Prophylactic or ameliorating agent for cachexia

Abstract

The present invention provides: a prophylactic or ameliorating agent for cachexia, which contains a phospholipid and / or a lysophospholipid as an active ingredient; and a method for screening for a substance capable of ameliorating cachexia. The prophylactic or ameliorating agent for cachexia according to the present invention is effective not only against cancer cachexia but also against cachexia induced by a disease other than cancer, can suppress the decrease in a skeletal muscle mass, can suppress fat atrophy, and can suppress the expression of inflammatory cytokines.

Description

Agents for preventing or improving cachexia

[0001] The present invention relates to an agent for preventing or ameliorating cachexia.

[0002] Cachexia refers to a state of debilitation due to poor nutritional status. In 2007, a consensus conference of European and American experts defined cachexia as "a syndrome of complex metabolic disorders caused by underlying disease and characterized by a loss of skeletal muscle mass, regardless of whether or not fat mass is lost. Clinical symptoms include weight loss in adults and growth retardation in children. Anorexia, inflammation, insulin resistance, and muscle protein breakdown are frequently observed. Cachexia is a pathological condition distinct from starvation, age-related muscle loss, depression, malabsorption, and hyperthyroidism, and increases the prevalence of these conditions" (Non-Patent Document 1). In particular, cachexia is characterized by a loss of skeletal muscle mass. Skeletal muscle weakness significantly impacts quality of life and prognosis, and has come to be known as sarcopenia.

[0003] Cachexia reduces exercise capacity and overall recovery, leading to treatment resistance in many diseases and the inability to receive treatment. It is well known that the progression of cancer patients to cachexia significantly impacts the prognosis of many cancer patients, particularly in the case of cancer treatment drugs, which have significant effects on normal tissues and side effects, by depriving them of the opportunity to receive cancer treatment. Of course, cachexia is not limited to cancer. It is also associated with many chronic wasting diseases, such as chronic heart failure, chronic obstructive pulmonary disease, chronic renal failure, chronic liver disease, chronic infections and sepsis (e.g., AIDS), autoimmune diseases such as rheumatoid arthritis, and diabetes. Therefore, cachexia can also lead to treatment resistance in patients with these diseases.

[0004] Currently, it is believed that the central cause of cachexia is the local or systemic increased expression of inflammatory cytokines (Non-Patent Document 2). However, the full picture of cachexia has not yet been clarified, and it is thought to be caused by a complex interplay of metabolic disorders that lead to tissue damage and increased catabolism due to persistent tissue inflammation, and reduced energy intake due to loss of appetite.

[0005] Along with elucidating the molecular mechanisms of cachexia from a basic medical perspective, there is a need to develop therapeutic drugs to suppress cachexia. Conventionally, drugs such as anti-inflammatory drugs, appetite-stimulating drugs (ghrelin receptor agonists), and selective androgen receptor modulators with anabolic effects have been used for cachexia patients, but their effectiveness is limited. Therefore, there is hope for the emergence of therapeutic drugs that can be expected to significantly improve the condition.

[0006] Evans WJ et al.: Cachexia: a new definition. Clinical Nutrition 2008; 27: 793-799. Cederholm T et al.: ESPEN guidelines on definitions and terminology of clinical nutrition. Clinical Nutrition 2017; 36: 49-64.

[0007] An objective of the present invention is to provide an agent for preventing or ameliorating cachexia that is effective not only for cancer cachexia but also for cachexia induced by diseases other than cancer, and to provide a method for screening for substances that ameliorate cachexia.

[0008] In order to solve the above-mentioned problems, the present invention encompasses the following inventions. [1] A preventive or ameliorating agent for cachexia, comprising a phospholipid and / or a lysophospholipid as an active ingredient. [2] The preventive or ameliorating agent according to [1] above, wherein the phospholipid is phosphatidylcholine or phosphatidylserine. [3] The preventive or ameliorating agent according to [1] above, wherein the lysophospholipid is lysophosphatidylcholine or lysophosphatidylserine. [4] The preventive or ameliorating agent according to any of [1] to [3] above, which is used to suppress loss of skeletal muscle mass. [5] The preventive or ameliorating agent according to any of [1] to [3] above, which is used to suppress lipoatrophy. [6] The preventive or ameliorating agent according to any of [1] to [3] above, which is used to suppress the expression of inflammatory cytokines. [7] The preventive or ameliorating agent according to any of [1] to [6] above, wherein the disease inducing cachexia is a chronic disease accompanied by inflammation. [8] The preventive or ameliorating agent according to [7], wherein the chronic disease accompanied by inflammation is a disease selected from the group consisting of cancer, chronic heart failure, chronic obstructive pulmonary disease, chronic renal failure, chronic liver disease, chronic infectious disease, sepsis, autoimmune disease, and diabetes. [9] A method for screening for a substance that improves cachexia, comprising: in an in vitro differentiation induction system from stem cells or progenitor cells to skeletal muscle cells, using a differentiation-inducing medium containing a culture supernatant of cancer cells, selecting a test substance that promotes myotube formation when the test substance is added to the differentiation-inducing medium and cultured in comparison to when the test substance is not added.

[10] A method for screening for a substance that improves cachexia, comprising: in an in vitro differentiation induction system from stem cells or progenitor cells to adipocytes, using a differentiation-inducing medium containing a culture supernatant of cancer cells, selecting a test substance that promotes differentiation into adipocytes when the test substance is added to the differentiation-inducing medium and cultured in comparison to when the test substance is not added.

[0009] The present invention provides an agent for preventing or ameliorating cachexia that is effective not only for cancer-related cachexia but also for cachexia induced by diseases other than cancer, and also provides a method for screening for substances that ameliorate cachexia.

[0010]

[0033] Figure 1 shows the effects of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) on the inhibition of myotube formation by a cachectic substance contained in the culture supernatant of colon cancer cells (Colon26) in an in vitro differentiation induction system from myoblasts (C2C12) to skeletal muscle cells. Figure 2 shows the effects of LPC on the inhibition of differentiation by a cachectic substance contained in the culture supernatant of colon cancer cells (Colon26) in an in vitro differentiation induction system from mesenchymal cells (OP9) to adipocytes. Figure 3 shows the results of intravenous administration of PC to mice in which colon cancer cells (Colon26) were subcutaneously transplanted to form tumors, and the weights of the gastrocnemius and tibialis muscles were measured 36 days after transplantation. PC was intravenously administered to mice that had been subcutaneously transplanted with colon cancer cells (Colon26) to form tumors, and RNA was extracted from the muscle 36 days after transplantation, and the expression levels of the MuRF-1 gene and Atrogin-1 gene, which induce muscle breakdown, were measured by quantitative PCR. PC was intravenously administered to mice that had been subcutaneously transplanted with colon cancer cells (Colon26) to form tumors, and the weight of epididymal adipose tissue was measured 36 days after transplantation. PC was intravenously administered to mice that had been subcutaneously transplanted with colon cancer cells (Colon26) to form tumors, and RNA was extracted from epididymal adipose tissue 36 days after transplantation, and the expression level of the Leptin gene, which maintains adipose tissue, was measured by quantitative PCR. PC was intravenously administered to mice that had been subcutaneously transplanted with colon cancer cells (Colon26) to form tumors, and the survival rate was calculated. PC, LPC, or a PC / LPC mixture was orally administered to mice that had been subcutaneously implanted with colon cancer cells (Colon26) to form tumors, and the gastrocnemius and tibialis muscles were excised and weighed 24 days after implantation. Figure 9 shows the results of hematoxylin-eosin staining of tissue sections prepared from the gastrocnemius muscles whose weights were measured in Figure 8. Figure 9 shows the results of measuring the circumference of 50 muscle fiber bundles per mouse in each group in the tissue images of Figure 9.13 shows the results of quantitative PCR assay of the expression levels of the MuRF-1 gene and Atrogin-1 gene, which induce muscle breakdown, extracted from RNA from the muscle tissues weighed in FIG. 8 . 14 shows the results of serum IL-6 concentration measurement in blood collected from the mice used in the experiment in FIG. 8 on day 24 after colon cancer cell transplantation. 15 shows the results of blood glucose measurement on days 0, 7, and 31 after oral administration of two types of PC / LPC mixtures to streptozocin (STZ)-induced type 1 diabetes model mice starting on day 7 after STZ administration. 16 shows the results of weight measurement of the gastrocnemius and tibialis muscles on day 31 after STZ administration in the experiment in FIG. 13 . 17 shows the results of quantitative PCR assay of the expression levels of the MuRF-1 gene and Atrogin-1 gene, which induce muscle breakdown, extracted from RNA from the muscle tissues weighed in FIG. 14 . PC, LPC, or a PC / LPC mixture was orally administered to mice in which colon cancer cells (Colon26) were subcutaneously transplanted to form tumors, and 24 days after transplantation, laparotomy was performed under anesthesia to observe the appearance of the epididymal adipose tissue. This figure shows the results of measuring the weight of epididymal adipose tissue excised and weighed after the external observation in Figure 16 . This figure shows the results of hematoxylin-eosin staining of tissue sections prepared from the epididymal adipose tissue whose weight was measured in Figure 17 . This figure shows the results of measuring the perimeter of adipocytes (50 cells per mouse per group) in the tissue images in Figure 18 . This figure shows the results of measuring oncostatin M (OMS) levels in serum from blood collected 24 days after colon cancer cell transplantation in the mice used in the experiment in Figure 16 . This figure shows the results of measuring the IL-6 concentration in the culture supernatant of colon cancer cells (Colon26) treated with PC or phosphatidylserine (PS).

[0011] [Agent for preventing or ameliorating cachexia] The present invention provides an agent for preventing or ameliorating cachexia (hereinafter referred to as "the agent of the present invention") containing a phospholipid and / or a lysophospholipid as an active ingredient. The agent of the present invention may contain only a phospholipid, only a lysophospholipid, or both a phospholipid and a lysophospholipid.

[0012] The phospholipid used in the improving agent of the present invention may be a diacylglycerophospholipid or a sphingophospholipid. Examples of diacylglycerophospholipids include phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol. Examples of sphingophospholipids include sphingomyelin. Phospholipids may be used alone or in combination of two or more. They may also be formulated in the form of a composition primarily composed of phospholipids. Examples of compositions primarily composed of phospholipids include PC-98N (manufactured by Kewpie Corporation, phosphatidylcholine content approximately 95%). Phospholipids may be extracted from natural sources or chemically synthesized. Phospholipids extracted from natural sources can be obtained from animal or plant materials such as egg yolk, soybean, and rapeseed. Phospholipids may be hydrogenated, if necessary.

[0013] The phospholipid used in the improving agent of the present invention is not particularly limited, but phosphatidylcholine (PC) can be preferably used.Phosphatidylcholine (also called lecithin) is not particularly limited as long as it has a structure in which fatty acids are ester-bonded to the C1 and C2 positions of glycerin and phosphocholine is ester-bonded to the C3 position of glycerin.Examples of phosphatidylcholine include egg yolk lecithin, soybean lecithin, soybean phosphatidylcholine, dioctanoyl phosphatidylcholine, dinonanoyl phosphatidylcholine, didecanoyl phosphatidylcholine, diundecanoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoleoyl phosphatidylcholine, di ... Examples include phosphatidylcholine, dilinoleoylphosphatidylcholine, dieicosapentaenoylphosphatidylcholine, didocosahexaenoylphosphatidylcholine, dierucoylphosphatidylcholine, (1-myristoyl-2-palmitoyl)phosphatidylcholine, (1-palmitoyl-2-myristoyl)phosphatidylcholine, (1-oleoyl-2-palmitoyl)phosphatidylcholine, and (1-palmitoyl-2-oleoyl)phosphatidylcholine.

[0014] Furthermore, phosphatidylserine (PS) can be preferably used as the phospholipid used in the improving agent of the present invention. The phosphatidylserine is not particularly limited as long as it has a structure in which a fatty acid is ester-bonded to the C1 and C2 positions of glycerin and phosphoserine is ester-bonded to the C3 position. Examples of phosphatidylcholine include dioctanoylphosphatidylserine, dinonainoylphosphatidylserine, didecanoylphosphatidylserine, diundecanoylphosphatidylserine, dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, distearoylphosphatidylserine, dipalmitoleoylphosphatidylserine, dioleoylphosphatidylserine, dilin ... Examples of phosphatidylserine include oleoyl phosphatidylserine, dieicosapentaenoyl phosphatidylserine, didocosahexaenoyl phosphatidylserine, dierucoyl phosphatidylserine, (1-myristoyl-2-palmitoyl) phosphatidylserine, (1-palmitoyl-2-myristoyl) phosphatidylserine, (1-oleoyl-2-palmitoyl) phosphatidylserine, and (1-palmitoyl-2-oleoyl) phosphatidylserine.

[0015] Lysophospholipids are those obtained by removing one fatty acid from the above-mentioned phospholipids. The lysophospholipid used in the improving agent of the present invention may be a monoacylglycerophospholipid obtained by removing one of the fatty acids ester-bonded to the hydroxyl group of the glycerol skeleton from a diacylglycerophospholipid, or a lysosphingophospholipid obtained by removing one of the fatty acids amide-bonded to the amino group of the sphingosine skeleton. Examples of monoacylglycerophospholipids include lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidylinositol. Examples of lysosphingophospholipids include lysosphingomyelin and sphingosine-1-phosphate. The lysophospholipid may be used alone or in combination of two or more. Furthermore, the lysophospholipid may be formulated in the form of a composition primarily composed of a lysophospholipid. An example of a composition containing lysophospholipids as a main component is LPC-1 (manufactured by Kewpie Corporation, lysophosphatidylcholine content: approximately 95%). Lysophospholipids can be obtained by treating the phospholipids with an enzyme such as phospholipase or ceramide deacylase. Alternatively, lysophospholipids can be obtained by directly treating animal or plant raw materials with an enzyme such as phospholipase or ceramide deacylase, followed by extraction with a solvent such as ethanol. Lysophospholipids may be hydrogenated as necessary.

[0016] The lysophospholipid used in the improving agent of the present invention is not particularly limited, but lysophosphatidylcholine (LPC) or lysophosphatidylserine (LPS) can be preferably used. Examples of lysophosphatidylcholine include those obtained by removing one fatty acid from the various phosphatidylcholines exemplified above. Examples of lysophosphatidylserine include those obtained by removing one fatty acid from the various phosphatidylserines exemplified above.

[0017] As used herein, "preventing cachexia" means suppressing the worsening of a subject's condition who shows signs of cachexia, and "ameliorating cachexia" means improving the condition of a subject who has already developed cachexia. Symptoms of cachexia include loss of skeletal muscle mass, fat atrophy, weight loss, growth retardation, loss of appetite, anemia, edema, inflammation, fatigue, general weakness, malaise, pain, nausea, vomiting, depression, anxiety, changes in taste, changes in smell, etc. The improving agent of the present invention has been confirmed to suppress loss of skeletal muscle mass and fat atrophy caused by cachexia, and to suppress the expression of inflammatory cytokines (e.g., IL-6, oncostatin M, etc.) caused by cachexia (see Examples).

[0018] Diseases that induce cachexia include, but are not limited to, cancer, heart disease, vascular disease, kidney disease, liver disease, lung disease, gastrointestinal disease, urinary tract disease, infectious diseases, immune diseases, diabetes, central nervous system diseases such as Parkinson's disease, amyloidosis, psychiatric diseases, endocrine diseases, etc. Diseases that induce cachexia may also be chronic diseases accompanied by inflammation. Examples of chronic diseases accompanied by inflammation include cancer, chronic heart failure, chronic obstructive pulmonary disease, chronic renal failure, chronic liver disease, chronic infectious diseases, sepsis, autoimmune diseases, diabetes, etc.

[0019] The improving agent of the present invention can be used as a medicine. When the improving agent of the present invention is used as a medicine, it can be formulated by appropriately blending pharma- ceutically acceptable carriers or additives with phospholipids and / or lysophospholipids as active ingredients according to a known method for producing pharmaceutical preparations (e.g., the method described in the Japanese Pharmacopoeia). Specifically, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules), troches, syrups, liquids, emulsions, suspensions, controlled-release preparations (e.g., immediate-release preparations, sustained-release preparations, sustained-release microcapsules, etc.), aerosols, films (e.g., orally disintegrating films, oral mucosal patch films, etc.), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), drip infusions, transdermal preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories, etc.), pellets, nasal preparations, gastric preparations, enteral preparations, pulmonary preparations (inhalants), eye drops, and other oral or parenteral preparations are exemplified. The blending ratio of the carrier or additive can be appropriately set based on the range commonly used in the pharmaceutical field. The carriers or additives that can be added are not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0020] Examples of additives that can be incorporated into tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, and gum arabic; excipients such as crystalline cellulose; bulking agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavorings such as peppermint, saffron oil, and cherry. When the dosage unit is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be prepared according to standard formulation procedures (e.g., dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and the like, which may be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50, etc.). As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. Furthermore, the liquid may be blended with a buffer (e.g., phosphate buffer, sodium acetate buffer, etc.), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc.

[0021] The improving agent of the present invention can be implemented as a food or beverage. Examples of the food or beverage include nutritional supplements, health foods, functional foods, foods for the sick, foods for infants, and foods for the elderly. The form of the food or beverage is not particularly limited, and examples include supplements, beverages, processed foods, confectioneries, dairy products, and oil-and-fat processed foods. Examples of supplements include fine granules, tablets, granules, powders, capsules (including soft capsules and hard capsules), chewable tablets, syrups, drinks, natural liquid diets, semi-digested nutritional foods, elemental nutritional foods, gastric nutrients, and enteral nutrients. Examples of beverages include tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks. Examples of processed foods include bread, noodles, pasta, ham, bacon, sausages, and kamaboko (fish cake). Examples of confectioneries include candy, jam, chewing gum, ice cream, snacks, cookies, biscuits, cakes, wafers, sweet rolls, chocolate, and Japanese sweets. Dairy products include processed milk, fermented milk, butter, cheese, yogurt, etc. Oil and fat processed foods include margarine, mayonnaise, shortening, whipped cream, dressing, etc.

[0022] The improving agent of the present invention can be implemented as a preparation in which phospholipids and / or lysophospholipids are encapsulated in cyclic oligosaccharides. Examples of cyclic oligosaccharides include cyclodextrin. The improving agent of the present invention can also be implemented as a self-emulsifying emulsion preparation. A self-emulsifying emulsion contains an oily component, a polyhydric alcohol, water, and an emulsifier, and generates a nanoscale oil-in-water emulsion upon contact with body fluids such as blood or digestive fluids. The use of a self-emulsifying emulsion improves the bioavailability of the phospholipids and / or lysophospholipids, making it preferable for the effects of the present invention to be more easily achieved. The self-emulsifying emulsion can be prepared by conventional methods. For example, a method can be used in which phospholipids and / or lysophospholipids and a polyhydric alcohol are uniformly mixed and stirred, and then an oil or fat is added and mixed.

[0023] The oily component used in the self-emulsifying emulsion is not particularly limited as long as it does not adversely affect the human body. Examples include vegetable oils such as soybean oil, sesame oil, rapeseed oil, safflower oil, olive oil, castor oil, corn oil, cottonseed oil, rice bran oil, sunflower oil, grapeseed oil, and wheat germ oil, and medium-chain triglycerides (MCTs). The content of the oily component is not particularly limited, and may be, for example, 0.1% by mass or more and 35% by mass or less based on the total weight of the formulation. Within this range, nanoscale oil-in-water emulsions are easily formed upon contact with body fluids such as blood and digestive fluids, making it easier to achieve the effects of the present invention.

[0024] The polyhydric alcohol used in the self-emulsifying emulsion is not particularly limited as long as it does not adversely affect the human body. Examples include 1,3-butanediol, 1,3-propanediol, 3-methyl-1,3-butanediol, propylene glycol, dipropylene glycol, polypropylene glycol, butylene glycol, 1,2-pentanediol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, polyoxyethylene glycol, polyglycerols such as glycerin, diglycerin, and tetraglycerin, xylitol, maltitol, pentaerythritol, sorbitol, and trehalose. Among these, glycerin, propylene glycol, and butylene glycol are preferred due to their high safety to the human body. The content of the polyhydric alcohol is not particularly limited, but, for example, if it is 20% by mass or more but 99% by mass or less based on the entire formulation, it is likely to form a nanoscale oil-in-water emulsion upon contact with body fluids such as blood and digestive fluids, making it easier to achieve the effects of the present invention.

[0025] Phospholipids and lysophospholipids are components present in living organisms and have a history of being administered to humans as active pharmaceutical ingredients or additives, and therefore have low toxicity and can be safely administered to humans and other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.). The content of the active ingredient in the formulation is appropriately determined depending on the dosage form, administration method, carrier, etc., but the phospholipid and / or lysophospholipid can be added in a proportion of typically 0.01 to 100% by mass, preferably 0.1 to 95% by mass, based on the total amount of the formulation.

[0026] The dosage of phospholipids and / or lysophospholipids varies depending on the subject, symptoms, route of administration, etc., but in the case of oral administration, for example, for a human weighing approximately 60 kg, it is generally about 0.01 to 25,000 mg, preferably about 0.1 to 2,500 mg, and more preferably about 0.5 to 500 mg per day. In the case of parenteral administration, the daily dosage varies depending on the patient's condition, symptoms, administration method, etc., but for example, in the case of injections, it is usually about 0.01 to 100 mg, preferably about 0.01 to 50 mg, and more preferably about 0.01 to 20 mg per kg of body weight.

[0027] The present invention includes the following inventions: A method for preventing or ameliorating cachexia, which comprises administering an effective amount of a phospholipid and / or lysophospholipid to a mammal; A phospholipid and / or lysophospholipid for use in preventing or ameliorating cachexia; Use of a phospholipid and / or lysophospholipid for preventing or ameliorating cachexia; Use of a phospholipid and / or lysophospholipid for producing an agent for preventing or ameliorating cachexia.

[0028] [Method for screening for substances that improve cachexia] The present invention provides a method for screening for substances that improve cachexia. A first embodiment of the screening method of the present invention is a method comprising using a differentiation-inducing medium containing a culture supernatant of cancer cells in an in vitro system for inducing differentiation from stem cells or progenitor cells to skeletal muscle cells, and selecting a test substance that promotes myotube formation when the test substance is added to the differentiation-inducing medium and cultured in comparison to when the test substance is not added. A second embodiment of the screening method of the present invention is a method comprising using a differentiation-inducing medium containing a culture supernatant of cancer cells in an in vitro system for inducing differentiation from stem cells or progenitor cells to adipocytes, and selecting a test substance that promotes differentiation into adipocytes when the test substance is added to the differentiation-inducing medium and cultured in comparison to when the test substance is not added.

[0029] The test substance to be subjected to the screening method of the present invention is not particularly limited, and examples thereof include natural compounds, organic compounds, inorganic compounds, nucleic acids, nucleic acid oligos, proteins, peptides, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic unicellular extracts, and animal cell extracts. Furthermore, the test substance may be an expression product of a compound library, a nucleic acid oligo library, a peptide library, or a gene library. The test substance may be a novel substance or a known substance. These test substances may form a salt. The salt of the test substance is preferably a salt with a physiologically acceptable acid or base.

[0030] The cancer cell culture supernatant used in the screening method of the present invention is not particularly limited as long as it is a culture supernatant of cancer cells cultured by a standard method. It is preferable that the culture supernatant be obtained by culturing cancer cells seeded under normal conditions until they become roughly confluent (about 80% or more). The type of cancer cells used is not particularly limited, but it is preferable to use cancer cells of the same type as the stem or progenitor cells used.

[0031] The stem cells or progenitor cells used in the first embodiment may be any cells that can differentiate into skeletal muscle cells, such as skeletal muscle stem cells (satellite cells) and myoblasts. The medium for inducing differentiation into skeletal muscle cells can be selected from known media that can be used to induce differentiation of stem cells or progenitor cells into skeletal muscle cells. For example, a medium containing horse serum, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), etc. can be used.

[0032] In a first embodiment, stem cells or progenitor cells are induced to differentiate into skeletal muscle cells by adding a test substance to a differentiation-inducing medium containing a culture supernatant of cancer cells (test substance group), and a test substance that promotes myotube formation compared to a control group to which the test substance was not added is selected as a cachexia-ameliorating substance. Myotube formation can be evaluated, for example, by observing the cultured cells under a microscope.

[0033] The stem cells or progenitor cells used in the second embodiment may be any cells that can differentiate into adipocytes, such as mesenchymal stem cells and preadipocytes. The medium for inducing differentiation into adipocytes can be selected from known media that can be used to induce differentiation of stem cells or progenitor cells into adipocytes. For example, a medium containing insulin, dexamethasone, etc. can be used.

[0034] In a second embodiment, stem cells or progenitor cells are induced to differentiate into adipocytes by adding a test substance to a differentiation-inducing medium containing cancer cell culture supernatant (test substance group), and a test substance that promotes adipocyte differentiation compared to a control group without the test substance is selected as a cachexia-ameliorating substance. Promotion of adipocyte differentiation can be evaluated, for example, by staining cultured cells with Oil Red O and counting the number of positive cells.

[0035] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0036] Example 1: Effects of PC and LPC in an in vitro model of cachectic substance-induced skeletal muscle mass loss. 1-1 Experimental method. (1) Cells used: Mouse myoblasts (C2C12, RIKEN cell bank) and mouse colon cancer cells (Colon26, RIKEN cell bank) were used. The culture medium used was DMEM (Sigma-Aldrich Japan) or RPMI (Sigma-Aldrich Japan) containing 10% fetal bovine serum (Gibco), 100 units / mL penicillin G (Sigma-Aldrich Japan), and 100 μg / mL streptomycin (Sigma-Aldrich Japan).

[0037] (2) Preparation of Colon26 cell culture supernatant: 5 x 10 Colon26 cells 4 cells / cm 2 Cells were seeded onto 10-mm culture dishes at a concentration of 100 μM each and cultured at 37°C in 5% CO2 until 80%-90% confluent. The medium was then replaced with medium containing 16:0 lysophosphatidylcholine (LPC), 18:0 LPC, 16:0 phosphatidylcholine (PC), or 18:0 PC (all Avanti Polar Lipids) at a final concentration of 25 μM, or medium without LPC or PC, and cultured for an additional 24 hours. The cells were washed with PBS and then replaced with serum-free RPMI medium for an additional 48 hours. The medium was collected and centrifuged at 3000 rpm for 20 minutes at 4°C to precipitate cellular components. The supernatant was sterilized by filtration using a 0.22 μm sterile filter (Merck Millipore) and stored at -80°C until use.

[0038] (3) C2C12 Cell Culture: C2C12 cells were cultured in a 6-well culture plate until they reached 80%-90% confluence. The medium was replaced with DMEM containing horse serum (Gibco, final concentration 2%) or DMEM containing 50% horse serum, each of the five culture supernatants prepared in (2), and the cells were cultured for 9 days to induce differentiation into skeletal muscle cells. Cell morphology was observed under a phase-contrast microscope on day 9.

[0039] 1-2 Results The results are shown in Figure 1. (A) Control: Cultured in horse serum-containing DMEM medium. (B) 50% CM: Cultured in horse serum-containing DMEM medium containing 50% Colon26 cell culture supernatant. (C) 50% CM + 16 LPC 25 μM: Cultured in horse serum-containing DMEM medium containing 50% Colon26 cell culture supernatant supplemented with 16:0 LPC. (D) 50% CM + 18 LPC 25 μM: Cultured in horse serum-containing DMEM medium containing 50% Colon26 cell culture supernatant supplemented with 18:0 LPC. (E) 50% CM + 16 PC 25 μM: Cultured in horse serum-containing DMEM medium containing 50% Colon26 cell culture supernatant supplemented with 16:0 PC. (F) 50% CM + 18 PC 25 μM: Cultured in horse serum-containing DMEM medium containing 50% Colon26 cell culture supernatant supplemented with 18:0 PC. These results were obtained by culturing cells in DMEM medium containing 50% horse serum. In the control (A), myotube formation from skeletal muscle cells differentiated from myoblasts was observed, but in the medium containing Colon26 cell culture supernatant (B), myotube formation was suppressed. In the medium containing Colon26 cell culture supernatant with LPC (C) and (D), and in the medium containing Colon26 cell culture supernatant with PC (E) and (F), the inhibition of differentiation from myoblasts to skeletal muscle cells was prevented, and myotube formation was observed, although to varying degrees.

[0040] These results revealed that the factors secreted by cancer cells contain cachexia substances that inhibit the differentiation of myoblasts into skeletal muscle cells. In other words, it was revealed that the cachexia substances secreted by cancer cells induce skeletal muscle mass loss, and this culture system was understood to be an in vitro model of skeletal muscle mass loss caused by cachexia substances. Furthermore, using this culture system, it was confirmed that when cancer cells were stimulated with LPC or PC, the expression of skeletal muscle mass loss-inducing factors (cachexia substances) was suppressed.

[0041] Example 2: Effect of LPC in an in vitro model of cachexia-induced adipocyte atrophy 2-1 Experimental method (1) Cells used: OP9 cells (RIKEN cell bank), which are mesenchymal cells, were used. The culture medium used was MEM medium (Sigma-Aldrich Japan) containing 20% ​​fetal bovine serum (Gibco), 100 units / mL penicillin G (Sigma-Aldrich Japan), 100 μg / mL streptomycin (Sigma-Aldrich Japan), and 2 mM L-glutamine.

[0042] (2) Cultivation of OP9 cells 7 × 10 OP9 cells 3 cells / cm 2 OP9 cells were seeded onto a 96-well culture plate at a concentration of 100 μg / mL and cultured until confluent. When the cells reached confluence, the adipocyte differentiation medium was changed to adipocyte differentiation medium containing MEM medium containing 100 units / mL penicillin G and 100 μg / mL streptomycin, 15% KnockOut SR (Invitrogen), 10% Colon26 cell culture supernatant prepared in Example 1(2), or 10% Colon26 cell culture supernatant supplemented with 16:0 LPC to promote adipocyte differentiation. The medium was changed every four days, and the cells were cultured for seven days. On day 7, the OP9 cells were fixed with 4% paraformaldehyde (Wako) for 30–60 minutes, washed twice with 60% isopropanol, stained with Oil Red O Solution (Cayman Chemical) for 20 minutes, and then observed under a microscope.

[0043] 2-2 Results The results are shown in Figure 2. (A) Control: Cells cultured in adipocyte differentiation medium. (B) 10% CM: Cells cultured in adipocyte differentiation medium containing 10% Colon26 cell culture supernatant. (C) 10% CM + 16:0 LPC 25 μM: Cells cultured in adipocyte differentiation medium containing 10% Colon26 cell culture supernatant supplemented with 16:0 LPC. In (A) Control, adipocytes stained with Oil Red O were observed, while in (B) Colon26 cell culture supernatant, adipocyte differentiation was inhibited. In (C) Colon26 cell culture supernatant supplemented with LPC, the inhibition of adipocyte differentiation was prevented, and adipocytes stained with Oil Red O were observed. These results demonstrate that molecules secreted by cancer cells include cachectic substances that inhibit the differentiation and maintenance of adipocytes, and that LPC inhibits the secretion of cachectic substances.

[0044] Example 3: Effect of PC administration on skeletal muscle mass loss in cancer-bearing mice 3-1 Experimental method (1) Animals used and cancer cell transplantation Male BalB / c mice (SLC) aged 8 to 9 weeks were used. Colon26 cells were cultured in the same manner as in Example 1 until they reached 80% to 90% confluence, and then 1 × 10 6 The cells were suspended in PBS to a concentration of 100 μL / mL. 100 μL of this cell suspension was subcutaneously implanted into the right upper abdomen of each mouse. The mice were weighed every 3 or 4 days to observe their overall condition, and the maximum diameter (a) and minimum diameter (b) of the tumor were measured to determine the tumor size [1 / 2(a × b)]. 2 )mm 3 ] was calculated.

[0045] (2) Preparation of Phosphatidylcholine (PC) Hydrogenated PC (HSPC; Avanti Polar Lipids) was dissolved in ethanol to a concentration of 50 mM and stored at −20° C. until use.

[0046] (3) Grouping and Administration Schedule Three groups were established: a control group (without cancer cells transplanted), a PBS-treated group (with cancer cells transplanted and treated with PC), and a PC-treated group (with cancer cells transplanted and treated with PC). One week after Colon26 cell transplantation, at least five mice per group were randomly assigned to receive either PC or PBS treatment. PC and PBS treatment were administered intravenously every 3 or 4 days. PC was administered at a dose of 3 mg / kg body weight, diluted in PBS, and administered at 100 μL per mouse. PBS treatment was administered at 100 μL per mouse.

[0047] (4) Muscle Weight Measurement and Quantitative PCR. Mice were euthanized 36 days after Colon26 cell transplantation, and the gastrocnemius and tibialis muscles were removed and weighed. 20 mg of muscle tissue was homogenized using a High Power Homogenizer ASG50 (AS ONE Corporation), and total RNA was extracted using an RNeasy Fibrous Mini Kit (Qiagen). cDNA was prepared from the total RNA using a PrimeScript RT reagent kit (Takara) and subjected to quantitative PCR (qPCR). The following primer sets were used to detect the MuRF-1 and Atrogin-1 genes, which are known to induce muscle breakdown. TB Green Premix Ex Taq II (Takara) was used as the qPCR reagent, and the Light Cycler 96 system (Roche) was used as the real-time PCR system. Primer set for amplifying the mouse GAPDH gene: sense 5'-TGATACAGTCGGGGTGAGTG-3' (SEQ ID NO: 1) anti-sense 5'-AAGATGGTGATGGGCTTCCCG-3' (SEQ ID NO: 2) Primer set for amplifying the mouse MuRF-1 gene: sense 5'-ACGGAAACGACCTCCAGACATG-3' (SEQ ID NO: 3) anti-sense 5'-TACCAAGCCTGTGGTCATCCTG-3' (SEQ ID NO: 4) Primer set for amplifying the mouse Atrogin-1 gene: sense 5'-CTTCTCGACTGCCATCCTGGAT-3' (SEQ ID NO: 5) anti-sense 5'-TCTTTTGGGCGATGCCACTCAG-3' (SEQ ID NO: 6)

[0048] 3-2 Results The results of muscle weight measurements are shown in Figure 3. (A) shows the weight of the gastrocnemius muscle, and (B) shows the weight of the tibialis muscle. In the vehicle-treated group (PBS), muscle weights of both the gastrocnemius and tibialis muscles decreased, indicating that cachexia-induced loss of skeletal muscle mass was induced. On the other hand, in the PC-treated group (PC), muscle weights of both the gastrocnemius and tibialis muscles did not decrease, indicating that cachexia-induced loss of skeletal muscle mass was not induced.

[0049] The results of quantitative PCR are shown in Figure 4. (A) shows the results for the MuRF-1 gene, and (B) shows the results for the Atrogin-1 gene. In the vehicle-treated group (PBS), expression of both the MuRF-1 and Atrogin-1 genes was elevated, but in the PC-treated group (PC), expression of both genes was at normal mouse levels, consistent with the muscle weight results.

[0050] Example 4: Effect of PC Administration on Survival Rate and Fat Atrophy in Cancer-Bearing Mice 4-1 Experimental Method Colon26 cells were transplanted into mice using the same method as in Example 3. One week later, mice were divided into three groups: a control group (not transplanted with cancer cells), a group treated with PBS (n = 20), and a group treated with PC (n = 20). PC or PBS was administered according to the same administration schedule as in Example 3. During the administration period, mice were observed daily for general condition, and the number of deaths was counted to calculate survival rates. Surviving mice were euthanized 36 days after Colon26 cell transplantation, and epididymal adipose tissue was excised and weighed (n = 8 per group). Total RNA was extracted from less than 100 mg of epididymal adipose tissue using the same method as in Example 3, and cDNA was prepared and subjected to qPCR (n = 2 per group). The same reagents and equipment were used as in Example 3. The following primer set was used to detect the leptin gene, which is known to play a role in maintaining adipose tissue. The primer set used for amplifying the mouse GAPDH gene was the same as that used in Example 3. Primer set used for amplifying the mouse leptin gene: sense 5'-TGAGTTTGTCCAAGATGGACC-3' (SEQ ID NO: 7) anti-sense 5'-GCCATCCAGGCTCTCTGG-3' (SEQ ID NO: 8)

[0051] 4-2 Results Figure 5 shows the results of measuring epididymal adipose tissue weight 36 days after cancer cell transplantation. Adipose tissue weight decreased in the vehicle-treated group (PBS), indicating the induction of cachexia-induced lipoatrophy. In contrast, adipose tissue weight did not decrease in the PC-treated group (PC), indicating the inhibition of lipoatrophy. The results of quantitative PCR are shown in Figure 6. Leptin gene expression was suppressed in the vehicle-treated group (PBS), which induced lipoatrophy, but in the PC-treated group (PC), which inhibited lipoatrophy, leptin gene expression was at the same level as in normal mice. The results of survival rate are shown in Figure 7. The PC-treated group (PC) had an improved survival rate compared to the vehicle-treated group (PBS).

[0052] Example 5: Effect of administration of PC, LPC, or a PC / LPC mixture on tumor-bearing mice (1) 5-1 Experimental method (1) Animals used, tumor cell transplantation, and group assignment Colon26 cells were transplanted into mice using the same method as in Example 3. One week later, the mice were divided into six groups: a tumor cell-untransplanted group (control), a tumor cell transplantation solvent-administered group (PBS), a tumor cell transplantation group A (PC A), a tumor cell transplantation group B (PC B), a tumor cell transplantation group C (PC C), and a tumor cell transplantation group D (PC D). At least five mice were randomly assigned to each group.

[0053] (2) Preparation and administration schedule of PC, LPC, and PC / LPC mixture (self-emulsifying emulsion). PC was phosphatidylcholine extracted from egg yolk (egg yolk lecithin PC-98N, Kewpie Corporation). LPC was lysophosphatidylcholine extracted and purified from phospholipase-treated egg yolk. Group A received a PC:LPC mixture at a ratio of 1:2 (self-emulsifying emulsion), Group B received a PC:LPC mixture at a ratio of 1:4 (self-emulsifying emulsion), Group C received only PC, and Group D received only LPC. The PC and LPC mixture was prepared by mixing PC, LPC, and propylene glycol uniformly and then adding medium-chain fatty acid oil (Coconard RK, Kao Chemical Co.) while stirring. The mass ratio of each component of the mixture for Group A was PC:LPC:medium-chain fatty acid oil:propylene glycol = 16.7:33.3:5.0:45.0. The mass ratio of each component of the mixture for Group B was PC:LPC:medium-chain fatty acid oil:propylene glycol = 10.0:40.0:5.0:45.0. The total dose of PC and LPC was 3 mg / kg, and 100 μL was orally administered every 3 or 4 days using a mouse probe (FCR & Bio Inc.) starting 1 week after cancer cell transplantation.

[0054] (3) Muscle Weight Measurement, Muscle Tissue Analysis, Quantitative PCR, and Serum IL-6 Measurement. Twenty-four days after Colon26 cell transplantation, mice were anesthetized and transcardially bled using a Microtainer serum separator tube (BD). After euthanasia, the gastrocnemius and tibialis muscles were removed, weighed, and stored in liquid nitrogen. Gastrocnemius muscles from three randomly selected mice per group were thawed, fixed in 4% paraformaldehyde for 48 hours, and embedded in paraffin. 4-μm tissue sections were prepared and stained with hematoxylin and eosin (Wako). The specimens were photographed at 20x magnification using a VS-200 research slide scanner (Olympus). Image analysis was performed using QuPath and Fiji (Image J) software. The circumference of 50 muscle fiber bundles per mouse was measured (n = 3). Expression levels of MuRF-1 and Atrogin-1 genes were measured by quantitative PCR using the same method as in Example 3. The collected blood was left at room temperature for 30 minutes and then centrifuged at 9,000 rpm for 10 minutes to collect serum. IL-6 concentrations in the serum were measured using the LEGEND MAX Mouse IL-6 ELISA Kit (BioLegend).

[0055] 5-2 Results (1) Muscle Weight Figure 8 shows the results of muscle weight measurements. (A) shows the gastrocnemius weight, and (B) shows the tibialis weight. In the vehicle-treated group (PBS), muscle weights of both the gastrocnemius and tibialis muscles decreased, indicating that cachexia-induced skeletal muscle mass loss was induced. In contrast, in groups A, B, C, and D, muscle weights of both the gastrocnemius and tibialis muscles did not decrease, indicating that cachexia-induced skeletal muscle mass loss was suppressed.

[0056] (2) Muscle tissue images and muscle fiber circumference Figure 9 shows the tissue images of the gastrocnemius muscle, and the analysis results comparing the muscle fiber circumference measured from the tissue images are shown in Figure 10. Muscle fiber atrophy due to cachexia was suppressed in groups A, B, C, and D.

[0057] (3) Quantitative PCR The results of quantitative PCR are shown in Figure 11. (A) shows the results for the MuRF-1 gene, and (B) shows the results for the Atrogin-1 gene. In the vehicle-treated group (PBS), expression of the MuRF-1 gene and Atrogin-1 gene, which induce muscle breakdown, was increased. On the other hand, expression of both genes was suppressed in groups A, B, C, and D.

[0058] (4) Serum IL-6 Concentration The results of serum IL-6 concentration are shown in Figure 12. In the vehicle-treated group (PBS), serum IL-6 concentration increased significantly. On the other hand, the increase in serum IL-6 concentration was suppressed in groups A, B, C, and D. These results demonstrate that the anti-cachexia effects of PC, LPC, and the PC / LPC mixture are not limited to muscle or adipose tissue, but also extend to systemic inflammation.

[0059] Example 6: Effect of PC / LPC Mixture Administration on Type 1 Diabetes Model Mice 6-1 Experimental Method (1) Animals and Type 1 Diabetes Model Mice Eight-week-old male C57BL / 6J mice (SLC) were used. Mice were acclimatized by pre-breeding for at least one week and then fasted for 14 hours to induce starvation. Streptozocin (STZ) was dissolved in 0.1 M cryopreserved citrate buffer (pH 4.5) and administered intraperitoneally at 150 mg / kg within 15 minutes. Control mice received the same volume of citrate buffer intraperitoneally. Body weight was measured every three or four days, and the overall condition was observed. Blood glucose levels were measured using a lab glucometer (Fora) on day 7 after STZ administration. Mice with blood glucose levels of 250 mg / dL or higher were used as diabetic models for subsequent experiments.

[0060] (2) Grouping and Administration Schedule Mice were divided into four groups: a non-diabetic group (control), a diabetic vehicle-treated group (PBS), diabetic group A (PC A), and diabetic group B (PC B). Group A received a PC:LPC mixture at a ratio of 1:2, and group B received a PC:LPC mixture at a ratio of 1:4 (see Example 5). The total dose of the PC / LPC mixture was 3 mg / kg, and 100 μL was orally administered every 3 or 4 days from day 7 to day 31 after STZ administration using a mouse probe (F.C.R. & Bio).

[0061] (3) Blood glucose measurement, muscle weight measurement, and quantitative PCR. On day 31 after STZ administration, blood glucose levels were measured using a lab glucometer (Fora) in the same manner as on day 7. Mice were then euthanized, and the gastrocnemius and tibialis muscles were removed and weighed. Expression levels of MuRF-1 and Atrogin-1 genes were measured by quantitative PCR using the same method as in Example 3.

[0062] 6-2 Results (1) Blood Glucose Levels The blood glucose measurement results are shown in Figure 13. The blood glucose levels of the non-diabetic group (control) mice remained at approximately 200 mg / dL, while the blood glucose levels of the diabetic vehicle-administered group (PBS) mice remained at approximately 600 mg / dL. The blood glucose levels of the diabetic group A (PC A) and diabetic group B (PC B), which were orally administered a PC / LPC mixture, were at the same level as the diabetic vehicle-administered group (PBS), with no significant difference.

[0063] (2) Muscle Weight The results of muscle weight measurements are shown in Figure 14. (A) shows the weight of the gastrocnemius muscle, and (B) shows the weight of the tibialis muscle. In the vehicle-treated group (PBS), muscle weights of both the gastrocnemius and tibialis muscles were reduced, whereas the degree of muscle weight reduction was less in the diabetic A group (PC A) and diabetic B group (PC B), which were orally administered the PC / LPC mixture.

[0064] (3) Quantitative PCR The results of quantitative PCR are shown in Figure 15. (A) shows the results for the MuRF-1 gene, and (B) shows the results for the Atrogin-1 gene. In the vehicle-treated group (PBS), the expression of the MuRF-1 gene and Atrogin-1 gene, which induce muscle breakdown, was increased, whereas in diabetic groups A (PC A) and B (PC B), which were orally administered a PC / LPC mixture, the expression of both genes was suppressed.

[0065] Example 7: Effect of Administration of PC, LPC, or a PC / LPC Mixture on Cancer-Bearing Mice (2) 7-1 Experimental Method (1) Adipose Tissue Weight Measurement and Adipose Tissue Analysis The same experiment as in Example 5 was performed. Mice were anesthetized 24 days after Colon26 cell transplantation, and blood was collected from the heart using a Microtainer serum separator tube (BD). After euthanasia, the mice were photographed for their epididymal adipose tissue appearance. The epididymal adipose tissue was then excised, weighed, and stored in liquid nitrogen. Epididymal adipose tissue from three randomly selected mice per group was thawed, fixed in 4% paraformaldehyde for 48 hours, and embedded in paraffin. 4 μm tissue sections were prepared and stained with hematoxylin and eosin (Wako). The specimens were photographed at 20x magnification using a VS-200 research slide scanner (Olympus). Image analysis was performed using QuPath and Fiji (Image J) software to measure the perimeter of 50 adipocytes per mouse (n = 3). Blood samples were left at room temperature for 30 minutes and then centrifuged at 9000 rpm for 10 minutes to collect serum. Serum oncostatin M (OSM) concentrations were measured using a Mouse Oncostatin M (OSM) Quantikine ELISA Kit (R&D Systems).

[0066] 7-2 Results (1) Appearance and weight of adipose tissue The appearance of epididymal adipose tissue is shown in Figure 16, and the results of measuring adipose tissue weight are shown in Figure 17. In the vehicle-treated group (PBS), adipose tissue weight was reduced, and fat atrophy due to cachexia was induced. On the other hand, in groups A, B, C, and D, the loss of adipose tissue weight due to cachexia was suppressed.

[0067] (2) Adipose tissue image and adipocyte perimeter Figure 18 shows a histological image of epididymal adipose tissue. Figure 19 shows the results of measuring the perimeter of individual adipocytes. In groups A, B, C, and D, the decrease in lipid droplets within adipocytes was suppressed, and the decrease in adipocyte perimeter was also suppressed. These results demonstrated that fat atrophy due to cachexia is due to the suppression of the decrease in lipid droplets within adipocytes.

[0068] (3) Serum Oncostatin M (OSM) Concentration The results for serum OSM concentration are shown in Figure 20. Serum OSM concentration increased in the vehicle-treated group (PBS), but the increase in serum OSM concentration was suppressed in groups A, B, C, and D. OSM is known to be a cytokine that promotes cancer cachexia, and PC, LPC, and the PC / LPC mixture were found to suppress cancer cachexia by inhibiting OSM secretion.

[0069] [Conclusion] These findings demonstrate that PC and LPC not only ameliorate cancer-induced cachexia, but also improve cachexia in other metabolic and inflammatory diseases such as diabetes.

[0070] Example 8: Effects of PC and PS on IL-6 expression enhanced by cachexia substances 8-1 Experimental method Colon26 cells were cultured at 5 × 10 4 cells / cm 2 The cells were seeded onto a 10 mm culture dish at a density of 100 μg / ml, and incubated at 37°C and 5% CO in the medium described in Example 1 until they reached 80% to 90% confluence. 2 The cells were cultured under the following conditions. 16:0 PC and 18:0 PC were added at a final concentration of 25 μM, and PS (Larodan) was added at a final concentration of 100 μg / ml. The cells were then cultured for 24 hours. After washing with PBS, the medium was replaced with serum-free RPMI medium (Wako) and cultured for an additional 48 hours. The medium was collected and centrifuged at 3000 rpm for 20 minutes at 4°C to precipitate cellular components. The supernatant was then filtered through a 0.22 μm sterile filter to remove the precipitate. The IL-6 concentration in the supernatant was measured using the LEGEND MAX Mouse IL-6 ELISA Kit (BioLegend).

[0071] 8-2 Results The results are shown in Figure 21. 16:0 PC, 18:0 PC, and PS were found to have the effect of directly suppressing IL-6 expression in cancer cells. This indicates that PC has the effect of suppressing cachexia regardless of the number of carbon atoms in the PC, and that not only PC but also phospholipids such as PS suppress cachexia.

[0072] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A preventive or ameliorating agent for cachexia, comprising at least one active ingredient selected from the group consisting of phosphatidylcholine, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylserine, and characterized by suppressing at least one cachectic symptom selected from the group consisting of skeletal muscle mass loss and fat atrophy.

2. The preventive or ameliorating agent according to claim 1, which suppresses the cachexia symptoms by suppressing an increase in serum IL-6 concentration.

3. The preventive or ameliorating agent according to claim 1, which suppresses the cachexia symptoms by suppressing an increase in serum oncostatin M concentration.

4. The preventive or ameliorating agent according to claim 1, which suppresses the cachexia symptoms by suppressing the expression of the MuRF-1 gene and the Atrogin-1 gene in muscle.

5. The preventive or ameliorative agent according to any one of claims 1 to 4, wherein the active ingredient is a mixture of phosphatidylcholine and lysophosphatidylcholine.

6. The preventive or ameliorative agent according to any one of claims 1 to 4, which is for oral administration.

7. The prophylactic or ameliorating agent according to any one of claims 1 to 4, wherein the disease that induces cachexia is a disease selected from the group consisting of cancer, chronic heart failure, chronic obstructive pulmonary disease, chronic renal failure, chronic liver disease, chronic infection, sepsis, autoimmune disease, and diabetes.