Patent Application
20230330159
A computer readable text file, entitled “SequenceListing.txt,” created on or about Mar. 24, 2023 with a file size of 2,358 bytes contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
The present invention relates to a composition for diagnosis or treatment of inflammatory disease comprising a Faecalibacterium spp. microorganism.
Inflammation occurs when an organism's immune system attempts to defend harmful substances such as invasive pathogens. Immunomodulatory mechanisms, including immunomodulatory cells, cytokines and apoptosis, regulate immune responses caused by an overreaction to defend against pathogens. The deficiency of the mechanisms manifests inflammatory diseases including inflammatory bowel disease (IBD). The inflammatory disease includes for example an inflammatory gastrointestinal disease selected from the group consisting of inflammatory bowel disease (Crohn's disease, ulcerative colitis), irritable bowel syndrome, chronic digestive disorder (coeliac disease), infectious colitis (for example, caused by C. difficile), Behcet's disease, other gastrointestinal-related diseases, and any combination thereof.
Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, characterized by the disrupted mucosal barrier, altered composition of intestinal microorganism and systemic biochemical abnormality. IBD is categorized into ulcerative colitis (UC) and Crohn's disease (CD) according to clinical features and inflammation localization in intestine. The accurate etiologic cause of IBD is not yet known, but it is known that an excessive immune response triggered by altered intestinal flora or environmental factors of pathogenic microorganisms is a main etiologic cause.
IBD is a disease caused by a complex and unknown etiologic cause, and treatment for IBD is very limited, and focuses on inducing remission and maintaining remission by improving symptoms rather than treatment. An optimal treatment method may be selected in consideration of the regional range of lesions, severity and clinical features, complications and the like.
Therapeutic agents of IBD show an effect through various mechanisms of action, and thus, they are being used step by step according to detailed clinical guidelines, and 5-aminosalicylate and corticosteroid are often used as primary therapy for mild to moderate IBD, and biological agents such as TNF-α inhibitors (e.g., infliximab, adalimumab and golimumab, etc.) are recommended as a secondary medicine for a patient who does not respond or fail to the primary treatment. However, patients who are administered with TNF-α inhibitor at a high dose in the early stage of disease is increasing sharply. The long-term use of a powerful immunosuppressive agent has a problem in serious side-effect, and thus, there is an ongoing clinical need for alternative therapies.
The relationship between IBD and intestinal flora has been confirmed in many studies would wide, and it has been reported that in IBD patients, microbiota dysbiosis characterized by deficiency of beneficial gut bacteria is shown, and the microbiota dysbiosis is being considered as immunopathogenesis of IBD.
An embodiment of the present invention is to provide a composition, kit or method for predicting a risk of developing inflammatory disease, specifically, inflammatory gastrointestinal disease, or diagnosing inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease using a Faecalibacterium spp. Microorganism, or for predicting or evaluating responsiveness of prevention, improvement or treatment to inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease, using the microorganism.
An embodiment of the present invention provides a composition for prevention, improvement or treatment of inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease, comprising at least one selected from the group consisting of; a microbial cell of a Faecalibacterium spp.; a culture of the microorganism; a lysate of the microorganism; and at least an extract selected from the group consisting of the microbial cell, culture and lysate. The composition may be a pharmaceutical or food composition.
In addition, the present invention relates to diagnosis of inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease, including for example, selection of a subject to be administered or evaluation of prevention, improvement or treatment efficacy of inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease.
An embodiment of the present invention provides a method, composition or kit for diagnosis of bowel disease comprising at least one selected from the group consisting of; a microbial cell of a Faecalibacterium spp.; a culture of the microorganism; a lysate of the microorganism; and at least an extract selected from the group consisting of the microbial cell, culture and lysate; a method, composition or kit for selecting a subject to which a microbial cell of a Faecalibacterium spp. and the like is administered; or a method, composition or kit for evaluating prevention, improvement or treatment efficacy of inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease by administering a microbial cell of a Faecalibacterium spp. into a subject.
An embodiment of the present invention relates to a composition for prevention, improvement or treatment of inflammatory disease, specifically, inflammatory gastrointestinal disease or inflammatory bowel disease, comprising at least one selected from the group consisting of anti-inflammatory agents, corticosteroids, immunomodulators, antibiotics, tumor necrosis factor inhibitors (tumor necrosis factor-α agent inhibitor, TNF-α inhibitor), Janus kinase inhibitors (e.g., tofacitinib, etc.), anti-integrin agents (e.g., vedolizumab, etc.), interleukin inhibitors (e.g., ustekinumab, etc.), and the like, in addition to at least one selected from the group consisting of; a microbial cell of a Faecalibacterium spp.; a culture of the microorganism; a lysate of the microorganism; and at least an extract selected from the group consisting of the microbial cell, culture and lysate. The example of the anti-inflammatory agent includes 5-aminosalicylate, sulfasalazine and olsalazine. The example of the steroids includes corticosteroids, glucocorticosteroids, prednisone, hydrocortisone, methylprednisolone, dexamethasone and adrenocorticotropic hormone (hereinafter, abbreviated as “ACTH”.). The example of the immunomodulator includes deglycolipidated, deglycolipidated Mycobacterium vaccae (hereinafter, abbreviated as “PVAC”.), anti-tumor necrosis factor receptor superfamily member 5 (hereinafter, abbreviated as “anti-CD40”.) ligands, anti-CD40, natalizumab (Antegren™), anti-vascular adhesion molecule 1 (hereinafter, abbreviated as “anti-VCAM1”.) and anti-intercellular adhesion molecule-1 (hereinafter, abbreviated as “anti-ICAM1”.). The example of the cytokine includes interleukin-10 (hereinafter, abbreviated as “IL-10”.). The example of the TNF antagonist includes infliximab (Remicade0), etanercept (EnbrelOO), adalimumab (Humira), and Certolizumab Pegol (hereinafter, abbreviated as “CDP870”.). An example of the Janus kinase inhibitor includes Tofacitinib (Xeljanz), and an example of the anti-integrin agent includes vedolizumab (KyntelesR), and an example of the interleukin inhibitor includes ustekinumab (Stelara pfs).
An embodiment of the present invention provides a composition, kit or method for selecting a subject to be administered with a Faecalibacterium spp. Specifically, the present invention predicts a risk of developing inflammatory gastrointestinal disease, for example, inflammatory bowel disease, or diagnoses gastrointestinal disease, for example, inflammatory bowel disease, or predicts or evaluates responsiveness of prevention, improvement or treatment to inflammatory gastrointestinal disease, for example, inflammatory bowel disease according to administration of the microorganism, by detecting Faecalibacterium cancerinhibens in a sample of a test subject, and accordingly, it may determine whether the subject can receive improvement or treatment therapy of gastrointestinal disease, for example, inflammatory bowel disease.
An embodiment of the present invention provides a composition, kit or method for evaluating efficacy or responsiveness of Faecalibacterium cancerinhibens in a subject administered with Faecalibacterium cancerinhibens, using a Faecalibacterium spp. Specifically, an embodiment of the present invention relates to a composition or kit for predicting a risk of developing gastrointestinal disease, for example, inflammatory bowel disease, or diagnosing gastrointestinal disease, for example, inflammatory bowel disease, or predicting or evaluating responsiveness of prevention, improvement or treatment to gastrointestinal disease, for example, inflammatory bowel disease, comprising an agent for detecting Faecalibacterium cancerinhibens in a biological sample of a test subject.
An additional embodiment of the present invention relates to a method for predicting a risk of developing inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease, or diagnosing inflammatory disease, specifically, inflammatory gastrointestinal disease, for example, inflammatory bowel disease, or predicting or evaluating responsiveness of prevention, improvement or treatment to gastrointestinal disease, for example, inflammatory bowel disease, comprising detecting a Faecalibacterium cancerinhibens in a biological sample of a test subject.
The present invention provides a method for prevention, improvement or treatment of disease or disorder associated with the reduced level of Faecalibacterium cancerinhibens compared to the level of Faecalibacterium cancerinhibens in a healthy control group, wherein the method includes determining a subject as having a reduced level of Faecalibacterium cancerinhibens, and administering a composition including Faecalibacterium cancerinhibens when the subject is determined to have a reduced level of Faecalibacterium cancerinhibens.
An embodiment of the present invention relates to an anti-inflammatory agent, comprising at least one selected from the group consisting of; a microbial cell of a Faecalibacterium spp.; a culture of the microorganism; a lysate of the microorganism; and at least an extract selected from the group consisting of the microbial cell, culture and lysate. The Faecalibacterium spp. has an anti-inflammatory activity.
The composition may be a pharmaceutical composition or food composition, and may include probiotics alone, or in combination with prebiotics.
Herein, the inflammatory disease may include various inflammations, and for example, it may be inflammatory gastrointestinal disease, preferably, inflammatory gastrointestinal disease selected from the group consisting of inflammatory bowel disease (Crohn's disease, ulcerative colitis), irritable bowel syndrome, chronic digestive disorder (coeliac disease), infectious colitis (for example, caused by C. difficile), Behcet's disease, other gastrointestinal-related diseases, and any combination thereof Otherwise, the inflammatory disease is inflammatory autoimmune disease, and herein, dysbiosis-balanced microbiota and low-grade mucosal inflammation may be related to an etiologic cause of disease, such as diabetes (types 1 and 2), asthma and atopic disorders.
In the present invention, the effect of improvement, treatment or prevention of immunomodulatory or inflammatory disease exhibited by the strain of present invention may be characterized by being induced by increase or decrease of cytokines involved in inflammation inhibition and immunomodulation, specifically, any one or more actions of decrease in secretion of IFN-γ, IL-8, IL-6 and TNF-α and the like, and increase in secretion of IL-10.
The role of IFN-γ is representatively NK cell activation, macrophage activation and increased expression of MHC and the like, and chronic exposure to IFN-γ is known to be involved in several non-infectious pathologies such as autoimmune disease and inflammatory disease.
When inflammation occurs, Neutrophil can migrate to intestinal lumen through tight junction between intestinal epithelial cells and can play a role in additionally exacerbating inflammation. In this stage, IL-8 as an inflammatory cytokine is an important factor playing a role in attracting such Neutrophil to the inflammatory site, and causing tissue damage by infiltrating Neutrophil into the lamina propria musoca and secreting inflammatory mediators.
IL-6 is secreted from various cells such as T lymphocytes, macrophages and the like to stimulate an immune response, and in this case, it may cause tissue damage leading to infection, trauma, particularly, burns or inflammation. In addition, it is known that IL-6 can play a role in accelerating inflammation by inducing Th17 cell differentiation and promoting secretion of RORγt or IL-17.
TNF-α, one of major factors causing inflammation, is mainly secreted from activated macrophages, and is secreted from various cells such as helper T cells, natural killer cells, and damaged neurons and the like. In addition, its most important role is regulation of immunocytes, which can induce production of IL-1 and IL-6, and is known to play a role in promoting an inflammatory response in various inflammatory diseases such as rheumatoid arthritis, Crohn's disease and ulcerative colitis, and the like.
In addition, IL-10 is an important immunomodulatory cytokine produced in various cells such as helper T cells, B cells, monocytes and the like, and its most important function is inhibition of an inflammatory response, and it has immunosuppressive and anti-inflammatory activities such as inhibiting production of pro-inflammatory cytokines including IL-6 and TNF-α, and the like.
Inflammatory bowel disease (IBD) is chronic inflammatory disease of the gastrointestinal tract characterized by diarrhea, bloody stool, abdominal pain and weight loss, and is chronic, recurrent inflammation of unknown etiology. IBD includes ulcerative colitis (UC) and Crohn's disease (CD), which are two major disease conditions, and more specifically, includes chronic or acute ulcerative colitis and Crohn's disease. UC mainly affects the mucosal layer of large intestine or colon. In contrast, CD is defined as transmural granulomatous inflammation that may include segments of small and large intestine.
The present invention provides an anti-inflammatory (probiotic) composition, for example, a functional food, neutraceuticals or a pharmaceutical composition containing probiotic bacteria, and a method for promoting survival of probiotics in the gastrointestinal tract.
In the present invention, the Faecalibacterium spp. may be for example, a bacterium comprising a 16S rDNA sequence having sequence identity of 98% or more, 98.5% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more with the nucleotide sequence of SEQ ID NO: 1, and may be a bacterium comprising a 16S rDNA gene comprising the nucleotide sequence of SEQ ID NO: 1, preferably, the Faecalibacterium cancerinhibens strain CLCC1 deposited with Accession number KCTC 13783BP. The microorganism or bacterium may have at least one characteristic selected from the group consisting of the following characteristics:
Faecalibacterium cancerinhibens of the present invention may have at least one characteristic selected from the above characteristics. For example, the strain having all the characteristics of (1) to (7) above, and Faecalibacterium cancerinhibens CLCC1 is a new species that has not been previously isolated and reported. The Faecalibacterium spp. strain was identified by the above method, named as Faecalibacterium cancerinhibens CLCC1, and deposited to Korea Research Institute of Bioscience and Biotechnology, Korean Collection for Type Cultures as Accession number of KCTC 13783BP.
More specifically, as a result of genome analysis of Faecalibacterium cancerinhibens strain CLCC1 and comparison with allied species, Faecalibacterium cancerinhibens strain CLCC1 shows an average nucleotide identity (ANI) value of 85.99% at a genome level with the closest allied species, Faecalibacterium prausnitzii, so it is a novel species that has not been previously isolated and reported.
More specifically, the anti-cancer activity according to the present invention may be an anti-cancer activity against colorectal cancer and/or liver cancer, and for example, it may be an activity to inhibit occurrence or progress of liver cancer and/or colorectal cancer. Specifically, it may delay tumor occurrence or inhibit the growth rate of tumor, and it may further have an activity to prevent cancer metastasis due to inhibition of the growth rate of tumor.
Faecalibacterium spp. according to the present invention is isolated from feces of healthy adult male human, the effect of microorganism to inhibit cancer occurrence and tumor growth in a human cancer cell line, a mouse model subcutaneously transplanted with the cancer cell line, or a mouse model having the corresponding tissue transplanted with the cancer cell line. Specifically, in an example of the present invention, Faecalibacterium cancerinhibens strain CLCC1 was isolated from feces of healthy adult males and was confirmed to have an effect of inhibiting tumor growth in mice which was induced for colorectal or liver cancer or transplanted with colorectal or liver cancer. The activity to inhibit the tumor growth may be measured by using for example, the tumor volume/size or tumor weight.
When the Faecalibacterium cancerinhibens provided by the present invention is administered to the animal model subcutaneously transplanted with the cancer cell line, compared to the control group not administered by the microorganism, the tumor growth inhibition rate according to the tumor weight may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more, and the tumor growth inhibition rate according to the tumor weight may be calculated by following Equation 1.
IR(%)=(1−(T1/C1)×100 [Equation 1]
In the Equation 1, IR is the tumor growth inhibition rate according to the tumor weight (IR), and T1 is the average tumor weight in each experimental group, and C1 is the average tumor weight in the negative control group.
When Faecalibacterium cancerinhibens provided by the present invention is administered to the animal model subcutaneously transplanted with a cancer cell line, compared to the control group not administered by the microorganism, the relative reduction rate of tumor volume may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more, and the tumor growth inhibition rate according to the tumor volume may be calculated by Equation 2 below.
Relative reduction rate of tumor volume (%)=(1−(T2/C2))×100 [Equation 2]
In the Equation 2, T2 is the average tumor volume in each experimental group and C2 is the average tumor volume in the negative control group.
When Faecalibacterium cancerinhibens strain CLCC1 is administered to a tumor animal model transplanted with cancer cell lines according to an example of the present invention, based on 100% of the tumor volume of the negative control group not administered with the microorganism, the tumor volume may be 90% or less, 85% or less, 80% or less, 77% or less, 70% or less, 67% or less, and the tumor volume may have a numerical range combining a value selected from the lower limit of 10% or more, 15% or more, 18% or more, and 20% or more, and a value selected from the upper limit of 90% or less, 85% or less, 80% or less, 77% or less, 70% or less, and 67% or less.
In case that the cancer is colorectal cancer, Faecalibacterium cancerinhibens strain CLCC1 may have an activity to reduce the tumor volume by a level of 10 to 90%, 10 to 85%, 10 to 80%, 10 to 77%, 10 to 70%, 10 to 67%, 20 to 90%, 20 to 80%, 20 to 70%, 20 to 67%, 30 to 90%, 30 to 80%, 30 to 70%, 30 to 67%, 40 to 90%, 40 to 80%, 40 to 70%, 40 to 67%, 50 to 90%, 50 to 80%, 50 to 70%, 50 to 67% or 60 to 67%, based on 100% of the tumor volume of the negative control group not administered with the microorganism, when the microorganism is administered.
Specifically, it was confirmed that the average tumor size at the day (Day 1) when the administration of the microorganism was started in a mouse subcutaneous tumor model using a human colorectal cancer cell line had a tumor volume of about 60 to 67% compared to the negative control group not administered with the microorganism, thereby confirmed that the effect of Faecalibacterium cancerinhibens on inhibiting the growth of colorectal cancer.
In case that the cancer is liver cancer, Faecalibacterium cancerinhibens CLCC1 provided by the present invention may have an activity to reduce the tumor volume by a level of 90% or less, 85% or less, 80% or less, 77% or less, 10 to 90%, 10 to 85%, 10 to 80%. 10 to 77%, 15 to 90%, 15 to 85%, 15 to 80%, 15 to 77%, 18 to 90%, 18 to 85%, 18 to 80%, or 18 to 77%, based on 100% of the liver tumor volume of the control group not administered with the microorganism, when the microorganism was administered.
In the tumor volume reduction of liver cancer according to the present invention, the volume of the liver cancer was 19.5% to 75.3%, and the average volume of the liver cancer was about 32.9% lower in the experimental groups orally administered with CLCC1, compared to the control group. Thus, it can be found that the orally administered CLCC1 microorganism has an activity to inhibit the occurrence and progression of the tumor in the liver cancer.
The Faecalibacterium cancerinhibens according to the present invention may have a characteristic showing a lipid production pattern different from Faecalibacterium prausnitzii which is a Faecalibacterium spp.
The Faecalibacterium cancerinhibens according to the present invention does not comprise 17:0 iso 3OH fatty acid. In an example of the present invention, as a result of analyzing the polar lipid content of Faecalibacterium cancerinhibens CLCC1 and Faecalibacterium prausnitzii, it was confirmed that CLCC1 additionally had PL3 which is absent in Faecalibacterium prausnitzii (
In an example of the present invention, as a result of comparing the fatty acid composition of Faecalibacterium cancerinhibens CLCC1 and Faecalibacterium prausnitzii by performing gas chromatography analysis, it could be seen that there was a difference in the composition of major fatty acids between two microorganisms. Specifically, 15:1 ω8c fatty acid, 19:0 cyclo 10c/19ω6 fatty acid, 20:1 ω9c fatty acid, 14:0 3OH/16:1 iso I fatty acid, 15:0 3OH fatty acid, 16:0 iso fatty acid and 16:0 iso 3OH fatty acid were detected only in the CLCC1 microorganism, but 17:0 iso 3OH fatty acid was detected only in Faecalibacterium prausnitzii ATCC 27768.
The Faecalibacterium cancerinhibens provided by the present invention has the high production of butyric acid among short chain fatty acids (SCFA). Specifically, the butyric acid production of Faecalibacterium cancerinhibens strain CLCC1 of the present invention may be 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 6.5 times or more, or 7 times or more, compared to the Faecalibacterium prausnitzii species, and specifically, it may be 2 to 50 times, 2 to 40 times, 2 to 30 times, 2 to 20 times, 2 to 15 times, 2 to 10 times, 5 to 50 times, 5 to 40 times, 5 to 30 times, 5 to 20 times, 5 to 15 times, or 5 to 10 times. In one specific embodiment, as a result of SCFA analysis of the culture supernatant of Faecalibacterium cancerinhibens strain CLCC1 and Faecalibacterium prausnitzii, the butyric acid content of Faecalibacterium cancerinhibens strain CLCC1 showed 7.8 times higher than that of Faecalibacterium prausnitzii (
The Faecalibacterium cancerinhibens strain according to the present invention forms a colony within 3 mm or within 2 mm and has a long rod-shaped cell shape when observed by optical microscope, when smeared and cultured on an agar medium. In an example of the present invention, Faecalibacterium cancerinhibens had a genome size of 2.9 Mbp, and the number of protein coding regions (CDS) was 2695, and the GC ratio was 56.10% and the number of rRNA genes was 21, and the total number of tRNA genes was 69. In Faecalibacterium cancerinhibens CLCC1 according to the present invention, the butyric acid (butanoic acid) was detected at a content 7.8 times higher than that of Faecalibacterium prausnitzii was detected (
In a specific embodiment of the present invention, the colitis model induced by DSS is historically similar to human ulcerative colitis (UC) in terms of changes and loss of crypt morphology, ulceration, inflammatory cell infiltration and the like, and is widely used in colitis models. The difference according to the dose by administering the Faecalibacterium cancerinhibens CLCC1 according to the present invention at a dose of 2×107 cells/head and 2×108 cells/head at a frequency of once/day for 24 days, the difference according to the administration period by administering it at a dose of 2×108 cells/head at a frequency of once/day for 10 days, and the effect on colitis improvement according to live and dead cells by administering it at a dose of dead cell 2×108 cells/head at a frequency of once/day for 24 hours were confirmed.
As a result, the body weight of the negative control group, in which colitis was induced with 3% DSS, decreased rapidly, and soft stools and diarrhea including bloody stools were observed, and the length of the large intestine was reduced, and histopathologically, the crypt structure was disrupted and Goblet cells involved in mucosal formation disappeared, and full-thickness inflammation occurred, and inflammatory cells were observed. In addition, in the negative control group, the expression of inflammatory cytokines, IFN-γ, TNF-α and IL-6 was increased.
Repeated administration of Faecalibacterium cancerinhibens CLCC1 living cells to an animal model of DSS-induced colitis in BALB/c mice reduced colitis-induced weight loss, improved the decrease in the length of the large intestine, and reduced tissue damage to the colorectal mucosa, and inhibited the expression of the inflammatory cytokines, IFN-γ, TNF-α, and IL-6, thereby having an effect on improvement of colitis. Specifically, the expression of the inflammatory cytokines, IFN-γ and IL-6 was statistically significantly reduced compared to the negative control group, and TNF-α also showed a tendency to decrease. A similar result was obtained for all measurements, without significant change according to the number of bacteria and duration of administration. The weight loss and length of the large intestine of the group administered with dead cell 2×108 cells/head 24 times was similar to that of the negative control group. In addition, the inflammatory cytokines, IFN-γ and IL-6 were reduced compared to the negative control group.
The composition for preventing, improving or treating an inflammatory gastrointestinal tract, for example, IBD according to the present invention may comprise a microbial cell itself, or be a cell-free form not comprising a microbial cell. The lysate means a lysate obtained by crushing the Faecalibacterium spp. microbial cells or a supernatant obtained by centrifugation of the lysate. Herein, unless otherwise stated, the Faecalibacterium spp. having an anti-cancer activity is used to mean one or more kinds selected from the group consisting of a microbial cell of the microorganism; a culture of the microorganism; a lysate of the microorganism; and an extract of one or more kinds selected from the group consisting of the microbial cell, culture and lysate.
The composition according to the present invention may comprise a lyophilized microbial cell. The lyophilization of the microbial cell of the microorganism may be carried out by a method known to those skilled in the art. Alternatively, the composition of the present invention may comprise a culture of a live microorganism. In an embodiment, the microorganism of the present invention may be a living cell, a dead cell, or a mixture thereof when the Faecalibacterium spp. itself comprises a microbial cell.
The composition for preventing, improving or treating IBD according to the present invention comprises the microorganism of the present invention in a therapeutically effective amount. The microorganism in a therapeutically effective amount is sufficient for exhibiting a beneficial effect to a patient. The bacterium or extract is formulated to be administered to a subject in a therapeutically effective amount, depending on for example, the type of subject, severity of disease and route of administration. For example, an appropriate daily dose of the bacterium for an adult human may be about 1×103 to about 1×1011 colony forming unit (CFU), for example, about 1×103 to about 1×1015 CFU, about 1×103 to about 1×1014 CFU, about 1×103 to about 1×1013 CFU, about 1×103 to about 1×1012 CFU, about 1×103 to about 1×1011 CFU, about 1×105 to about 1×1015 CFU, about 1×105 to about 1×1014 CFU, about 1×105 to about 1×1013 CFU, about 1×105 to about 1×1012 CFU, about 1×105 to about 1×1011 CFU, about 1×107 to about 1×1015 CFU, about 1×107 to about 1×1014 CFU, about 1×107 to about 1×1013 CFU, about 1×107 to about 1×1012 CFU, about 1×107 to about 1×1011 CFU, and the unit of the bacterium dose may be /g colony forming unit (CFU), that is, CFU/g, based on the total weight of the composition for preventing, improving or treating an inflammatory gastrointestinal tract, for example, IBD.
Administration of the bacterium may be by any appropriate route. For example, the specific Faecalibacterium spp. of the present invention may be administered to an animal (including humans) in an orally digestible form. In the case of the food composition or neutraceuticals, the bacterium may be simply comprised in a conventional food product or food supplement. A representative pharmaceutical formation includes capsules, microcapsules, tablets, granules, powders, troches, pills, suppositories, suspensions and syrups. In another embodiment, the composition is in a form for rectal administration to an animal (including humans) as rectal suppository or enema. An appropriate formulation may be prepared by a conventional method using conventional organic and inorganic additives. The amount of the active ingredient in the medical composition may be at a level that achieves the desired therapeutic effect.
The composition of the present invention may be encapsulated for transport of the Faecalibacterium spp. in the intestine. The encapsulation protects the composition until it is transported to the target site, for example, by rupture by chemical or physical stimuli, such as physical disruption that may be caused by changes in pressure, enzymatic activity or pH. Any appropriate method for encapsulation may be used. An illustrative encapsulation technology includes capture in porous matrix, attachment or adhesion to the surface of a solid carrier, aggregation or autoagglutination by a crosslinking agent, and mechanical blocking after microporous membranes or microcapsules.
The pharmaceutical composition for prevention, improvement or treatment of inflammatory disease, for example, inflammatory gastrointestinal disease according to the present invention comprises at least one selected from the group consisting of; a microbial cell of a Faecalibacterium spp.; a culture of the microorganism; a lysate of the microorganism; and at least an extract selected from the group consisting of the microbial cell, culture and lysate as an active ingredient, and may comprise an acceptable carrier, diluent or excipient. The acceptable carrier or diluent for therapeutic use is well known in the pharmaceutical field.
The dose of the pharmaceutical composition of the present invention may vary depending on the patient's age, body weight, gender, administration type, health condition and disease degree, and it may be administered in divided doses from once to several times a day at a certain time interval according to the judgement of the doctor or pharmacist. For example, based on the active ingredient content, the daily dose may be 0.1 to 500 mg/kg, preferably, 0.5 to 300 mg/kg. The above dose is an example of an average case, and the dose may be higher or lower depending on individual differences.
The composition of the present invention may be probiotics, and the probiotics may be mixed with at least one appropriate prebiotic compound. The prebiotic compound is generally a non-digestible carbohydrate such as an oligo- or polysaccharide or sugar alcohol, which is not degraded or absorbed in the upper digestive tract. Known prebiotics include commercially available products such as inulin and transgalacto-oligosaccharides. Synbiotics represent nutritional supplements that combine probiotics and prebiotics in a synergistic form.
An embodiment of the present invention relates to a food composition for prevention or improvement of inflammatory bowel disease, for example, a food composition or anti-cancer functional food composition for prevention or improvement of inflammatory bowel disease, comprising at least one selected from the group consisting of, a microbial cell of a Faecalibacterium spp.; a culture of the microorganism; a lysate of the microorganism; and at least an extract selected from the group consisting of the microbial cell, culture and lysate.
It is intended to include all foods, food additives, health functional foods, beverages and beverage additives, and the like in a conventional sense. Herein, the beverage means a generic term for a drink for thirst quenching or taste enjoyment and is intended to include functional beverages. The beverage may be in liquid, syrup and/or gel form.
As an active ingredient contained in the food composition, the content of the Faecalibacterium spp. is not particularly limited, suitably depending on the type of food, desired use, and the like, and for example, at least one selected from the group consisting of a microbial cell of the Faecalibacterium spp., a culture of the microorganism, a lysate of the microorganism and an extract of the microorganism, which are active ingredients of the total of the food, may be added in an amount of 0.00001% by weight to 100% by weight, 0.001% by weight to 99.9% by weight, 0.1% by weight to 99% by weight, more preferably, 1% by weight to 50% by weight, 0.01 to 15% by weight. For example, the food composition may be added in a ratio of 0.02 to 10 g, preferably, 0.3 to 1 g, based on 100 ml.
The composition of the present invention may be formulated. For example, food may provide nutritional benefits in addition to the therapeutic effect of the present invention as a nutritional supplement.
Herein, the term “prediction of risk” or “prediction of likelihood of occurrence” refers to determining whether a subject has a possibility of developing a specific disease, and it may be used clinically to delay or prevent the occurrence of a patient with a high risk of occurrence of a specific disease by special and appropriate management, or to decide on treatment by selecting the most appropriate treatment method. In addition, “diagnosis” means confirming the presence or characteristics of a pathological state, and for the purpose of the present invention, diagnosis may mean confirming the occurrence of a disease.
The “subject” refers to a subject for predicting a risk of occurrence of inflammatory bowel disease or diagnosis of cancer, predicting or evaluating treatment response to a Faecalibacterium spp., or with the possibility of administering the microorganism, or a subject in need of receiving an administration of the microorganism and monitoring the efficacy of the microorganism, and for example, it may be a subject suspected of being at risk of occurrence of inflammatory bowel disease, a subject receiving a diagnosis of inflammatory bowel disease, or a subject receiving treatment for anti-inflammatory bowel disease. The subject is preferably a human.
In the present description, a biological sample of a subject may include a cell extract, any organ, tissue, cell, saliva or feces, or the like isolated from a subject, such as a sample isolated from a mammal having inflammatory bowel disease or suspected of having inflammatory bowel disease. For example, the sample may include, without limitation, a cell or tissue from any part (including without limitation colon, stomach, feces, anus, rectum, and duodenum) of the gastrointestinal tract (for example, from biopsy or autopsy), a cell lysate of stomach obtained from a patient (human or animal), an experimental subject or experimental animal, or an excretion from a subject. Preferably, the sample may be feces.
In an embodiment of the present invention, detecting the Faecalibacterium cancerinhibens includes confirming the presence or absence of the microorganism in a sample, identifying the microorganism, measuring the level of the microorganism, and analyzing relative occupancy. Specifically, detection of Faecalibacterium cancerinhibens or measurement of the level of the microorganism in a sample from a test subject, may include performing using a nucleic acid sequence, amino acid sequence, metabolite, or the microorganism itself as a biomarker specific for the Faecalibacterium cancerinhibens, or evaluating the level of the microorganism by obtaining sequence information of the whole genome, or at least one specific gene in the microbial community present in the sample.
Specifically, after constructing a sequencing library using the PacBio sequencing library kit, whole genome sequencing may be performed using the PacBio RS II platform, and genome sequencing information may be analyzed using Whole Genome analysis pipeline of BIOiPLUG, our genome data analysis platform, and as a result, the whole genome size of the Faecalibacterium cancerinhibens strain CLCC1 is about 2.9 Mbp, and the number of protein coding regions (CDS) is 2695, and a region specific to Faecalibacterium cancerinhibens such as CDS region or Intron region or the like is selected and used as a biomarker.
For example, the detection of the microorganism may be performed by calculating relative abundance of microorganisms distinguished at the genus or species level with respect to the intestinal microbial community of the test subject and Faecalibacterium cancerinhibens in these microorganisms, and measuring the level of Faecalibacterium cancerinhibens.
The measurement of the level of the microorganism, may be the measurement of the ratio of the Faecalibacterium cancerinhibens strain from a sample of a subject, for example, intestine biopsis or a feces sample. The method for measuring the ratio of the strain may be appropriately selected and used by those skilled in the art according to the technical common sense of the art, and for example, it may be performed using a molecular method selected from the group consisting of quantitative polymerase chain reaction (qPCR), massively parallel sequencing, fluorescence in-situ hybridization (FISH), microarray and PCR-ELISA, but not limited thereto.
For example, it may determine that the test subject is at risk of developing bowel disease, when the test subject's level of the microorganism is lower than reference subject's level of the microorganism, by measuring the level of the Faecalibacterium cancerinhibens microorganism in the subject's sample and comparing it to the level of the microorganism in a reference subject.
The method may further comprise determining that the test subject is at risk of developing bowel disease, when the test subject's level of the microorganism is lower than the reference subject's level of the microorganism by measuring the Faecalibacterium cancerinhibens microorganism level in the test subject's sample and comparing the microorganism level with that of a reference subject.
By predicting a risk of developing cancer in advance by metagenome analysis using a human-derived sample according to the present invention, the risk group of cancer can be diagnosed and predicted early, and the onset time can be delayed or the onset can be prevented by appropriate management, and even after the onset, the incidence rate can be lowered and the therapeutic effect can be increased. In addition, by avoiding exposure to causative factors through metagenome analysis in patients diagnosed with cancer, the course of the cancer can be improved, or recurrence can be prevented.
The method for detecting Faecalibacterium cancerinhibens in a sample from a test subject or measuring the level of the microorganism will be described in more detail below.
As an embodiment of the present invention, a method for detecting the microorganism or measuring the level using g a specific gene, for example, a 16S rRNA gene sequence may be provided.
Specifically, the level of the microorganism may be preferably measured by determining the level of a specific nucleic acid sequence in the sample, and the nucleic acid sequence may be preferably a 16S rRNA gene sequence of one or more bacterium above, more preferably, a region of the 16S rRNA gene sequence, for example, at least one of variable regions V1 and/or V6 of the 16S rRNA gene sequence.
Accordingly, the detection of the microorganism may be carried out by obtaining the microbial species distinguished at the species level with respect to the intestinal microbial community of the test subject and the relative abundance of Faecalibacterium cancerinhibens in these microbial species, and measuring the relative level of Faecalibacterium cancerinhibens. It may comprise obtaining 16S rRNA genetic information from the genome information of the intestinal microorganism; and analyzing the 16S rRNA information of the intestinal microorganism and obtaining the abundance of the microbial species comprised in the intestinal microbial community of the test subject.
The genome information of the test subject may be genetic information stored in a storage medium, or genome information of a microorganism isolated from a sample of the test subject. The obtaining 16S rRNA genetic information, may be analyzing the 16S rRNA gene sequence of the extracted DNA, using a next-generation genome sequencing (NGS) platform.
The analyzing the 16S rRNA gene sequence of the extracted DNA may comprise performing PCR using a primer set capable of specifically amplifying a variable region of 16S rRNA, preferably, performing PCR using a primer set capable of specifically amplifying V3 to V4 regions of 16S rRNA, more specifically, performing PCR using a universal primer having the following sequence to produce an amplicon.
By analyzing the 16S rRNA information of the intestinal microorganism, the intestinal microbial community information which obtains the microbial species distinguished at the species level with respect to the intestinal microbial community of the test subject and the abundance of these microbial species may be analyzed.
The analyzing the microbial community, may comprise identification and classification of microorganisms at the genus or species level using 16S rRNA database and/or analysis of each microbial community population. The database used for identification and classification of the microorganisms may be appropriately selected and used by those skilled in the art if necessary, and for example, it may be at least one database selected from the group consisting of EzBioCloud, SILVA, RDP and Greengene, but not limited thereto.
The microbial community population may be expressed as a ratio (%) occupied by a specific microbial community in the total intestinal microbial flora. The ratio (%) occupied by the microbial community may be expressed as a percentage of the frequency of the number of 16S rRNA reads of a specific microorganism among the total number of sequencing reads. The specific microorganism is Faecalibacterium cancerinhibens provided by the present invention.
When which microorganism exists is to be known by performing microbial community analysis, the cost and time of analysis may be reduced by using an amplicon sequencing method that selectively amplifies only a marker gene for species identification and then analyzes the nucleotide sequence of the amplification product, the cost and time required for analysis may be reduced. For bacterial community analysis, the 16S rRNA gene is used as a marker gene, and this gene is present in all bacteria and appropriately comprises conserved regions and mutated regions (v1-v9), so it is suitable for phylogenetic analysis and ecological research.
As an additional embodiment of the present invention, a method is provided for detecting the microorganism or measuring the level using a biomarker specific for Faecalibacterium cancerinhibens.
In the present invention, as a biomarker specific for the Faecalibacterium cancerinhibens, it may comprise a nucleic acid sequence, an amino acid sequence and a metabolite. Accordingly, as an agent capable of detecting the biomarker of the present invention, a primer, a probe, an antisense oligonucleotide, an aptamer and an antibody and the like, which can specifically detect an organic biomolecule such as a protein, nucleic acid, lipid, glycolipid, glycoprotein or sugar (monosaccharide, disaccharide, oligosaccharide, etc.) which is specifically present in the corresponding microorganisms in a sample may be used.
As an embodiment, in the present invention, the agent capable of detecting the microorganisms may be a probe capable of detecting the corresponding microorganism, or a primer for amplifying the probe. It is preferred that the primer to specifically detects a specific probe gene of the corresponding microorganisms and does not specifically bind to a genome sequence of another microorganism.
One specific embodiment of the present invention relates to a probe, a primer set, a kit, a composition and a method for detecting Faecalibacterium cancerinhibens capable of specifically detecting Faecalibacterium cancerinhibens by distinguishing it from other microbial species or subspecies, and sensitively detecting Faecalibacterium cancerinhibens present in a small amount in a sample. An example of the probe for detecting Faecalibacterium cancerinhibens may be Site-specific DNA-methyltransferase (dam), but not limited thereto. In addition, the probe may be linked to a detection label. The detection label may be any chemical moiety that may be detected by any method known in the art. The primer of the present invention may also be modified using a label capable of directly or indirectly providing a detectable signal. The example of the label includes radioactive isotopes, fluorescent molecules, biotin, and the like. As a method for amplifying a sequence using a primer, various methods known in the art may be used.
In an embodiment of the present invention, the analyzing may perform at least one method selected from the group consisting of gel electrophoresis, capillary electrophoresis, sequencing, DNA chip, radioactivity measurement, fluorescence measurement and phosphorescence measurement.
The present invention relates to a composition for diagnosis or treatment of inflammatory disease, for example, inflammatory gastrointestinal disease, comprising a Faecalibacterium spp.
The present invention will be described in more detail by the following illustrative examples, but it is not intended that the scope of the present invention is not limited by the following examples.
1-1: Isolation of microorganism Faecalibacterium cancerinhibens strain CLCC1 was isolated by diluting and applying feces of a healthy 20s adult male in a reinforced clostridial media (RCM) medium and then culturing at a room temperature of 37° C. under an anaerobic condition.
Specifically, Faecalibacterium cancerinhibens CLCC1 has a characteristic of forming colonies of 2 to 3 mm at maximum and showing a long rod-shaped cell shape when observed with an optical microscope, when cultured in an RCM agar medium for 3 days.
1-2: Phylogenetic Analysis Using 16S rRNA Analysis
Faecalibacterium cancerinhibens strain CLCC1 isolated by the method of Example 1 was cultured in an RCM liquid medium at a room temperature of 37° C. under an anaerobic culture condition for 1 day, and then centrifugation was performed to obtain a microorganism. Using FastDNA SPIN Kit for Soil (MP Biomedicals), the total genome was isolated from the obtained microorganism.
After producing a sequencing library using PacBio sequencing library kit, the total genome sequencing was performed using PacBio RS II platform, and using Whole Genome analysis pipeline of BIOiPLUG, our genome data analysis platform, the genome sequencing information was analyzed.
The genome characteristics of Faecalibacterium cancerinhibens strain CLCC1 were shown. It was shown that the total genome size of Faecalibacterium cancerinhibens strain CLCC1 was about 2.9 Mbp, and the number of protein coding regions (CDS) was 2695, and the GC ratio (%) was 56.1%. It was shown that the total rRNA genes were 21 and the total tRNA genes were 69.
Phylogenetic analysis was performed by analyzing rRNA of Faecalibacterium cancerinhibens strain CLCC1 sequenced by the method. The 16S rRNA sequence of Faecalibacterium cancerinhibens strain CLCC1 was shown in SEQ ID NO: 1.
Specifically, the similarity analysis of 16S rRNA was performed using 16S-based identification for prokaryote pipeline of EzBioCloud database, and the phylogenetic analysis was conducted using MEGA program. In
1-3: Phylogenetic Analysis Using Core Genes
Phylogenetic analysis was performed through analysis between gene sequences using the genome sequence data obtained by the method of Example 2-1. Specifically, phylogenetic analysis was conducted using similarity of 92 genes (up-to-date bacterial core gene (UBCG)) which could be used as a taxonomic marker of bacteria. The 92 genes refer to genes provided by UBCG (up-to-date bacterial core gene) pipeline (Na, S. I., Kim, Y. O., Yoon, S. H., Ha, S. M., Baek, I. & Chun, J. (2018)). In
1-4: Relationship Analysis Using Average Nucleotide Identity (ANI)
Using the genome analysis data obtained by the method of Example 1-2, the relationship was analyzed by comparing ANI values of Faecalibacterium cancerinhibens strain CLCC1 and allied species.
Specifically, using Genome-based identification for prokaryote (TrueBAC ID) pipeline, an analysis platform provided by Chunlab, the genome similarity values were analyzed by comparing the total genome sequence of Faecalibacterium cancerinhibens strain CLCC1 and allied species.
In
As the result of decoding the genome of Faecalibacterium cancerinhibens strain CLCC1 and comparing with allied species, the strain CLCC1 showed an average nucleotide identity (ANI) value of 85.99% at a genome level with Faecalibacterium prausnitzil which is the closest allied species at the total genome level, and it can be found that it is a novel species not isolated and reported conventionally. The Faecalibacterium spp. strain identified by the above method was named Faecalibacterium cancerinhibens CLCC1.
2-1: Analysis of Fatty Acid Content
To analyze the molecular biological characteristics of Faecalibacterium cancerinhibens strain CLCC1, the fatty acid composition of Faecalibacterium cancerinhibens strain CLCC1 and Faecalibacterium prausnitzii was compared.
Specifically, Faecalibacterium cancerinhibens strain CLCC1 and Faecalibacterium prausnitzii strain were cultured in an RCM medium at a temperature of 37° C. under an anaerobic condition, respectively, and then using microbial cells of 40 mg, fatty acids were obtained according to the method of Miller (Miller, L. T. (1982) J. Clin. Microbiol. 18, 861-867).
In analysis of the fatty acids extracted, Agilent technologies 6890 Gas chromatography was used, and as a separation column, A30 m×0.320 mm×0.25 μm Crosslinked Methyl siloxane column (HP-1) was used. In
As shown in the Table 1, 15:1 ω8c fatty acid, 19:0 cyclo ω10c/19ω6 fatty acid, 20:1 ω9c fatty acid, 14:0 3OH/16:1 iso I fatty acid, 15:0 3OH fatty acid, 16:0 iso fatty acid and 16:0 iso 3OH fatty acid were detected only in strain CLCC1, and 17:0 iso 3OH fatty acid was detected only in Faecalibacterium prausnitzii ATCC 27768.
As the result of comparison analysis of the molecular biological characteristics, it could be confirmed that Faecalibacterium cancerinhibens strain CLCC1 was a different species from Faecalibacterium prausnitzii which is a known species included in Faecalibacterium spp. When comparing the fatty acid composition of two microorganisms, it could be found that there was a difference in the composition of major fatty acids between two microorganisms, and the corresponding result was shown in
2-2: Polar Lipid Analysis
To compare the polar lipid composition, thin layer chromatography (TLC) analysis was performed.
Specifically, the polar lipid was extracted from a microorganism lyophilized sample of 50 mg by the method of Minikin (Minikin, D. E., et al. (1984). J Microbial Meth 2, 233-241.). In addition, the primary development was performed in a solvent having a ratio of chloroform, methanol and water of 65:25:3.8(v/v) (chloroform:methanol:water=65:25:3.8(v/v)). Then, secondary development was formed in a solvent having a ratio of chloroform, methanol, acetate and water of 40:7.5:6:1.8(v/v) (chloroform:methanol:acetic acid:water=40:7.5:6:1.8(v/v)). After the secondary development, it was stained with 5% (w/v) ethanolic molybdatophosphoric acid.
In addition, when comparing the composition of the polar lipid, it was confirmed that Faecalibacterium cancerinhibens strain CLCC1 and Faecalibacterium prausnitzii had one phosphatidylglycerol (PG), two unknown unidentified phospholipids (PL1, PL2), and a plurality of unidentified lipids (L). In addition, it was confirmed that strain CLCC1 had unidentified phospholipids (PL3) not present in Faecalibacterium prausnitzii additionally, and the corresponding result was shown in
2-3: Short Chain Fatty Acid (SCFA) Analysis
To examine the difference of short chain fatty acid production patterns of Faecalibacterium prausnitzii and Faecalibacterium cancerinhibens CLCC1 microorganism, by requesting analysis to Korea Research Institute of Bio medical Science, the analysis of the short chain fatty acid distribution was performed.
Specifically, after culturing Faecalibacterium prausnitzii and Faecalibacterium cancerinhibens strain CLCC1 of the present invention in an RCM medium for 16 hours, cells were precipitated by centrifugation (4° C., 4,000 rpm, 10 min) to collect the supernatant. The collected supernatant was refrigerated after filtering with a 0.22 um filter. A GC-MS sample for SCFA analysis from the supernatant was prepared using the method of Takeshi Furuhashi (Takeshi Furuhashi, et al., Analytical Biochemistry, Volume 543, 2018, Pages 51-54), and 6890 series equipment of Agilent was used for the short chain fatty acid analysis using GC-MS.
As the result of comparison analysis of short chain fatty acids (SCFA) present in the culture supernatant of Faecalibacterium cancerinhibens CLCC1 and Faecalibacteriumprausnitzii, other known species of Faecalibacterium spp., in both samples, as SCFA, only butyric acid (butanoic acid) was detected. In addition, butyric acid (butanoic acid) at a content 7.8 times higher was detected in the Faecalibacterium cancerinhibens CLCC1 sample, compared to the Faecalibacterium prausnitzii sample. In
Furthermore, when looking at the ratio of butyric acid in each sample, 91.64% of the culture supernatant of Faecalibacterium cancerinhibens CLCC1 was butyric acid, and 76.40% of the culture supernatant of Faecalibacterium prausnitzii was butyric acid. The result of the short chain fatty acid analysis of Faecalibacterium cancerinhibens strain CLCC1 was shown in Table 2, and the result of the short chain fatty acid analysis of Faecalibacterium prausnitzii was shown in Table 3. Other substances indicated in Table 2 to Table 3 below were detected in the substance library database, but they were classified as noise signals that could not accurately identify the substances because of their very low matching rates (quality). In Table 2 and Table 3 below, RT means a retention time.
It can be predicted that Faecalibacterium cancerinhibens strain CLCC1 of the present invention exhibits an excellent anti-cancer activity, due to higher production of butyric acid among short chain fatty acids, compared to Faecalibacterium prausnitzii.
3-1: Test of Inhibitory Efficacy of Cancer Occurrence in Subcutaneous Tumor Model
To confirm the inhibiting ability of cancer occurrence of Faecalibacterium cancerinhibens strain CLCC1, the inhibitory efficacy of cancer occurrence was tested in a subcutaneous tumor model using a mouse liver cancer cell line. In 10 6-week-age male C57BL/6 mice, through a subcutaneous tumor transplantation experiment of a liver cancer cell line (Hep55.1c), liver cancer was caused, and during a process of liver cancer occurrence for 55 days, the effect of oral administration of Faecalibacterium cancerinhibens strain CLCC1 on the growth of liver cancer cells was examined. The liver cancer cell line Hep55.1c was mouse-derived hepatocellular carcinoma (hepatoma).
Specifically, Faecalibacterium cancerinhibens CLCC1 was orally administered 4 days before the mouse liver cancer cell line was transplanted into the mouse subcutaneous tissue. At Day 4 after oral administration, the mouse liver cancer cell line (Hep55.1c) was transplanted into the subcutaneous tissue of the right flank to make 2.0×106 cells.
For the subcutaneous tumor model using the mouse liver cancer cell line, Faecalibacterium cancerinhibens strain CLCC1 was orally administered once a day, 5 times a week, for 55 days. Then, from the day of transplantation (Day 0) to Day 55, the size of the liver cancer tumor (tumor volume) was measured using Magnetic Resonance (MR) imaging at a 7-day interval.
Faecalibacterium cancerinhibens strain CLCC1 was cultured in an RCM medium, and then concentrated and stored frozen using a PBS buffer and glycerol to a final glycerol concentration of 15% (v/v). In addition, after thawing immediately before administration to mice, 200 ul (2×108 cells) per animal was orally administered. As a control group, 10 mice not administered with strain CLCC1 were used. For the control group, 200 ul each of PBS and glycerol (15%, v/v) solution containing no strain was orally administered.
In Table 4 and Table 5 below, the result of the tumor size of the control group and test group in the test of the inhibitory efficacy of liver cancer occurrence of the isolated strain in the subcutaneous tumor model was shown. Table 4 is the result of the tumor volume change for the control group, and Table 5 is the result of the tumor size (volume) change of the experimental group administered with Faecalibacterium cancerinhibens strain CLCC1. In the tables, S. D means standard deviation.
.4
4.6
.3
0.7
2.98
7.3
.5
09
23.1
indicates data missing or illegible when filed
In
As the experimental result, it was confirmed that the liver cancer occurrence and progress were statistically significantly inhibited in the experimental group orally administered with CLCC1, compared to the control group. The tumor volume was averagely about from about 19.5% to 75.3%, on average, 32.9% lower, in the experimental group orally administered with CLCC1, compared to the control group, and thereby it was confirmed that in the experimental group administered with Faecalibacterium cancerinhibens strain CLCC1, the liver cancer occurrence was delayed, and the tumor growth progress rate was inhibited. At Day 4 which is the first day of measuring the tumor size after surgery, it can be confirmed that there is an inhibitory effect of cancer occurrence, as the tumor size is definitely small in the experimental group administered with Faecalibacterium cancerinhibens strain CLCC1, compared to the control group. Thus, it can be found that orally administered strain CLCC1 has an activity to inhibit occurrence and progress of liver cancer.
3-2: Test of Inhibitory Efficacy of Cancer Occurrence in Liver Transplantation Model
To examine the effect of Faecalibacterium cancerinhibens strain CLCC1 on the growth of liver cancer cells, in 4 8-week-age male C57BL/6 mice, through a liver transplantation experiment of a liver cancer cell line, liver cancer was caused, and during a process of liver cancer occurrence for 39 days, the effect of oral administration of Faecalibacterium cancerinhibens strain CLCC1 on the growth of liver cancer cells was examined.
Specifically, as the liver cancer cell line, Hep55.1c was used, and the epigastrium of the 8-week-old male C57BL/6 mice was opened, and the liver cancer cell line was transplanted into the left lobe of liver so as to be 5.0×105 cells. After a recovery period of 4 days after transplanting the tumor cell line into the liver, the tumor size was confirmed by MRI scanning, and 4 mice in which tumor was well formed were selected and used for a subsequent experiment.
For 39 days from the day of transplantation (Day 0), strain CLCC1 was prepared by the same method as Example 4, and orally administered to 4 mice that completed the transplantation operation, and strain CLCC1 was not administered to 4 control mice. For the control group, 200 ul each of PBS and glycerol (15%, v/v) solution containing no strain was orally administered. Thereafter, the tumor size and relative size and relative value of growth were periodically measured.
The tumor size was measured by magnetic resonance (MR) imaging at a 7-day interval, and the relative value of the tumor size was drawn by calculating a relative size to the tumor size at the day of transplantation. In
As a result, it was confirmed that the liver cancer occurrence and progress were statistically significantly inhibited in the experimental group orally administered with CLCC1, compared to the control group not administered with the isolated line. The result for inhibition of liver cancer occurrence and data of the change in the tumor size were shown in
The relative figure table (Relative growth; Fold) of the tumor size was shown in Table. 7, and the relative figure table (Relative growth; Delta) of the tumor growth is shown in Table 7.
.0
indicates data missing or illegible when filed
indicates data missing or illegible when filed
As can be confirmed in Table 6 and Table 7 and
4-1. Confirmation of Anti-Cancer Cancer in Subcutaneous Tumor Model
To confirm the inhibitory ability of cancer occurrence of Faecalibacterium cancerinhibens strain CLCC1, the inhibitory efficacy of cancer occurrence in a subcutaneous tumor model using a human colorectal cancer cell line was tested. As the colorectal cancer cell line, HCT-116 cells were used, and as the mouse, nude mice (CAnN.Cg-Foxn1nu/CrlOri, SPF) were used. The HCT-116 is a cancer cell line corresponding to human-derived colorectal carcinoma.
Specifically, the colorectal cancer cell line HCT-116 (2.5×107 cells/mL) was administered to the mice that had undergone a one-week purification period and transplanted by administering 0.2 mL/head subcutaneously to the right back of mice using a disposable syringe. When the size of cancer cells grew to about 85˜119 mm3 after cancer cell transplantation, 10 mice per group were classified into a negative control group not administered with CLCC1 and 3 experimental groups administered with strain CLCC1 by concentration. Specifically, the experimental group was divided into 3 groups of low, medium and high according to the concentration of strain CLCC1, and F. cancerinhibens strain CLCC1 was administered at a concentration of 2×106 cells/head for the low concentration, 2×107 cells/head for the medium concentration, and 2×108 cells/head for the high concentration. The administration of the strain was oral administration once a day for 36 days with a syringe. For the negative control group, only an excipient (PBS solution comprising glycerol 15% (v/v)) was administered. In Table 8 below, the information of the negative control group and each experimental group was shown.
(1) Tumor Size Change and Growth Inhibition Rate
To confirm the anti-colorectal cancer effect of F. cancerinhibens strain CLCC1, the change in the tumor size was confirmed. Specifically, once a day after starting the test of strain administration, general symptoms such as appearance, behavior, excretion and the like were observed, and the body weight was measured once a week, and the tumor size was measured, and the volume was calculated twice a week. In
The tumor volume (Tv) was calculated by the method of Equation 3 below by measuring the perpendicular width (W) and maximum length (L) of tumor, respectively.
Tv(mm3)×L(mm)×W2(mm2)×½ [Equation 3]
The result of confirming the change in the tumor volume throughout 36 days was shown in Table 9 and
)
9
6
indicates data missing or illegible when filed
Specifically, the average tumor size at the day (Day 1) of staring administration of the microorganism was same as 105 mm3 in all the 4 groups, but in 36 days, the average tumor volume was shown as 4330 mm3 in the negative control group, and on the other hand, the volume of 3000 mm3 or less was shown in all the experimental groups administered with strain CLCC1, and therefore, it was confirmed that it had the tumor volume of about 60 to 67%, compared to the control group, and thereby, the inhibitory effect of the colorectal cancer growth of the Faecalibacterium cancerinhibens was confirmed.
In addition, based on the result of weight measurement, the tumor growth inhibition rate (IR) was calculated by the method of Equation 1 below in each experimental group.
IR(%)=(1−(T1/C1))×100 [Equation 1]
In the Equation 1, T is the average tumor weight in each experimental group, and C is the average tumor weight in the negative control group.
In Table 10 and
In the negative control group (G1), the tumor weight extracted after autopsy was shown as about 3.21 g, but in the experimental groups, it was shown as 1.94, 2.17 and 1.95 g, respectively, at the low, medium and high concentrations, and therefore the weight of the tumor tissue was shown significantly low. On the other hand, the total body weight in each group did not show a significant difference between the control group and experimental groups.
The tumor growth inhibition rate was 39.6% in the experimental group at the low concentration, and the tumor growth inhibition rate was 32.4% in the experimental group at the medium concentration, and the tumor growth inhibition rate was 39.3% in the experimental group at the high concentration, and therefore, it was confirmed that the tumor growth was inhibited 30% or more in all the experimental groups.
(2) Necrosis and Apoptosis of Tumor Cells
H&E staining (Hematoxylin & Eosin staining) to see necrosis of tumor cells in tumor tissue and TUNEL assay to see apoptosis were performed by conducting an autopsy after completing administration of strain CLCC1 for 36 days.
In
In
In Table 12 below, the result of comparing the necrosis and apoptosis of tumor cells was shown. In the table below, depending on the degree of apoptosis, ± represents minimal, and + represents mild, and ++ represents moderate, and +++ represents marked, and ++++ represents severe. Below each symbol, the number of killed cells corresponding to the corresponding stage is indicated.
12.47 ± 2.34##
##p < 0.01, Significant difference from the negative control group (G1) by Steel's t-test.
As can be confirmed in the Table 12, the apoptosis ratio of about 10.33% was shown in the control group (Control, G1), and the apoptosis ratio similar to the control group was shown in the experimental group at the low concentration (G2), but the apoptosis of 12.4% or more was observed in the experimental groups at the medium concentration (G3) and high concentration (G4). Accordingly, it was confirmed that the apoptosis of tumor cells was increased by administration of the Faecalibacterium cancerinhibens microorganism.
4-2: Evaluation of Efficacy Using Colorectal Cancer Subcutaneous Tumor Model
To confirm the anti-cancer effect against colorectal cancer during administration of strain CLCC1, mice in which a colorectal cancer cell was subcutaneously transplanted were used, and a test was performed in Champion's oncology, a non-clinical commission testing institution in the United States. As the colorectal cancer cell, a mouse-derived colorectal cancer cell line, MC38 cell was used, and as the mouse strain, C57BL/6 was used. The MC38 cell is a cancer cell line corresponding mouse-derived colorectal carcinoma. The test group is as follows, and 10 mice were included per group.
Specifically, through a subcutaneous tumor transplantation experiment of MC38 cell line, the colorectal cancer cell line, in 6-week-old male C57BL/6 mice, colorectal cancer was caused, and the effect of the oral administration of Faecalibacterium cancerinhibens strain CLCC1 on the growth of colorectal cancer cells during the process of colorectal cancer occurrence was examined.
By the substantially same method as the preparation method of test formulations of the control group and experimental groups of Example 4-1, Faecalibacterium cancerinhibens strain CLCC1 of Example 1 was cultured in an RCM medium, and then concentrated and stored frozen to final glycerol concentration of 15% (v/v) using PBS buffer and glycerol. In addition, after hawing immediately before administration to mice, low concentration living cells at a content of 200 ul per animal (2×107 cells/dose) (Sample 1 corresponds to G3 of Example 4-1) and high concentration living cells (2×108 cells/dose) at a content of 200 ul per animal (2×108 cells) (Sample 2 corresponds to G4 of Example 4-1) were orally administered. As a control group, 10 mice not administered with strain CLCC1 were used.
Sample 3 was used as high concentration dead cells (2×108 cells/dose) by treating the prepared Sample 2 in an oven at a temperature of 100° C. for 2 hours.
Specifically, the test formulations of the control group and Samples 1 to 3 were administered once a day for 40 days in total by starting administration 2 weeks before inoculating cancer cells. Specifically, the test formulations of the control group and Samples 1 to 3 were orally administered once a day for 40 days, and after 14 days from the start date of administration, colorectal cancer cells (5×105 MC38 cells in 0.1 ml PBS) were inoculated to the subcutaneous tissue of the left flank, and after a one-week engraftment period, the tumor size was observed from the 22nd day to the 40th day for 18 days. Day 22, the start date of tumor observation was regarded as Day 0, and the size of the colorectal cancer tumor (tumor volume) was measured using Magnetic Resonance (MR) imaging for 18 days. The change in the tumor size was calculated by measuring the maximum length (L) and perpendicular width (W) of tumor for 22 days after engraftment of cancer cells and introducing them to the following Equation 3. The tumor volume measured for 18 days was shown in Table 13 and
Tv(mm3)×L(mm)×W2(mm2)×½ [Equation 3]
Relative reduction rate of tumor volume (%)=(1−T2/C2))×100 [Equation 2]
In Table 13 below, Mean means the average tumor volume and S.D. means standard deviation. Table 13 is the result of the tumor volume change in the colorectal cancer cell line-transplanted subcutaneous tumor model in which the isolated strain according to the control group and Samples 1 to 3 was administered.
As the result, the tumor volume change obtained in the groups administered with the strain of Samples 1 to 3 compared to the control group was shown in
It was confirmed that the tumor growth was statistically significantly inhibited even when dead cells of strain CLCC1 were administered. In addition, regarding the reduction of the tumor volume of Sample 1 and Sample 2 using living cells of strain CLCC1 strain but at different concentrations, it was confirmed that the anti-cancer activity was increased in a concentration-dependent manner under the same experimental condition.
In addition, after the day of administrating the test formulations, at an interval of 1 to 3 days, general symptoms such as appearance, body weight, behavior, excretion, and the like were observed. The test was performed for 40 days in total after administration of the test formulations, and it was observed for 18 days after engraftment of cancer cells. The measured body weight (Kg) of the experimental animals was shown in Table 14 below.
For the total body weight of the experimental animals, the body weight of mice corresponding to the control group tended to be lower than that of the test groups after engraftment of cancer cells, but there was no significant difference between the control group and the test groups overall.
5-1: Model Animal Preparation and Sample Administration
Female BALB/c mice (about 6 weeks old) were supplied from a supplier and an experiment was conducted after an adaptation period (about 7 days). After the adaptation period, for 7 days at the 3rd week of test substance administration, the normal control group was supplied with normal negative water, and all groups except the normal control group were provided with 3% DSS in a drinking bottle and freely supplied with water to induce colitis. Female BALB/c mice (6 weeks old) were subjected to a 6-day acclimatization period, and 6 mice (7 weeks old) per group were used in the present experiment. Test animals were conducted in a total of 6 groups, and the group composition is as follows.
Control groups were set as a normal control group (G1), a negative control group (G2) and a positive control group (G3). G1 and G2 were administered with 15% glycerol as an excipient from the start of the test, and G3 was administered with 200 mg/kg of Salicylazosulfapyridine (SASP). Test substance administration groups were divided into living cell groups (G4, G5) and a dead cell group (G7). The living cell groups, G4 and G5, and dead cell group (G7) were administered with CLCC1 once a day for 24 days from the start of the test to the end to confirm the preventive effect of CLCC1. Colitis was induced by putting 3% DSS in a drinking bottle and providing free water at the 3rd week of the test substance administration for 7 days.
In the present experiment, the weight loss evaluation, disease activity index (DAI) and large intestine length were measured, and histopathological examination of large intestine tissue was performed.
5-2: Weight Loss Evaluation
Body weight was measured twice/weekly from the start of administration to the end of the test, and it was measured before administration on each administration day. Based on the body weight measured before 3% DSS supply, the change in body weight was calculated as a percentage, and the result was shown in
When DSS was supplied, the body weight began to decrease in all groups (G2-G7) after 6 days compared to before water supply, and the reduction rate tended to increase until the end of the test. Weight loss is an important and potentially dangerous side effect of inflammatory bowel disease. Compared to G2, the reduction rate of the body weight was statistically significantly reduced in G4 and G5 groups. (*p<0.05 and **p<0.01, Independent t-test) G7 showed no difference from G2. Therefore, administration of the CLCC1 living cell was confirmed to improve body weight loss due to colitis.
5-3: DAI (Disease Activity Index)
DAI was scored according to body weight, stool consistency and presence of fecal blood from the day of 3% DSS administration and expressed as a sum. Specifically, the disease activity index was scored based on weight loss (for example, weight gain or maintenance within 1% compared to baseline (score 0); 1.5% weight loss (score 1); 5˜10% weight loss (score 2); 10˜15% weight loss (score 3); >15% weight loss (score 4)); stool consistency (for example, normal (score 0); loose stool (score 2); diarrhea (score 4)); and bleeding (for example, no bleeding (score 0), moderate bleeding (score 2), severe bleeding (score 4))).
The evaluation result was shown in
Therefore, it was confirmed that the DAI score was statistically significantly reduced compared to G2 due to administration of the CLCC1 living cell, and this means that symptoms such as body weight, stool condition, and presence or absence of blood in stool were improved.
5-4: Measurement of Large Intestine Length
After completing the test, the animals were anesthetized, performed laparotomy, and exsanguinated, and then the large intestine tissue was extracted by individual. The extracted large intestine was photographed and its length was measured. The measured result was shown in
Induced colitis with DSS reduced the length of the large intestine. The large intestine length of G2 was statistically significantly reduced compared to G1. (††p<0.01, Independent t-test) The large intestine length of the living cell administration groups G4 and G5 was reduced statistically significantly less than G2. (*p<0.05, Independent t-test) This meant that the living cell administration group inhibited large intestine atrophy compared to G2, thereby preventing or improving the decrease in length of the large intestine.
Therefore, it was confirmed that administration of the CLCC1 living cell inhibited colon atrophy and improved the decrease in length of the large intestine.
5-5: Analysis of Cytokines
RNA was extracted from the large intestine and the gene expression of IL-6, TNF-α and IFN-γ was analyzed by RT-qPCR.
The extracted large intestine tissue was washed with PBS and then stored in RNAlater (Lot No.: 00833281, Invitrogen, MA, U.S.A.). According to the manual of Total RNA extraction kit (Lot No.: 157031929, QIAGEN, Germany), total RNA was extracted. The extracted total RNA was quantified by measuring absorbance at 260 nm and 280 nm using a nucleic acid quantifier (IFNinite M200 pro, Tecan, Austria). RT-qPCR of TNF-α, IFN-γ and IL-6 was conducted using Real time PCR machine (CFX384, BIO-RAD, CA, U.S.A.) under the following condition.
The composition of the RT-qPCR mixture was shown in Table 16 below, and the RT-qPCR condition was shown in Table 17 below. As the RT-qPCR result, gene expression levels were compared using 2−ΔΔCt method.
The measured result was shown in
When colitis was induced by DSS, inflammatory cytokines (TNF-α, IL-17, IL-10, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3α, CXCL1 and IL-23, etc.) increased in expression, and anti-inflammatory cytokines (IL-4, IL-10, IL-13, IFN-α, TGF-β, etc.) decreased in expression. Among them, in the present experiment, the expression levels of IFN-γ, IL-6, and TNF-α as inflammatory cytokines were measured.
In all the living cell administration groups (G4-G5), compared to the negative control group (G2), the levels of IFN-γ, and IL-6 were statistically significantly low. The TNF-α level was statistically significantly lower than G2 in the CLCC1 high-concentration living cell administration group (G5). (*p<0.05, Independent t-test) Therefore, it was confirmed that administration of the CLCC1 living cell lowered the level of inflammatory cytokines such as IFN-γ, IL-6 and TNF-α.
5-6: Histopathological Examination
The extracted tissue was fixed in 10% neutral formalin. The extracted and fixed large intestine tissue was cut into tissue sections with a thickness of 4 to 5 μm, sectioned and attached to slides. After H&E staining, it was sealed with permount (Fisher Scientific, NJ, U.S.A.), and it was observed under a microscope (CKX41, Olympus, Japan), and photographing was conducted.
In the large intestine mucosa of the test group G2, the crypt structure was lost, and goblet cells secreting mucus were lost, and transmural inflammation occurred. A large number of red blood cells and white blood cells were observed in the tissue. In G3˜G5, it was observed that the crypt structure was maintained and inflammation was reduced. The observed result was shown in
6-1: Model Animal Preparation and Sample Administration
Female BALB/c mice (about 6 weeks old) were supplied from a supplier and an experiment was conducted after an adaptation period (about 7 days). After the adaptation period, for 7 days at the 3rd week of test substance administration, the normal control group was supplied with normal negative water, and all groups except the normal control group were provided with 3% DSS in a drinking bottle and freely supplied with water to induce colitis. Female BALB/c mice (6 weeks old) were subjected to a 6-day acclimatization period, and 6 mice (7 weeks old) per group were used in the present experiment. Test animals were conducted in a total of 4 groups, and the group composition is as follows.
Control groups were set as a normal control group (G1), a negative control group (G2) and a positive control group (G3), and the treatment group (test substance administration group 4) was set as living cell group G6, respectively. G1 and G2 were administered with 15% glycerol as an excipient from the start of the test, and G3 was administered with 200 mg/kg of Salicylazosulfapyridine (SASP). The test substance administration group G6 was administered with CLCC1 once a day for 10 days from the time DSS induced colitis as a living cell group to see the therapeutic effect of CLCC1. Colitis was induced by putting 3% DSS in a drinking bottle and providing free water at the 3rd week of the start of test substance administration for 7 days.
By the substantially same method as Example 5, the weight loss evaluation, disease activity index (DAI) and large intestine length were measured, and histopathological examination of large intestine tissue was performed.
6-2: Weight Loss Evaluation
By the substantially same method as Example 5, body weight was measured twice/weekly from the start of administration to the end of the test, and it was measured before administration on each administration day. Based on the body weight measured before 3% DSS supply, the change in body weight was calculated as a percentage, and the result was shown in
When DSS was supplied, the body weight began to decrease in all groups (G2-G7) after 6 days compared to before water supply, and the reduction rate tended to increase until the end of the test. Weight loss is an important and potentially dangerous side effect of inflammatory bowel disease. Compared to G2, the reduction rate of the body weight was statistically significantly reduced in G4 and G5 groups. (*p<0.05 and **p<0.01, Independent t-test) Therefore, administration of the CLCC1 living cell was confirmed to improve body weight loss due to colitis.
6-3: DAI (Disease Activity Index)
By the substantially same method as Example 5, DAI gave scores, and the evaluation result was shown in
Therefore, it was confirmed that the DAI score was statistically significantly reduced compared to G2 due to administration of the CLCC1 living cell, and this means that symptoms such as body weight, stool condition, and presence or absence of blood in stool were improved.
6-4: Measurement of Large Intestine Length
By the substantially same method as Example 5, the large intestine tissue of the experimental animals was extracted, and the extracted large intestine was photographed and the length was measured. The measured result was shown in
The large intestine length of G2 was statistically significantly reduced compared to G1. (††p<0.01, Independent t-test) The large intestine length of the living cell administration group G6 was reduced statistically significantly less than G2. (*p<0.05, Independent t-test) This meant that the living cell administration group inhibited large intestine atrophy compared to G2, thereby preventing or improving the decrease in length of the large intestine.
Therefore, it was confirmed that administration of the CLCC1 living cell inhibited colon atrophy and improved the decrease in length of the large intestine.
6-5: Cytokines Analysis
By the substantially same method as Example 5, RNA was extracted from the large intestine and the gene expression of IL-6, TNF-α and IFN-γ was analyzed by RT-qPCR. The measured result was shown in
When colitis was induced by DSS, inflammatory cytokines (TNF-α, IL-17, IL-10, IL-2, IFN-γ, IL-6, IL-12, IL-25, IL-33, IL-8, MCP-1, MIP-3a, CXCL1 and IL-23, etc.) increased in expression, and anti-inflammatory cytokines (IL-4, IL-10, IL-13, IFN-α, TGF-β, etc.) decreased in expression. Among them, in the present experiment, the expression levels of IFN-γ, IL-6, and TNF-α as inflammatory cytokines were measured.
In all the living cell administration group (G6), compared to the negative control group (G2), the levels of IFN-γ, and IL-6 were statistically significantly low. The TNF-α level was statistically significantly lower than G2 in the CLCC1 high-concentration living cell administration group (G6). (*p<0.05, Independent t-test) Therefore, it was confirmed that administration of the CLCC1 living cell lowered the level of inflammatory cytokines such as IFN-γ, IL-6 and TNF-α.
6-6: Histopathological Examination
By the substantially same method as Example 5, the sections of the extracted and fixed large intestine tissue were observed with a (CKX41, Olympus, Japan), and photographing was conducted.
In the large intestine mucosa of the test group G2, the crypt structure was lost, and goblet cells secreting mucus were lost, and transmural inflammation occurred. A large number of red blood cells and white blood cells were observed in the tissue. In G6, it was observed that the crypt structure was maintained and inflammation was reduced. The observed result was shown in
7-1: Culture of HT-29 and RAWN 264.7 Cell Lines
HT-29 (Korean Cell Line Bank, KCLB 30038) and RAW 264.7 (Korean Cell Line Bank, KCLB 40071) cells were cultured in RPMI 1640 medium in which 10% FBS (Fetal bovine serum), penicillin (100 μg/mL), and streptomycin (100 μg/mL) were added under the 37° C., 5% CO2 conditions, and subculturing was carried out once per 2˜3 days.
7-2: Culturing and Recovering of Strain
F. prausnitzii A2-165 used in the present experiment was obtained from DSMZ (DSMZ collection, Braunschweig, Germany) (DSM No 17677).
The used Faecalibacterium spp. microorganisms, F. cancerinhibens CLCC1 and F. prausnitzii A2-165 are obligate anaerobic strains, and all the culturing processes were carried out in an anaerobic chamber, and cultured in media of mRCM, and activated by subculture at 18 to 23 hour intervals twice in total, and the used in the experiment. The obtained culturing solution was centrifuged under the condition of 3200×g, 5 min, and the supernatant except for precipitate was recovered and used in the experiments of Example 7 and Example 8.
7-3: Efficacy Evaluation for IL-8 in HT-29 Cells
Evaluation of Immuno-Modulatory Properties Using HT-29 Cells was performed (ELISA).
Specifically, using human large intestine epithelial cells, HT-29, after culturing them in a 24-well plate by 5×10{circumflex over ( )}4 Cells/well, one day before co-culture with the supernatant of CLCC1 and A2-165, it was changed to a medium which 10% FBS was comprised in RPMI1640. On the day of co-culture, to the RPMI1640 (+10% FBS) medium, CLCC1 and A2-165 supernatant or media of mRCM was added to be 10%. After treating TNF-α (5˜10 ng/ml; Peprotech, NJ) in a 37° C., 5% CO2 incubator for 6 hours, culture was performed. All samples were performed in triplicate. After the co-culture, the culture supernatant was collected, and Interleukin-8 (IL-8) was measured by ELISA (Invitrogen, USA). The result was expressed in pg/ml, and normalization was performed using the IL-8 reference value using only one treated only media of mRCM as a negative control.
The secretion amount of human IL-8 from the HT-29 cells analyzed by ELISA was shown in
This indicates that F. cancerinhibens CLCC1 can reduce an inflammatory response increased by TNF-α, and in addition, it means that it can be utilized as a therapeutic agent of inflammatory bowel disease, by inhibiting infiltration of the intestinal mucosa by Neutrophil and secretion of inflammatory mediators (
7-4: Efficacy Evaluation for IL-6 and TNF-α in RAW 264.7 Cells
Evaluation of Immuno-Modulatory Properties Using RAW 264.7 was performed. (ELISA)
Specifically, using a mouse macrophage-derived cell line, RAW 264.7, after culturing them in a 24-well plate by 5×10{circumflex over ( )}4 Cells/well, one day before co-culture with the supernatant of F. cancerinhibens CLCC1 and A2-165, it was changed to a medium which 10% FBS was comprised in RPMI1640. On the day of co-culture, to the RPMI1640 (+10% FBS) medium, CLCC1 and A2-165 supernatant or media of mRCM was added to be 10%. After treating LPS (100 ng/ml; Sigma, L4391) in a 37° C., 5% CO2 incubator for 6 hours, culture was performed. All samples were performed in triplicate. After the co-culture, the culture supernatant was collected, and Interleukin-6 (IL-6) and TNF-α were measured by ELISA (Invitrogen, USA). The result was expressed in pg/ml, and normalization was performed using the IL-6 and TNF-α reference values using only one treated only media of mRCM as a negative control.
The secretion amount of mouse IL-6 and TNF-α from the RAW 264.7 cells analyzed by ELISA was shown in
This indicates that CLCC1 can reduce an inflammatory response increased by LPS, and improvement of inflammation can be expected by inhibiting secretion of RORγT or IL-17 according to Th17 cell differentiation which can be induced by IL-6, and in addition, it meant that it can be utilized as a therapeutic agent of inflammatory bowel disease, by involving in expression of inflammation-related cytokines through regulation of various immunocytes in TNF-α (
8-1: HPBMC Culture
HPBMC (Lonza, 4W-270) was cultured in LGM-3™ (Lonza, CC-3211) medium in which 10% FBS (Fetal bovine serum) was added under the 37° C., 5% CO2 condition, thereby conducting an experiment.
8-2: Efficacy Evaluation for Human IL-10 in HPBMC
Evaluation of Immuno-Modulatory Properties Using HPBMC was performed.
Specifically, after culturing Human Peripheral Blood Mononuclear Cells (HPBMC) in a 12-well plate by 1×106 Cells/well, the supernatants of CLCC1 and A2-165 and PBS were added to be 10%. After the reaction in a 37° C., 5% CO2 incubator for 24 hours, culture was performed. All samples were performed in triplicate. After the co-culture, the culture supernatant was collected, and Interleukin-10 (IL-10) was measured by ELISA (Invitrogen, USA). The result was expressed in pg/ml, and normalization was performed using the IL-10 reference value using only one treated only PBS as a negative control.
The result of the analyzed efficacy for human IL-10 in HPBMC was shown in
This indicates that F. cancerinhibens CLCC1 can exert an anti-inflammatory action by involving in expression of IL-10 among anti-inflammation-related cytokines in human peripheral blood mononuclear cells, and this means that it can be utilized as a therapeutic agent of inflammatory bowel disease (
8-3: Efficacy Evaluation for IL-6 in HPBMC
Evaluation of Immuno-Modulatory Properties Using HPBMC was performed.
Specifically, after culturing Human Peripheral Blood Mononuclear Cells (HPBMC) in a 24-well plate by 1×106 Cells/well, the CLCC1 supernatant and PBS were added to be 10%. After treating LPS (100 ng/ml; Sigma, L4391) in a 37° C., 5% CO2 incubator for 6 hours, culture was performed. All samples were performed in triplicate. After the co-culture, the culture supernatant was collected, and Interleukin-6 (IL-6) was measured by ELISA (Invitrogen, USA). The result was expressed in pg/ml, and normalization was performed using the IL-6 reference value using only one treated only PBS as a negative control.
The result of the analyzed efficacy for human IL-6 in HPBMC was shown in
This indicates that F. cancerinhibens CLCC1 can inhibit an inflammatory response by involving in expression of IL-6 among inflammation-related cytokines in human peripheral blood mononuclear cells, and this means that it can be utilized as a therapeutic agent of inflammatory bowel disease (
9-1: Preparation of Samples and Primers
The strain genomic DNA was prepared using DNA prep kit (e.g., MO BIO's PowerSoil DNA Isolation Kit).
Through analysis using blastn program, it was confirmed that Site-specific DNA-methyltransferase (dam) was specific to F. cancerinhibens CLCCL as it was not found in other microorganisms. In order to confirm F. cancerinhibens CLCCL-specific detection more easily and quickly and accurately in real time, based on the dam gene sequence, real-time PCR amplification reaction was performed on the sample using a primer pair consisting of forward and reverse primers.
9-2: Detection Using Curve Value (Ct Value)
Real-time PCR amplification reaction was performed using Bio-rad's CFX96 Real-time PCR system (Bio-Rad laboratories, Inc., USA), and the real-time PCR amplification reaction was conducted in a total amount of 20 μl with a real-time PCR mixed solution containing SYBR premix Ex Taq (2×) (TAKARA Bio, Inc., Japan) and 10 pM of each of the above primers. The amount of genomic DNA of the strains added to the real-time PCR mixed solution was about 25 ng. The real-time PCR amplification reaction was denaturing at 95° C. for 30 seconds, repeating 40 cycles at 95° C. for 5 seconds, and at 60° C. for 30 seconds, and then reacting at 95° C. for 15 seconds. Thereafter, it was increased by 0.5° C. per 5 seconds to 65˜95° C. to derive a melting curve and a melting peak. Relative Fluorescence Units (RFU) were measured according to the number of cycles.
The curve value (Ct value) was specifically confirmed only in the sample comprising F. cancerinhibens CLCCL DNA, and from the above result, it could be seen that the primer set and probe of the present invention could specifically detect F. cancerinhibens.
9-3: Quantitative Analysis
Using a Site-specific DNA-methyltransferase (dam) gene, using a substance (e.g., plasmid, or genomic DNA) for which the amount of Faecalibacterium cancerinhibens was already measured, a standard curve was set, and then it was utilized to measure the concentration.
For the amount of total bacteria, after extracting the genome of all microorganisms in the fecal sample using a 16S universal primer, like this, using a substance of which amount was already measured (e.g., plasmid, genomic DNA), a standard curve was set, and then it was utilized to measure the concentration.
For relative quantification, a value of dividing the F. cancerinhibens CLCCL amount calculated above by the amount of total bacteria was used.
The data used in the analysis is data obtained by performing the shotgun metagenome sequencing method on fecal microorganisms, and among the data used in the analysis, in the case of Colorectal Cancer and the healthy group compared to Colorectal Cancer group (data of which SampleId starts with ERR among healthy controls), open data published as a parper (https://gut.bmj.com/content/66/1/70) were downloaded, and in case of other ulcerative colitis, Crohn's disease and healthy group data, fecal microorganism shotgun metagenome sequence data collected independently through joint research were used.
Each sample data was analyzed by K-mer exact matching method (Korean Patent Publication No. 10-2020-0027900) for a core gene which calculates abundance of microbial species at the species level, thereby obtaining a profile of the abundance of microbial species in the sample. For analysis of the abundance of the F. cancerinhibens CLCCL strain, first, in order to confirm that it is distinguished from the existing genome at the species level, ANI (Average Nucleotide Frequency) analysis was performed with the genome of Faecalibacterium prausnitzii ATCC 27768, it was confirmed that it was a novel species with a significantly low value of 85.74 compared to the standard similarity of 95 at the species level, and therefore, it was added to the reference microbial genome database.
For each sample, the distribution in each disease group and healthy group was compared using the reference database comprising the genome of Faecalibacterium cancerinhibens CLCC1. The experimental result was shown in Table 19 and
The CLCC1 strain according to the present invention showed significantly different results between the healthy group and ulcerative colitis group, and the healthy group and Crohn's disease, and this shows that disease groups can be classified by the difference in the amount of distribution while residing as an intestinal microorganism.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0126243 | Sep 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/013152 | 9/27/2021 | WO |
