Composition for preventing or treating cachexia and muscle loss

Abstract

The present invention provides a pharmaceutical composition and a food composition for preventing or treating cachexia and muscle loss, the compositions comprising an ionic compound that includes a calcium cation, a first anion, and a second anion, wherein the first anion and the second anion are different from each other, each independently being an anion of ascorbic acid, dichloroacetic acid, a branched-chain amino acid, or a derivative thereof.

Description

Composition for the prevention or treatment of cachexia and muscle wasting

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cachexia and muscle loss, which comprises a calcium-based ionic compound as an active ingredient, and a food composition comprising the active ingredient.

[0002] Cachexia is a complex syndrome caused by diseases such as cancer, tuberculosis, diabetes, AIDS, chronic obstructive pulmonary disease, multiple sclerosis, congestive heart failure, hemophilia, hypopituitarism, or liver cirrhosis. Cachexia is not curable or reversible with nutritional supplementation alone, and it causes loss of appetite and persistent muscle wasting, leading to debilitation or death.

[0003] The prevalence of cachexia is approximately 10% in patients with heart failure, but more than 50% in cancer patients. Approximately 70% of patients with gastrointestinal cancer experience cachexia symptoms, and more than 80% of patients with terminal cancer, regardless of the type of cancer, are known to experience cachexia symptoms. Cancer treatments include surgery, radiation therapy, and chemotherapy. In terminal stages, when invasive treatment becomes difficult, radiation therapy or chemotherapy is applied, which lowers the patient's nutritional status and immune function, leading to the development or accelerated worsening of cachexia. The mortality rate of cachexia in patients with chronic obstructive pulmonary disease is approximately 20%, and in patients with heart failure, it is approximately 30%. However, the mortality rate of cachexia in cancer patients is up to 80%, garnering attention as much as the importance of treating cachexia as treating the underlying disease.

[0004] Because the precise mechanisms underlying cachexia remain unclear, existing treatments include increasing nutrient intake or using appetite stimulants like megestrol acetate. However, these treatments often result in temporary weight gain followed by subsequent weight loss, or weight gain without improvement in physical performance. Long-term use also presents side effects such as erectile dysfunction, uterine bleeding, thromboembolism, edema, hyperglycemia, adrenal insufficiency, and hypertension. Therefore, there is a pressing need for a completely new treatment approach based on an understanding of the mechanisms underlying cachexia and muscle loss.

[0005] The present invention takes into account the mechanism of occurrence and aggravation of muscle loss, which is a major symptom of cachexia, and, taking into account that cachexia is a multifactorial disease, aims to provide an effective ingredient that acts in a multimodal manner and a composition for preventing or treating cachexia and muscle loss, including the same.

[0006] In addition, the present invention aims to provide a food composition comprising the above-mentioned effective ingredient.

[0007] One aspect of the present invention provides a pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising an ionic compound, wherein the ionic compound comprises a calcium cation, a first anion, and a second anion, wherein the first anion and the second anion are different from each other and are each independently an anion of ascorbic acid, dichloroacetic acid, a branched-chain amino acid, or a derivative thereof.

[0008] The branched-chain amino acid may be at least one selected from the group consisting of Leucine, Isoleucine, and Valine.

[0009] The above pharmaceutical composition may be administered at a dosage of 20 to 4800 mg / kg / day of the ionic compound.

[0010] The pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solvent, and the degree of dissociation of the ionic compound may be from 1 to 50%.

[0011] The pharmaceutical composition may further comprise one or more active agents selected from the group consisting of appetite stimulants, anticancer agents, anti-inflammatory agents, antibacterial agents, antifungal agents, antiviral agents, immunomodulators, steroid agents, anticoagulants, anticonvulsants, antidepressants, antioxidants, and vitamins.

[0012] The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, excipients and diluents.

[0013] The pharmaceutical composition may be formulated as a liquid, powder, oral preparation, injection, infusion, aerosol, tablet, capsule, pill, depot or suppository.

[0014] The pharmaceutical composition may be administered orally, intravenously, subcutaneously, intramuscularly, or mucosally.

[0015] The above cachexia and muscle wasting may be caused by cancer, tuberculosis, diabetes, AIDS, chronic obstructive pulmonary disease, multiple sclerosis, congestive heart failure, hemophilia, hypopituitarism, or cirrhosis.

[0016] The cancer may be selected from the group consisting of lung cancer, breast cancer, colon cancer, stomach cancer, liver cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, and melanoma.

[0017] The above muscle loss may be caused by sarcopenia, atony, muscular atrophy, muscular dystrophy, amyotrophic lateral sclerosis, or myasthenia gravis.

[0018] Another aspect of the present invention provides a food composition comprising an ionic compound, wherein the ionic compound comprises a calcium cation, a first anion, and a second anion, wherein the first anion and the second anion are different from each other and are each independently an anion of ascorbic acid, dichloroacetic acid, a branched-chain amino acid, or a derivative thereof.

[0019] The above food composition may be a health functional food.

[0020] The composition according to the present invention provides a multimodal effect of preventing, improving or treating cachexia and muscle loss by simultaneously suppressing (1) catabolism due to energy metabolism, (2) disruption of muscle homeostasis, and (3) muscle loss due to inflammatory factors.

[0021] The above composition can suppress appetite loss in a subject of administration, maintain or increase body weight, reduce body weight change, reduce or increase muscle loss, and improve muscle function according to the above multimodal method.

[0022] Figure 1 shows the results of a cytotoxicity test on C2C12 pre-differentiation cells of (a) calcium salt of Manufacturing Example 1, (b) calcium salt of Manufacturing Example 2, and (c) calcium salt of Manufacturing Example 3.

[0023] Figure 2 shows the results of a cytotoxicity test on C2C12 cells after differentiation of (a) calcium salt of Manufacturing Example 1, (b) calcium salt of Manufacturing Example 2, and (c) calcium salt of Manufacturing Example 3.

[0024] Figure 3(a) is a crystal violet staining image of the root canal of the normal control group (UCM), (b) is a cancer cell culture medium treatment group (CCM), and (c) is a result of measuring the root canal diameter of (a) and (b) using Image J.

[0025] Figure 4 shows the results of Western blot of markers associated with muscle degradation and differentiation in the normal control group (UCM) and cancer cell culture medium treatment group (CCM).

[0026] Figure 5 is a graph showing the relative expression levels of (a) MuRF-1, (b) Atrogin-1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 4.

[0027] Figure 6 is a graph showing (a) the Western blot results and (b) the relative expression level of the target marker PDK-4 according to the calcium salt treatment concentration of Manufacturing Example 1.

[0028] Figure 7 is a graph showing (a) the Western blot results and (b) the relative expression level of the target marker Calpain 3 according to the calcium salt treatment concentration of Manufacturing Example 1.

[0029] Figure 8 shows the Western blot results of the target marker according to the calcium salt treatment concentration of Manufacturing Example 2.

[0030] Figure 9 is a graph showing the relative expression levels of target markers (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results of Figure 8.

[0031] Figure 10 shows the Western blot results of the target marker according to the calcium salt treatment concentration of Manufacturing Example 3.

[0032] Figure 11 is a graph showing the relative expression levels of target markers (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results of Figure 10.

[0033] Figure 12 is a graph showing (a) the Western blot result and (b) the relative expression level of the target marker PDK-4 when treating myotube cells with cancer cell culture medium (CCM), (c) the Western blot result and (d) the relative expression level of the target marker Calpain 3, and (e) the Western blot result and (f) the relative expression level of the target marker GDF-15.

[0034] Figure 13(a) is a crystal violet staining image of the C2C12 cell line of the normal control group (UCM), (b) is an image of the group treated with WY-14643, a PDK-4 activator, and (c) is the result of measuring the diameter of the myotubes of (a) and (b) using Image J.

[0035] Figure 14 is a graph showing (a) Western blot results and (b) relative expression levels of target markers (PDK-4, MuRF1, Atrogin1, MyHC, Myogenin) in the normal control group (UCM) and WY-14643 treatment group used in Figure 13.

[0036] Figure 15(a) is a crystal violet staining image of the C2C12 cell line as a normal control group (UCM), (b) is an image of the group transfected with scrambled siRNA as a vehicle control group, (c) is an image of the group transfected with Calpain 3 siRNA, and (d) is the result of measuring the diameter of the myotubes of (a) to (c) using Image J.

[0037] Figure 16 is a graph showing (a) Western blot results and (b) relative expression levels of target markers (Calpain 3, MuRF1, Atrogin1, MyHC, Myogenin) in the normal control group (UCM), vehicle control group (scrambled siRNA), and Calpain 3 siRNA treatment group used in Figure 15.

[0038] Figure 17(a) is a crystal violet staining image of the C2C12 cell line of the normal control group (UCM), the cancer cell culture medium treatment group (CCM), and the experimental group treated with calcium salt (Asc-Ca-DCA) of Preparation Example 1 in CCM, and (b) is the result of measuring the diameter of the root canal of (a) using Image J.

[0039] Figure 18 shows the Western blot results of C2C12 cell lines in a normal control group (UCM), a cancer cell culture medium treatment group (CCM), and an experimental group treated with calcium salt (Asc-Ca-DCA) of Preparation Example 1 in CCM.

[0040] Figure 19 is a graph showing the relative expression levels of target markers (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 18.

[0041] Figure 20 shows the Western blot results of the C2C12 cell line in the normal control group (UCM), the cancer cell culture medium treatment group (CCM), and the experimental group treated with calcium salt (Asc-Ca-Leu) of Preparation Example 2 in CCM.

[0042] Figure 21 is a graph showing the relative expression levels of target markers (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 20.

[0043] Figure 22 shows the Western blot results of the C2C12 cell line in the normal control group (UCM), the cancer cell culture medium treatment group (CCM), and the experimental group treated with calcium salt (DCA-Ca-Leu) of Preparation Example 3 in CCM.

[0044] Figure 23 is a graph showing the relative expression levels of target markers (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 22.

[0045] Figure 24(a) is a crystal violet staining image of C2C12 cell lines of a normal control group (UCM), a cancer cell culture medium treatment group (CCM), an experimental group treated with calcium salt (Asc-Ca-DCA) of Preparation Example 1 in CCM, a group treated with ascorbic acid (Asc) in CCM, a group treated with dichloroacetic acid (DCA) in CCM, and a group treated with a combination of ascorbic acid and dichloroacetic acid in CCM, and (b) is the result of measuring the root canal diameter of each group in (a) using Image J.

[0046] Figure 25 is a Western blot result for MuRF1, Atrogin1, MyHC, and Myogenin as target markers in the treatment group of Figure 24.

[0047] Figure 26 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 25.

[0048] Figure 27 shows the Western blot results for Calpain 3, PDK-4, and GDF-15 as target markers in the treatment group of Figure 24.

[0049] Figure 28 is a graph showing the relative expression levels of (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results of Figure 27.

[0050] Figure 29 shows the results of Western blot analysis for MuRF1, Atrogin1, MyHC, and Myogenin as target markers in the normal control group (UCM), cancer cell culture medium treatment group (CCM), experimental group treated with calcium salt (Asc-Ca-Leu) of Preparation Example 2 in CCM, group treated with ascorbic acid (Asc) in CCM, group treated with leucine (Leu) in CCM, and group treated with a combination of ascorbic acid and leucine in CCM.

[0051] Figure 30 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 29.

[0052] Figure 31 shows the results of Western blot analysis for Calpain 3, PDK-4, and GDF-15 as target markers in the normal control group (UCM), the cancer cell culture medium treatment group (CCM), the experimental group treated with calcium salt (Asc-Ca-Leu) of Preparation Example 2 in CCM, the group treated with ascorbic acid (Asc) in CCM, the group treated with leucine (Leu) in CCM, and the group treated with a combination of ascorbic acid and leucine in CCM.

[0053] Figure 32 is a graph showing the relative expression levels of (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results of Figure 31.

[0054] Figure 33 shows the results of Western blot analysis for MuRF1, Atrogin1, MyHC, and Myogenin as target markers in the normal control group (UCM), cancer cell culture medium treatment group (CCM), experimental group treated with calcium salt (DCA-Ca-Leu) of Preparation Example 3 in CCM, group treated with dichloroacetic acid (DCA) in CCM, group treated with leucine (Leu) in CCM, and group treated with a combination of dichloroacetic acid and leucine in CCM.

[0055] Figure 34 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 33.

[0056] Figure 35 shows the results of Western blot analysis for Calpain 3, PDK-4, and GDF-15 as target markers in the normal control group (UCM), cancer cell culture medium treatment group (CCM), experimental group treated with calcium salt (DCA-Ca-Leu) of Preparation Example 3 in CCM, group treated with dichloroacetic acid (DCA) in CCM, group treated with leucine (Leu) in CCM, and group treated with a combination of dichloroacetic acid and leucine in CCM.

[0057] Figure 36 is a graph showing the relative expression levels of (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results of Figure 35.

[0058] Figure 37 is (a) a crystal violet staining image of the root canal of a normal control group (UCM), a cancer cell culture medium-treated group (CCM), and an experimental group treated with dexamethasone (Dex) in UCM, and (b) is the result of measuring the root canal diameter size of (a) using Image J.

[0059] Figure 38 shows the results of Western blot for MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15 as target markers in the normal control group (UCM), cancer cell culture medium treatment group (CCM), and experimental group treated with dexamethasone (Dex) in UCM.

[0060] Figure 39 shows the results of Western blot for MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15 as target markers in the normal control group (UCM), the experimental group treated with dexamethasone in UCM (Dex), and the experimental group treated with calcium salt (Asc-Ca-DCA) of Preparation Example 1 in Dex.

[0061] Figure 40 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, (d) Myogenin, (e) Calpain 3, (f) PDK-4, and (g) GDF-15 from the Western blot results of Figure 39.

[0062] Figure 41 shows the results of Western blot for MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15 as target markers in the normal control group (UCM), the experimental group treated with dexamethasone in UCM (Dex), and the experimental group treated with calcium salt (Asc-Ca-Leu) of Preparation Example 2 in Dex.

[0063] Figure 42 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, (d) Myogenin, (e) Calpain 3, (f) PDK-4, and (g) GDF-15 from the Western blot results of Figure 41.

[0064] Figure 43 shows the results of Western blot for MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15 as target markers in the normal control group (UCM), the experimental group treated with dexamethasone in UCM (Dex), and the experimental group treated with calcium salt (DCA-Ca-Leu) of Preparation Example 3 in Dex.

[0065] Figure 44 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, (d) Myogenin, (e) Calpain 3, (f) PDK-4, and (g) GDF-15 from the Western blot results of Figure 43.

[0066] In the following Figures 45 to 53, G1 represents a normal control group, G2 represents an excipient control group, G3 represents an oral administration group of megestrol (125 mpk), G4 represents an oral administration group of Manufacturing Example 1 (200 mpk), G5 represents an intravenous administration group of Manufacturing Example 1 (100 mpk), and G6 represents an intravenous administration group of Manufacturing Example 1 (200 mpk).

[0067] Figure 45 shows the results of Western blot for MuRF1, Atrogin1, MyHC, and Myogenin as target markers in mouse muscle tissue.

[0068] Figure 46 is a graph showing the relative expression levels of (a) MuRF1, (b) Atrogin1, (c) MyHC, and (d) Myogenin from the Western blot results of Figure 45.

[0069] Figure 47 is a graph showing the results of Western blot for Calpain 3 and PDK-4 as target markers in mouse muscle tissue, and the relative expression levels of (b) Calpain 3 and (c) PDK-4 from the Western blot results.

[0070] Figure 48 is a graph showing the concentrations of (a) IL-1β and (b) lactic acid measured in mouse blood.

[0071] Figure 49 is a graph showing the weight loss inhibition effect in mice (a) over time and (b) according to the experimental group.

[0072] Figures 50 (a) to (f) are images showing cross-sections of the gastrocnemius of mice in experimental groups G1 to G6, respectively, and (g) is a graph summarizing the cross-sectional areas from (a) to (f).

[0073] Figures 51 (a) to (f) are images showing cross-sections of the quadriceps of mice in experimental groups G1 to G6, respectively, and (g) is a graph summarizing the cross-sectional areas from (a) to (f).

[0074] Figures 52 (a) to (f) are images showing cross-sections of the soleus of mice in experimental groups G1 to G6, respectively, and (g) is a graph summarizing the cross-sectional areas from (a) to (f).

[0075] Figure 53(a) shows the results of the Rotarod-treadmill test of a mouse, and (b) shows the results of the Wire hanging test of a mouse.

[0076] Hereinafter, each component of the present invention will be described in more detail so that a person having ordinary skill in the art to which the present invention pertains can easily practice it; however, this is only an example, and the scope of the rights of the present invention is not limited by the following contents.

[0077] The term "comprises" as used herein is used to list materials, compositions, devices, and methods useful in the present invention, but is not limited to the listed examples.

[0078] As used herein, the terms "about" and "substantially" are used to mean a range or approximation of a numerical value or degree, taking into account inherent manufacturing and material tolerances, and are used to prevent infringers from unfairly exploiting the disclosure, which includes precise or absolute numerical values ​​provided to aid understanding of the present invention.

[0079] The term "subject" as used herein may mean a mammal exhibiting one or more of the symptoms of cachexia and muscle wasting.

[0080] The term "treatment" as used herein means any act of administering to a subject a composition according to one embodiment of the present invention to improve or benefit at least one of the symptoms of cachexia and muscle loss.

[0081]

[0082] One specific example of the present invention provides a pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising an ionic compound, wherein the ionic compound comprises a calcium cation, a first anion, and a second anion, wherein the first anion and the second anion are different from each other and are each independently an anion of ascorbic acid, dichloroacetic acid, a branched-chain amino acid, or a derivative thereof.

[0083] An ionic compound is a compound in which ions with opposite charges form an ionic bond due to electrostatic forces, and can exhibit electrical neutrality. Specifically, the ionic compound is Ca 2+ Two anions selected from the group consisting of ascorbic acid, dichloroacetic acid, branched-chain amino acids, and derivatives thereof may be ionically bonded to each other on both sides of an ion (calcium ion). Preferably, the ionic compound may be a compound in which two different anions selected from the group consisting of ascorbic acid, dichloroacetic acid, and branched-chain amino acids are ionically bonded to each other on both sides of a calcium ion. For example, the ionic compound may include a calcium salt of ascorbic acid and dichloroacetic acid; a calcium salt of ascorbic acid and a branched-chain amino acid; or a calcium salt of dichloroacetic acid and a branched-chain amino acid.

[0084] The branched-chain amino acid (BCAA) may include one or more selected from the group consisting of leucine, isoleucine, and valine. The branched-chain amino acid may contribute to the synthesis and conversion of proteins. Preferably, the branched-chain amino acid may be leucine. For example, the ionic compound may include a calcium salt of ascorbic acid and leucine, or a calcium salt of dichloroacetic acid and leucine.

[0085] The above ionic compound provides the following three mechanisms for cachexia and muscle wasting simultaneously, thereby providing preventive, ameliorating or therapeutic effects on cachexia and muscle wasting, which are multifactorial diseases:

[0086] (1) A pharmaceutical composition containing the above ionic compound can prevent weight loss caused by unnecessary energy consumption by inhibiting catabolic action due to energy metabolism.

[0087] For example, in cancers that cause cachexia and muscle wasting, cancer cells can perform energy metabolism through oxidative phosphorylation, which requires oxygen, and glycolysis, which does not use oxygen. In particular, cancer cells primarily use glycolysis to survive in hypoxic environments where normal cells cannot survive, and the mitochondrial-initiated apoptosis control process is disabled. When cancer cells' glycolysis is increased, glycogen is broken down in the liver into lactic acid. Lactate can be converted to pyruvate by lactate dehydrogenase (LDH), and pyruvate can then be converted to oxaloacetate and malate, and then to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase. Fructose-1,6-biphosphate is produced from phosphoenolpyruvate, which is then converted to fructose-6-phosphate, which is then converted to glucose via glucose-6-phosphate. This series of processes is called gluconeogenesis, and a total of 4 ATP and 2 GTP are required to produce one glucose molecule. Consequently, the hyperactivity of cancer cells induces hyperactivity of gluconeogenesis, which leads to unnecessary energy waste in the body and causes weight loss.

[0088] Ascorbic acid (L-ascorbic acid; Asc) contained in the above ionic compound is a substance widely known as vitamin C. Initially discovered, it was used to prevent and treat scurvy. However, as other functions, such as its strong reducing power and involvement in the production of certain neurotransmitters, were discovered, it is now used in various fields such as pharmaceuticals, food, and cosmetics. Ascorbic acid has excellent electron donating ability, so it not only has excellent antioxidant action, removal of active oxygen, and anti-aging ability, but it can also act as a coenzyme for various enzymes in the body. In particular, ascorbic acid can act as a coenzyme for an enzyme called prolyl hydroxylase. Prolyl hydroxylase can be activated by ascorbic acid to hydroxylate and remove the proline residue of the HIF-1α protein. HIF-1α protein is a subunit of the heterodimer HIF-1 (hypoxia inducible factor-1), which is involved in the control of energy metabolism and angiogenesis. Because cancer cells consume a lot of energy and nearby oxygen, they require oxygen supply through the angiogenic function of HIF-1α protein. Therefore, the reduction of HIF-1α protein due to the administration of ascorbic acid creates an environment unfavorable for cancer cell growth, which provides an inhibitory effect on tumor growth. At the same time, it can prevent weight loss by suppressing the mechanism of gluconeogenesis and reducing unnecessary energy consumption.

[0089] Dichloroacetic acid (DCA), included in the above ionic compound, is known to have a therapeutic effect on metabolic diseases caused by mitochondrial abnormalities when provided in salt form. Specifically, cancer cells are in a state where pyruvate is not converted to acetyl-CoA because the hypoxia-inducible factor HIF-1 promotes the expression of pyruvate dehydrogenase kinase (PDK). Sodium dichloroacetate can activate oxidative phosphorylation within the mitochondria by increasing the activity of pyruvate dehydrogenase (PDH) through inhibition of pyruvate dehydrogenase kinase (PDK), thereby converting pyruvate to acetyl-CoA. The administration of the above ionic compound to provide dichloroacetate can inhibit the glycolytic process through oxidative phosphorylation activity in the mitochondria of cancer cells, thereby inhibiting lactate release and gluconeogenesis, and can also induce apoptotic cell death of cancer cells. This process can be referred to as mitochondrial reforming, which allows cancer cells to perform normal mitochondrial functions. The above ionic compound can suppress cancer cell growth through mitochondrial reforming and, by changing the environment surrounding the cancer, inhibit gluconeogenesis and reduce blood glucose concentration, thereby suppressing weight loss.

[0090] Leucine (Leu), a branched-chain amino acid contained in the above ionic compound, is an essential amino acid used in protein biosynthesis. It is not synthesized in the body and must be supplied through the diet. Leucine is known to be involved in muscle protein synthesis, including skeletal muscle, and acts as an important regulatory factor in translation initiation during protein synthesis. In particular, leucine can act on the insulin signaling pathway. Specifically, leucine can act on pancreatic cells to directly promote insulin secretion or suppress glucagon secretion, thereby reducing blood glucose concentration. Insulin activates the insulin receptor and can activate the Akt signaling protein through the insulin signaling pathway. Activated Akt activates the TSC1 / 2 complex, and the TSC1 / 2 complex can activate mTORC1 (mammalian target of rapamycin complex 1), which controls protein synthesis, thereby activating protein synthesis. When insulin resistance occurs, the insulin signaling pathway fails to sufficiently activate the Akt signaling protein, which also inactivates mTORC1. Leucine, together with insulin, participates in mTORC1 activation, regulating blood glucose levels and contributing to protein synthesis. mTORC1 interacts with metabolic proteins such as AMPK, and leucine can participate in AMPK activation, inducing GLUT4 translocation to the cell membrane and promoting glucose uptake into cells. The ionic compound activates the blood glucose reduction mechanism through the supply of leucine, contributing to protein synthesis and suppressing weight loss.

[0091] Calcium ions contained in the above ionic compound can affect the calcium homeostasis of cancer cells and induce cancer cell death. Specifically, administration of the pharmaceutical composition may cause calcium accumulation in the mitochondria of cancer cells, and excessive reactive oxygen species may be generated within the cancer cells, leading to apoptotic cell death. Calcium ions can form a direct bond with α-KGDC (ketoglutarate dehydrogenase complex) and act as a regulatory factor for the TCA cycle. The above ionic compound excessively increases the calcium ion concentration within cancer cells through the supply of calcium, which may induce metabolic disorders in mitochondria through the activity of endonucleases and proteases, thereby promoting the release of cytochrome c. The release of cytochrome c activates caspase-9, which in turn activates caspases 3 and 7, leading to apoptosis of cancer cells.

[0092] A pharmaceutical composition comprising the above ionic compound can act as a multimodal preventive or therapeutic agent for cachexia and muscle loss, by compositely contributing to one or more of apoptosis of cancer cells, reduction of unnecessary energy consumption, activation of a blood glucose reduction mechanism, and activation of protein synthesis by calcium ions and first and second anions selected from the above-mentioned anions.

[0093] The effects of treatment with a pharmaceutical composition containing the above ionic compound can be assessed by checking the expression level of PDK as a target marker. Specifically, when a pharmaceutical composition containing the above ionic compound is administered, the expression level of PDK-4 (PDK4) may be reduced in subjects suffering from cachexia or muscle loss. A decrease in PDK-4 expression indicates the activation of oxidative phosphorylation within mitochondria, which may imply a decrease in unnecessary metabolism in the body and a decrease in blood glucose concentration.

[0094] (2) A pharmaceutical composition containing the above ionic compound can maintain muscle homeostasis and prevent a decrease in muscle mass.

[0095] Loss of muscle mass refers to a decrease in the size of muscle cells and muscle tissue that occurs in various catabolic conditions such as abnormalities in related genes, hormonal imbalances, severe injury, sepsis, cancer, and aging.

[0096] Ascorbic acid contained in the above ionic compound can reduce the expression level of GDF15 (growth differentiation factor 15, GDF-15), an indicator related to cachexia. GDF15 is known as a marker related to obesity and diabetes, but recently, as it has been found that the GDF15 level is higher in patients with cachexia compared to healthy people in clinical trials, the expression of GDF15 is attracting attention as being related to cachexia. Since GDF15 level can increase by HIF-1, the decrease in HIF-1 due to the administration of ascorbic acid can reduce the expression level of GDF15, thereby providing a preventive or therapeutic effect for cachexia. In addition, dichloroacetic acid contained in the above ionic compound can normalize (reform) the mechanism of mitochondria, thereby reducing the expression level of GDF, and when administered together with ascorbic acid, can induce a synergistic effect for the preventive or therapeutic effect for cachexia.

[0097] Dichloroacetic acid, contained in the above ionic compound, can inhibit gluconeogenesis by reducing blood glucose concentration. While the exact mechanism of muscle mass loss is unknown, the Cori cycle is believed to be involved. When anaerobic glucose breakdown occurs in muscle cells, lactic acid can be produced through this process. Lactate then travels to the liver, where it is converted into glucose through gluconeogenesis, which can then be supplied to muscle cells as an energy source. As previously described, gluconeogenesis leads to unnecessary energy waste and muscle loss. Dichloroacetate can also inhibit the Cori cycle mechanism, thereby preventing muscle loss. In muscle tissue and myotube cells of animals exhibiting muscle mass loss, PDK-4 may be overexpressed compared to normal tissue and cells. In addition to its metabolic regulatory function, dichloroacetate can reduce the expression level of overexpressed PDK-4 specifically in muscle that is affected by muscle loss, thereby providing a preventive or therapeutic effect against muscle loss.

[0098] In addition, as mentioned above, it is known that there is a correlation between muscle loss and the expression level of GDF15, and it has been confirmed that mitochondrial dysfunction acts as a major mediating mechanism for the correlation. Mitochondrial dysfunction can be caused by defects in the internal electron transport system or damage to mitochondria due to oxidative stress, and the expression level of GDF15 can increase as the inflammatory response due to oxidative stress increases. Dichloroacetate can improve mitochondrial dysfunction through mitochondrial reforming and reduce the expression level of GDF15. Specifically, PGC-1α, a transcription factor related to mitochondrial function, regulates cellular energy metabolism, and dichloroacetate can affect PGC-1α and activate AMPK (AMP-activated protein kinase), which is mechanistically related to PGC-1α, thereby regulating the energy status and metabolism within the cell. In this way, dichloroacetate affects the mitochondrial remodeling mechanism linked to PDK and PDH and factors affecting energy metabolism and mitochondrial function, thereby contributing to the normalization of mitochondrial function in a complex manner. Through this, it can provide a preventive or therapeutic effect on diseases related to cachexia and muscle loss by reducing the expression level of GDF15.

[0099] Leucine, a branched-chain amino acid included in the above ionic compound, can provide protein synthesis and cell growth effects by participating in the IGF-1 / PI3K / Akt mechanism related to muscle synthesis. Insulin-like growth factor-1 (IGF-1) is a hormone with a similar structure to insulin and is involved in body growth, maintenance, and metabolism. IGF-1 contributes to mTORC1 activation through the PI3K / Akt signaling pathway. Leucine can activate mTORC1 through the insulin transmission pathway and the IGF / PI3K / Akt mechanism, thereby reducing blood glucose concentration and activating protein synthesis.

[0100] Calcium ions contained in the above ionic compound can improve the structure and function of muscles by increasing the expression of calpain 3, which is associated with muscle expression. Calpain 3 is a type of calcium-dependent non-lysosomal cysteine ​​protease, and its decreased expression is known to cause muscle loss. Calpain 3 controls the structure of proteins within tissues or fibroblasts and plays an important role in the growth and shape maintenance of muscle fibers. Genetic mutations related to calpain 3 can cause muscle diseases such as Duchenne muscular dystrophy, which causes a deficiency of dystrophin, a protein within muscle fibers, resulting in loss of muscle strength and motor ability, and limb girdle muscular dystrophy (LGMD), which causes muscle weakness due to degeneration of muscle tissue and damage to muscle fibers. The above ionic compound can provide a preventive or therapeutic effect for cachexia and muscle loss, as well as muscle-related diseases, by increasing the expression level of calpain 3, a marker that directly affects the above diseases, through the supply of calcium. In addition, leucine contained in the above ionic compound can increase the expression of calpain 3 through the activation of mTORC1, and when administered together with calcium ions, can induce a synergistic effect for the prevention or treatment of cachexia and muscle loss.

[0101] A pharmaceutical composition comprising the above ionic compound can act as a multimodal preventive or therapeutic agent for cachexia and muscle loss, by complexly contributing to one or more of the following: regulation of the expression level of a muscle loss-related marker, reduction of blood glucose concentration, and activation of protein synthesis, with calcium ions and a first anion and a second anion selected from the above-mentioned anions.

[0102] The effect of treatment with a pharmaceutical composition containing the above ionic compound can be evaluated by checking at least one of the expression levels of GDF15, PDK-4, and calpain 3 as target markers.

[0103] Specifically, when a pharmaceutical composition containing the above ionic compound is administered, the expression level of GDF15 may be shown to decrease in subjects suffering from cachexia or muscle loss. GDF15 is known to exhibit a mechanism of suppressing appetite and regulating body weight by transmitting signals to the nervous system through a receptor called GFRAL (GDNF Family Receptor Alpha-Like). The appetite suppression mechanism of GDF15-GFRAL can be divided into the following three steps: first, the expression of GDF15 increases due to the aforementioned mitochondrial dysfunction; second, the increased GDF15 specifically binds to GFRAL and transmits signals to a specific part of the brainstem including the NTS (Nucleus Tractus Solitarius); and third, the brainstem that received the signals regulates various neurotransmitters and suppresses appetite. Ascorbic acid and / or dichloroacetic acid included in the above ionic compound can provide a fundamental therapeutic effect on appetite suppression by reducing the expression level of GDF15, which is the first step as the starting point of the above mechanism.

[0104] Specifically, when a pharmaceutical composition containing the ionic compound is administered, the expression level of PDK-4 may be reduced in a subject suffering from cachexia or muscle loss.

[0105] Specifically, when a pharmaceutical composition containing the ionic compound is administered, the expression level of calpain 3 may be increased in subjects suffering from cachexia or muscle loss. Calpain 3 may affect the mechanism related to calcium-induced muscle protein degradation and may affect muscle loss by affecting c-FLIP (Caspase-8 Long Isoform), a protein involved in the apoptosis process. Calpain 3 is expressed in muscle cells and can inhibit the degradation of c-FLIP, thereby inhibiting the function of caspase-8. Inhibition of caspase-8 leads to inhibition of apoptosis, thereby preventing muscle loss and maintaining the structural stability of muscle cells. Calpain 3 can prevent muscle loss not only through c-FLIP but also through interaction with myostatin, a protein associated with muscle loss. The ionic compound may provide a preventive effect against muscle loss by increasing the expression level of calpain 3.

[0106] (3) A pharmaceutical composition containing the above ionic compound can prevent a decrease in muscle mass caused by an inflammatory factor.

[0107] Proinflammatory cytokines can be identified regardless of cancer type and are known to be a major cause of muscle loss. Specifically, inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) can induce cachexia and muscle loss. For example, tumor necrosis factor can activate the ubiquitin-proteasome system (UPS), which degrades cytoplasmic proteins, thereby promoting muscle protein degradation. Furthermore, it can increase oxidative stress, damaging muscle cells. Furthermore, it can decrease the expression of stress proteins, thereby reducing muscle protein production and regeneration.

[0108] Ascorbic acid contained in the above ionic compound suppresses the secretion of inflammatory cytokines and simultaneously acts as a low-toxicity metabolic anticancer agent that directly inhibits cancer cell growth, thereby providing a preventive or therapeutic effect on cachexia and muscle loss induced by inflammatory factors. Ascorbic acid can suppress the secretion of cytokines by inhibiting NF-κB and COX2, which are representative factors that mediate and regulate inflammatory responses. Specifically, ascorbic acid can suppress the secretion of cytokines by inhibiting the signal transduction pathway of NF-κB. Ascorbic acid's inhibition of the NF-κB signal transduction pathway can also prevent muscle loss by inhibiting the mechanism of GDF15. COX (cyclooxygenase) catalyzes the conversion of prostaglandins, which are major mediators of inflammatory responses, to arachidonic acid, and COX1 is expressed at a constant level in the body and produces prostaglandins that are essential for vasodilation, maintaining blood flow, and protecting the gastric mucosa. In contrast, COX2 is rarely expressed in normal tissues, but is induced by inflammatory factors, cell growth factors, and tumor-promoting factors, instantly producing large amounts of prostaglandins and triggering various inflammatory responses. COX2 is overexpressed in cancer cells, promoting their proliferation and metastasis and inhibiting apoptosis. Ascorbic acid can inhibit the mechanism of COX2, preventing the worsening of cachexia and muscle loss.

[0109] Dichloroacetic acid contained in the above ionic compound can reduce the release of lactic acid in the body, thereby reducing the expression of cytokines. Lactic acid can modulate signaling pathways that regulate the expression of various enzymes involved in cytokines and other immune responses through the activation of NF-κB and HIF-1. The above ionic compound can suppress the cytokine mechanism by supplying dichloroacetic acid to reduce the release of lactic acid, thereby preventing a decrease in protein production.

[0110] Leucine, a branched-chain amino acid contained in the above ionic compound, can prevent muscle loss by inhibiting the secretion of inflammatory cytokines such as tumor necrosis factor, IL-6, and IFNγ. Leucine can suppress the phosphorylation level of NF-κB, thereby inhibiting cytokine secretion. The above ionic compound can prevent protein production decline by supplying leucine and suppressing the cytokine mechanism.

[0111] A pharmaceutical composition comprising the above ionic compound can act as a multimodal preventive or therapeutic agent for cachexia and muscle loss by inhibiting the secretion of inflammatory cytokines by the first anion and the second anion.

[0112] The effect of treatment with a pharmaceutical composition containing the ionic compound may be evaluated by checking at least one of the expression level of GDF15, the expression level of cytokines, and the amount of lactic acid in the blood as target markers. Specifically, when the pharmaceutical composition containing the ionic compound is administered, the expression level of GDF15 in a subject suffering from cachexia or muscle loss may be shown to decrease. When the pharmaceutical composition containing the ionic compound is administered, the expression level of cytokines in a subject suffering from cachexia or muscle loss may be shown to decrease. When the pharmaceutical composition containing the ionic compound is administered, the amount of lactic acid in the blood of a subject suffering from cachexia or muscle loss may be shown to decrease.

[0113] The ionic compound may be provided at a dosage of 20 to 4800 mg / kg / day. Specifically, the dosage of the ionic compound may be 500 mg / kg / day or more and 4500 mg / kg / day or less, 1000 mg / kg / day or more and 4000 mg / kg / day or less, 1500 mg / kg / day or more and 3500 mg / kg / day or less, or 2000 mg / kg / day or more and 3000 mg / kg / day or less. Preferably, the dosage of the ionic compound may be 20 mg / kg / day or more and 2000 mg / kg / day or less. Within the dosage range of the ionic compound described above, an effective preventive or therapeutic effect on cachexia and muscle loss can be provided without cytotoxicity. A more specific dosage of the ionic compound within the above-described range may be determined depending on the type of the ionic compound, the age, weight, health, sex, sensitivity to the ionic compound of the subject, the time of administration of the pharmaceutical composition, the route of administration, the excretion rate, the treatment period, or the type and amount of drugs used simultaneously.

[0114] The frequency of administration of the above ionic compound may be once daily, but is not limited thereto. The dosage within the above-described range may be divided and administered multiple times.

[0115] The pharmaceutical composition may further comprise the ionic compound and a pharmaceutically acceptable aqueous solvent. The aqueous solvent may be, but is not limited to, distilled water, saline solution, an injection solution, or a buffer solution.

[0116] For example, the pharmaceutical composition may be a composition for oral administration, and may be prepared by adding 20 to 4800 mg of the ionic compound to 1 mL of distilled water.

[0117] As a specific example, the pharmaceutical composition for oral administration can be prepared by adding 160 mg of an ionic compound including a calcium cation, an ascorbic acid anion, and a dichloroacetic acid anion to 1 mL of distilled water.

[0118] As a specific example, the pharmaceutical composition for oral administration can be prepared by adding 200 mg of an ionic compound including a calcium cation, an ascorbic acid anion, and an anion of a branched-chain amino acid to 1 mL of distilled water.

[0119] As a specific example, the pharmaceutical composition for oral administration can be prepared by adding 480 mg of an ionic compound including a calcium cation, a dichloroacetic acid anion, and an anion of a branched-chain amino acid to 1 mL of distilled water.

[0120] For example, the pharmaceutical composition may be a composition for intravenous administration, and may be prepared by adding 20 to 4800 mg of the ionic compound to 1 mL of an injection solution.

[0121] As a specific example, the pharmaceutical composition for intravenous administration can be prepared by adding 80 mg of an ionic compound containing calcium cation, ascorbic acid anion, and dichloroacetic acid anion to 1 mL of an injection solution.

[0122] As a specific example, the pharmaceutical composition for intravenous administration can be prepared by adding 100 mg of an ionic compound including a calcium cation, an ascorbic acid anion, and an anion of a branched-chain amino acid to 1 mL of an injection solution.

[0123] As a specific example, the pharmaceutical composition for oral administration can be prepared by adding 240 mg of an ionic compound containing a calcium cation, a dichloroacetic acid anion, and an anion of a branched-chain amino acid to 1 mL of an injection solution.

[0124] The ionic compound may be mixed with the aqueous solvent to dissociate at least a portion of the ionic bonds. The degree of dissociation of the ionic compound may be 1 to 50%. The ionic compound may maintain its chemical structure without dissociating by at least 1% within the pharmaceutical composition, and may maintain the chemical structure even after administration to a subject to provide the multimodal effect described above. The pharmaceutical composition comprising the ionic compound may include an ionic compound dissociated in the aqueous solvent and an ionic compound dispersed in the aqueous solvent without dissociating. The pharmaceutical composition may have a superior preventive or therapeutic effect on cachexia and muscle loss compared to an aqueous solution containing the same content of cations and anions that may be included in the ionic compound of the present invention.

[0125] The pharmaceutical composition may include the ionic compound and one or more pharmaceutically acceptable active agents. Specifically, the active agents may include, but are not limited to, appetite stimulants, anticancer agents, anti-inflammatory agents, antibacterial agents, antifungal agents, antiviral agents, immunomodulators, steroids, anticoagulants, anticonvulsants, antidepressants, antioxidants, and vitamins. That is, the ionic compound may be used in combination with the active agent.

[0126] For example, the appetite stimulant may be, but is not limited to, megestrol, cyproheptadine, dronabinol, or metoclopramide.

[0127] For example, the anticancer drugs include Imatinib, 5-FU (5-Florouracil), Irinotecan, Sunitinib, Oxaliplatin, Cisplatin, Paclitaxel, Lapatinib, Trastuzumab (Herceptin), Gefitinib, Erlotinib, Methotrexate, Carboplatin, Docetaxel, Everolimus, Sorafenib, Carbonic anhydrase inhibitors, Monocarboxylate transporter inhibitors, Pumpro Pembrolizumab, atezolizumab, PD-1 anticancer drugs, nivolumab, PARP-1 (Poly(ADP-ribose) polymerase 1) inhibitor, PARP-2 (Poly(ADP-ribose) polymerase 2) inhibitor, olaparib, rucaparib, niraparib, bevacizumab, or VEGF inhibitor may be selected from the group consisting of, but are not limited to, PT anticancer drugs such as oxaliplatin, cisplatin, and carboplatin. PT anticancer drugs are known to increase GDF15 levels in subjects and induce cachexia depending on their use. A pharmaceutical composition containing the above ionic compound, when used in combination with the PT series anticancer drug, can suppress weight loss and appetite loss, which are side effects of the anticancer drug, while maintaining activity against tumor cells as an effect inherent to the anticancer drug.

[0128] For example, the anti-inflammatory agent may be an NSAID such as, but not limited to, salicylic acid, ibuprofen, dexibuprofen, naproxen, ketoprofen, indomethacin, diclofenac, piroxicam, or meloxicam.

[0129] For example, the antibacterial agent may be, but is not limited to, benzylpenicillin, ampicillin, streptomycin, chloramphenicol, or tetracycline.

[0130] For example, the antifungal agent may be, but is not limited to, nystatin, amphotericin B, or trichomycin.

[0131] For example, the antiviral agent may be, but is not limited to, oseltamivir, acyclovir, ganciclovir, sofosbuvir, or remdesivir.

[0132] For example, the immunomodulator may be, but is not limited to, adalimumab, infliximab, etanercept, or cyclosporine.

[0133] For example, the steroid may be, but is not limited to, hydrocortisone, prednisone, triamcinolone, or fluticasone.

[0134] In addition, the active agents may further include anticoagulants such as warfarin and heparin; anticonvulsants such as carbamazepine and phenytoin; antidepressants such as selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs); antioxidants such as coenzyme Q10 and alpha lipoic acid; and vitamins such as vitamins A, B, C, D, and E.

[0135] The pharmaceutical composition may include one or more selected from pharmaceutically acceptable carriers, excipients, and diluents. For example, the carriers, excipients, and diluents may be, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The carriers, excipients, and diluents may be included within a range capable of securing an effective concentration of the ionic compound.

[0136] The pharmaceutical composition may be formulated as a liquid, powder, oral preparation, injection, infusion, aerosol, tablet, capsule, pill, depot, or suppository. The pharmaceutical composition may further include additives necessary for formulation, such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and excipients.

[0137] The pharmaceutical composition may be administered orally, intravenously, subcutaneously, intramuscularly, or mucosally. Preferably, the pharmaceutical composition may be administered orally or intravenously. For oral administration, the pharmaceutical composition may be formulated into the aforementioned liquid formulations, oral preparations, etc., and for intravenous administration, it may be formulated into the aforementioned injections, infusions, etc.

[0138] The cachexia and muscle loss may be caused by causes such as cancer, tuberculosis, diabetes, AIDS, chronic obstructive pulmonary disease, multiple sclerosis, congestive heart failure, hemophilia, hypopituitarism, or cirrhosis. Preferably, the cachexia and muscle loss may be caused by cancer. The cancer may be selected from the group consisting of lung cancer, breast cancer, colon cancer, stomach cancer, liver cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, and melanoma. The pharmaceutical composition provides a multimodal preventive or therapeutic effect for cachexia and muscle loss caused by the above causes.

[0139] The above muscle loss may be caused by causes such as sarcopenia, atony, muscular atrophy, muscular dystrophy, amyotrophic lateral sclerosis, or myasthenia gravis. The pharmaceutical composition provides a multimodal preventive or therapeutic effect for muscle loss caused by the above causes.

[0140]

[0141] Another specific example of the present invention provides a food composition comprising an ionic compound, wherein the ionic compound comprises a calcium cation, a first anion, and a second anion, wherein the first anion and the second anion are different from each other and are each independently an anion of ascorbic acid, dichloroacetic acid, a branched-chain amino acid, or a derivative thereof. In the food composition, the ionic compound is described as in the preceding specific example.

[0142] The above food composition may be a health functional food.

[0143] Functional health foods, also known as foods for special health use (FoSHU), are foods that are processed to effectively exhibit bioregulatory functions in addition to providing nutrition, or that exhibit medical benefits. For example, these functional health foods can be provided in various forms, such as processed foods like canned foods and retort packs, or tablets, capsules, powders, granules, liquids, and pills.

[0144] The food composition may further include commonly used food additives. For example, the food composition may include preservatives, antioxidants, colorants, coloring agents, seasonings, sweeteners, flavorings, leavening agents, emulsifiers, thickeners, film-forming agents, gum-forming agents, antifoaming agents, and the like.

[0145]

[0146] Hereinafter, the present invention will be described in more detail through specific manufacturing examples, examples, and experimental examples. The following manufacturing examples, examples, and experimental examples are intended to illustrate the present invention, and the present invention is not limited to the following manufacturing examples, examples, and experimental examples.

[0147] Manufacturing Example 1. Manufacturing of calcium salt of ascorbic acid and dichloroacetic acid

[0148] An ascorbic acid solution was prepared by dissolving 176 mg of ascorbic acid in 125 mL of distilled water at room temperature. A dichloroacetic acid solution was prepared by dissolving 129 mg of dichloroacetic acid in 125 mL of distilled water. The ascorbic acid solution was slowly added to the dichloroacetic acid solution while stirring. 105 mg of calcium carbonate (CaCO3) was slowly added to the mixed solution, and the mixture was stirred at room temperature for 30 minutes. The reaction temperature was gradually increased to 60°C while stirring until CO2 was no longer generated. The mixture was dried using a rotary evaporator and a vacuum oven, unreacted substances were removed with diethyl ether, and then filtered, dried, and pulverized to obtain powdered ascorbic acid and calcium salt of dichloroacetic acid. All of the above processes were performed under a nitrogen atmosphere, and the calcium salt was referred to as Asc-Ca-DCA or DCA-Ca-Asc.

[0149] Manufacturing Example 2. Manufacturing of calcium salts of ascorbic acid and leucine

[0150] At room temperature, 1000 mg of ascorbic acid and 745 mg of leucine were dissolved in 15 mL of distilled water in a round flask and stirred for 10 minutes to prepare a mixed solution of ascorbic acid and leucine. 568 mg of calcium carbonate (CaCO3) was slowly added to the mixed solution, and the mixture was stirred at room temperature for 30 minutes. The reaction temperature was gradually increased to 60°C and stirred until CO2 no longer evolved. The mixture was concentrated and dried using a rotary evaporator and a vacuum oven, and unreacted substances were removed with diethyl ether. The resulting mixture was filtered, dried, and pulverized to obtain calcium salts of ascorbic acid and leucine in powder form. All of the above processes were performed under a nitrogen atmosphere, and the calcium salts were referred to as Asc-Ca-Leu or Leu-Ca-Asc.

[0151] Manufacturing Example 3. Manufacturing of calcium salt of dichloroacetic acid and leucine

[0152] At room temperature, 1000 mg of dichloroacetic acid and 1018 mg of leucine were dissolved in 15 mL of distilled water in a round flask and stirred for 10 minutes to prepare a mixed solution of ascorbic acid and leucine. 777 mg of calcium carbonate (CaCO3) was slowly added to the mixed solution, and the mixture was stirred at room temperature for 30 minutes. The reaction temperature was gradually increased to 60°C and stirred until CO2 no longer evolved. The mixture was concentrated and dried using a rotary evaporator and a vacuum oven, and unreacted substances were removed with diethyl ether. The mixture was then filtered, dried, and pulverized to obtain a calcium salt of dichloroacetic acid and leucine in powder form. All of the above processes were performed under a nitrogen atmosphere, and the calcium salt was referred to as DCA-Ca-Leu or Leu-Ca-DCA.

[0153]

[0154] Experimental Examples 1-1 and 1-2. Confirmation of Cytotoxicity of Ionic Compounds

[0155] Experimental Example 1-1. Cytotoxicity of C2C12 cells (primary cells) before differentiation

[0156] C2C12 cell line was seeded at 1.0 x 10 in a 96-well plate. 4 After dispensing cells / well, they were cultured for one day in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2. When the cell density reached approximately 70%, the calcium salts of Preparation Examples 1 to 3 were added to each well at eight predetermined concentrations, diluted by half, and cytotoxicity was confirmed. After culturing for 72 hours, MTT solution was treated and cultured in a dark incubator for 3 hours. After removing the supernatant, 100 ㎕ of DMSO was treated per well, and cytotoxicity was confirmed by measuring the optical density (OD) at 560 nm. The above experiment was repeated three times, and the cell viability is shown in Figures 1 (a) to (c).

[0157] Referring to (a) of Fig. 1, the calcium salt of Preparation Example 1 exhibited toxicity such that cell viability decreased by about 50% starting from a treatment concentration of 1000 μM. Referring to (b) of Fig. 1, the calcium salt of Preparation Example 2 exhibited toxicity such that cell viability decreased by about 50% starting from a treatment concentration of 100 μM. Referring to (c) of Fig. 1, the calcium salt of Preparation Example 3 did not exhibit toxicity up to a treatment concentration of 1200 μM.

[0158] Experimental Example 1-2. Cytotoxicity of C2C12 cells (myotube cells) after differentiation.

[0159] C2C12 cell line was seeded at 1.5 x 10 in 96-well plates. 4 After dispensing cells / well, they were cultured for one day in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. The calcium salts of Preparation Examples 1 to 3 were added to each well at eight predetermined concentrations, diluted by half, and cytotoxicity was confirmed. After culturing for 72 hours, MTT solution was treated and cultured in a dark incubator for 3 hours. After removing the supernatant, 100 ㎕ of DMSO was treated per well, and cytotoxicity was confirmed by measuring the OD at 560 nm. The above experiment was repeated three times, and the cell viability is shown in Fig. 2 (a) to (c).

[0160] Referring to (a) of Fig. 2, the calcium salt of Preparation Example 1 exhibited toxicity such that cell viability decreased by about 50% starting from a treatment concentration of 500 μM. Referring to (b) of Fig. 2, the calcium salt of Preparation Example 2 exhibited toxicity such that cell viability decreased by about 50% starting from a treatment concentration of 500 μM. Referring to (c) of Fig. 2, the calcium salt of Preparation Example 3 did not exhibit toxicity up to a treatment concentration of 1200 μM.

[0161] Experimental Examples 2-1 to 2-3. Preparation of a cell model in a cachexia state.

[0162] Experimental Example 2-1. Confirmation of Differentiation Conditions for Mouse Myogenic Cell Line (C2C12)

[0163] Mouse myogenic cell line C2C12 was seeded at 4.0 x 10 in 6-well plates. 5 After dispensing cells / well, they were cultured for one day in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. When the cell density reached approximately 80%, the cells were washed with DPBS and the medium was replaced with differentiation medium (DMEM) containing 2% horse serum, and cultured for 3 days.

[0164] Experimental Example 2-2. Preparation of cancer cell culture medium (cancer-cell conditioned medium; CCM)

[0165] Mouse colon cancer cell line CT26 was seeded at 2.0 x 10 in a 100-mm culture plate. 6 After dispensing cells / well, the cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 48 h. The medium was then replaced with serum-free medium, and the medium was collected 24 h later. The collected medium was centrifuged (1500 rpm, 3 min) to remove cell debris and finally collected using a 0.22-μm filter.

[0166] Experimental Example 2-3. Production of a cell model in a cachexia state.

[0167] After replacing the differentiation medium, C2C12 cells that had differentiated into myotube cells were treated with a 1:1 mixture of differentiation medium and prepared cancer cell culture medium (CCM), and cultured for 72 hours. In order to compare the diameter of the myotubes with that of the normal control group, crystal violet staining was performed, and each image was confirmed and shown in Fig. 3. Fig. 3 (a) is an image of the normal control group (unconditioned medium; UCM), and (b) is an image of the CCM. The scale bar inserted in Fig. 3 (a) and (b) indicates a horizontal length of 50 μm. After the staining, the myotube diameter was measured with Image J and is shown in Fig. 3 (c). Referring to Fig. 3, a decrease in myotube cells was confirmed in the CCM.

[0168] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, and Myogenin, included in the treatment group (CCM) and the normal control group (UCM), were analyzed through Western blot, and the results are shown in Fig. 4, and the relative expression levels (fold change; FC) for each marker are shown in Fig. 5 (a) to (d).

[0169] Referring to Fig. 3(c), the diameter of the myotubes in the treatment group (CCM) was reduced to approximately 50% of that in the normal control group (UCM). Referring to Figs. 5(a) to (d), the expression of muscle degradation markers MuRF1 and Atrogin1 was confirmed to have increased, while the expression of muscle differentiation markers MyHC and Myogenin was confirmed to have decreased.

[0170] Experimental Examples 3-1 to 3-2. Confirmation of target marker expression according to calcium salt treatment of Manufacturing Example 1.

[0171] Experimental Example 3-1. Confirmation of changes in PDK-4 expression levels

[0172] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5After dispensing cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. After confirming that the cells had differentiated, the differentiation medium and cancer cell culture medium (CCM) were mixed in a 1:1 ratio to form a control group, and at the same time, the calcium salt (Asc-Ca-DCA) of Preparation Example 1 was added at three predetermined concentrations to set up a test group. After culturing for 72 hours, the cells were harvested and analyzed for the target marker PDK-4 by Western blotting, and the results are summarized in Fig. 6.

[0173] Figure 6(a) shows the results of a Western blot, and Figure 6(b) is a graph summarizing the PDK-4 expression level from the Western blot results. Referring to Figure 6, the expression level of PDK-4 increased in the control group (CCM) treated only with cancer cell culture medium, but it was confirmed that expression was suppressed by treatment with Asc-Ca-DCA.

[0174] Experimental Example 3-2. Confirmation of changes in calpain 3 expression level

[0175] The experiment was performed in the same manner as Experimental Example 3-1 above, except that Calpain 3 was analyzed as a target marker for Western blot, and the results are summarized in Fig. 7.

[0176] Figure 7(a) is a Western blot result, and Figure 7(b) is a graph summarizing the expression level of Calpain 3 from the Western blot result. Referring to Figure 7, the expression level of Calpain 3 decreased in the control group (CCM) treated only with cancer cell culture medium, but it was confirmed that the decrease in expression was suppressed by treatment with Asc-Ca-DCA.

[0177] Experimental Examples 4-1 to 4-3. Confirmation of target marker expression according to calcium salt treatment of Manufacturing Example 2.

[0178] Experimental Example 4-1. Confirmation of changes in the expression level of Calpain 3.

[0179] Except for using the calcium salt of Manufacturing Example 2, the target marker Calpain 3 was analyzed in the same manner as Experimental Example 3-1 and the results are summarized in Figures 8 and 9.

[0180] Experimental Example 4-2. Confirmation of changes in PDK-4 expression levels

[0181] Except for analyzing PDK-4 as a target marker for Western blot, the analysis was performed in the same manner as Experimental Example 4-1 and the results are summarized in Figures 8 and 9.

[0182] Experimental Example 4-3. Confirmation of Changes in GDF-15 Expression Levels

[0183] Except for analyzing GDF-15 as a target marker for Western blot, the analysis was performed in the same manner as Experimental Example 4-1 and is summarized in Figures 8 and 9.

[0184] Figure 8 shows the results of Western blot, and Figure 9 is a graph summarizing the expression levels of (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results. Referring to Figure 9(a), the expression level of Calpain 3 decreased in the control group (CCM) treated only with cancer cell culture medium, but it was confirmed that the decrease in expression was suppressed by treatment with Asc-Ca-Leu. Referring to Figures 9(b) and (c), the expression levels of PDK-4 and GDF-15 increased in the control group (CCM) treated only with cancer cell culture medium, but it was confirmed that the increase in expression was suppressed by treatment with Asc-Ca-Leu.

[0185] Experimental Examples 5-1 to 5-3. Confirmation of target marker expression according to calcium salt treatment of Manufacturing Example 3.

[0186] Experimental Example 5-1. Confirmation of changes in the expression level of Calpain 3.

[0187] Except for using the calcium salt of Manufacturing Example 3, the target marker Calpain 3 was analyzed in the same manner as Experimental Example 3-1, and the results are summarized in Figures 10 and 11.

[0188] Experimental Example 5-2. Confirmation of changes in PDK-4 expression levels.

[0189] Except for analyzing PDK-4 as a target marker for Western blot, the analysis was performed in the same manner as Experimental Example 3-1 and is summarized in Figures 10 and 11.

[0190] Experimental Example 5-3. Confirmation of changes in GDF-15 expression levels

[0191] Except for analyzing GDF-15 as a target marker for Western blot, the analysis was performed in the same manner as Experimental Example 4-1 and is summarized in Figures 10 and 11.

[0192] Figure 10 shows the results of Western blot, and Figure 11 is a graph summarizing the expression levels of (a) Calpain 3, (b) PDK-4, and (c) GDF-15 from the Western blot results. Referring to Figure 11(a), the expression level of Calpain 3 decreased in the control group (CCM) treated only with cancer cell culture medium, but it was confirmed that the decrease in expression was suppressed by treatment with DCA-Ca-Leu. Referring to Figures 11(b) and (c), the expression levels of PDK-4 and GDF-15 increased in the control group (CCM) treated only with cancer cell culture medium, but it was confirmed that the increase in expression was suppressed by treatment with DCA-Ca-Leu.

[0193] Experimental Examples 6-1 and 6-2. Confirmation of the Correlation Between Target Markers and the Induction of Cachexia

[0194] Experimental Example 6-1. Confirmation of changes in target markers following treatment with cancer cell culture medium (CCM) in C2C12 myotube cells.

[0195] PDK-4, a target gene of Forkhead box protein O (FOXO), which is closely related to energy metabolism and a key protein degradation mechanism in muscle cell, is known to be overexpressed when subjects exhibit cachexia symptoms. We examined whether the expression of PDK-4 increased compared to the normal control group when cancer cachexia was created by treating myotube cells with cancer cell culture medium (CCM).

[0196] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5 After dispensing cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for an additional 3 days. After confirming differentiation, the differentiation medium and cancer cell culture medium (CCM) were mixed in a 1:1 ratio and cultured for 72 hours. The cells were harvested and analyzed by Western blotting, and the results are summarized in Figure 12 (a) and (b).

[0197] In the same way, Calpain 3, known to be underexpressed in muscle in cancer cachexia, was also analyzed through Western blotting and summarized in Figure 12 (c) and (d). In the same way, GDF-15, known to be overexpressed in muscle in cancer cachexia, was also analyzed through Western blotting and summarized in Figure 12 (e) and (f).

[0198] Experimental Example 6-2. Confirmation of Cancer Cachexia Induction Based on Changes in Target Marker Expression

[0199] Target markers PDK-4 and Calpain 3 are known to be overexpressed and underexpressed, respectively, in cancer cachexia. We investigated whether increased PDK-4 expression induced cancer cachexia and whether changes in muscle breakdown and differentiation markers were identical to those in cachexia.

[0200] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5 After dividing into cells / well, they were cultured for one day in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2. When the cell density reached approximately 80%, the medium was replaced and cultured for 3 more days to establish a normal control group, and at the same time, WY-14643, an activator of PDK-4, was treated at a concentration of 50 μM to establish a test group. After culturing for 72 hours, the cells were stained with crystal violet staining, and each image is shown in Fig. 13. Fig. 13 (a) is an image of the normal control group (UCM), and (b) is an image of the activator-treated group (WY-14643). The scale bars inserted in Fig. 13 (a) and (b) represent a horizontal length of 50 μm. After the staining, the myotube diameter was measured using Image J, and is shown in Fig. 13 (c). Referring to Figure 13, it was confirmed that when treated with WY-14643, a PDK-4 activator, the diameter of myotube cells decreased, inducing a state similar to cancer cachexia.

[0201] Next, the expression of PDK-4, MuRF1, Atrogin1, MyHC, and Myogenin in the normal control group (UCM) and the activator-treated group (WY-14643) was analyzed by Western blotting, and the results are shown in (a) and (b) of Fig. 14. Referring to Fig. 14, it was confirmed that in the activator WY-14643-treated group, PDK-4, MuRF1, and Atrogin1 were overexpressed, while MyHC and Myogenin were underexpressed, inducing a state similar to cancer cachexia.

[0202] Experimental Example 6-3. Confirmation of Cancer Cachexia Induction Based on Changes in Target Marker Expression

[0203] We confirmed whether a cancer cachexia state was induced in a situation where the expression level of Calpain 3 was reduced, and whether the changes in muscle degradation and differentiation markers were the same as in the cachexia state.

[0204] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5 After dividing into cells / well, they were cultured for one day in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days to establish a normal control group. At the same time, they were transfected by treating 100 nM each of Calpain 3 siRNA and vehicle control scrambled siRNA. The transfection was performed using lipofectamine™ 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. After culturing for 72 hours, the cells were stained with crystal violet staining, and the respective images are shown in Figure 15. Figure 15 (a) is an image of the normal control group (UCM), (b) is an image of the vehicle control group (scrambled siRNA), and (c) is an image of the Calpain 3 siRNA treatment group. The scale bar inserted in (a) to (c) of Fig. 15 represents a horizontal length of 50 μm. After the staining, the myotube diameter was measured using Image J and is shown in Fig. 15(d). Referring to Fig. 15, it was confirmed that the myotube diameter decreased with statistical significance compared to the control group under the condition of Calpain 3 knockdown by Calpain 3 siRNA transfection, confirming that it was induced in a state similar to cancer cachexia.

[0205] Next, the expression of Calpain 3, MuRF1, Atrogin1, MyHC, and Myogenin in the normal control group (UCM), vehicle control group (scrambled siRNA), and Calpain 3 siRNA treatment group was analyzed by Western blotting, and the results are shown in (a) and (b) of Fig. 16. Referring to Fig. 16, it was confirmed that Calpain 3 expression decreased, MuRF1 and Atrogin1 were overexpressed, and MyHC and Myogenin were underexpressed according to Calpain 3 siRNA transfection, inducing a state similar to cancer cachexia.

[0206] Experimental Example 7. Confirmation of the COX2 inhibitory effect of the calcium salt of Manufacturing Example 1.

[0207] The calcium salt of Manufacturing Example 1 was mixed with DMSO and added to 200 mM Tris-HCl buffer (pH 8.0, containing 6 μM EDTA, and 10 μM hematin) (the concentration of DMSO in the final buffer was 1%). At this time, the concentration of the calcium salt contained in the buffer was 10 μM. 34 U / ml of human recombinant cyclooxygenase COX-2 expressed in insect Sf21 cells as an enzyme was further added to the buffer and pre-reacted at 25°C for 15 minutes. Additionally, 3 μM of arachidonic acid and 100 μM of Ampliflu Red were further added and reacted at 25°C for 3 minutes. The amount of Resofurin produced during the reaction was measured by spectrofluorimetry at 535 nm / 590 nm and the inhibitory effect on COX2 was confirmed by comparing it with the calcium salt-untreated group.

[0208] The calcium salt (Asc-Ca-DCA) of Manufacturing Example 1 was confirmed to have a 74% inhibitory effect on COX2, and was judged to be able to provide a preventive or therapeutic effect on cachexia and muscle loss.

[0209] Experimental Examples 8-1 to 8-2. Confirmation of the inhibitory effect of the calcium salt of the manufacturing example on the reduction of C2C12 root canal differentiation.

[0210] Experimental Example 8-1. Measurement of the diameter of C2C12 myotube cells

[0211] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5 After dividing into cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. After confirming that the cells had differentiated, the differentiation medium and cancer cell culture medium (CCM) were mixed in a 1:1 ratio to form a control group, and at the same time, the calcium salt of Manufacturing Example 1 was added at three predetermined concentrations to set up a test group. After culturing for 72 hours, they were stained with crystal violet staining, and each image is summarized in Fig. 17(a). The scale bar inserted in Fig. 17(a) represents 50 μm in horizontal length. The myotube diameter was measured using Image J from the above images, and the change in myotube cell diameter is summarized in Fig. 17(b).

[0212] Referring to Figure 17, the diameter of myotube cells treated with cancer cell culture medium (CCM) to create cancer cachexia conditions was reduced by approximately 50% compared to the normal control group (UCM), and in the test group treated with calcium salt (Asc-Ca-DCA) of Manufacturing Example 1, the decrease in diameter of myotube cells was statistically significantly suppressed from 10 μM or higher.

[0213] Experimental Example 8-2. Identification of C2C12 Myotube Cell Degradation and Differentiation Markers

[0214] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5After dividing into cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. After confirming that the cells had differentiated, the differentiation medium and cancer cell culture medium (CCM) were mixed in a 1:1 ratio and treated to form a control group, and at the same time, the calcium salts of Preparation Examples 1 to 3 were treated at a preset concentration to set the test group. After culturing for 72 hours, the cells were harvested and analyzed by Western blot, and the results are summarized in Fig. 18 (Preparation Example 1), Fig. 20 (Preparation Example 2), and Fig. 22 (Preparation Example 3).

[0215] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, and Myogenin, included in each experimental group, were analyzed through Western blot, and the relative expression levels of each marker are shown in Fig. 19 (Preparation Example 1), Fig. 21 (Preparation Example 2), and Fig. 23 (Preparation Example 3).

[0216] Referring to Figures 18 to 23, the muscle degradation markers MuRF1 and Atrogin1 were suppressed from increasing their expression due to cancer cell culture medium (CCM) by the calcium salt treatment of Preparation Examples 1 to 3, respectively, and the muscle differentiation markers MyHC and Myogenin were suppressed from decreasing their expression due to cancer cell culture medium (CCM).

[0217] Experimental Examples 9-1 to 9-3. Comparison of the effects of calcium salts of the manufacturing examples and ascorbic acid, dichloroacetic acid, and combinations of ascorbic acid and dichloroacetic acid.

[0218] Experimental Example 9-1. Measurement of the diameter of C2C12 root canal cells

[0219] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5After dividing into cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. After confirming that the cells had differentiated, the differentiation medium and cancer cell culture medium (CCM) were mixed in a 1:1 ratio and treated to form a control group, and at the same time, the calcium salt (Asc-Ca-DCA) of Preparation Example 1, ascorbic acid, dichloroacetic acid, and a combination of ascorbic acid and dichloroacetic acid were treated at set concentrations to set the test group. After culturing for 72 hours, the image stained with crystal violet staining is summarized in Fig. 24(a). The scale bar inserted in Fig. 24(a) represents 50 μm in horizontal length. The above image was used to measure the diameter of the root canal using Image J, and the change in the diameter of the root canal cells was confirmed, which is shown in Figure 24(b).

[0220] Referring to Figure 24, the diameter of myotube cells was found to increase when treated with ascorbic acid, dichloroacetic acid, or a combination of ascorbic acid and dichloroacetic acid compared to cancer cell culture medium (CCM). However, when treated with the calcium salt of Manufacturing Example 1, it was confirmed to increase to a level similar to that of the normal control group (UCM).

[0221] Experimental Example 9-2. Identification of C2C12 Myotube Cell Degradation and Differentiation Markers

[0222] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5After dividing into cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. After confirming that the cells had differentiated, the differentiation medium and cancer cell culture medium (CCM) were mixed in a 1:1 ratio and treated to form a control group, and at the same time, the calcium salt (Asc-Ca-DCA) of Preparation Example 1, ascorbic acid, dichloroacetic acid, and a combination of ascorbic acid and dichloroacetic acid were treated at set concentrations to set the test group. After culturing for 72 hours, the cells were harvested and subjected to Western blot analysis, which is summarized in Figs. 25 and 27.

[0223] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15, included in each experimental group were analyzed through Western blot, and the relative expression levels of each marker are shown in Figures 26 and 28.

[0224] Referring to Figures 25 to 28, the muscle degradation markers MuRF1 and Atrogin1 showed a superior effect of suppressing the increase in expression in the calcium salt (Asc-Ca-DCA) treatment group of Preparation Example 1 compared to other experimental groups, and the muscle differentiation markers MyHC and Myogenin showed a superior effect of suppressing the decrease in expression in the calcium salt treatment group of Preparation Example 1 compared to other experimental groups. The calcium salt treatment group of Preparation Example 1 showed a higher expression level of Calpain 3 compared to other experimental groups, and the expression of PDK-4 and GDF-15 was suppressed.

[0225] Experimental Example 9-3. Identification of C2C12 Myotube Cell Degradation and Differentiation Markers

[0226] The experiment was conducted in the same manner as Experimental Example 9-2, except that the test groups were set by treating the calcium salt (Asc-Ca-Leu), ascorbic acid, leucine, and a combination of ascorbic acid and leucine of Manufacturing Example 2 at set concentrations. After culturing for 72 hours, the cells were harvested and subjected to Western blot analysis, which is summarized in Figures 29 and 31.

[0227] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15, included in each experimental group were analyzed through Western blot, and the relative expression levels of each marker are shown in Figures 30 and 32.

[0228] Referring to Figures 29 to 32, the muscle degradation markers MuRF1 and Atrogin1 showed a superior effect of suppressing the increase in expression in the calcium salt (Asc-Ca-Leu) treatment group of Preparation Example 2 compared to other experimental groups, and the muscle differentiation markers MyHC and Myogenin showed a superior effect of suppressing the decrease in expression in the calcium salt treatment group of Preparation Example 2 compared to other experimental groups. The calcium salt treatment group of Preparation Example 2 showed a higher expression level of Calpain 3 compared to other experimental groups, and the expression of PDK-4 and GDF-15 was suppressed.

[0229] Experimental Example 9-4. Identification of C2C12 Myotube Cell Degradation and Differentiation Markers

[0230] The experiment was conducted in the same manner as Experimental Example 9-2, except that the test groups were set by treating the calcium salt (DCA-Ca-Leu), dichloroacetic acid, leucine, and the group combining dichloroacetic acid and leucine of Manufacturing Example 3 at set concentrations. After culturing for 72 hours, the cells were harvested and subjected to Western blot analysis, which is summarized in Figures 33 and 35.

[0231] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, Myogenin, Calpain 3, PDK-4, and GDF-15, included in each experimental group were analyzed through Western blot, and the relative expression levels for each marker are shown in Figures 34 and 36.

[0232] Referring to Figures 33 to 36, the muscle degradation markers MuRF1 and Atrogin1 showed a superior effect of suppressing the increase in expression in the calcium salt (DCA-Ca-Leu) treatment group of Preparation Example 3 compared to other experimental groups, and the muscle differentiation markers MyHC and Myogenin showed a superior effect of suppressing the decrease in expression in the calcium salt treatment group of Preparation Example 3 compared to other experimental groups. The calcium salt treatment group of Preparation Example 3 showed a higher expression level of Calpain 3 compared to other experimental groups, and the expression of PDK-4 and GDF-15 was suppressed.

[0233] Experimental Examples 10-1 to 10-3. Effect of the calcium salt of the manufacturing example on sarcopenia.

[0234] Experimental Example 10-1. Preparation of a sarcopenia cell model

[0235] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5 After dividing into cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for 3 more days. After confirming that the cells had differentiated, the experimental group was set by treating them with dexamethasone (Dex) at a set concentration. After culturing for 72 hours, the image stained with crystal violet staining is summarized in Fig. 37(a). The scale bar inserted in Fig. 37(a) represents 50 μm in horizontal length. The image was used to measure the myotube diameter using Image J to confirm the change in myotube cell diameter, which is shown in Fig. 37(b).

[0236] Referring to Figure 37, a decrease in myotube diameter was confirmed starting from a treatment concentration of 1 μM dexamethasone. Since no statistically significant difference was confirmed from the 1 μM concentration treatment group at concentrations above the above concentration, a treatment concentration of 1 μM dexamethasone was established as the experimental condition.

[0237] Experimental Example 10-2. Confirming Target Marker Changes in a Sarcopenia Cell Model

[0238] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5 After dispensing cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37℃ and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for an additional 3 days. After confirming differentiation, the normal control group (UCM) was treated with 1 μM dexamethasone and cultured for 72 hours. The cells were harvested and Western blotting for related markers was performed, which is shown in Figure 38.

[0239] As a result of the analysis of Figure 38, the expression levels of muscle breakdown markers MuRF1 and Atrogin1 increased in the dexamethasone-treated group, and the expression levels of muscle differentiation markers MyHC and Myogenin decreased, confirming that the expression of related markers changes along with muscle loss when dexamethasone is treated. In addition, by confirming an increase in the expression of PDK4, a decrease in the expression of Calpain 3, and an increase in the expression of GDF15 in the dexamethasone-treated group, it was confirmed that the above target markers are also involved in the main mechanism in the sarcopenia model.

[0240] Experimental Example 10-3. Confirmation of the effectiveness of the calcium salt in the manufacturing example.

[0241] C2C12 cell line was seeded at 4.0 x 10 in 6-well plates. 5After dispensing cells / well, they were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for one day. When the cell density reached approximately 80%, they were replaced with differentiation medium and cultured for an additional 3 days. After confirming that differentiation had occurred, dexamethasone was treated at 1 μM and the calcium salts of Preparation Examples 1 to 3 were treated at the set concentrations to set the experimental groups. After culturing for 72 hours, the cells were harvested and analyzed by Western blot, and the results are summarized in Fig. 39 (Preparation Example 1), Fig. 41 (Preparation Example 2), and Fig. 43 (Preparation Example 3).

[0242] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, Myogenin, and Calpain 3, PDK-4, and GDF-15, included in each experimental group, were analyzed through Western blot, and the relative expression levels of each marker are shown in Fig. 40 (Preparation Example 1), Fig. 42 (Preparation Example 2), and Fig. 44 (Preparation Example 3).

[0243] Referring to Figures 39 to 44, the expression increase of muscle degradation markers MuRF1 and Atrogin1 due to dexamethasone treatment was suppressed by the calcium salt treatment of Preparation Examples 1 to 3, and the expression decrease of muscle differentiation markers MyHC and Myogenin due to dexamethasone treatment was suppressed. In addition, a decrease in the expression of PDK-4, an increase in the expression of Calpain 3, and a decrease in the expression of GDF15 were confirmed in the calcium salt treatment groups of Preparation Examples 1 to 3.

[0244] Experimental Example 11. Confirmation of blood drug concentration and pharmacokinetic factors according to intravenous (IV) and oral (PO) administration in white rats (SD rats).

[0245] Calcium salt (Asc-Ca-DCA) of Preparation Example 1 was administered intravenously (200 mg / kg, once daily) or orally (200, 500 mg / kg, once daily or 1000 mg / kg twice daily) to white rats for a total of 6 days. Blood was collected from each white rat at different times after administration (0 to 48 hours), centrifuged, and the plasma obtained was analyzed for the blood concentration of the administered drug and the resulting pharmacokinetic factors (PK parameters) using LC / MS / MS analysis, which are summarized in Table 1 below.

[0246] Asc-Ca-DCAIVPOPKparameters200 mg / kg200 mg / kg500 mg / kg2000 mg / kg(1000 mg / kg, BID)1 day6 days1 day6 days1 day6 days1 day6 daysAUC last (㎍·h / mL)19.777.9838.7834.62353.65147.85889.77575.25C max (㎍ / mL)30.242.7023.916.2666.4421.28130.9277.20T max (h)0.250.000.500.001.500.001.750.00t 1 / 2 (h)0.272.720.592.724.145.585.643.11

[0247]

[0248] Referring to Table 1 above, the blood concentrations of calcium salts were well analyzed in the test groups administered Manufacturing Example 1, regardless of the route and concentration of administration, allowing for specific numerical values ​​for pharmacodynamic factors to be identified. From this, it can be concluded that the calcium salt according to Manufacturing Example 1 will persist in the subject's body for a certain period of time and provide the aforementioned multimodal effects.

[0249] Experimental Examples 12-1 to 12-6. In vivo tests on mice

[0250] Experimental Example 12-1. Identification of Muscle Degradation or Differentiation Markers in Mouse Muscle Tissue

[0251] A syngeneic model was created by subcutaneously transplanting the CT26 mouse colon cancer cell line into Balb / c mice, and then the calcium salt (Asc-Ca-DCA) of Preparation Example 1 was administered for a total of 21 days. The mice were sacrificed, and at autopsy, the quadriceps skeletal muscle was removed, pulverized, and proteins were extracted using RIPA lysis buffer, and the results were analyzed by Western blot and summarized in Figure 45.

[0252] Next, markers related to muscle breakdown and differentiation, MuRF1, Atrogin1, MyHC, and Myogenin, included in each experimental group, were analyzed through Western blot, and the relative expression levels of each marker are shown in Figures 46(a) to (d).

[0253] In Figures 45 and 46, G1 is a normal control group without tumor, G2 is a vehicle control group that administered only vehicle (injection water or saline) to the cachexia model, G3 is a megestrol oral administration group (125 mpk) that administered megestrol to the cachexia model, G4 is a preparation example 1 oral administration group (200 mpk) that administered preparation example 1 to the cachexia model, G5 is a preparation example 1 intravenous administration group (100 mpk) that administered preparation example 1 to the cachexia model, and G6 is a preparation example 1 intravenous administration group (200 mpk) that administered preparation example 1 to the cachexia model.

[0254] Referring to Figures 45 and 46, it was confirmed that G4 to G6 treated with Manufacturing Example 1 showed a decrease in the expression levels of muscle degradation markers MuRF1 and Atrogin1, and an increase in the expression levels of muscle differentiation markers MyHC and Myogenin, compared to the excipient control group G2. G4 to G6 treated with Manufacturing Example 1 showed an excellent effect of decreasing Atrogin1 expression and increasing Myogenin expression compared to G3 treated with megestrol, a cachexia treatment agent, and the effect of regulating MuRF1 and MyHC expression was confirmed to be at a similar level.

[0255] Experimental Example 12-2. Identification of Target Markers in Mouse Muscle Tissue

[0256] A syngeneic model was created by subcutaneously transplanting the CT26 mouse colon cancer cell line into Balb / c mice, and then the calcium salt (Asc-Ca-DCA) of Preparation Example 1 was administered for a total of 21 days. The mice were sacrificed, and at autopsy, the quadriceps skeletal muscles were removed, pulverized, and proteins were extracted using RIPA lysis buffer, and the results were summarized in Figure 47(a).

[0257] Next, Calpain 3 and PDK-4 included in each experimental group were analyzed through Western blot, and the relative expression levels for each marker are shown in Figures 47(b) and (c).

[0258] In Figure 47, G1 is the normal control group, G2 is the excipient control group, G3 is the oral administration group of megestrol (125 mpk), G4 is the oral administration group of Manufacturing Example 1 (200 mpk), G5 is the intravenous administration group of Manufacturing Example 1 (100 mpk), and G6 is the intravenous administration group of Manufacturing Example 1 (200 mpk).

[0259] Referring to Figure 47, it was confirmed that G4 to G6 treated with Manufacturing Example 1 had an increased expression level of Calpain 3 and a decreased expression level of PDK-4 compared to the excipient control group G2. Compared to G3 treated with megestrol, a cachexia treatment agent, intravenously administered G6 had an excellent effect of increasing the expression level of Calpain 3, and G4 to G6 had an excellent effect of decreasing the expression level of PDK-4.

[0260] Experimental Example 12-3. Measurement of Cytokines and Lactic Acid in Mouse Blood

[0261] A syngeneic model was created by subcutaneously transplanting the CT26 mouse colon cancer cell line into Balb / c mice, and then the calcium salt (Asc-Ca-DCA) of Preparation Example 1 was administered for a total of 21 days. The mice were sacrificed, and the serum collected at autopsy was used to analyze IL-1β and lactic acid, and the results are summarized in Figures 48(a) and (b).

[0262] In Figure 48, G1 is the normal control group, G2 is the excipient control group, G3 is the oral administration group of megestrol (125 mpk), G4 is the oral administration group of Manufacturing Example 1 (200 mpk), G5 is the intravenous administration group of Manufacturing Example 1 (100 mpk), and G6 is the intravenous administration group of Manufacturing Example 1 (200 mpk).

[0263] Referring to Figure 48, G4 to G6 treated with Manufacturing Example 1 were confirmed to have decreased IL-1β and lactic acid compared to the excipient control group G2. Compared to group G3 treated with megestrol, a cachexia treatment, G4 to G6 were confirmed to have similar levels of IL-1β and lactic acid reduction effects.

[0264] Experimental Example 12-4. Confirmation of the weight loss suppression effect of calcium salt treatment of Preparation Example 1 in an animal model of cancer cachexia.

[0265] A syngeneic model was created by subcutaneously transplanting the CT26 mouse colon cancer cell line into Balb / c mice, and then the calcium salt (Asc-Ca-DCA) of Manufacturing Example 1 was administered for a total of 21 days, and the change in body weight was analyzed and summarized in Figures 49(a) and (b).

[0266] In Figure 49, G1 is the normal control group, G2 is the excipient control group, G3 is the oral administration group of megestrol (125 mpk), G4 is the oral administration group of Manufacturing Example 1 (200 mpk), G5 is the intravenous administration group of Manufacturing Example 1 (100 mpk), and G6 is the intravenous administration group of Manufacturing Example 1 (200 mpk).

[0267] Referring to Figure 49, the body weight loss rate of the excipient control group G2 compared to the normal control group G1 on the 17th day was approximately 8.4%, and on the 20th day it was approximately 13.3% (p < 0.01 or p < 0.0001). In the case of G6, the body weight increased by approximately 6% on the 17th day and 13% on the 20th day compared to G2 (p < 0.05).

[0268] Experimental Example 12-5. Pathological Analysis of Mouse Muscle Tissue Treated with Calcium Salt of Preparation Example 1

[0269] A syngeneic model was created by subcutaneously transplanting the CT26 mouse colon cancer cell line into Balb / c mice, and then the calcium salt (Asc-Ca-DCA) of Preparation Example 1 was administered for a total of 21 days. The mice were sacrificed, and three types of skeletal muscles (gastrocnemius, quadriceps, soleus) were extracted during autopsy and subjected to histopathological analysis. Cross-sectional images of the muscle tissue were obtained, and the area of ​​the cross-section (muscle fiber cross-sectional area; CSA) was measured, and the results are summarized in Figures 50 to 52.

[0270] In Figures 50 to 52, G1 represents the normal control group, G2 represents the excipient control group, G3 represents the oral megestrol administration group (125 mpk), G4 represents the oral administration group of Manufacturing Example 1 (200 mpk), G5 represents the intravenous administration group of Manufacturing Example 1 (100 mpk), and G6 represents the intravenous administration group of Manufacturing Example 1 (200 mpk). The scale bars inserted in (a) to (f) of Figures 50 to 52 represent a horizontal length of 100 μm.

[0271] Referring to Figures 50 to 52, it was confirmed that G4 to G6 treated with Manufacturing Example 1 had an increased muscle tissue cross-sectional area compared to the excipient control group G2, and a similar level to the group G3 treated with megestrol, a cachexia treatment agent.

[0272] Experimental Example 12-6. Behavioral Evaluation of Mice Treated with Calcium Salt of Manufacturing Example 1

[0273] A syngeneic model was created by subcutaneously implanting the CT26 mouse colon cancer cell line into Balb / c mice. Rotarod latency tests were performed once a week before and after test substance administration. The animals were carefully placed on a Rotarod treadmill (JD-A-07RA5, BS Technolab Inc., Korea), and the rotational speed was increased at regular intervals from 4 rpm to 40 rpm, and measurements were made for 300 seconds, which are summarized in Figure 53(a).

[0274] In addition, a wire hanging test was also conducted once a week, in which the animal was placed on the top of a wire cage, then turned over and hung over the home cage, and the waiting time until the animal fell was recorded and summarized in Figure 53(b).

[0275] Referring to Figure 53(a), in the Rotarod-treadmill test, the excipient control group G2 showed a value that was reduced by about 23% compared to the normal control group G1, while G6 showed a value that was improved by about 24% compared to G2.

[0276] Referring to Figure 53(b), in the wire hanging test, G4 to G6 showed values ​​that were 5 to 10% improved compared to the excipient control group G2, and showed levels similar to those of the positive control group G3 administered with megestrol.

Claims

1. A pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising an ionic compound, The above ionic compound comprises a calcium cation, a first anion and a second anion, A pharmaceutical composition for preventing or treating cachexia and muscle loss, wherein the first anion and the second anion are different from each other and are each independently an anion of ascorbic acid, dichloroacetic acid, branched-chain amino acid (BCAA) or a derivative thereof.

2. In paragraph 1, A pharmaceutical composition for preventing or treating cachexia and muscle loss, wherein the branched-chain amino acid is at least one selected from the group consisting of leucine, isoleucine, and valine.

3. In paragraph 1, A pharmaceutical composition for preventing or treating cachexia and muscle wasting, wherein the ionic compound is administered at a dosage of 20 to 4800 mg / kg / day.

4. In paragraph 1, The pharmaceutical composition further comprises a pharmaceutically acceptable aqueous solvent, A pharmaceutical composition for preventing or treating cachexia and muscle loss, wherein the degree of dissociation of the ionic compound is 1 to 50%.

5. In paragraph 4, A pharmaceutical composition for preventing or treating cachexia and muscle loss, further comprising at least one active agent selected from the group consisting of appetite stimulants, anticancer agents, anti-inflammatory agents, antibacterial agents, antifungal agents, antiviral agents, immunomodulators, steroids, anticoagulants, anticonvulsants, antidepressants, antioxidants, and vitamins.

6. In paragraph 1, A pharmaceutical composition for preventing or treating cachexia and muscle loss, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier, excipient and diluent.

7. In paragraph 1, A pharmaceutical composition for preventing or treating cachexia and muscle wasting, wherein the pharmaceutical composition is formulated as a liquid, powder, oral preparation, injection, infusion, aerosol, tablet, capsule, pill, depot or suppository.

8. In paragraph 1, The pharmaceutical composition is a pharmaceutical composition for preventing or treating cachexia and muscle loss, which is administered orally, intravenously, subcutaneously, intramuscularly or mucosally.

9. In paragraph 1, A composition for preventing or treating cachexia and muscle loss, wherein the cachexia and muscle loss are caused by cancer, tuberculosis, diabetes, AIDS, chronic obstructive pulmonary disease, multiple sclerosis, congestive heart failure, hemophilia, hypopituitarism, or cirrhosis.

10. In paragraph 9, A composition for preventing or treating cachexia and muscle loss, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, colon cancer, stomach cancer, liver cancer, brain cancer, pancreatic cancer, thyroid cancer, skin cancer, bone marrow cancer, lymphoma, uterine cancer, cervical cancer, ovarian cancer, kidney cancer, and melanoma.

11. In paragraph 1, A composition for preventing or treating cachexia and muscle loss, wherein the muscle loss is caused by sarcopenia, atony, muscular atrophy, muscular dystrophy, amyotrophic lateral sclerosis, or myasthenia gravis.

12. A food composition comprising an ionic compound, The above ionic compound comprises a calcium cation, a first anion and a second anion, A food composition wherein the first anion and the second anion are different from each other and are each independently an anion of ascorbic acid, dichloroacetic acid, a branched-chain amino acid or a derivative thereof.

13. In paragraph 12, The above food composition is a health functional food.

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