Composition for preventing, alleviating or treating cachexia, comprising lactobacillus reuteri ATG-f4 strain

Abstract

The present invention relates to a composition containing a Lactobacillus reuteri ATG-F4 strain as an active ingredient for preventing, alleviating, or treating side effects of cachexia or chemotherapy.

Description

Composition for preventing, improving or treating cachexia comprising Lactobacillus reuteri ATG-F4 strain

[0001] The present invention relates to a composition for preventing, improving or treating cachexia comprising the Lactobacillus reuteri ATG-F4 strain, and more particularly, to a composition containing the Lactobacillus reuteri ATG-F4 strain as an active ingredient, which can be used for preventing, improving or treating cachexia or side effects of anticancer chemotherapy.

[0002] Cancer cachexia and anorexia are a multifactorial syndrome characterized by persistent loss of skeletal muscle mass in cancer patients, which is not reversible by regular nutritional support, and progressive functional impairment.

[0003] Unlike patients with other chronic diseases, cancer patients are prone to not only cachexia but also the side effects of various cancer treatments. Head and neck cancer and gastrointestinal cancers, in particular, experience cachexia more frequently than cancers in other areas, as they are organs through which food passes or that control digestion and absorption. Some reports indicate that cachexia affects 50-80% of patients, and the mortality rate due to cachexia reaches 20-30%. While cancer cachexia can manifest as sarcopenia due to age or temporary fasting, it is characterized by increased catabolic responses due to various cytokines, as well as inflammatory responses and changes in carbohydrate, protein, and fat metabolism, leading to muscle loss and weight loss despite normal food intake. These changes are often accompanied by nutritional restrictions due to treatments such as chemotherapy, radiation, or surgery, which can lower treatment response rates and hinder effective treatment progress, ultimately leading to a significant decline in survival and quality of life.

[0004] Therefore, proper understanding and correction of cachexia can improve quality of life by increasing compliance with treatment and reducing secondary side effects.

[0005] Loss of appetite and weight loss, relatively common in cancer patients, can be a symptom of cachexia, as well as side effects from surgery, chemotherapy, and radiation therapy. However, unlike a typical fasting state, cachexia undergoes a completely different mechanism, driven by metabolic changes, cytokines, and various substances secreted by patients and tumors involved in appetite mechanisms. This resulting cachexia can negatively impact a patient's quality of life, survival rate, and prognosis. Various medications have been studied to treat cachexia, but the results have not yet been satisfactory. Therefore, prospective studies involving a larger patient population are needed.

[0006] Accordingly, the inventors of the present invention completed the present invention by conducting various studies on methods for improving cachexia using lactic acid bacteria and confirming that Lactobacillus reuteri ATG-F4 exhibits excellent effects in improving and treating cachexia.

[0007] The purpose of the present invention is to provide a composition for preventing, improving or treating cachexia containing Lactobacillus reuteri ATG-F4 as an active ingredient.

[0008] Another object of the present invention is to provide a pharmaceutical composition or food composition for preventing, improving or treating cachexia containing Lactobacillus reuteri ATG-F4 as an active ingredient.

[0009] Another object of the present invention is to provide a composition for preventing, improving or treating side effects of anticancer chemotherapy, containing Lactobacillus reuteri ATG-F4 as an active ingredient.

[0010] The problems to be solved by the present invention are not limited to the problem(s) mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0011] In order to solve the above-mentioned purpose, the present invention provides a composition for preventing, improving or treating cachexia, which contains as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0012] In addition, the present invention provides a pharmaceutical composition for preventing, improving or treating cachexia, which contains as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0013] In addition, the present invention provides a food composition for preventing, improving or treating cachexia, which contains as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0014] In addition, the present invention provides a composition for preventing, improving or treating side effects of anticancer chemotherapy, containing as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0015] The above composition may increase the survival rate decreased due to administration of an anticancer drug.

[0016] The above composition may increase the amount of fat reduced due to administration of an anticancer drug.

[0017] The above composition may increase the strength of the jaw joint that has been reduced due to administration of an anticancer drug.

[0018] The composition may reduce inflammation increased by administration of an anticancer drug.

[0019] The above composition may inhibit phosphorylation of NF-κB (Nuclear factor-κB), an inflammation-related factor increased due to administration of an anticancer drug.

[0020] The above composition may reduce damage to colon tissue increased due to administration of an anticancer drug.

[0021] The above composition may restore the amount of occludin or claudin protein expression in colon tissue that has decreased due to administration of an anticancer drug.

[0022] The above composition may reduce the concentration of TNF-α or IL-6 in the blood increased due to administration of an anticancer agent.

[0023] The above composition may reduce blood lipopolysaccharide (LPS) increased due to administration of an anticancer agent.

[0024] The above composition may reduce diarrhea symptoms caused by anticancer drug administration.

[0025] A composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention is effective in preventing, improving or treating cachexia or side effects of anticancer chemotherapy.

[0026] In addition, the composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has the effect of increasing the survival rate decreased due to administration of an anticancer agent.

[0027] In addition, the composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has an effect of suppressing weight loss, muscle and fat loss caused by anticancer drug administration.

[0028] In addition, the composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has the effect of increasing atrophied muscle fibers due to anticancer drug administration, inhibiting NF-κB phosphorylation, reducing muscle atrophy protein MuRF1, and increasing mitochondrial-related proteins PGC-1α, NRF-1, and mt TFA.

[0029] In addition, a composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has an effect of alleviating damage to colon tissue caused by administration of an anticancer agent.

[0030] In addition, the composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has the effect of restoring the amount of occludin or claudin protein expression in colon tissue decreased due to administration of an anticancer agent.

[0031] In addition, a composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has an effect of reducing the concentration of TNF-α or IL-6 in the blood increased due to administration of an anticancer agent.

[0032] In addition, a composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has an effect of reducing blood lipopolysaccharide (LPS) increased due to administration of an anticancer agent.

[0033] In addition, a composition comprising the Lactobacillus reuteri ATG-F4 strain according to the present invention has the effect of reducing diarrhea symptoms caused by anticancer drug administration.

[0034] Figure 1 shows the survival rate after administration of Lactobacillus reuteri ATG-F4 (low, medium, and high doses) for 5-FU-induced anticancer drug side effects according to one embodiment of the present invention.

[0035] FIG. 2a is a graph showing tumor weight after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, FIG. 2b is a graph showing body weight-tumor weight after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, FIG. 2c is a graph showing changes in body weight excluding tumor after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, FIG. 2d is a graph showing changes in calf muscle mass after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, and FIG. 2e is a graph showing changes in calf muscle mass after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention. This is a graph showing the change in fat weight after administration of Lactobacillus reuteri ATG-F4 (low, medium, and high doses), and Fig. 2f is a graph showing the change in grip strength after administration of Lactobacillus reuteri ATG-F4 (low, medium, and high doses) for 5-FU-induced anticancer drug side effects according to one embodiment of the present invention. The 5-FU was 50 mg / kg, and the low, medium, and high doses of Lactobacillus reuteri ATG-F4 were 1.0 x 10 in that order. 8 , 1.0 x 10 9 , 1.0 x 10 10 Administered as CFU / day (one-way ANOVA Dunnett's p<0.001:***, p<0.01:**, p<0.05:* vs LLC_5-FU).

[0036] FIG. 3a is a photograph of quadriceps femoris (QF) muscle fibers after administration (low, medium, high doses) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, FIG. 3b is a graph showing the size of quadriceps femoris (QF) muscle fibers after administration (low, medium, high doses) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention (one-way ANOVA Dunnett's p<0.001:***, p<0.01:** vs LLC_5-FU), FIG. 3c is a graph showing the expression of inflammation-related proteins (p-NF-κB / NF-κB) in QF muscles after administration (high doses) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, and FIG. 3d is a graph showing the expression of inflammation-related proteins (p-NF-κB / NF-κB) in QF muscles after administration (high doses) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention This is a graph showing the expression of muscle atrophy-related protein (MuRF1) after administration (high dose) of Lactobacillus reuteri ATG-F4 for side effects, and Fig. 3e is a graph showing the expression of mitochondrial-related proteins PGC-1, NRF-1, and mt TFA after administration (high dose) of Lactobacillus reuteri ATG-F4 for side effects of 5-FU-induced anticancer drugs according to an embodiment of the present invention. The 5-FU was 30 mg / kg, and the low, medium, and high doses of Lactobacillus reuteri ATG-F4 were 4.0 x 10 in that order. 8 , 4.0 x 10 9 , 4.0 x 10 10 administered as CFU / day (Fig. 3c-e: one-way ANOVA Dunnett's p<0.001:***, p<0.01:**, p<0.05:* vs LLC_5-FU).

[0037] FIG. 4a is a H&E staining photograph of colon tissue after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, FIG. 4b is a comparison of colon tissue inflammation index reading scores after administration of Lactobacillus reuteri ATG-F4 (low, medium, high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, and FIG. 4c is a graph showing the expression levels of occludin and claudin proteins after administration of Lactobacillus reuteri ATG-F4 (high dose) for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention.

[0038] FIG. 5a is a graph showing the blood TNF-α concentration after administration (low, medium, high doses) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, FIG. 5b is a graph showing the blood IL-6 concentration after administration (low, medium, high doses) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention, and FIG. 5c is a graph showing the blood lipopolysaccharide (LPS) concentration after administration (high dose) of Lactobacillus reuteri ATG-F4 for 5-FU-induced anticancer drug side effects according to an embodiment of the present invention.

[0039] FIG. 6a shows the survival rate after administration of Lactobacillus reuteri ATG-F4 for FOLFIRI-induced anticancer drug side effects according to one embodiment of the present invention, and FIG. 6b shows the survival rate after administration of Lactobacillus reuteri ATG-F4 for FOLFOX-induced anticancer drug side effects according to one embodiment of the present invention.

[0040] [Revised 19.11.2024 under Rule 91] Fig. 7a is a graph showing the results of stool consistency scores after administration of Lactobacillus reuteri ATG-F4 for FOLFIRI-induced anticancer drug side effects according to an embodiment of the present invention, and Fig. 7b is a graph showing the results of stool consistency scores after administration of Lactobacillus reuteri ATG-F4 for FOLFOX-induced anticancer drug side effects according to an embodiment of the present invention. Fig. 8 shows a survival rate experiment schedule according to administration of Lactobacillus reuteri ATG-F4 for 5-FU-induced cachexia symptoms according to an embodiment of the present invention. Fig. 9 shows an efficacy evaluation experiment schedule according to administration of Lactobacillus reuteri ATG-F4 for 5-FU-induced cachexia symptoms according to an embodiment of the present invention. Fig. 10 shows a graph showing the results of stool consistency scores after administration of Lactobacillus reuteri ATG-F4 for FOLFIRI-induced and FLFOX-induced cachexia symptoms according to an embodiment of the present invention. This shows the survival rate experimental schedule according to the administration of Lactobacillus reuteri ATG-F4.

[0041] It should be noted that in the following description, only the parts necessary for understanding the embodiments of the present invention are described, and the description of other parts will be omitted to the extent that it does not distract from the gist of the present invention.

[0042] The terms and words used in this specification and claims described below should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted with meanings and concepts that conform to the technical idea of ​​the present invention based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0043] Therefore, the embodiments described in this specification and the configurations illustrated in the drawings are merely preferred embodiments of the present invention and do not represent all of the technical ideas of the present invention. Therefore, it should be understood that there may be various equivalents and modified examples that can replace them at the time of filing this application.

[0044] Hereinafter, the present invention will be described in detail.

[0045] In the present invention, “pharmaceutical composition” means a drug used for the purpose of diagnosing, treating, alleviating, managing or preventing a disease in animals, including humans.

[0046] In the present invention, the term "food composition" includes fresh foods distributed as is from agricultural, livestock, forestry, and marine products, and processed foods manufactured or processed using their raw materials to enhance storage properties, nutritional properties, etc. In addition, the "food composition" of the present invention includes a "health functional food composition," and the term "health functional food composition" means a food manufactured or processed using raw materials or ingredients having functionality useful to the human body, and includes all forms such as functional foods, nutritional supplements, health foods, and food additives.

[0047] The present invention provides a composition for preventing, improving or treating cachexia, containing as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0048] In addition, the present invention provides a pharmaceutical composition for preventing, improving or treating cachexia, which contains as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0049] In addition, the present invention provides a food composition for preventing, improving or treating cachexia, which contains as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0050] In addition, the present invention provides a composition for preventing, improving or treating side effects of anticancer chemotherapy, containing as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

[0051] The physiological / genetic information on the above Lactobacillus reuteri ATG-F4 (Lactobacillus reuteriATG-F4, Accession No. KCTC13717BP) strain is described in Korean Patent No. 10-1951919 (Title of the invention: Novel Lactobacillus reuteri ATG-F4 strain having dopamine secretion enhancing function, composition for preventing or treating mental illness containing the same, Applicant: A2Gen Co., Ltd., Registration date: February 19, 2019).

[0052] In the present invention, “strain culture” means a culture solution itself cultured in a medium, for example, a liquid medium, a supernatant (filtrate) obtained by filtering and / or centrifuging the culture solution to remove the strain, etc.

[0053] In the present invention, the "culture extract, concentrate, and dried product" may undergo centrifugation or filtration to remove the liquid culture medium in the culture and recover only the concentrated fungal cells, but the present invention is not limited thereto. In addition, the concentrated fungal cells may be dried, frozen, or lyophilized using conventional methods to preserve their activity.

[0054] The Lactobacillus reuteri ATG-F4 strain, cells of the strain, culture of the strain, extract, concentrate and dried product of the culture may be added to the composition of the present invention in an amount of 0.001 to 100 wt%.

[0055] In the present invention, "cachexia" refers to a severe systemic weakness that can be seen in the terminal stages of cancer, tuberculosis, diabetes, acquired immunodeficiency syndrome (AIDS), etc., and frequently appears in patients with digestive cancers such as gastric cancer, esophageal cancer, pancreatic cancer, and colon cancer, as well as lung cancer. It shows symptoms such as decreased appetite, loss of weight and physical strength due to loss of muscle and fat, anemia, lethargy, and indigestion, and in particular, it shows a state in which weight is lost even when normal food intake is consumed. When cachexia occurs, the patient shows a low response to anticancer chemotherapy or radiotherapy, which lowers the quality of life of the patient, shortens life expectancy, and causes death due to weight loss in 10 to 20% of all cancer patients.

[0056] In the present invention, the "cachexia" may include, but is not limited to, cancer cachexia, AIDS cachexia, chronic obstructive pulmonary disease cachexia, multiple sclerosis cachexia, or congestive heart failure cachexia, and may specifically be "cancer cachexia" or "anticancer drug-induced cachexia."

[0057] In the present invention, "anticancer chemotherapy" means a systemic treatment method that uses drugs used for cancer treatment, i.e., anticancer drugs, to prevent or kill cancer cells from growing, and "side effects of anticancer chemotherapy" have different degrees of severity in the manifestation of each symptom depending on the type of anticancer chemotherapy, but the main adverse effects include loss of appetite, nausea, vomiting, diarrhea, stomatitis, bone marrow toxicity (reduction of white blood cells and granulocytes), skin damage, hair loss, nerve damage, interstitial pneumonia, liver damage, kidney damage, heart damage, etc.

[0058] The above pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the strain of the present invention or its culture with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0059] The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject being treated, the specific disease or pathological condition being treated, the severity of the disease or pathological condition, the route of administration, and the prescriber's judgment. Determining the dosage based on these factors is within the skill of those skilled in the art, and the composition may be administered once daily or in multiple divided doses. The above dosage does not limit the scope of the present invention in any way.

[0060] The pharmaceutical composition of the present invention can be administered to mammals, such as rats, livestock, and humans, via various routes. All modes of administration are conceivable, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebrovascular injection. The strain of the present invention exhibits minimal toxicity and side effects, making it a safe drug for long-term prophylactic use.

[0061] In an embodiment of the present invention, in order to confirm the preventive, ameliorating or therapeutic efficacy of Lactobacillus reuteri ATG-F4 on cachexia / anticancer chemotherapy side effects, cachexia / anticancer chemotherapy side effects were induced by administering anticancer drugs such as 5-FU, FOLFIRI or FOLFOX to cancer-induced mice, and Lactobacillus reuteri ATG-F4 according to the present invention was administered to the 5-FU-induced cachexia / anticancer chemotherapy side effects or FOLFIRI / FOLFOX-induced cachexia / anticancer chemotherapy side effects models, and the following changes in cachexia / anticancer chemotherapy side effects symptoms were confirmed.

[0062] The composition of the present invention is characterized by increasing the survival rate reduced due to the administration of an anticancer agent. In an example of the present invention, when Lactobacillus reuteri ATG-F4 was administered to a 5-FU or FOLFIRI / FOLFOX-induced cachexia / chemotherapy side effect model, it was confirmed that the survival rate increased and the survival period was prolonged (Fig. 1, Fig. 6a-b).

[0063] The composition of the present invention is characterized by increasing body weight, muscle mass, and fat mass decreased due to anticancer drug administration. In an example of the present invention, when Lactobacillus reuteri ATG-F4 was administered to a 5-FU-induced cachexia / chemotherapy side effect model, it inhibited 5-FU-induced body weight loss (Figs. 2b-c) and 5-FU-induced muscle mass and fat mass decrease (Figs. 2d-e) without affecting 5-FU's tumor growth inhibition (Fig. 2a).

[0064] The composition of the present invention is characterized by increasing grip strength decreased due to anticancer drug administration. In an example of the present invention, Lactobacillus reuteri ATG-F4 was administered to a 5-FU-induced cachexia / chemotherapy side effect model, and as a result, grip strength decreased due to 5-FU was increased (Fig. 2f).

[0065] The composition of the present invention is characterized by reducing inflammation increased by anticancer drug administration. The inflammation may be any one of muscle tissue inflammation, colon tissue inflammation, or blood inflammation.

[0066] The composition of the present invention is characterized by reducing muscle tissue inflammation increased due to anticancer drug administration, and more specifically, is characterized by inhibiting phosphorylation of NF-κB (Nuclear factor-κB), an inflammation-related factor increased in muscle tissue due to anticancer drug administration. In an embodiment of the present invention, the composition of the present invention increased atrophied muscle fibers due to anticancer drug administration in muscle tissue, inhibited NF-κB phosphorylation, decreased muscle atrophy protein MuRF1, and increased mitochondrial-related proteins PGC-1α, NRF-1, and mt TFA. Specifically, when Lactobacillus reuteri ATG-F4 was administered to a 5-FU-induced cachexia / chemotherapy side effect model, the size of quadriceps femoris (QF) muscle fibers that had been atrophied due to chemotherapy administration increased (Fig. 3a-b), NF-κB phosphorylation (p-NF-κB / NF-κB) that had been increased due to chemotherapy administration was suppressed (Fig. 3c), muscle atrophy protein MuRF1 that had been increased due to chemotherapy administration was decreased (Fig. 3d), and mitochondria-related proteins PGC-1α, NRF-1, and mt TFA that had been decreased due to chemotherapy administration were increased (Fig. 3e).

[0067] In addition, the composition of the present invention is characterized by reducing inflammation of colon tissue increased due to administration of an anticancer drug, and more specifically, it is characterized by reducing damage to colon tissue increased due to administration of an anticancer drug. In an embodiment of the present invention, the composition of the present invention confirmed damage to colon tissue (inflammatory cell infiltration, epithelial cell change, mucosal structure change in H&E staining) in a 5-FU-induced cachexia / chemotherapy side effect model caused by administration of an anticancer drug (Fig. 4a), and as a result of administering Lactobacillus reuteri ATG-F4, damage to colon tissue was reduced (Fig. 4b). In addition, the composition of the present invention is characterized by restoring the amount of occludin or claudin protein expression decreased in colon tissue due to administration of an anticancer drug. In an example of the present invention, when Lactobacillus reuteri ATG-F4 was administered to a 5-FU-induced cachexia / chemotherapy side effect model, it was confirmed that the expression level of occludin or claudin, which are tight junction or barrier-related proteins of colon tissue, was restored (Fig. 4c).

[0068] In addition, the composition of the present invention is characterized by reducing blood inflammation increased due to administration of an anticancer drug. More specifically, it is characterized by reducing the concentration of blood TNF-α or IL-6 increased due to administration of an anticancer drug, and / or reducing the concentration of blood lipopolysaccharide (LPS) increased due to administration of an anticancer drug. In an example of the present invention, when Lactobacillus reuteri ATG-F4 was administered to a 5-FU-induced cachexia / anticancer chemotherapy side effect model, it was confirmed that the blood concentration of TNF-α and IL-6, which are inflammatory cytokines that increased due to administration of an anticancer drug, decreased (Fig. 5a-b). In addition, in an example of the present invention, when Lactobacillus reuteri ATG-F4 was administered to a 5-FU-induced cachexia / chemotherapy side effect model, it was confirmed that the blood inflow of intestinal lipopolysaccharide (LPS), an endotoxin, was reduced (Fig. 5c).

[0069] The composition of the present invention is characterized by reducing diarrhea symptoms caused by anticancer drug administration. In an example of the present invention, when Lactobacillus reuteri ATG-F4 was administered to a FOLFIRI and FOLFOX-induced cachexia / chemotherapy side effect model, it was confirmed that diarrhea symptoms were reduced as the stool consistency score was lowered (Fig. 7a-b).

[0070] Anticancer drugs are broadly classified into cytotoxic chemotherapy drugs, targeted anticancer drugs, and immunotherapy drugs based on their mechanism of action. Among these, cytotoxic chemotherapy drugs directly attack cancer cells that divide more rapidly than normal cells and exhibit anticancer effects. The types include alkylating agents (oxaliplatin, cisplatin, cyclophosphamide, iphosphide, bendamustine, melphalan, carboplatin, busulfan, dacarbazine, temozolomide), antimetabolites (fluorouracil, capecitabine, doxifluridine, tegafur, cytarabine, azacitidine, decitamine, enocitabine, methotrexate, pemetrexed, pralatrexate, cladribine, clofarabine, fludarabine, mergaptopurine), DNA rotator cuff inhibitors (irinotecan, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, etoposide, topotecan), and microtubule inhibitors. There are inhibitors (cabazitaxel, paclitaxel, docetaxel, vinblastine, vincristine, vinorelbine) and others (bleomycin, hydroxyurea, mitomycin C), and the above chemotherapeutic agents have similar enzymatic mechanisms of action, mainly through DNA damage or inhibition of DNA synthesis.

[0071] As described above, in the examples of the present invention, the Lactobacillus reuteri ATG-F4 strain of the present invention was confirmed to be effective in preventing, improving, or treating cachexia / chemotherapy side effects induced by single 5-FU treatment, and was also confirmed to be effective in preventing, improving, or treating cachexia / chemotherapy side effects induced by combination anticancer drugs FOLFIRI and FOLFOX (both 5-FU-based anticancer drugs). Therefore, the Lactobacillus reuteri ATG-F4 strain of the present invention is effective in preventing, improving, or treating cachexia / chemotherapy side effects induced by anticancer drugs, and in particular, when considering the type of mechanism of action of cytotoxic chemotherapeutic agents, it is expected to reduce the side effects of anticancer drugs related to alkylating agents, antimetabolites, and DNA polymerase inhibitors. In addition, since the Lactobacillus reuteri ATG-F4 strain of the present invention also showed an effect on cachexia / anticancer chemotherapy side effects induced by the combination anticancer drugs FOLFIRI and FOLFOX, it is expected that the Lactobacillus reuteri ATG-F4 strain of the present invention will also reduce the side effects of anticancer drugs resulting from combination anticancer chemotherapy that uses two or more drugs simultaneously to increase the anticancer effect.

[0072] Targeted anticancer drugs selectively attack cancer cells, and immunotherapy drugs have the advantage of having fewer side effects than chemotherapy drugs because the immune cells better recognize and attack cancer cells. However, they take a long time to take effect and have the disadvantage of developing resistance, so they are often used simultaneously with chemotherapy drugs. Considering that chemotherapy drugs cause side effects by directly attacking and killing cancer cells but also attacking normal cells, it seems that the symptoms of cachexia and the side effects of chemotherapy are caused by chemotherapy drugs rather than targeted anticancer drugs and immunotherapy drugs. In other words, the Lactobacillus reuteri ATG-F4 strain of the present invention is expected to have the effect of preventing, improving, or treating cachexia or side effects of chemotherapy caused by the combined (or simultaneous) use of chemotherapy drugs with targeted anticancer drugs and / or immunotherapy drugs.

[0073] Hereinafter, the present invention will be described in detail through examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0074] Experimental Example 1: Increased survival rate and alleviation of cachexia symptoms in 5-FU-induced cachexia.

[0075] [Experimental Method]

[0076] [Revised on 19.11.2024 under Article 91 of the Rules] Prior to all animal experiments conducted in this experimental example, the Institutional Animal Care and Use Committee (IACUC) of A2Gen Co., Ltd. approved the animal experiments as appropriate procedures (Approval Number: ATG-IACUC-RDSP-220321), and guidance on ethical animal experiments was provided. The animals used in this experiment were 5-week-old (Fig. 8) or 7-week-old (Fig. 9) C57BL / j mice purchased from Raon Bio (Seoul, Korea). Lewis lung carcinoma cells (LLC, ATCC, Manassas, VA, USA) were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum).

[0077] Mice were housed in a cage maintained at a constant room temperature of 23 ± 2°C, with controlled temperature and humidity and a 12:12 light / dark cycle. An acclimation period of at least 7 days was provided to allow the mice to adapt to the new environment and diet, and water and rodent food (Purina) intake were not restricted.

[0078] After the acclimation period, 0.1 mL of sterile saline was injected subcutaneously (SC) into the right flank of 10 mice in the normal control group. The remaining mice received 5 × 10 5 LLC cells were inoculated subcutaneously (SC). Fourteen days after inoculation, LLC-treated mice were divided into five groups, ensuring that tumor sizes were similar.

[0079] [Revised 19.11.2024 by Rule 91] First, the experimental schedule and group settings related to survival rate were as shown in Fig. 8 and Table 2. ATG-F4 was administered in three doses (1.0 x 10 8 , 1.0 x 10 9 , 1.0 x 10 10CFU) was mixed in PBS and administered orally, and 5-Fluorouracil (5-FU) at 50 mg / kg was injected intraperitoneally in a 3-day cycle.

[0080] [Correction pursuant to Rule 91, November 19, 2024]

[0081]

[0082]

[0083] [Revised 19.11.2024 according to Rule 91] Second, the experimental schedule and group setting related to the ATG-F4 efficacy evaluation were as shown in Fig. 9 and Table 4. ATG-F4 was administered in three doses (4.0 x 10 8 , 4.0 x 10 9 , 4.0 x 10 10 CFU) was mixed in PBS and administered orally, and 30 mg / kg of 5-Fluorouracil (5-FU) was injected intraperitoneally in a 3-day cycle.

[0084] [Correction pursuant to Rule 91, November 19, 2024]

[0085]

[0086]

[0087] Tumor volume was measured in three dimensions using calipers and calculated using the following formula (tumor volume = (height x width x length) x 0.5 cm) 3 ). Whole body fat-free weight and calf fat-free weight were measured using DXA (model iNSiGHT VET DXA, Osteosys, Korea). Grip strength tests were performed the day before dissection. On the day of autopsy, blood was collected from the posterior vena cava after CO2 euthanasia. The collected blood was centrifuged at 4000 rpm for 10 minutes to extract serum. The quadriceps femoris (QF) muscle was separated from the right lower calf and weighed. The colon was stored in a -70℃ freezer until analysis after collection.

[0088] [Analysis method]

[0089] 1. Western blot analysis

[0090] QF muscle or colon tissues were placed in RIPA buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40) containing a protease inhibitor cocktail (Millipore, USA) and minced with scissors. The supernatant was collected after centrifugation at 14,000 rpm at 4°C for 10 min. The protein concentration in the supernatant was measured using the BCA assay (Thermo Fisher, USA). Protein extracts (40 μg) were separated using 8% or 10% polyacrylamide mini gels and transferred to PVDF membranes (Bio-Rad, USA). The transferred PVDF membrane was incubated overnight at 4°C in SuperBlock (PBS) Blocking buffer (pH 7.4) containing Kathon™ Anti-microbial Agent and primary antibodies MuRF1 (1:1000; Mybioscience), p-NF-κB (1:1000; CST), NF-κB (1:1000; CST), PGC-1α (1:1000; Abcam), mt TFA (1:1000; Abcam), NRF-1 (1:1000; Abcam), Occludin (1:1000; Thermo fisher), Claudin (1:1000; Thermo fisher), and β-actin (1:1000; Cell signaling). PVDF membranes were washed four times with 0.1% Tween TBS, placed in 0.1% Tween TBS buffer containing 3% BSA (Bovogen, USA), and incubated with goat anti-rabbit IgG HRP-conjugated secondary antibody (Bio-Rad, USA) for 1 h at room temperature. After thorough washing with 0.1% Tween TBS, the immunostained bands were confirmed by ECL (Bio-Rad, USA).Confirmation of target protein is available at ChemiDoc. TM Imaging System (Bio-Rad, USA) was used, and band intensity was quantified using Image LabTM software (Bio-Rad, USA).

[0091] 2. Grip strength test

[0092] Grip strength (Biosep, Bio GS3) was measured the day before dissection. All four paws were placed on a steel rail, and the tail was pulled with a constant force to measure tension. Each individual was measured nine times.

[0093] 3. Histology

[0094] Colon and quadriceps femoris (QF) muscles were isolated and fixed in 10% formalin for more than 48 hours at room temperature. The fixed tissues were subjected to standard tissue processing procedures such as trimming, dehydration, paraffin embedding, and thinning to prepare specimens for histopathological examination, followed by hematoxylin and eosin (H&E) staining. Histopathological changes were observed through H&E slide photographs at 400x magnification using an optical microscope (Olympus BX53, Japan). Muscle fiber size was quantified on the H&E slides. Three areas were selected from one tissue slide, and a total of more than 30 muscle fibers were quantified and compared. Colon tissues were quantified using three inflammation-related indices: inflammatory cell infiltrate, epithelial change, and mucosal architecture.

[0095] 4. Endotoxin analysis

[0096] Serum endotoxin levels were measured using ToxinSensor TMAnalysis was performed using the Chromogenic LAL Endotoxin Assay Kit (Cat.No.: L00350).

[0097] 5. Cytokine analysis

[0098] Serum IL-6 and TNF-α levels were measured using commercial kits (mouse IL-6 ELISA max standard set, BioLegend).

[0099] [result]

[0100] Referring to Figure 1, 1.0 x 10 10 It was confirmed that the survival period was extended by 2 days by administering CFU of ATG-F4.

[0101] Referring to Figures 2a to 2f, ATG-F4 did not change the anticancer effect of 5-FU (Figure 2a), but suppressed weight loss caused by the anticancer drug (Figures 2b and 2c), and increased calf muscle mass (Figure 2d), fat mass (Figure 2e), and grip strength (Figure 2f).

[0102] Referring to FIGS. 3a to 3e, ATG-F4 increased the muscle fiber size reduced by 5-FU in QF muscle tissue (FIGS. 3a, 3b), suppressed inflammation-related proteins (p-NF-κB / NF-κB) in QF muscle (FIG. 3c), suppressed muscle atrophy-related proteins (MuRF1) (FIG. 3d), and activated mitochondria-related proteins (PGC-1α, NRF-1, mt TFA) (FIG. 3e).

[0103] Referring to Figures 4a to 4c, ATG-F4 in colon tissue inhibited the increase in inflammation caused by 5-FU (Figures 4a, 4b) and increased the expression of barrier-related proteins (Occludin, Claudin) in colon tissue (Figure 4c).

[0104] Referring to Figures 5a to 5c, the inflammatory cytokines TNF-α and IL-6 in the blood were suppressed (Figures 5a, 5b), and the influx of intestinal LPS into the blood was reduced (Figure 5c).

[0105] Experimental Example 2: Confirmation of increased survival rate and improvement of diarrhea symptoms in cachexia induced by FOLFIRI and FOLFOX.

[0106] [Experimental Method]

[0107] Prior to all animal experiments conducted in this experimental example, animal experiments were approved as appropriate procedures by the Institutional Animal Care and Use Committee (IACUC) of A2Gen Co., Ltd. (Approval Number: ATG-IACUC-RDSP-230427), and guidance on ethical animal testing was provided. The animals used in this experiment were 5-week-old Balb / c nude mice purchased from Raon Bio (Seoul, Korea).

[0108] HCT-116 cells (human colon cancer cell line, Korea Cell Line Bank) were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum).

[0109] Mice were housed in a cage maintained at a constant room temperature of 23 ± 2°C, with controlled temperature and humidity and a 12:12 light / dark cycle. An acclimation period of at least 7 days was provided to allow the mice to adapt to the new environment and diet, and water and rodent food (Purina) intake were not restricted.

[0110] [Revised 19.11.2024 by Rule 91] The experimental schedule and group settings related to survival rate were as shown in Figure 10 and Table 6. After the acclimatization period, 1.0 x 10 6HCT-116 cells were subcutaneously administered into the flanks of mice and tumors were grown for 10 days. The tumors were cut into 1.5 mm in length x width x height and fixed to the sigmoid colon of the mouse using surgical sutures. At this time, the sigmoid colon was slightly scratched with a razor blade to help the tumor settle well. After the tumor was transplanted into the sigmoid colon, a recovery period of 7 days was allowed. After that, FOLFIRI (Leucovorin 90 mg / kg, 5-FU 50 mg / kg, Irinotecan 24 mg / kg) and FOLFOX (Oxaliplatin 6 mg / kg, 5-FU 50 mg / kg, Leucovorin 90 mg / kg) were administered intraperitoneally, and 4.0 x 10 10 ATG-F4 was administered orally daily at 10 CFU. Survival rates were observed after administration. Stool consistency scores (assessment of improvement in diarrhea symptoms, Table 7) were performed 30 minutes after the second dose of FOLFIRI and FOLFOX, respectively.

[0111] [Correction pursuant to Rule 91, November 19, 2024]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] [result]

[0118] Referring to Figures 6a and 6b, 1.0 x 10 10 It was confirmed that the survival period of the FOLFIRI and FOLFOX groups was significantly prolonged by administering CFU of ATG-F4.

[0119] Referring to Figures 7a and 7b, 1.0 x 10 10 By administering CFU of ATG-F4, the stool consistency scores in FOLFIRI and FOLFOX were lowered, confirming that diarrhea symptoms were reduced to the same level as in the normal group.

[0120] Although specific examples of a composition for preventing, improving or treating cachexia or side effects of anticancer chemotherapy comprising the Lactobacillus reuteri ATG-F4 strain according to one embodiment of the present invention have been described so far, it is obvious that various modifications are possible within the scope of the present invention.

[0121] Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined not only by the claims described below but also by equivalents of the claims.

[0122] That is, it should be understood that the above-described embodiments are exemplary in all respects and not restrictive, and the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0123] The present invention provides a composition for preventing, improving or treating cachexia or side effects of anticancer chemotherapy, containing Lactobacillus reuteri ATG-F4 strain as an active ingredient.

[0124] [Accession number]

[0125] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0126] Accession number: KCTC13717BP

[0127] Date of acceptance: 20181115

[0128]

Claims

1. A composition for preventing, improving or treating cachexia, containing as an active ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

2. A pharmaceutical composition for preventing, improving or treating cachexia, containing as an active ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

3. A food composition for preventing, improving or treating cachexia, containing as an effective ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

4. A composition for preventing, improving or treating side effects of anticancer chemotherapy, containing as an active ingredient at least one selected from the group consisting of a strain of Lactobacillus reuteri ATG-F4 (accession number KCTC13717BP), a cell of the strain, a culture of the strain, an extract, a concentrate and a dried product of the culture.

5. In any one of paragraphs 1 to 4, A composition characterized by increasing the survival rate decreased due to administration of an anticancer drug.

6. In any one of paragraphs 1 to 4, A composition characterized by increasing the amount of fat reduced due to administration of an anticancer drug.

7. In any one of paragraphs 1 to 4, A composition characterized by increasing the strength of the jaw joint that has decreased due to administration of an anticancer drug.

8. In any one of paragraphs 1 to 4, A composition characterized by reducing inflammation increased due to administration of an anticancer drug.

9. In any one of paragraphs 1 to 4, A composition characterized by reducing diarrhea symptoms caused by anticancer drug administration.

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