Pharmaceutical composition containing fecal microbiota

Abstract

To provide a pharmaceutical composition suitable for prevention or treatment of an inflammatory bowel disease, particularly ulcerative colitis, through fecal microbiota transplantation.SOLUTION: The pharmaceutical composition of the present invention comprises an intestinal bacterium belonging to the genus Agathobacter, and preferably further comprises an intestinal bacterium belonging to at least one taxon selected from the group consisting of the genus Alistipes, the Christensenellaceae R-7 group, and the Oscillospiraceae UCG-002.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition containing enterobacteria belonging to a specific classification, an agent for improving inflammatory bowel disease, and a medicine or food containing the agent for improving inflammatory bowel disease. [Background technology]

[0002] Ulcerative colitis (UC), a type of inflammatory bowel disease (IBD), is a diffuse, nonspecific inflammation of unknown cause that primarily affects the mucosa, forming erosions and ulcers. It has been designated as an intractable disease by the Ministry of Health, Labor and Welfare. The number of patients is increasing annually, estimated to be over 200,000, making it the most common designated intractable disease in Japan. In recent years, the emergence of new drug therapies has dramatically improved the effectiveness of treatment, but the long-term prognosis remains unclear, and there is a need for a fundamental treatment method with fewer side effects and a different mechanism of action (Non-Patent Document 1).

[0003] Fecal microbiota transplantation therapy (also known as "FMT" or "fecal transplant therapy") has shown high therapeutic efficacy against refractory Clostridium difficile infectious enteritis and has already been put into practical use in Europe and the United States. However, its therapeutic efficacy against other diseases remains unclear, and numerous studies are currently being conducted. Various studies on fecal transplantation therapy have also been conducted for inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, but it has been reported that there are significant differences in therapeutic efficacy depending on the method used. Therefore, there is a need to elucidate the factors that indicate effectiveness and establish better treatment methods (Non-Patent Document 2).

[0004] In fecal transplant therapy for ulcerative colitis, the present inventors have conducted clinical studies on "antibiotic-assisted gut flora transplantation therapy (A-FMT)," in which antibiotics are administered as a pretreatment before transplantation, with the aim of more effectively reconstructing the gut flora and enhancing the efficacy of the transplant. These clinical studies have revealed the short- and long-term effectiveness of A-FMT for ulcerative colitis, and that the gut bacteria of the phylum Bacteroidetes are related to the therapeutic effect and the disease progression of ulcerative colitis (Non-Patent Documents 3 and 4).

[0005] Meanwhile, attempts have been made to isolate and identify bacterial species that are effective in fecal transplant therapy for inflammatory bowel diseases such as ulcerative colitis, and as a result, there are patent documents that disclose methods for preparing effective bacterial isolates and pharmaceutical compositions containing the obtained bacterial isolates (Patent Document 1).

[0006] Fecal transplantation therapy can be an effective treatment for inflammatory bowel diseases such as ulcerative colitis. However, little is known about the types of intestinal bacteria that are transplanted and established in the patient's intestine during fecal transplantation therapy, resulting in therapeutic effects. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2022-541278 [Non-patent literature]

[0008] [Non-Patent Document 1] Cai Z. et al., Front Med (Lausanne). 2021 Dec 20:8:765474 [Non-patent document 2] Zhang Z. et., Front Med (Lausanne). 2022; 9: 1068567 [Non-patent document 3] Ishikawa D. et al., Inflamm Bowel Dis, 2018, 24(12):2590-2598 [Non-patent document 4] Okahara K. et al., J Clin Med, 2020, 9(6):1650 Summary of the Invention [Problem to be solved by the invention]

[0009] The objective of the present invention is to screen intestinal bacteria that are deemed to be effective in treating ulcerative colitis by fecal transplant therapy, to clarify which bacterial species are effective, and to provide a means for preventing or treating ulcerative colitis. [Means for solving the problem]

[0010] As a result of extensive research, the inventors of the present invention have found that enterobacteria belonging to the genus Agathobacter are involved in improving the symptoms of patients with ulcerative colitis, and have demonstrated that the above-mentioned problem can be solved. That is, by administering a pharmaceutical composition containing enterobacteria belonging to the genus Agathobacter to patients with ulcerative colitis, it becomes possible to improve the symptoms.

[0011] More specifically, the present application provides the following aspects to solve the above-mentioned problems. [1] A pharmaceutical composition comprising an enterobacterium belonging to the genus Agathobacter. [2] The pharmaceutical composition according to [1], wherein the intestinal bacteria are obtained from human feces. [3] The pharmaceutical composition according to claim [1] or [2], wherein the composition is used for the prevention or treatment of inflammatory bowel disease. [4] The pharmaceutical composition according to any one of [1] to [3], wherein the inflammatory bowel disease is ulcerative colitis. [5] The pharmaceutical composition according to any one of [1] to [4], further comprising an enterobacteria belonging to at least one classification selected from the group consisting of the genus Alistipes, the family Christensenellaceae R-7 group, and the family Oscillospiraceae UCG-002. [6] An agent for improving inflammatory bowel disease, comprising an intestinal bacterium belonging to the genus Agatobacter that has an anti-inflammatory effect. [7] The improving agent described in [6], wherein the intestinal bacteria are obtained from human feces. [8] The improving agent according to [6] or [7], wherein the inflammatory bowel disease is ulcerative colitis. [9] The composition according to any one of [6] to [8], further comprising an enterobacteria belonging to at least one classification selected from the group consisting of the genus Alistipes, the family Christensenellaceae R-7 group, and the family Oscillospiraceae UCG-002.

[10] A pharmaceutical product containing the improving agent according to any one of [6] to [9].

[11] A food product containing the improving agent according to any one of [6] to [9]. [Effects of the Invention]

[0012] According to the present invention, by administering a pharmaceutical composition containing enterobacteria belonging to the genus Agathobacter to a patient suffering from ulcerative colitis, it is possible to improve the symptoms of the patient. The present invention also provides an agent for improving ulcerative colitis, which contains enterobacteria belonging to the genus Agathobacter, and a medicine or food containing the agent. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the bacterial abundance of Agatobacter bacteria in the feces of donors (HC) and ulcerative colitis patients (UC) before fecal transplantation (FMT). The vertical axis represents the log2(value+1)-transformed value of the bacterial count per 10,000, and the horizontal axis represents the HC or UC population. [Figure 2]Figure 2 shows the amount of Alistipes bacteria present in the feces of donors (HC) and ulcerative colitis patients (UC) before fecal transplantation. The vertical and horizontal axes are the same as those in Figure 1. [Figure 3] Figure 3 shows the amount of enterobacteria belonging to the R-7 group of Christensenellaceae present in the feces of donors (HCs) and ulcerative colitis patients (UCs) before fecal transplantation. The vertical and horizontal axes are the same as those in Figure 1. [Figure 4] Figure 4 shows the amount of enterobacteria belonging to the family Oscillospiraceae UCG-002 present in the feces of donors (HC) and ulcerative colitis patients (UC) before fecal transplantation. The vertical and horizontal axes are the same as those in Figure 1. [Figure 5] Figure 5 shows the amount of Agatobacter bacteria in the feces of responders and non-responders after FMT. The vertical axis represents the log2(value+1)-transformed value of the bacterial count per 10,000, and the horizontal axis represents responders or non-responders. [Figure 6] 6 is a graph showing the amount of enterobacteria belonging to the genus Alistipes present in the feces of responders and non-responders after FMT. The vertical and horizontal axes are the same as those in FIG. 5. [Figure 7] Figure 7 shows the amount of enterobacteria belonging to the R-7 group of the Christensenellaceae family present in the feces of responders and non-responders after FMT. The vertical and horizontal axes are the same as those in Figure 5. [Figure 8] 8 is a graph showing the amount of enterobacteria belonging to the family Oscillospiraceae UCG-002 present in the feces of responders and non-responders after FMT. The vertical and horizontal axes are the same as those in FIG. 5. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below. <Pharmaceutical Composition>

[0015] The pharmaceutical composition of the present invention contains enterobacteria belonging to the genus Agathobacter. Enterobacteria belonging to the genus Agatobacter are a type of butyrate-producing bacteria, anaerobic bacteria that produce butyrate as one of their major metabolic products. Butyrate-producing bacteria are present in the intestinal tract and oral cavity of animals, including humans. In the intestinal tract, butyrate-producing bacteria utilize indigestible carbohydrates, such as dietary resistant starch and plant-derived polysaccharides, as their primary growth substrates to produce butyrate. It is also believed that there is a pathway for butyrate-producing bacteria to secondary-use the degradation products, such as oligosaccharides and monosaccharides, produced by the metabolism of indigestible carbohydrates by bacteria with high polysaccharide degradation activity, or intermediate fermentation products, such as lactic acid and succinic acid, to produce butyrate.

[0016] The enterobacteria belonging to the genus Agathobacter used in the present invention may be any species of the genus Agathobacter that exhibits an anti-inflammatory effect in the intestine, including, for example, Agathobacter lectalis and Agathobacter ruminis. Specifically, the enterobacteria belonging to the genus Agathobacter used in the present invention may be identified as an enterobacterium of the genus Agathobacter by 16S rRNA gene sequence analysis.

[0017] In one embodiment of the pharmaceutical composition of the present invention, the intestinal bacterium belonging to the genus Agatobacter, which is an active ingredient in the pharmaceutical composition, is obtained from human feces. As described above, enterobacteria belonging to the genus Agathobacter are present in the intestinal tracts of not only humans but also other animals, and although those derived from any animal can be used, those derived from humans are preferred in terms of compatibility with humans, etc. Furthermore, enterobacteria belonging to the genus Agathobacter are normally present in the intestine and may be collected from the intestine by any method, or may be collected from the intestine and grown by culturing using an appropriate method. However, from the perspective of ease of collection, it is preferable to collect and use those present in feces.

[0018] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition is used for the prevention or treatment of inflammatory bowel disease. Inflammatory bowel disease (IBD) is a general term for a group of diseases characterized by chronic and recurrent intestinal inflammation, and includes primarily ulcerative colitis and Crohn's disease.

[0019] In one embodiment of the pharmaceutical composition of the present invention, the inflammatory bowel disease is ulcerative colitis. As described in the "Background Art" section above, ulcerative colitis ("UC") is a diffuse, nonspecific inflammation of unknown cause that primarily affects the mucosa, causing erosions and ulcers. The ulcerative colitis targeted for prevention or treatment by the pharmaceutical composition of the present invention may be of any of the following types: proctitis, distal colitis, left-sided colitis, and pancolitis, and the severity may be mild, moderate, or severe.

[0020] In one embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition further comprises an enterobacterium belonging to at least one species selected from the group consisting of the genus Alistipes, the family Christensenellaceae R-7 group, and the family Oscillospiraceae UCG-002.

[0021] Alistipes is a genus of Gram-negative, rod-shaped, anaerobic bacteria in the phylum Bacteroidetes. When bacteria from this genus colonize the human gastrointestinal tract, they confer protective effects against colitis (intestinal inflammation), autism, and cirrhosis (liver fibrosis). The intestinal bacteria of the genus Alistipes used herein may be any species of the genus Alistipes that exhibits an anti-inflammatory effect in the intestine, including, for example, Alistipes indistinctus, Alistipes massiliensis, Alistipes onderdonkii, Alistipes putredinis, and Alistipes shahii. Specifically, the intestinal bacteria belonging to the genus Alistipes used in the present invention include those identified as intestinal bacteria of the genus Alistipes by 16S rRNA gene sequence analysis.

[0022] It has been reported that the relative abundance of gut bacteria in the Christensenellaceae family is inversely correlated with human body mass index (BMI) and is associated with health conditions such as obesity and inflammatory bowel disease (BMC Biology, volume 17, Article number: 83 (2019) (https: / / bmcbiol.biomedcentral.com / articles / 10.1186 / s12915-019-0699-4)). The enterobacteria belonging to the Christenseneraceae R-7 group used herein can be any bacterium belonging to the Christenseneraceae R-7 group, as long as it exhibits an anti-inflammatory effect in the intestine. Specifically, the enterobacteria belonging to the Christenseneraceae R-7 group used in the present invention can be identified as belonging to the Christenseneraceae R-7 group by 16S rRNA gene sequence analysis.

[0023] The gut bacteria of the Oscillospiraceae family UCG-002 have been observed as part of a microbial community associated with health risks in certain studies (Nature Cancer, volume 5, pages 187-208 (2024) (https: / / www.nature.com / articles / s43018-023-00669-x)). The intestinal bacteria of the Oscillospiraceae family UCG-002 used herein may be any bacteria belonging to the Oscillospiraceae family UCG-002 family that exhibits an anti-inflammatory effect in the intestine. Specifically, the intestinal bacteria belonging to the Oscillospiraceae family UCG-002 used in the present invention include those identified as Oscillospiraceae family UCG-002 by 16S rRNA gene sequence analysis.

[0024] The pharmaceutical composition of the present invention can be applied (eg, administered, ingested, or inoculated) to humans as it is or together with conventional components.

[0025] The application form of the pharmaceutical composition of the present invention is not particularly limited, and it can be used in any application form, such as oral administration, enteral administration, rectal administration using a suppository or the like, or administration using a colonoscope.

[0026] The pharmaceutical composition of the present invention can be formulated by known pharmaceutical methods, for example, into capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated formulations, pellets, pastes, syrups, suspensions, emulsions, suppositories, etc., for oral or parenteral use.

[0027] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a pharmacologically acceptable carrier, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0028] In addition, the pharmaceutical composition of the present invention may contain other anti-inflammatory ingredients depending on its form, purpose, etc., as long as the effects of the present invention are not impaired. Examples of such anti-inflammatory ingredients include vitamin C, squalane, niacin, niacinamide, long-chain hyaluronic acid, placenta extract, sorbitol, chitin, chitosan, various plant extracts, etc. The amount of these ingredients to be added is not limited as long as the effects of the present invention are not impaired.

[0029] In preparing these formulations, particularly those intended for oral administration, it is preferable to combine the pharmaceutical composition of the present invention with a composition that enables efficient delivery to the large intestine.

[0030] The composition or method that enables delivery to the large intestine is not particularly limited, and known compositions or methods can be appropriately adopted. For example, a pH-sensitive composition, more specifically, an enteric polymer that releases its encapsulated contents when the pH becomes alkaline after passing through the stomach, can be used. Furthermore, when a pH-sensitive composition is used in formulation, it is preferable that the polymer has a degradation threshold of pH 6.8 to 7.5. This range corresponds to the pH shift to the alkaline side that occurs in the distal part of the stomach, and is therefore suitable for delivery to the large intestine.

[0031] Furthermore, compositions that enable delivery to the large intestine include compositions that ensure delivery to the large intestine by delaying the release of the contents for 3 to 5 hours, which roughly corresponds to the small intestinal transit time. Furthermore, formulations for delayed release compositions include formulations with a hydrogel shell that hydrates and expands upon contact with gastrointestinal fluid, thereby effectively releasing the contents. Furthermore, delayed release metering units include drug-containing compositions with a drug coating or selective coating material.

[0032] Examples of the selective coating material include biodegradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and / or enzymatically degradable polymers. Coating materials suitable for efficiently delaying release are not particularly limited, and examples thereof include cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

[0033] Further, compositions that enable delivery to the large intestine include bioadhesive compositions that specifically adhere to the large intestine mucosa (e.g., the polymers described in U.S. Pat. No. 6,368,586), and compositions that incorporate protease inhibitors to protect biological preparations, in particular, from degradation by proteolytic enzyme activity in the digestive tract.

[0034] Furthermore, systems that enable delivery to the large intestine include pressure-change-triggered delivery systems, such as those that release contents by utilizing pressure changes in the distal part of the stomach resulting from gas production by bacterial fermentation. Examples of such systems include, but are not limited to, capsules that contain contents dispersed in a suppository base coated with a hydrophobic polymer (e.g., ethyl cellulose).

[0035] Furthermore, examples of systems that enable delivery to the large intestine include delivery systems that are specifically degraded by enzymes present in the large intestine (e.g., carbohydrate hydrolases, carbohydrate reductases). Such systems are not particularly limited, but more specifically include systems that utilize food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

[0036] In the pharmaceutical composition of the present invention, the blending ratio of the enterobacteria having an anti-inflammatory effect to the composition varies depending on the form of the composition, etc. For example, in the case of an oral administration formulation, it is preferably 10 5pieces / g~10 12 pieces / g, more preferably 10 6 pieces / g~10 11 The range of bacterial counts per gram is given below.

[0037] The application (e.g., administration, ingestion, inoculation) of the pharmaceutical composition of the present invention is not particularly limited as long as it is an effective amount, and generally, the number of enterobacteria having an anti-inflammatory effect as an active ingredient is preferably 10 per day. 5 ~10 pieces 12 pcs, more preferably 10 6 ~10 pieces 11 The above dosage is preferably administered once a day or in divided doses of two or more (for example, two to three times a day), and can be increased or decreased as appropriate depending on age, pathological condition, and symptoms.

[0038] Those skilled in the art can easily determine the appropriate dosage of the enterobacteria with anti-inflammatory activity to be administered to a subject without undue experimentation. Usually, a doctor will determine the appropriate dosage for each individual patient, and the dosage will depend on various factors, including the activity of the strain used, the metabolic stability and duration of action of the strain, age, body weight, overall health, sex, diet, method and frequency of administration, excretion rate, drug combinations, the severity of the specific condition, and the treatment the individual is undergoing. The dosages disclosed herein are exemplary of standard cases. Of course, there may be individual cases where higher or lower dosage ranges are appropriate, and such cases are also encompassed by the present invention.

[0039] The pharmaceutical composition of the present invention can be administered to patients by a commonly used administration method depending on the form of the composition, particularly by oral administration in the case of a capsule, or by nasogastric tube, nasoduodenal tube, upper endoscopy, small intestinal balloon endoscopy, more preferably colonoscopy, or enema.

[0040] The pharmaceutical composition of the present invention exerts a therapeutic effect when administered to patients with ulcerative colitis. To achieve a greater therapeutic effect, it is desirable to administer antimicrobial therapy to the patient before FMT to reduce the patient's intestinal flora, thereby performing FMT afterward, thereby performing antimicrobial-assisted gut flora transplantation therapy (A-FMT). The antibacterial agent used in A-FMT can be any antibacterial agent that is normally administered orally, but it is particularly desirable to use a combination of the three antibacterial agents amoxicillin, fosfomycin, and metronidazole (AFM), or vancomycin and neomycin, and more preferably a combination of the three antibacterial agents amoxicillin, fosfomycin, and metronidazole. There is no problem if the administration of antibiotics is completed before FMT and the patient's intestinal flora has been reduced, but it is preferable to orally administer antibiotics to the patient for two weeks, for example, up until two days before FMT. <Agent for treating inflammatory bowel disease>

[0041] The agent for improving inflammatory bowel disease of the present invention contains an intestinal bacterium belonging to the genus Agatobacter that has an anti-inflammatory effect. In one embodiment of the agent of the present invention, the intestinal bacteria belonging to the genus Agathobacter, which is the active ingredient in the agent, are obtained from human feces. In one embodiment of the improving agent of the present invention, the inflammatory disease is ulcerative colitis. Furthermore, in one embodiment of the improving agent of the present invention, the improving agent further contains enterobacteria belonging to at least one class selected from the group consisting of the genus Alistipes, the family Christensenellaceae R-7 group, and the family Oscillospiraceae UCG-002.

[0042] The improving agent of the present invention can be used as an active ingredient in medicines or foods. The improving agent of the present invention can be applied to humans (e.g., administered, ingested, or inoculated) as is or together with conventional ingredients. <Pharmaceuticals>

[0043] The pharmaceutical of the present invention contains the above-mentioned inflammatory bowel disease-improving agent as an active ingredient, and by administering it to patients with inflammatory bowel disease, particularly ulcerative colitis, or people suspected of having the disease, the disease can be treated or prevented.

[0044] Various matters relating to the pharmaceutical product of the present invention overlap with the description of the "pharmaceutical composition" above, and therefore will be omitted here. <Food>

[0045] The food of the present invention contains the above-mentioned inflammatory bowel disease-improving agent as an active ingredient, and by administering it to people at risk of inflammatory bowel disease, particularly ulcerative colitis, the disease can be prevented. The food of the present invention can be, for example, a health food, a functional food, a nutritional supplement, or a supplement, and can also be a health functional food such as a food labeled to reduce the risk of disease (e.g., a food for specified health uses, a nutritionally functional food, a food with functional claims, etc.), or a food for sick people.

[0046] The formulation may be in any form, such as powder, granules, tablets, capsules, liquid, paste, or jelly, but when using fecal-derived enterobacteria, it is particularly desirable to use capsules.

[0047] In the case of the food of the present invention, the amount of intestinal bacteria having an anti-inflammatory effect to be added to the food varies depending on the form of the food, etc., but a person skilled in the art can appropriately set the amount of intestinal bacteria having an anti-inflammatory effect to be used, while predicting the effect based on known information, animal tests, etc. The amount of intestinal bacteria having an anti-inflammatory effect that can be set in this way is preferably 10 5 pieces / g~10 12 pieces / g, more preferably 10 6 pieces / g~10 11 The range of bacterial counts per gram is given below.

[0048] Furthermore, in the case of the food of the present invention, the improving agent of the present invention can be provided as a food additive for improving inflammatory diseases, either alone or in combination with other ingredients. When the improving agent of the present invention is used as a food additive, the content of the intestinal bacteria having an anti-inflammatory effect in the food additive, the amount of the food additive added to the food, etc. can be appropriately set so that the intestinal bacteria having an anti-inflammatory effect in the food to which it is added can satisfy the above-mentioned content.

[0049] The food of the present invention can be produced by a production technique known in the art. One or more ingredients effective in improving the condition of the large intestine through their anti-inflammatory effect may be added to the food. Furthermore, the food may be made multifunctional by combining it with other ingredients or other functional foods that exhibit functions other than anti-inflammatory. <Preparation of enteric bacteria>

[0050] The enterobacteria used in the present invention can be obtained or prepared by any of the following methods. (1) Obtaining effective bacteria from humans (2) Obtaining from sales companies, mail banks, etc. (3) Cultivation and propagation of the product obtained by (1) or (2)

[0051] These will be explained in order below. (1) Obtaining effective bacteria from humans (Acquisition of effective bacteria)

[0052] As described above, enterobacteria that can be used as active ingredients in the pharmaceutical composition and improving agent of the present invention are enterobacteria belonging to the genus Agatobacter, enterobacteria belonging to the genus Alistipes, enterobacteria belonging to the R-7 group of the Christensenellaceae family, and enterobacteria belonging to the UCG-002 family of the Oscillospiraceae family. The effectiveness of enterobacteria belonging to each of these groups in preventing or treating ulcerative colitis was demonstrated by examining the intestinal flora of donors (healthy individuals) who provided feces for FMT and of ulcerative colitis (UC) patients who underwent FMT, as described in the Examples below. These enterobacteria, which serve as active ingredients in pharmaceutical compositions effective in FMT, can be obtained, for example, as follows.

[0053] (Isolation and culture from human feces) Target bacteria can be isolated from human feces by culturing them in selective media or media supplemented with specific selective agents. For example, enteric bacteria belonging to the genus Agatho- bacterium are grown in selective media or on specific substrates, and then the desired bacteria are isolated using highly specific antibodies. (2) Obtaining from sales companies, mail banks, etc.

[0054] The enterobacteria that can be used as the active ingredient of the pharmaceutical composition and improving agent of the present invention can be purchased and used if they are commercially available from a company, etc. For example, they can also be obtained from libraries that already contain many isolated strains, such as those at the National Institute of Technology and Evaluation, National Biotechnology Center (NBRC), and the RIKEN BioResource Research Center (BRC). (3) Cultivation and propagation of the product obtained by (1) or (2)

[0055] In the pharmaceutical composition and improving agent of the present invention, the enterobacteria obtained by the method (1) or (2) can be used as an active ingredient of the pharmaceutical composition and improving agent as they are, but it is usually desirable to culture and grow them by an appropriate method before use. Alternatively, the cultured and grown enterobacteria may be concentrated and used in a live state. (Preservation treatment such as drying)

[0056] When administered or ingested as a medicine or food, it is necessary to ensure a viable cell count before administration. When storing bacteria to ensure a viable cell count, they may be stored at temperatures as low as -20°C or lower, in which case it is preferable to use a cryoprotectant. Furthermore, when powdering bacteria to ensure a viable cell count, the freeze-drying method is used, in which case it is preferable to use a freeze-drying protectant to ensure a viable cell count.

[0057] Examples of cryoprotectants or lyoprotectants include various sugars (sucrose, fructose, lactose, mannitol, etc.), glycerol, polyethylene glycol (PEG), trehalose, glycine, glucose, dextran, erythritol, etc. Among these, glycerol is particularly preferred as a cryoprotectant, and trehalose is particularly preferred as a lyoprotectant. <Evaluation of efficacy against ulcerative colitis>

[0058] The effect on ulcerative colitis can be evaluated, for example, by Lichtiger's index (CAI). The CAI is an index created to evaluate the effectiveness of cyclosporine. Eight items of subjective and objective symptoms are scored and the total score (0-21) is used to determine the effectiveness of cyclosporine. A score of 3 or less is considered "remission," and a score of 10 or less for two consecutive days is considered "effective." (Journal of the Japanese Society of Coloproctology, 64:807-816, 2011)

[0059] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. [Example]

[0060] <Test Method> (Research subjects) Of 97 patients with active ulcerative colitis (UC), 49 who met the following two criteria were selected as study subjects and treated with antibiotic-assisted gut flora transplantation therapy (A-FMT). Data from these 49 patients were analyzed. 1) Pre-treatment, post-treatment, and donor stool samples were obtained. 2) Each patient received A-FMT treatment from a different donor. These patients were aged 16 years or older and had been diagnosed with active UC with a Lichtiger's index (CAI) of 4 or higher or a Mayo score (MES) of 1 or higher. Patients with UC who had not achieved clinical remission despite appropriate treatment, including 5-aminosalicylic acid (5-ASA), immunomodulatory agents, corticosteroids, and / or biologics, were also included in the study.

[0061] (A-FMT protocol) The A-FMT method was performed based on the method described in the following paper. (1) Ishikawa D. et al., Inflamm Bowel Dis, 2017, 23:116-125 (2) Ishikawa D. et al., Inflamm Bowel Dis, 2018, 24:2590-2598

[0062] A brief description of this method is as follows. First, patients were orally administered three antibiotics (AFM)—amoxicillin (1500 mg / day), fosfomycin (3000 mg / day), and metronidazole (750 mg / day)—for two weeks until two days before undergoing gut microbiota transplantation therapy (FMT). Then, stool samples were collected from donors via colonoscopy. Fresh or frozen stool samples were suspended in saline, diluted, and filtered to obtain a bacterial suspension. After intestinal cleansing using a standard polyethylene glycol solution (Moviprep®; EA Pharma, Tokyo, Japan), patients underwent total colonoscopy. Approximately 200–400 mL of the bacterial suspension described above was administered via colonoscopy from the patient's cecum to the transverse colon, preferably within 6 hours of donor stool sample collection. Approximately two-thirds of the bacterial suspension was instilled into the cecum and ascending colon, and the remaining portion into the transverse colon. After the procedure was completed, patients were administered scopolamine to slow intestinal transit and allow sufficient time for donor bacteria to colonize. Patients were permitted to continue treatment, and their probiotic intake was adjusted throughout the study. No changes in medication dosage or initiation of new treatments were permitted.

[0063] (clinical evaluation) Disease activity was assessed at colonoscopy, which was performed at the time of FMT after AFM administration. Clinical characteristics were observed 4 weeks after FMT. For short-term evaluation, clinical characteristics of UC were assessed using the Lichtiger's index (CAI) before treatment, after antibiotic combination therapy, and 4 weeks after treatment. A CAI score of 9 or less and an improvement of 3 or more points after 4 weeks of treatment was defined as "effective." "Remission" was defined as a further improvement of the CAI to 3 or less points. "Non-recurrence" was defined as an increase in the CAI or intensification of treatment 12 months after treatment, which included an increase in dosage or a switch to a new treatment in the effective group. Endoscopic evaluation was performed using the Mayo score (MES). For long-term evaluation of short-term responders, clinical scores were assessed at outpatient visits every 1-2 months. Patients who could not visit our hospital were interviewed by telephone and mail every 3-6 months.

[0064] (Fecal sampling and DNA extraction) Each patient provided three types of fecal samples: pre-FMT, post-FMT, and donor, for a total of 147 samples. The collected fecal samples were immediately suspended in TE (Tris-EDTA) buffer containing 10% glycerol and stored in a -80°C freezer. DNA extraction from the collected fecal suspension was performed as follows: 100 μL of fecal suspension was transferred to a tube containing beads. Lysis Solution F (1000 μL) was added, and the mixture was homogenized at 1500 rpm for 2 minutes using a Shake Master® Neo. The homogenized sample was incubated at 65°C for 10 minutes, then centrifuged at 12,000 × g for 2 minutes to separate the supernatant, which was then collected. DNA was purified from the collected supernatant using the Lab-Aid 824s DNA Extraction Kit (ZEESEN).

[0065] (Amplicon sequencing) Amplicon sequencing was performed on the purified DNA using the following procedure. The concentration of the DNA solution was measured using Synergy LX (BioTek) and the QuantiFlour dsDNA System (Promega). The library was prepared by step-tailed PCR. The primers used were 16S rRNA V3 / V4 region: 341f-805r. The primer sequences are as follows: Forward:ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-CCTACGGGNGGCWGCAG Reverse:GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-NNNNN-GACTACHVGGGTATCTAATCC PCR reactions were performed in a 20 μL volume containing the following components: 1.0 μL each of 10 μM forward and reverse primers, 1 μL of 1 ng / μL template DNA, 0.2 μL of TaKaRa Ex Taq HS (10x), and nuclease-free water to a final volume of 13.2 μL. A dNTP mix (2.5 μM each) (TaKaRa) was also added to a final volume of 1.6 μL. The PCR program consisted of an initial denaturation step at 94°C for 2 minutes, followed by 25–35 cycles of amplification using the following conditions: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds. A final extension step was performed at 72°C for 5 minutes. The concentration of the prepared libraries was measured using a Synergy H1 (BioTek) and QuantiFlour dsDNA System. The quality of the prepared libraries was confirmed using a Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies). The base sequence was determined using a MiSeq System and MiSeq Reagent Kit v3 (Illumina) under 2 × 300 bp conditions.

[0066] [Table 1]

[0067] (16S rRNA gene sequence analysis) Sequence analysis was performed using a bioinformatics pipeline for 16S rRNA sequencing. This pipeline consists of several steps, including quality control, sequence trimming, merging, and taxonomic classification. Specifically, the cutadapt tool (version 4.1) was used to trim adapter sequences from raw sequence reads, the sickle tool (version 1.33) was used to perform quality control on the trimmed reads, and the FLASH tool (version 1.2.11) was used to merge overlapping paired-end reads into long contigs. Subsequent sequence analysis was performed using Qiime2 (version 2022.8.0), up to the construction of a phylogenetic tree.

[0068] (statistical analysis) Differences in bacterial abundance between the UC (ulcerative colitis) and HC (healthy donor) groups, and between the responder and non-responder groups, were calculated using ANCOM-BC (version 2.0.2). P values ​​were obtained from two-tailed Z tests using the test statistic W. For the comparison of UC versus HC, the q-value threshold was set at 0.1 only if there were ≥5 samples with a value for either of the two groups being compared, whereas for the comparison of responder versus non-responder groups, a p-value threshold of 0.1 was set. <Reference example>

[0069] Specific microbial characteristics associated with reproducible disease phenotypes were evaluated at the genus level in the UC (ulcerative colitis) group and HC (healthy control) group. When the relative abundance of each genera was compared between UC and HC, significant differences were observed in several genera (Figures 1 to 4). Furthermore, when evaluated at the genus level between the remission and non-response groups, significant differences were observed in several genera between transplant recipients and donors (Figures 5 to 8). The bacteria that were significantly more abundant in all groups, including HC, the remission group of transplant recipients, and the remission group of donors, were Agatobacter. (Agathobacter) Genus, Alistipes (Alistipes) Genus, Christensenella (Christennellaceae) Family R-7 group, and Oscillospira (Ocillospiraceae) There were four groups in the UCG-002 department.

[0070] When we looked at the genus Agathobacter, we found the following: 1.Comparing HC and UC, differences were observed in the genus Agatho- bacter. 2. In fact, when comparing donors with high levels of Agatho- bacterium and those with low levels, it was found that the preparation from a donor with high levels of Agatho- bacterium was more likely to result in remission.

[0071] <Prescription example> Prepare a buffer for suspending the feces. To protect the bacterial cells during freezing, mix 50 g of glycerol with 500 mL of saline. To ensure that stool complies with the standards, a visual inspection and sampling for parasite testing are carried out, and a stool donation ID is assigned. The stool is suspended in twice the volume of buffer (200 mL for 100 g of stool), the intestinal bacterial components are extracted, and the solution is filtered through a filter mesh to remove impurities. Multiple solutions obtained from the same donor are mixed and 200 mL of the solution is dispensed into a designated extraction container. The solution in the container is used as one dose of FMT. [Industrial Applicability]

[0072] The present invention provides a pharmaceutical composition containing enterobacteria belonging to the genus Agatobacter and an agent for improving inflammatory bowel diseases, thereby making it possible to prevent or treat inflammatory bowel diseases such as ulcerative colitis, and can be used in the fields of pharmaceuticals and foods.

Claims

1. A pharmaceutical composition comprising an enterobacterium belonging to the genus Agathobacter.

2. 2. The pharmaceutical composition of claim 1, wherein the intestinal bacteria are obtained from human feces.

3. 3. The pharmaceutical composition according to claim 1, which is used for the prevention or treatment of inflammatory bowel disease.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the inflammatory bowel disease is ulcerative colitis.

5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising an enterobacterium belonging to at least one species selected from the group consisting of the genus Alistipes, the family Christensenellaceae R-7 group, and the family Oscillospiraceae UCG-002.

6. An agent for improving inflammatory bowel disease, comprising an intestinal bacterium belonging to the genus Agatobacter that has an anti-inflammatory effect.

7. The improving agent according to claim 6, wherein the intestinal bacteria are obtained from human feces.

8. The ameliorating agent according to claim 6 or 7, wherein the inflammatory bowel disease is ulcerative colitis.

9. The composition according to any one of claims 6 to 8, further comprising an enterobacterium belonging to at least one classification selected from the group consisting of the genus Alistipes, the family Christensenellaceae R-7 group, and the family Oscillospiraceae UCG-002.

10. A pharmaceutical product containing the improving agent according to any one of claims 6 to 9.

11. A food product containing the improving agent according to any one of claims 6 to 9.

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