fucosyl lactose (O-α-L-fucopyranosyl-(1→2)-O-β-D-galactopyranosyl-(1→4)-D-glucose, also known as "2FL"), which is the most abundant in breast milk, as well as the major acidic HMOs, 3'-sialyl lactose (O-(N-acetyl-α-neuraminosyl)-(2→3)-O-β-D-galactopyranosyl-(1→4)-D-glucose, also known as "3SL") and 6'-sialyl lactose (O-(N-acetyl-α-neuraminosyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-Glucose, also known as "6SL"). HMOs have been reported to reach the intestines without being metabolized by human digestive enzymes and to exert various physiological activities and functions.

[0005] Patent Document 1 claims that 3SL and 6SL have a certain inhibitory effect on the reduction of intestinal barrier function caused by pathogenic E. coli in Caco2 cells. Specifically, in an in vitro test, when pathogenic E. coli was added to Caco2 cells without the addition of HMO, the transepithelial electrical road (TEER) value decreased (negative control). However, when Caco2 cells were cultured at 37°C for 1 hour with 2FL, 3-Fucosyllactose (hereinafter also referred to as "3FL"), 3SL, and 6SL respectively, and then pathogenic E. coli was added, the TEER values ​​in the group with 3SL and the group with 6SL were higher than the TEER values ​​in the negative control. Caco2 cells are known to be a cell line with a phenotype similar to that of small intestinal absorptive epithelium (Non-Patent Document 5).

[0006] Non-patent document 5 claims that 2FL, 3FL, 6SL, lacto-N-tetraose (hereinafter also referred to as "LNT"), and lacto-N-triose II (hereinafter also referred to as "LNT2") have an inhibitory effect on the decline in intestinal barrier function when inflammation is induced by the co-addition of TNF-α and IFN-γ at specific concentrations. Specifically, in an in vitro test, when Caco2 cells were cultured for 24 hours after adding 2FL, 3FL, 3SL, 6SL, LNT, and LNT2 respectively, inflammation was induced by co-adding 100 ng / mL of tumor necrosis factor (TNF-α) and 10 ng / mL of interferon-γ (IFN-γ), it was found that 20 hours after co-addition, the reduction in TEER value due to the above inflammation was suppressed when 2FL, 3FL, 6SL, LNT, and LNT2 were added, while no such suppression of TEER value reduction was observed when 3SL was added. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Specification of Chinese Patent No. 113519849 [Non-patent literature]

[0008] [Non-Patent Document 1] Suzuki, Yasuo. "I. The rapidly increasing number of inflammatory bowel disease patients in Japan and their metabolic aspects (gastrointestinal)." Journal of the Japanese Society of Internal Medicine 108.4 (2019): 666-672. [Non-Patent Document 2] "Diagnostic Criteria and Treatment Guidelines for Ulcerative Colitis and Crohn's Disease." Ministry of Health, Labour and Welfare Scientific Research Grant, Intractable Disease Policy Research Project, "Survey and Research on Intractable Inflammatory Bowel Disorders" (Hisamatsu Group), FY2023 Collaborative Research Report, prepared March 2024. [Non-Patent Document 3] Nalle SC, et al. “Intestinalbarrier loss as a critical pathogenic link between inflammatory bowel diseaseand graft-versus-host disease.” Mucosal Immunol. 2015 Jul; 8(4):720-30.2015.40. Epub 2015 May 6. [Non-Patent Document 4] Fukuda, Tomohiro et al. "Treatment Strategies for Inflammatory Bowel Disease Centered on Biological Agents." Journal of the Japanese Society of Gastroenterology 115.3 (2018): 272-282. [Non-Patent Document 5] Boll, Erik Juncker, et al. “Human milk oligosaccharides differentially support gut barrier integrity and enhance Th1 and Th17 cell effector responses in vitro.” Frontiers in Immunology 15 (2024): 1359499. [Non-Patent Document 6] Devriese, Sarah, et al. “T84 monolayers are superior to Caco-2 as a model system of colonocytes.” Histochemistry and cell biology 148.1(2017): 85-93. [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a composition that can suppress the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease. [Means for solving the problem]

[0010] This disclosure provides, for example, the inventions described in [1] to

[34] below. [1] An agent containing human milk oligosaccharides that suppresses the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease. [2] The inhibitor according to [1], wherein the reduction in barrier function is TNF-α independent. [3] The inhibitor according to [1] or [2], wherein the reduction in barrier function is promoted by IFN-γ. [4] The inhibitor according to any one of [1] to [3], wherein the human milk oligosaccharide is sialyl lactose. [5] The inhibitor according to any one of [1] to [4], wherein the human milk oligosaccharide is 3'-sialyl lactose. [6] The inhibitor according to any one of [1] to [5], wherein the intestinal epithelium is the epithelium of the large intestine. [7] The inhibitor according to any one of [1] to [6], wherein the inflammatory bowel disease is Crohn's disease, ulcerative colitis, intestinal Behçet's disease, or simple ulcer. [8] A treatment, preventive or ameliorative agent for non-infectious inflammatory bowel disease, containing human milk oligosaccharides. [9] The therapeutic, prophylactic or ameliorative agent according to [8], wherein the inflammatory bowel disease is a disease accompanied by a TNF-α-independent impairment of the intestinal epithelial barrier function.

[10] The therapeutic, prophylactic or ameliorative agent according to [8] or [9], wherein the inflammatory bowel disease is a disease accompanied by impaired barrier function of the intestinal epithelium induced by IFN-γ.

[11] The therapeutic, preventive, or ameliorative agent according to any one of [8] to

[10] , wherein the human milk oligosaccharide is sialyl lactose.

[12] The therapeutic, preventive, or ameliorative agent according to any one of [8] to

[11] , wherein the human milk oligosaccharide is 3'-sialyl lactose.

[13] The treatment, prevention, or improvement agent according to any one of [8] to

[12] , wherein the inflammatory bowel disease is an inflammatory bowel disease of the large intestine.

[14] The treatment, prevention or improvement agent according to any one of [8] to

[13] , wherein the inflammatory bowel disease is Crohn's disease, ulcerative colitis, intestinal Behçet's disease, or simple ulcer.

[15] A method for suppressing the reduction of the intestinal epithelial barrier function in non-infectious inflammatory bowel disease, which includes administering human milk oligosaccharide to a subject or the subject ingesting it.

[16] Human milk oligosaccharide for use in a therapeutic method for suppressing the reduction of the intestinal epithelial barrier function in non-infectious inflammatory bowel disease.

[17] Use of human milk oligosaccharide in a non-therapeutic method for suppressing the reduction of the intestinal epithelial barrier function in non-infectious inflammatory bowel disease.

[18] Use of human milk oligosaccharide for the manufacture of an agent for suppressing the reduction of the intestinal epithelial barrier function in non-infectious inflammatory bowel disease.

[19] The method, human milk oligosaccharide or use according to any one of

[15] to

[18] , wherein the reduction of the barrier function is TNF-α-independent.

[20] The method, human milk oligosaccharide or use according to any one of

[15] to

[19] , wherein the reduction of the barrier function is promoted by IFN-γ.

[21] The method, human milk oligosaccharide or use according to any one of

[15] to

[20] , wherein the human milk oligosaccharide is sialyllactose.

[22] The method, human milk oligosaccharide or use according to any one of

[15] to [~21], wherein the human milk oligosaccharide is 3'-sialyllactose.

[23] The inhibitor according to any one of

[15] to

[22] , wherein the intestinal epithelium is the epithelium of the large intestine.

[24] The method, human milk oligosaccharide or use according to any one of

[15] to

[23] , wherein the inflammatory bowel disease is Crohn's disease, ulcerative colitis, intestinal Behcet's disease or simple ulcer.

[25] A method for treating, preventing or improving non-infectious inflammatory bowel disease, which includes administering human milk oligosaccharide to a subject or the subject ingesting it.

[26] Human milk oligosaccharide for use in a method for treating non-infectious inflammatory bowel disease or in a therapeutic method for prevention.

[27] Use of human milk oligosaccharide in a non-therapeutic method for preventing or improving non-infectious inflammatory bowel disease. Use of human milk oligosaccharides for the manufacture of a therapeutic, prophylactic or ameliorating agent for non-infectious inflammatory bowel disease.

[29] The method, human milk oligosaccharide or use according to any one of

[25] to

[28] , wherein the inflammatory bowel disease is a disease associated with a reduction in the barrier function of the intestinal epithelium that is independent of TNF-α.

[30] The method, human milk oligosaccharide or use according to any one of

[25] to

[29] , wherein the inflammatory bowel disease is a disease associated with a reduction in the barrier function of the intestinal epithelium promoted by IFN-γ.

[31] The method, human milk oligosaccharide or use according to any one of

[25] to

[30] , wherein the human milk oligosaccharide is sialyllactose.

[32] The method, human milk oligosaccharide or use according to any one of

[25] to

[31] , wherein the human milk oligosaccharide is 3'-sialyllactose.

[33] The method, human milk oligosaccharide or use according to any one of

[25] to

[32] , wherein the inflammatory bowel disease is an inflammatory bowel disease in the large intestine.

[34] The method, human milk oligosaccharide or use according to any one of

[25] to

[33] , wherein the inflammatory bowel disease is Crohn's disease, ulcerative colitis, intestinal Behçet's disease or simple ulcer.

Advantages of the Invention

[0011] According to the present invention, a composition capable of suppressing a reduction in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease can be provided. The reduction in the barrier function may be independent of TNF-α, and the intestinal epithelium may be the epithelium of the large intestine.

[0012] Also according to the present invention, a composition capable of treating, preventing or ameliorating non-infectious inflammatory bowel disease can be provided. The inflammatory bowel disease may be a disease associated with a reduction in the barrier function of the intestinal epithelium that is independent of TNF-α, and the inflammatory bowel disease may be an inflammatory bowel disease in the large intestine.

Brief Description of the Drawings

[0013] [Figure 1]This figure shows the TEER values ​​of the cell layers formed by human colon cancer-derived intestinal epithelial cells when cultured in a medium containing IFN-γ and / or TNF-α (IFN-γ treatment group, TNF-α treatment group, or IFN-γ and TNF-α (IFN-γ+TNF-α) treatment group), or in a medium not containing either IFN-γ or TNF-α (control group). In Figure 1, the data are shown as mean ± standard error, and "*" indicates that the p-value for the control group in Dunnett's test after one-way ANOVA is less than 0.05. [Figure 2] This figure shows the TEER values ​​of the cell layers formed by human colon cancer-derived intestinal epithelial cells when cultured in a medium containing IFN-γ and 3SL or 6SL (IFN-γ and 3SL (IFN-γ+3SL) treatment group, or IFN-γ and 6SL (IFN-γ+6SL) treatment group), a medium containing IFN-γ (IFN-γ treatment group), or a medium not containing any of IFN-γ, 3SL, or 6SL (control group). The data in Figure 2 are shown as mean ± standard error, and "**" indicates that the p-value for the IFN-γ treatment group in Dunnett's test after one-way ANOVA is less than 0.01. [Modes for carrying out the invention]

[0014] The embodiments of this disclosure will be described in detail below.

[0015] <The drug related to this disclosure> The inhibitor of impaired intestinal epithelial barrier function in non-infectious inflammatory bowel disease and the therapeutic, preventive, or ameliorative agent for non-infectious inflammatory bowel disease related to this disclosure contain human milk oligosaccharides. Hereinafter, the "inhibitor of impaired intestinal epithelial barrier function in non-infectious inflammatory bowel disease related to this disclosure" and the "therapeutic, preventive, or ameliorative agent for non-infectious inflammatory bowel disease related to this disclosure" will be collectively referred to as the "agents related to this disclosure."

[0016] In this specification, non-infectious inflammatory bowel disease refers to inflammatory bowel disease that develops due to causes other than infection by pathogenic bacteria. Examples of non-infectious inflammatory bowel diseases include Crohn's disease, ulcerative colitis, intestinal Behçet's disease, and simple ulcers. The causes of Crohn's disease, ulcerative colitis, intestinal Behçet's disease, and simple ulcers are not always clear, but they can be attributed to causes other than infection by pathogenic bacteria, such as genetic factors, mental stress, diet, or abnormalities in the immune system. Crohn's disease, ulcerative colitis, intestinal Behçet's disease, and simple ulcers can also be referred to as non-infectious Crohn's disease, non-infectious ulcerative colitis, non-infectious intestinal Behçet's disease, and non-infectious simple ulcers, respectively.

[0017] In this specification, the barrier function of the intestinal epithelium refers to the function of preventing intestinal bacteria, pathogens, or harmful substances in the intestines from entering the tissue from the surface of the intestinal tract by a barrier formed by tight junctions between intestinal epithelial cells in the intestinal epithelium. The agents of this disclosure can suppress the decline of the barrier function of the intestinal epithelium by, for example, suppressing the destruction of the above barrier (destruction of tight junctions, cell death of intestinal epithelial cells, etc.) or promoting the repair of the destroyed barrier.

[0018] Here, "suppression of the decline in intestinal epithelial barrier function in non-infectious inflammatory bowel disease" can be determined by measuring the suppression of the decline in the barrier function of the cell layer formed by intestinal epithelial cells due to inflammatory cytokines. For example, human colon cancer-derived intestinal epithelial cells T84 cells are placed on a cell insert of a Transwell plate (Corning) in a size of 0.7-1.3 × 10⁶. 5Seeds are seeded to a density of cells per well and cultured at 37°C and 5% CO2 for 5-7 days. Then, the culture medium in the cell insert is replaced with a medium containing the test substance (test medium) and cultured at 37°C and 5% CO2 for 22-26 hours. Subsequently, the culture medium outside the cell insert is replaced with a medium containing 5 ng / mL of IFN-γ and cultured at 37°C and 5% CO2 for 44-52 hours. After that, the TEER value of the formed cell layer is measured. If the measured TEER value is larger than the TEER value measured using the same procedure except that a culture medium without the test substance was used instead of the test medium (for example, if the average TEER value of the former (n=3~5) is 1.05 times, 1.1 times, 1.2 times, or 1.3 times larger than the average TEER value of the latter (n=3~5); or if the TEER value of the former is statistically significantly larger than the TEER value of the latter), then the test substance can be judged to "suppress the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease."

[0019] The TEER value is the unit membrane area resistance (Ω·cm) calculated from the current flowing when a constant voltage is applied to a cell layer. 2 ) is a commonly used indicator for evaluating barrier function. The TEER value can be measured, for example, by the following method. First, the electrical resistance value of the cell layer on the wells of the cell insert in the Transwell plate (electrical resistance value of the well in which cells are seeded) is obtained using the Millicell® ERS-2 (manufactured by Millipore), a resistance measurement system. Then, the electrical resistance value (electrical resistance value of the blank well) is obtained using the same procedure as above, except that no cells are present on the wells of the cell insert. Next, the TEER value is calculated from both obtained electrical resistance values ​​using the following formula. Note that 0.33 (cm) in the following formula 2 ) represents the culture area of ​​the well mentioned above. TEER value (Ω·cm) 2 ) = ([Electrical resistance of the well where cells were seeded (Ω)] - [Electrical resistance of the blank well (Ω)]) × 0.33 (cm 2 ) …(formula)

[0020] The TEER value is a common indicator for evaluating barrier function. A high TEER value indicates high barrier function, while a low TEER value indicates low barrier function.

[0021] The agent relating to this disclosure can suppress the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease (NBC), and as a result, can treat, prevent, or improve NBC. Specifically, by ingesting or administering the agent relating to this disclosure to a subject suffering from NBC, NBC can be treated or improved. "Treatment of NBC" means, for example, improvement of symptoms or disease, prevention or delay of worsening of symptoms or disease, or prevention or delay of progression of symptoms or disease. "Improvement of NBC" means, for example, improvement of symptoms or disease, prevention or delay of worsening of symptoms or disease, or prevention or delay of progression of symptoms or disease. "Treatment of NBC" is particularly done by therapeutic methods, and "improvement of NBC" is particularly done by non-therapeutic methods. Furthermore, by ingesting or administering the agent relating to this disclosure to a subject who does not suffer from NBC, NBC can be prevented. "Prevention of non-infectious inflammatory bowel disease" may mean, for example, preventing and / or delaying the onset of symptoms or disease, or reducing the likelihood of developing symptoms or disease. "Prevention of non-infectious inflammatory bowel disease" may be achieved by therapeutic or non-therapeutic means.

[0022] Here, HMOs may be used in therapeutic or non-therapeutic methods. "Therapeutic methods" may mean, for example, a concept that includes medical procedures, and more specifically, a concept that includes treatments to the human body. "Non-therapeutic methods" may mean, for example, a concept that does not include medical procedures, and more specifically, a concept that does not include treatments to the human body. Examples of non-therapeutic purposes include health promotion or cosmetic purposes.

[0023] In non-infectious inflammatory bowel disease, impaired intestinal epithelial barrier function is caused by inflammatory cytokines such as IFN-γ and TNF-α. The agents according to this disclosure can preferably suppress TNF-α-independent impaired intestinal epithelial barrier function, IFN-γ-induced impaired intestinal epithelial barrier function, and TNF-α-independent and IFN-γ-induced impaired intestinal epithelial barrier function, respectively.

[0024] Therefore, in the inhibitor of impaired intestinal epithelial barrier function in non-infectious inflammatory bowel disease according to this disclosure, the impaired barrier function may be TNF-α independent, or promoted by IFN-γ, or TNF-α independent and promoted by IFN-γ. Furthermore, in the therapeutic, preventive, or ameliorative agent for non-infectious inflammatory bowel disease according to this disclosure, the impaired intestinal epithelial barrier function associated with the inflammatory bowel disease may be TNF-α independent impaired intestinal epithelial barrier function, IFN-γ promoted impaired intestinal epithelial barrier function, or TNF-α independent and IFN-γ promoted impaired intestinal epithelial barrier function. Furthermore, in the therapeutic, preventive, or ameliorative agent for non-infectious inflammatory bowel disease according to this disclosure, the inflammatory bowel disease may be inflammatory bowel disease that is resistant to treatment with anti-TNF-α antibodies.

[0025] The agent relating to this disclosure can suppress the decline in the barrier function of the intestinal epithelium, and the intestinal epithelium may be the epithelium of the small intestine or the epithelium of the large intestine, preferably the epithelium of the large intestine. The epithelium of the large intestine may be the epithelium of the rectum or the epithelium of the colon, preferably the epithelium of the colon.

[0026] Therefore, in the agent for inhibiting the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease according to this disclosure, the intestinal epithelium may be the epithelium of the small intestine or the epithelium of the large intestine, or the epithelium of the colon. Furthermore, in the agent for the treatment, prevention or improvement of non-infectious inflammatory bowel disease according to this disclosure, the inflammatory bowel disease may be inflammatory bowel disease in the small intestine or the large intestine, or inflammatory bowel disease in the large intestine, or inflammatory bowel disease in the colon.

[0027] The HMO is not particularly limited as long as it is an oligosaccharide found in breast milk, but examples include neutral HMOs or acidic HMOs, specifically 3SL, 6SL, 2FL, 3FL, lactodifucotetraose, lacto-N-fucopentaose I, LNT, lacto-N-neotetraose, LNT2, N-acetyllactosamine, or lactose-N-biose I. As the HMO, sialyl lactose is preferred, 3SL or 6SL is more preferred, and 3SL is even more preferred. Sialyl lactose is an acidic oligosaccharide formed by the bonding of N-acetylneuraminic acid and lactose. The agent according to this disclosure may contain one type of HMO alone, or it may contain a combination of two or more types of HMOs.

[0028] The agent relating to this disclosure contains an HMO, but the HMO may form a salt in the agent. The salt of the HMO may be a pharmaceutically acceptable salt. Examples of such salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; salts with organic acids such as acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, and p-toluenesulfonic acid; salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with amino acids such as arginine. When the HMO is 3SL or 6SL, it is preferable that the 3SL or 6SL is a free form that does not form a salt, or a sodium salt, and more preferably a sodium salt.

[0029] While HMOs or their salts may exist in polymorphic forms, in the agents relating to this disclosure, HMOs or their salts may be a single substance or a mixture of any of these crystalline forms. In the agents relating to this disclosure, HMOs or their salts may be amorphous. Furthermore, in the agents relating to this disclosure, HMOs or their salts may be anhydrous or solvates (especially hydrates).

[0030] HMOs or their salts can be produced by known methods. Known methods include chemical synthesis, microbial synthesis, and purification from whey. For example, sialyl lactose can be produced by the chemical synthesis method described in International Publication No. 2013 / 185780, the microbial synthesis method described in International Publication Nos. 2017 / 152918 and 2001 / 004341, and the purification from whey described in International Publication No. 2010 / 106320. Commercially available HMOs or their salts may also be used.

[0031] The agents described herein may be applied to humans and other mammals, but humans are preferred. Examples of other mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cattle, horses, goats, and monkeys, and the agents described herein can be used as feed, which is a form of food. Humans may be adults, elderly, children, infants, or newborns.

[0032] The agents relating to this disclosure can be administered orally or parenterally, and are preferably administered orally. When the agents relating to this disclosure are administered orally, the HMOs are more readily accessible to the intestinal tract, thus further suppressing the deterioration of the intestinal epithelial barrier function. Examples of parenteral administration include enteral administration, rectal administration, or intranasal administration.

[0033] Ingestion may be voluntary (i.e., ad libitum) or compulsory (i.e., forced ingestion). Administration may also be compulsory, and may involve supplying the agent relating to this disclosure to a subject, thereby allowing the subject to ad libitum intake of HMOs.

[0034] The content of HMOs in free form in the agent relating to this disclosure is not particularly limited, based on the total amount of the agent, but may be, for example, 0.01% to 90% by mass, or 0.1% to 80% by mass.

[0035] The dosage or intake of the agent relating to this disclosure is not particularly limited, but for example, for a subject weighing 60 kg, the daily dosage or intake in terms of free HMOs may be 50 mg to 30 g, and more preferably 100 mg to 10 g.

[0036] The number of times the agent relating to this disclosure is administered (ingested) is not particularly limited, but for example, it may be once to three times a day.

[0037] The duration of administration (ingestion) of the agent relating to this disclosure is not particularly limited, but may be, for example, from 1 day to 10 years, from 3 days to 1 year, or from 1 week to 6 months. Furthermore, the agent relating to this disclosure may be administered (ingested) throughout the subject's lifetime, or for a period of time in the subject's lifetime. HMOs may be administered (ingested), for example, until the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease is suppressed. The agent relating to this disclosure may be administered (ingested), for example, daily, or once every few days. In particular, the agent relating to this disclosure may be administered (ingested) daily. The dose (ingestion) of the agent relating to this disclosure at each administration (ingestion) may be constant or may vary.

[0038] The inhibitor of impaired intestinal epithelial barrier function in non-infectious inflammatory bowel disease relating to this disclosure may be a food or beverage composition or a pharmaceutical composition. The therapeutic agent for non-infectious inflammatory bowel disease relating to this disclosure may be a pharmaceutical composition. The preventive agent for non-infectious inflammatory bowel disease relating to this disclosure may be a food or beverage composition or a pharmaceutical composition. The ameliorative agent for non-infectious inflammatory bowel disease relating to this disclosure may be a food or beverage composition. Hereinafter, the agent relating to this disclosure that is a food or beverage composition will also be referred to as the "food or beverage composition relating to this disclosure," and the agent relating to this disclosure that is a pharmaceutical composition will also be referred to as the "pharmaceutical composition relating to this disclosure."

[0039] (Food and drink composition) The food and beverage compositions relating to this disclosure are not particularly limited as long as they contain HMOs. The food and beverage compositions may be provided in any form, such as liquid (syrup, etc.), paste, solid (capsule, tablet, granule, lozenge, etc.), or powder.

[0040] Food and beverage compositions may be, for example, food and beverages themselves, or materials used in the manufacture of food and beverages. Examples of such materials include seasonings, food additives, and other food ingredients. Specific examples of food and beverage compositions include wheat flour products, instant foods, processed agricultural products, processed marine products, processed livestock products, dairy products (fermented milk, cheese, infant formula, etc.), oils and fats, basic seasonings, compound seasonings, frozen foods, confectionery, beverages, and other commercially available food and beverages. Specific examples of food and beverage compositions include health foods, functional foods, enteral nutrition foods, foods for special dietary uses, health functional foods (foods for specified health uses, nutrient function foods, foods with functional claims, etc.), and nutritional supplements. Food and beverage compositions may also include supplements, such as tablet-shaped supplements.

[0041] The food and beverage compositions relating to this disclosure can be manufactured, for example, by combining an HMO with an additional component. The operation of combining an HMO with an additional component is also referred to as "addition of HMO." The method of manufacturing the food and beverage compositions relating to this disclosure is not particularly limited. Except for the addition of HMO, the food and beverage compositions relating to this disclosure can be manufactured, for example, using the same raw materials as ordinary food and beverages and using the same methods as ordinary food and beverages. The same applies when the food and beverage compositions relating to this disclosure are manufactured as materials used in the manufacture of food and beverages. The addition of HMO may be carried out at any stage of the manufacturing process of the food and beverage composition. The addition of HMO may be carried out, for example, during or after the manufacture of the food and beverage composition. That is, for example, the food and beverage compositions relating to this disclosure may be manufactured by adding HMO to a pre-prepared food or beverage.

[0042] Furthermore, other food and beverage compositions can be manufactured using the food and beverage compositions relating to this disclosure. That is, for example, if the food and beverage compositions relating to this disclosure are provided as materials used in the manufacture of food and beverages (e.g., seasonings, food additives, or other food and beverage ingredients), other food and beverage compositions may be manufactured by adding the food and beverage compositions relating to this disclosure. Such other food and beverage compositions are also examples of food and beverage compositions relating to this disclosure. For the addition of the food and beverage compositions relating to this disclosure in the manufacture of food and beverage compositions, the addition of HMOs in the manufacture of food and beverage compositions described above can be used.

[0043] The food and beverage compositions relating to this disclosure may be provided and sold as food and beverages labeled with intended uses (including health uses), such as suppressing the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease, preventing non-infectious inflammatory bowel disease, or improving non-infectious inflammatory bowel disease. The food and beverage compositions relating to this disclosure may be provided and sold as food and beverages labeled with intended uses.

[0044] "Display" includes all actions taken to inform consumers of the above-mentioned uses, and any expression that can evoke or infer the above-mentioned uses constitutes a "display," regardless of the purpose, content, object, or medium of the display. In particular, the display may be carried out using expressions that allow consumers to directly recognize the above-mentioned uses.

[0045] Specifically, examples of indications include transferring, delivering, displaying for transfer or delivery, or importing products relating to the food and beverage composition related to this disclosure or products with the above-mentioned uses described on the packaging; displaying or distributing advertisements, price lists, or transaction documents relating to the products with the above-mentioned uses described on them; or providing information containing these materials by electromagnetic means (such as the Internet). Examples of indications include indications on packaging, containers, catalogs, brochures, promotional materials (POP, etc.) at sales sites, or other documents.

[0046] Examples of labeling include labels for health foods, functional foods, enteral nutrition foods, foods for special dietary uses, health functional foods (foods for specified health uses, nutrient function foods, foods with functional claims, etc.), or nutritional supplements. Preferably, the labeling may be approved by the government or other authorities (for example, labels approved under various systems established by the government and made in accordance with such approval). Examples of labels approved by the government or other authorities include labels approved by the Consumer Affairs Agency of Japan. Examples of labels approved by the Consumer Affairs Agency include labels approved under the health functional food (foods for specified health uses, nutrient function foods, foods with functional claims, etc.) system and similar systems. Specifically, examples of labels approved by the Consumer Affairs Agency of Japan include labels as foods for specified health uses, labels as conditionally specified health foods, labels indicating an effect on the structure and function of the body, labels indicating a reduction in disease risk, and labels indicating scientifically based functionality. More specifically, the labels approved by the Consumer Affairs Agency of Japan include those for which the product is designated as a Food for Specified Health Uses (especially those for health purposes) as defined in the Cabinet Office Ordinance concerning the Permission for Special Use Labeling under the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009), and similar labels.

[0047] The HMO content in the food and beverage composition relating to this disclosure, as well as the amount and frequency of intake of the food and beverage composition relating to this disclosure, may be the same as those of the agent relating to this disclosure.

[0048] (Pharmaceutical composition) Examples of pharmaceutical compositions relating to this disclosure include pharmaceuticals or quasi-drugs, and are not particularly limited as long as they contain HMOs.

[0049] The pharmaceutical composition relating to this disclosure may be formulated into any desired dosage form. The dosage form of the pharmaceutical composition relating to this disclosure is not particularly limited. The dosage form of the pharmaceutical composition relating to this disclosure can be appropriately selected according to various conditions such as the method of administration. The pharmaceutical composition relating to this disclosure may be for oral administration or for parenteral administration. As mentioned above, since HMOs easily reach the intestinal tract, oral administration is preferred for the pharmaceutical composition relating to this disclosure, as this further suppresses the deterioration of the barrier function of the intestinal epithelium. In the case of oral administration, examples of dosage forms include solid preparations such as powders, granules, tablets, and capsules, as well as liquid preparations such as solutions, syrups, suspensions, and emulsions. In the case of parenteral administration, examples of dosage forms include suppositories or ointments.

[0050] The method of formulation is not particularly limited. Formulation can be carried out, for example, by known methods depending on the dosage form. Physiologically acceptable additives can be used in formulation. Examples of additives include various organic and inorganic components. Specifically, examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, pH adjusters, colorants, diluents, surfactants, or solvents. These additives can be appropriately selected, for example, depending on various conditions such as the dosage form.

[0051] Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, and carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

[0052] In addition to the above-mentioned excipients, other examples of binders include gelatin, polyvinylpyrrolidone, and macrogol.

[0053] In addition to the above-mentioned excipients, disintegrants include, for example, croscarmellose sodium, carboxymethyl starch sodium, or chemically modified starch or cellulose derivatives such as cross-linked polyvinylpyrrolidone.

[0054] Examples of lubricants include talc; stearic acid; metal stearate salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as beecam and gayl wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylate salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acid such as anhydrous silicic acid and silicic acid hydrate; and starch derivatives.

[0055] Examples of stabilizers include para-hydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid, etc.

[0056] Examples of flavoring and odor-modifying agents include sweeteners, acidulants, and flavorings.

[0057] The HMO content in the pharmaceutical composition relating to this disclosure, as well as the dosage, frequency of administration, and duration of administration of the pharmaceutical composition relating to this disclosure, may be the same as those of the agent relating to this disclosure described above.

[0058] <Method related to this disclosure> The methods relating to this disclosure for suppressing the impaired barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease, and for treating, preventing, or improving non-infectious inflammatory bowel disease, include administering them to or having them ingested by HMOs. Hereinafter, "the methods relating to this disclosure for suppressing the impaired barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease" and "the methods relating to this disclosure for treating, preventing, or improving non-infectious inflammatory bowel disease" will be collectively referred to as "the methods relating to this disclosure."

[0059] The administration (ingestion) conditions in the method relating to this disclosure (e.g., target recipient, dosage, frequency of administration, duration of administration, etc.) can be any of the conditions described above as the administration (ingestion) conditions for the agent relating to this disclosure, without limitation.

[0060] HMOs may be administered (ingested) to a subject as is, for example, or they may be prepared as an agent relating to this disclosure, such as a food or beverage composition or a pharmaceutical composition containing HMOs, and administered (ingested) to a subject. The food or beverage composition may be the food or beverage composition relating to this disclosure, and the pharmaceutical composition may be the pharmaceutical composition relating to this disclosure. That is, one aspect of the method relating to this disclosure may include administering the agent relating to this disclosure to a subject or having the subject ingest it. HMOs may be administered (ingested) alone, or they may be administered (ingested) in combination with additional components. Additional components include food or beverages, animal feed, pharmaceuticals, and components contained therein. The additional components may be the aforementioned components other than HMOs that the food or beverage composition or pharmaceutical composition relating to this disclosure may contain.

[0061] <Compositions related to this disclosure> One aspect of this disclosure relates to a composition containing human milk oligosaccharides for suppressing the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease. Specific embodiments of this composition can be applied without limitation to the embodiments described above as specific embodiments of the agent for suppressing the decline in the barrier function of the intestinal epithelium in non-infectious inflammatory bowel disease according to this disclosure.

[0062] One aspect of this disclosure relates to a composition containing human milk oligosaccharides for the treatment, prevention, or improvement of non-infectious inflammatory bowel disease. Specific embodiments of the composition may be applied without limitation to the embodiments described above as specific embodiments of the agent for the treatment, prevention, or improvement of non-infectious inflammatory bowel disease relating to this disclosure.

[0063] <HMO or use of HMO related to this disclosure> One aspect of the present disclosure relates to HMOs for use in a therapeutic method for suppressing the decline of the barrier function of the intestinal epithelium in non-infectious inflammatory bowel diseases. One aspect of the present disclosure relates to the use of HMOs in a non-therapeutic method for suppressing the decline of the barrier function of the intestinal epithelium in non-infectious inflammatory bowel diseases. One aspect of the present disclosure relates to the use of HMOs for the manufacture of an agent for suppressing the decline of the barrier function of the intestinal epithelium in non-infectious inflammatory bowel diseases. As specific embodiments of these HMOs and their uses, the above-described embodiments described as specific embodiments of the agent and / or the method according to the present disclosure can be applied without limitation.

[0064] One aspect of the present disclosure relates to HMOs for use in a method for treating non-infectious inflammatory bowel diseases or a therapeutic method for prevention. One aspect of the present disclosure relates to the use of HMOs in a non-therapeutic method for preventing or improving non-infectious inflammatory bowel diseases. One aspect of the present disclosure relates to the use of HMOs for the manufacture of an agent for treating, preventing or improving non-infectious inflammatory bowel diseases. As specific embodiments of these HMOs and their uses, the above-described embodiments described as specific embodiments of the agent and / or the method according to the present disclosure can be applied without limitation.

Examples

[0065] The present invention will be described in more detail with reference to the following examples.

[0066] In Examples 1 and 2, the evaluation of the barrier function was performed by the method described in the following <Evaluation of Barrier Function>. Also, in Examples 1 and 2, the T84 cells used were the T84 cells prepared by the method described in the following <Preparation of T84 Cells>.

[0067] <Evaluation of Barrier Function> The TEER value was used to evaluate the barrier function of the cell layer. First, the electrical resistance value (the electrical resistance value of the well seeded with cells) of the cell layer on the well of the cell insert of a Transwell plate (manufactured by Corning) was obtained using a Millicell (registered trademark) ERS-2 (manufactured by Millipore), which is a resistance measurement system. Also, an electrical resistance value (the electrical resistance value of the blank well) was obtained in the same procedure as the measurement of the above electrical resistance value, except that there were no cells on the well of the cell insert. Next, the TEER value was obtained by substituting the two obtained electrical resistance values into the following formula. Note that 0.33 (cm 2 ) in the following formula is the culture area of the above well. TEER value (Ω·cm 2 ) = ([Electrical resistance value of the well seeded with cells (Ω)] - [Electrical resistance value of the blank well (Ω)]) × 0.33 (cm 2 ) … (Formula)

[0068] For each treatment group, the mean value ± standard error of the TEER value was determined. Also, the significance test between treatment groups was performed by analyzing through Dunnett's test after one-way analysis of variance. When the calculated significance level (p-value) was less than 0.05, it was determined that there was a significant difference between treatment groups.

[0069] <Preparation of T84 cells> Human colon cancer-derived intestinal epithelial cells, T84 cells, were obtained from the European Collection of Authenticated Cell Cultures (ECACC). T84 cells are a cell line established from human colon cancer and have been confirmed to exhibit a colon-like phenotype, such as the expression of colon markers (Non-Patent Document 5). T84 cells were passaged 10 to 20 times using Dulbecco's modified Eagle's medium F-12 (DMEM / F-12; Thermo Fisher Scientific) containing 10% by volume of fetal bovine serum (manufactured by Sigma) and 1% by volume of penicillin-streptomycin (manufactured by Thermo Fisher Scientific) as a maintenance medium. Thereafter, T84 cells were detached using a 0.05% by mass trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), and T84 cells were recovered as a cell suspension using the above maintenance medium. Using a cell strainer (manufactured by Falcon), cell clumps in the above cell suspension were made uniform. Thereafter, T84 cells were seeded onto the cell insert of a transwell plate (manufactured by Corning) so as to have 1.0 × 10 5 cells / well, 1 mL of the maintenance medium was added outside the cell insert, and the cells were left standing in an incubator at 37°C and 5% by volume CO2 and used in Examples 1 and 2 described below.

[0070] [Example 1: Evaluation of the effect of IFN-γ and / or TNF-α on the barrier function of T84 cells] The T84 cells left standing in the incubator in the above <Preparation of T84 cells> were cultured in the incubator for 6 days. Thereafter, the wells of the cell insert of the above T84 cells were grouped. Specifically, the TEER value of the cell layer of the T84 cells in each well was measured using the method described in <Evaluation of Barrier Function> above. The T84 cells in each well were then divided into the treatment groups listed in Table 1 below, ensuring that no statistically significant difference was observed in the obtained TEER values. Subsequently, the cells were cultured for 24 hours in an incubator at 37°C and 5% CO2, and the culture medium outside the cell inserts of each treatment group was replaced with the maintenance medium containing IFN-γ (Fujifilm Wako Pure Chemical Industries, Ltd.) and / or TNF-α (ThermoFisher Scientific Corporation) at the concentrations listed in Table 1 below. However, in the control group, the culture medium was replaced with the maintenance medium that did not contain either IFN-γ or TNF-α. After that, the cells were cultured for 20 hours in an incubator at 37°C and 5% CO2 by volume, and the TEER value was measured using the method described in <Evaluation of Barrier Function> above. These tests were performed with n=4. The results are shown in Figure 1.

[0071] [Table 1]

[0072] As shown in Figure 1, in each of the experimental groups A to C, the mean TEER values ​​of the IFN-γ-treated group and the IFN-γ+TNF-α-treated group were lower than those of the control group. In each of the experimental groups A to C, the mean TEER value of the IFN-γ-treated group was similar to that of the IFN-γ+TNF-α-treated group. Also, in each of the experimental groups A to C, the mean TEER value of the TNF-α-treated group was similar to that of the control group. From the above, it was shown that IFN-γ (especially IFN-γ at 1-30 ng / mL) causes a decrease in the barrier function of T84 cells, while TNF-α at 0.1-3 ng / mL does not cause a decrease in the barrier function of T84 cells. Therefore, it was revealed that in human colon-derived T84 cells, a TNF-α-independent decrease in barrier function is caused by IFN-γ.

[0073] [Example 2: Evaluation of the effect of human milk oligosaccharides on the reduction of barrier function in T84 cells] The T84 cells that had been left standing in the incubator in the above <Preparation of T84 cells> were cultured in the same incubator for 5 days. Then, the T84 cells in the cell inserts were grouped by well. Specifically, the TEER value of the cell layer of the T84 cells in each well was measured by the method described in the above <Evaluation of Barrier Function>, and the T84 cells in each well were grouped into the treatment groups described in Table 2 below so that no statistically significant difference occurred in the obtained TEER values. Then, the medium in the cell inserts of each treatment group was replaced with the above maintenance medium in the control group and the IFN-γ treatment group, the above maintenance medium containing 10, 20, or 30 mg / mL of 3SL sodium salt in the IFN-γ + 3SL treatment group, and the above maintenance medium containing 10, 20, or 30 mg / mL of 6SL sodium salt in the IFN-γ + 6SL treatment group, respectively. Then, they were cultured in an incubator at 37°C and 5% CO2 for 24 hours, and the medium outside the cell inserts was replaced with the above maintenance medium containing 5 ng / mL of IFN-γ in the IFN-γ treatment group, the IFN-γ + 3SL treatment group, and the IFN-γ + 6SL treatment group, and the above maintenance medium without IFN-γ in the control group. Then, after culturing in an incubator at 37°C and 5% CO2 for 48 hours, the TEER value was measured by the method described in the above <Evaluation of Barrier Function>. These tests were performed with n = 4. The results are shown in Figure 2.

[0074]

Table 2

[0075] As shown in Figure 2, the mean TEER value in the IFN-γ-treated group was lower than that in the control group. This is consistent with Figure 1, indicating that IFN-γ causes a decrease in the barrier function of T84 cells. Furthermore, as shown in Figure 2, when comparing the mean TEER values ​​of the IFN-γ-treated group and the IFN-γ+3SL-treated group (3SL treatment concentration: 10, 20, or 30 mg / mL), the IFN-γ+3SL-treated group (3SL treatment concentration: 30 mg / mL) showed the highest value, followed by the IFN-γ+3SL-treated group (3SL treatment concentration: 20 mg / mL), followed by the IFN-γ+3SL-treated group (3SL treatment concentration: 10 mg / mL), and the IFN-γ-treated group showed the lowest value. Furthermore, when comparing the average TEER values ​​of the IFN-γ-treated group and the IFN-γ+6SL-treated group (6SL treatment concentration: 10, 20, or 30 mg / mL), the IFN-γ+6SL-treated group (6SL treatment concentration: 30 mg / mL) showed the highest value, followed by the IFN-γ+6SL-treated group (6SL treatment concentration: 20 mg / mL), followed by the IFN-γ+6SL-treated group (6SL treatment concentration: 10 mg / mL), and the IFN-γ-treated group showed the lowest value. Therefore, it was revealed that 3SL and 6SL, and especially 3SL, can suppress the decline in barrier function of human colon-derived T84 cells (particularly TNF-α-independent barrier function decline, and even more specifically IFN-γ-induced barrier function decline).

Claims

1. A non-infectious agent containing human milk oligosaccharides that suppresses the decline in the barrier function of the intestinal epithelium in inflammatory bowel disease.

2. The inhibitor according to claim 1, wherein the reduction in barrier function is TNF-α independent.

3. The inhibitor according to claim 1, wherein the aforementioned decrease in barrier function is promoted by IFN-γ.

4. The inhibitor according to any one of claims 1 to 3, wherein the human milk oligosaccharide is sialyl lactose.

5. The inhibitor according to claim 4, wherein the human milk oligosaccharide is 3'-sialyl lactose.

6. The inhibitor according to any one of claims 1 to 3, wherein the intestinal epithelium is the epithelium of the large intestine.

7. The inhibitor according to any one of claims 1 to 3, wherein the inflammatory bowel disease is Crohn's disease, ulcerative colitis, intestinal Behçet's disease, or simple ulcer.

8. A treatment, preventive, or ameliorative agent for non-infectious inflammatory bowel disease, containing human milk oligosaccharides.

9. The therapeutic, preventive, or ameliorative agent according to claim 8, wherein the inflammatory bowel disease is a disease accompanied by a TNF-α-independent impairment of the intestinal epithelial barrier function.

10. The therapeutic, preventive, or ameliorative agent according to claim 8, wherein the inflammatory bowel disease is a disease accompanied by impaired barrier function of the intestinal epithelium induced by IFN-γ.

11. The therapeutic, preventive, or ameliorative agent according to any one of claims 8 to 10, wherein the human milk oligosaccharide is sialyl lactose.

12. The therapeutic, preventive, or ameliorative agent according to claim 11, wherein the human milk oligosaccharide is 3'-sialyl lactose.

13. The therapeutic, preventive, or ameliorative agent according to any one of claims 8 to 10, wherein the inflammatory bowel disease is an inflammatory bowel disease in the large intestine.

14. The therapeutic, preventive, or ameliorative agent according to any one of claims 8 to 10, wherein the inflammatory bowel disease is Crohn's disease, ulcerative colitis, intestinal Behçet's disease, or simple ulcer.

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