Methods for Treating Chronic Inflammatory Diseases

Abstract

To provide a method of treating chronic inflammatory diseases such as Alzheimer's disease, Parkinson's disease, atherosclerosis, stroke.SOLUTION: The invention relates to a method of treating chronic inflammatory diseases in subjects in need thereof by administering calcium lactate as an active agent. The calcium lactate can be provided in a pharmaceutical, food, or nutrient composition.SELECTED DRAWING: None

Description

[Background technology]

[0001] 2. Background of the Invention Chronic inflammatory diseases (CIDs) are defined as pathological conditions characterized by chronic inflammation, which is defined as a prolonged and persistent proinflammatory state (1). CIDs include many common and rare CIDs, such as respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, pneumonitis, and pulmonary fibrosis) (2), eye diseases (e.g., keratitis and age-related macular degeneration) (3), autoimmune diseases (4), nephropathy (5), neurodegenerative diseases (e.g., Alzheimer's disease, stroke, Parkinson's disease, and multiple sclerosis) (6), cardiovascular diseases (e.g., atherosclerosis, arteriosclerosis, and myocarditis) (7), metabolic disorders (e.g., diabetes and obesity) (8), musculoskeletal diseases (e.g., rheumatoid arthritis and osteoporosis) (9, 10), periodontal diseases (e.g., pulpitis and periodontitis) (11), gastrointestinal diseases (e.g., nonalcoholic fatty liver disease, gastroenteritis, and chronic inflammatory bowel disease) (12), and skin disorders (e.g., psoriasis and atopic dermatitis) (13, 14).

[0002] Epidemiological studies have shown that CID is the leading cause of death worldwide and has been increasing for the past 30 years. It is estimated that by 2030, 171 million people in the United States will suffer from CID (15). There are various types of anti-inflammatory drugs to treat inflammation, including aspirin, antihistamines, COX-2 inhibitors, corticosteroids, and nonsteroidal anti-inflammatory drugs (NSAlDs). However, the long-term administration of such existing drugs is limited by side effects and temporary efficacy. Summary of the Invention

[0003] BRIEF SUMMARY OF THE INVETION Disclosed herein is a method of treating a chronic inflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0004] Disclosed herein is a method for treating Alzheimer's disease, Parkinson's disease, atherosclerosis, and / or stroke, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0005] Also disclosed is a method for treating multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease (NAFLD) and / or rheumatoid arthritis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.In some embodiments, the method is for treating NAFLD, and NAFLD is non-alcoholic steatohepatitis (NASH).

[0006] Also disclosed is a method of treating pulpitis and / or periodontitis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0007] Also disclosed is a method of treating inflammatory bowel disease (IBD), comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. In some embodiments, the IBD is Crohn's disease or ulcerative colitis.

[0008] Further disclosed is a method for treating spinal cord injury, cerebral hemorrhage, myocardial infarction, keratitis, and / or diabetic retinopathy, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0009] Disclosed herein is a method for treating asthma, pulmonary fibrosis, obesity, gastroenteritis, chronic inflammatory bowel disease, and / or atopic dermatitis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.Disclosed herein is a method for treating chronic obstructive pulmonary disease, pneumonitis, keratitis, atherosclerosis, arteriosclerosis, myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis, and / or psoriasis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.Disclosed herein is a method for treating Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease, sepsis, and / or osteoporosis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0010] In some embodiments, calcium lactate is administered in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, excipient and / or diluent. In some embodiments, the pharmaceutical composition is formulated into a liquid, powder, aerosol, injection, infusion, patch, capsule, pill, tablet, depot, or suppository.

[0011] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of calcium lactate as an active agent and a pharma- ceutically acceptable polysaccharide, polymer, lipid, or combination thereof. In some embodiments, the pharmaceutical composition comprises calcium lactate and a polysaccharide. In some embodiments, the weight ratio of calcium lactate to polysaccharide is 1:<0.2 to 1:5. In some embodiments, the weight ratio of calcium lactate to polysaccharide is 1:<0.2. In some embodiments, the weight ratio of calcium lactate to polysaccharide is 1:0.2 to 1:5.

[0012] In some embodiments, the pharmaceutical composition further comprises a polymer and / or lipid. In some embodiments, the pharmaceutical composition further comprises a polymer and lipid, the weight ratio of the polymer to the lipid being 1:0.1 to 1:50. In some embodiments, the weight ratio of the calcium lactate to the polymer and / or lipid is at least 1:5. In some embodiments, the weight ratio of the calcium lactate to the polymer and / or lipid is 1:5 to 1:30.

[0013] In some embodiments, the pharmaceutical composition comprises calcium lactate and a polymer and / or lipid. In some embodiments, the pharmaceutical composition comprises a polymer and lipid, the weight ratio of the polymer to the lipid being 1:0.1 to 1:50. In some embodiments, the weight ratio of the calcium lactate to the polymer and / or lipid is at least 1:5. In some embodiments, the weight ratio of the calcium lactate to the polymer and / or lipid is 1:5 to 1:30.

[0014] In some embodiments, the composition is short acting. In some embodiments, the composition is long acting. In some embodiments, the composition is an injectable composition.

[0015] In some embodiments, the polysaccharide is a cellulose derivative, pectin, hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen, alginate, alginic acid, or a combination thereof.

[0016] In some embodiments, the polymer is a poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic-lactic acid (PLGA), or a combination thereof.

[0017] In some embodiments, the lipid is a mono- or tri-fatty acid glycerol ester or polyethylene glycol, a polyethylene glycol ester of a vegetable oil, a fatty acid propylene glycol ester, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa oil, cottonseed oil, sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenated oil, oleic acid, linoleic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, or a combination thereof.

[0018] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and at least about 40% of the active agent is released after 6 hours.

[0019] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and at least about 60% of the active agent is released after 12 hours.

[0020] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and at least about 80% of the active agent is released after 24 hours.

[0021] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and at least about 90% of the active agent is released after 48 hours.

[0022] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and releases less than about 40% of the active agent after 24 hours.

[0023] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and releases less than about 60% of the active agent after 48 hours.

[0024] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and less than about 80% of the active agent is released after 72 hours.

[0025] In some embodiments, the pharmaceutical composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and less than about 90% of the active agent is released after 144 hours.

[0026] In some embodiments, the pharmaceutical composition is contained in a sterile glass or polyolefin container.

[0027] In some embodiments, the calcium lactate is coated with a pharma- ceutically acceptable enteric coating. In some embodiments, the enteric coating comprises hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or combinations thereof. In some embodiments, the weight ratio of calcium lactate to the enteric coating is 10:0.5 to 1:1.5. In some embodiments, in an in vitro dissolution test involving the USP Paddle method at 37°C and a paddle speed of 50 rpm, the composition is placed in 0.1 HCl for 120 minutes, followed by adjustment to pH 6.8 with phosphate buffer, and less than about 20% of the active agent is released after 30 minutes. In some embodiments, in an in vitro dissolution test involving the USP Paddle method at 37° C. and a paddle speed of 50 rpm, the composition is placed in 0.1N HCl for 120 minutes, and then adjusted to pH 6.8 with a phosphate buffer, and less than 30% of the active agent is released after 60 minutes. In some embodiments, in an in vitro dissolution test involving the USP Paddle method at 37° C. and a paddle speed of 50 rpm, the composition is placed in 0.1N HCl for 120 minutes, and then adjusted to pH 6.8 with a phosphate buffer, and less than 50% of the active agent is released after 120 minutes. In some embodiments, in an in vitro dissolution test involving the USP Paddle method at 37° C. and a paddle speed of 50 rpm, the composition is placed in 0.1N HCl for 120 minutes, and then adjusted to pH 6.8 with a phosphate buffer, and less than 10% of the active agent is released after 120 minutes.

[0028] In some embodiments, the calcium lactate is provided in a food or nutritional composition that comprises calcium lactate. In some embodiments, the food or nutritional composition is an injectable nutritional supplement. [The present invention 1001] 1. A method for treating Alzheimer's disease, Parkinson's disease, atherosclerosis, and / or stroke, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. [The present invention 1002] 1. A method for treating multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease (NAFLD), and / or rheumatoid arthritis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. [The present invention 1003] The method of the present invention 1002 for treating NAFLD, wherein NAFLD is non-alcoholic steatohepatitis (NASH). [The present invention 1004] 1. A method for treating pulpitis and / or periodontitis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. [The present invention 1005] 1. A method for treating inflammatory bowel disease (IBD), comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. [The present invention 1006] The method of the present invention 1005, wherein the IBD is Crohn's disease or ulcerative colitis. [The present invention 1007] 1. A method for treating spinal cord injury, cerebral hemorrhage, myocardial infarction, keratitis, and / or diabetic retinopathy, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. [The present invention 1008] The method of any of claims 1001 to 1007, wherein the calcium lactate is administered in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, excipient, or diluent. [The present invention 1009] The method of any of claims 1001-1008, wherein the pharmaceutical composition is formulated into a liquid, powder, aerosol, injection, infusion, patch, capsule, pill, tablet, depot, or suppository. [The present invention 1010] The method of any of claims 1001-1008, wherein the pharmaceutical composition comprises a therapeutically effective amount of calcium lactate as an active agent and a pharma- ceutically acceptable polysaccharide, polymer, lipid, or combination thereof. [The present invention 1011] The method of claim 10, wherein the pharmaceutical composition comprises calcium lactate and a polysaccharide. [The present invention 1012] The method of claim 1011, wherein the weight ratio of calcium lactate to polysaccharide is 1:<0.2 to 1:5. [The present invention 1013] The method of claim 1011, wherein the weight ratio of calcium lactate to polysaccharide is 1:<0.2. [The present invention 1014] The method of the present invention, wherein the weight ratio of calcium lactate to polysaccharide is 1:0.2 to 1:5. [The present invention 1015] The method of any one of claims 1010 to 1014, further comprising a polymer and / or a lipid. [The present invention 1016] The method of claim 1015, further comprising a polymer and a lipid, the weight ratio of polymer to lipid being from 1:0.1 to 1:50. [The present invention 1017] The method of any of claims 1015 to 1016, wherein the weight ratio of calcium lactate to polymer and / or lipid is at least 1:5. [The present invention 1018] The method of claim 1017, wherein the weight ratio of calcium lactate to polymer and / or lipid is 1:5 to 1:30. [The present invention 1019] The method of the present invention 1010, comprising calcium lactate and a polymer and / or lipid. [The present invention 1020] 1019. The method of claim 1019, comprising a polymer and a lipid, wherein the weight ratio of polymer to lipid is 1:0.1 to 1:50. [The present invention 1021] The method of any of claims 1019 to 1020, wherein the weight ratio of calcium lactate to polymer and / or lipid is at least 1:5. [The present invention 1022] The method of any of claims 1019 to 1021, wherein the weight ratio of calcium lactate to polymer and / or lipid is 1:5 to 1:30. [The present invention 1023] The method of any one of claims 1010 to 1022, wherein the composition is short-acting. [The present invention 1024] The method of any one of claims 1010 to 1022, wherein the composition is long-acting. [The present invention 1025] The method of any one of claims 1010 to 1024, wherein the composition is an injectable composition. [The present invention 1026] The method of any of claims 1010 to 1025, comprising a polysaccharide that is a cellulose derivative, a pectin, a hyaluronic acid, a starch, a guar gum, a chitosan, a gelatin, a collagen, an alginate, an alginic acid, or a combination thereof. [The present invention 1027] The method of any of claims 1010 to 1026, comprising a polymer that is a poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic-lactic acid (PLGA), or a combination thereof. [The present invention 1028] 8. The method of any of claims 1010-1027, comprising a lipid which is a mono- or tri-fatty acid glycerol ester or polyethylene glycol, a polyethylene glycol ester of a vegetable oil, a fatty acid propylene glycol ester, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa oil, cottonseed oil, sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenated oil, oleic acid, linoleic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, or a combination thereof. [The present invention 1029] Any of the methods of inventions 1010-1023 and 1025-1029, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 40% of the active agent is released after 6 hours. [The present invention 1030] Any of the methods of inventions 1010-1023 and 1025-1029, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 60% of the active agent is released after 12 hours. [The present invention 1031] The method of any of claims 1010-1023 and 1025-1030, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37°C at 300 rpm, and at least about 80% of the active agent is released after 24 hours. [The present invention 1032] Any of the methods of inventions 1010-1023 and 1025-1031, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 90% of the active agent is released after 48 hours. [The present invention 1033] Any of the methods of inventions 1010-1012, 1014-1022, and 1024-1028, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and wherein less than about 40% of the active agent is released after 24 hours. [The present invention 1034] Any of the methods of inventions 1010-1012, 1014-1022, 1024-1028, and 1033, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm at 37° C. in 200 ml of aqueous medium having a pH of 6.8 at 300 rpm, and wherein less than about 60% of the active agent is released after 48 hours. [The present invention 1035] Any of the methods of inventions 1010-1012, 1014-1022, 1024-1028, and 1033-1034, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and wherein less than about 80% of the active agent is released after 72 hours. [The present invention 1036] Any of the methods of inventions 1010-1012, 1014-1022, 1024-1028, and 1033-1035, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and wherein less than about 90% of the active agent is released after 144 hours. [The present invention 1037] The method of any one of claims 1010 to 1036, wherein the composition is contained in a sterile glass or polyolefin container. [The present invention 1038] The method of any of claims 1001 to 1008, wherein the calcium lactate is coated with a pharma- ceutically acceptable enteric coating. [The present invention 1039] The method of claim 1038, wherein the enteric coating comprises hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or combinations thereof. [The present invention 1040] The method of any of claims 1038 to 1039, wherein the weight ratio of calcium lactate to the enteric coating is 10:0.5 to 1:1.5. [The present invention 1041] Any of the methods of claims 1038 to 1040, wherein in an in vitro dissolution test including the USP Paddle Method at 37°C and a paddle speed of 50 rpm, when the composition is placed in 0.1 HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than about 20% of the active agent is released after 30 minutes. [The present invention 1042] Any of the methods of claims 1038 to 1041, wherein in an in vitro dissolution test including the USP Paddle Method at 37°C and a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than 30% of the active agent is released after 60 minutes. [The present invention 1043] Any of the methods of claims 1038 to 1042, wherein in an in vitro dissolution test including the USP Paddle Method at 37°C and a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than 50% of the active agent is released after 120 minutes. [The present invention 1044] Any of the methods of claims 1038 to 1043, wherein in an in vitro dissolution test including the USP Paddle Method at 37°C and a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than 10% of the active agent is released after 120 minutes. [The present invention 1045] The method of any of claims 1001 to 1007, wherein the calcium lactate is provided in a food or nutritional composition comprising calcium lactate. [The present invention 1046] The method of claim 1045, wherein the composition is an injectable nutritional supplement. [Brief description of the drawings]

[0029] [Figure 1]Human leukemia mononuclear cells in vitro experiment. Western blot results showed that inflammatory factors (NF-κb) were reduced by 2.5 mM calcium lactate treatment in THP-1 cells (human leukemia mononuclear cells) under hypoxic conditions. [Diagram 2] In vitro experiments on human leukemic monocytes differentiated into macrophages. Western blot results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in THP-1 cells (M0 macrophages) differentiated with PMA (phorbol-12-myristate-13-acetate) under normoxic and hypoxic conditions. [Diagram 3] Western blot results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in interferon gamma (IFN-γ) and lipopolysaccharide (LPS)-differentiated THP-1 cells (M1 macrophages) under normoxic and hypoxic conditions. [Figure 4] Western blot results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in interleukin-4 (IL-4) and interleukin-10 (IL-10)-differentiated THP-1 cells (M2 macrophages) under normoxic and hypoxic conditions. [Diagram 5] Liver fibrosis in vitro experiment. Western blot results showed that hypoxia-inducible factor-1α (HIF-1α) was decreased by 2.5 mM calcium lactate treatment in LX-2 cells (human hepatic stellate cells) under hypoxic conditions. [Figure 6] Human endothelial cell in vitro experiment. Western blot results showed that 2.5 mM calcium lactate treatment reduced hypoxia mediator (lactate dehydrogenase A) in human umbilical vein endothelial cells under hypoxic conditions. [Figure 7] Human fibroblast in vitro experiment. Western blot results showed that 2.5 mM calcium lactate treatment increased lactate dehydrogenase B in CCD-18Co cells (human colon fibroblasts) under hypoxic conditions. [Figure 8] Brain CID in vitro experiments. Western blot results showed that 2.5 mM calcium lactate treatment reduced hypoxia-mediating factors in SK-N-SH cells (brain epithelium) under normoxic and hypoxic conditions. [Figure 9] Liver CID in vitro experiment. Western blot results showed that 2.5 mM calcium lactate treatment altered hypoxia-mediated factors in HepG2 cells (hepatic epithelium) under hypoxic conditions. [Figure 10] Western blot results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (TLR-4) in HepG2 cells (hepatic epithelium) under hypoxic conditions. [Figure 11] Ocular CID in vitro experiments. Western blot results showed that 2.5 mM calcium lactate treatment reduced hypoxia-mediating factors in ARPE-19 cells (retinal pigment epithelium) under normoxic and hypoxic conditions. [Figure 12] Immunocytochemistry results showed that 2.5 mM calcium lactate treatment reduced hypoxia-inducible factor-1α (HIF-1α) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions. [Figure 13] Immunocytochemistry results showed that lactate dehydrogenase A was decreased by 2.5 mM calcium lactate treatment in ARPE-19 cells (retinal pigment epithelium) under normoxic conditions. [Figure 14] Immunocytochemistry results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (nuclear factor kappa B) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions. [Figure 15] Immunocytochemistry results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (Toll-like receptor 4) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions. [Figure 16] Immunocytochemistry results showed that 2.5 mM calcium lactate treatment increased lactate dehydrogenase B in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions. [Figure 17] Immunocytochemistry results showed that 2.5 mM calcium lactate treatment reduced drusen marker (apolipoprotein E) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions. [Figure 18] Western blot results showed that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions. [Figure 19] The photograph shows that there is no toxicity from 2.5 mM calcium lactate treatment in ARPE-19 cells (retinal pigment epithelium). Calcium lactate plays only a key role in the metabolic changes of injured epithelial cells. [Figure 20] Liver CID (NASH) in vivo experiment. Blood chemistry results showed that 2mg / kg calcium lactate treatment significantly decreased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). **P<0.001 vs. methionine-choline deficient (MCD). Results are mean ± SD. [Figure 21] Immunohistochemistry results show that 2 mg / kg calcium lactate treatment prevented fatty degeneration in the liver. [Figure 22] Immunohistochemistry results show that the 2 mg / kg calcium lactate treated group, like the control group, lacked lipid droplets in the liver, whereas the methionine-choline deficient (MCD) group did. [Figure 23] Immunohistochemistry results show that the 2 mg / kg calcium lactate treatment group had reduced immune cell infiltration in the liver, similar to the control group, while the methionine-choline deficient (MCD) group had increased immune cell infiltration. [Figure 24] Immunohistochemistry results show that in the 2 mg / kg calcium lactate treated group, similar to the control group, there is no fibrosis in the liver, whereas it is present in the methionine-choline deficient (MCD) group. [Diagram 25]Western blot results showed that inflammatory factors (NF-κB) were reduced in retinal pigment epithelium tissues by treatment with 2.5 mM calcium lactate. [Figure 26] Immunohistochemistry results showed that treatment with 2.5 mM calcium lactate regenerated retinal pigment epithelium with morphology similar to that of normal control epithelium. [Figure 27] Fluorescence image of lesions in the inner layer of the choroid on a flat mount due to laser-induced age-related macular degeneration (AMD). [Figure 28] Fluorescence images of lesions and neovascularization in the inner layer of the choroid on flat mounts due to laser-induced age-related macular degeneration (AMD). [Figure 29] Experimental scheme for establishing Alzheimer's disease and stroke. [Diagram 30] Representative images of lipopolysaccharide (LPS)-induced brain injury. Contralateral: normal area. Ipsilateral: LPS injection. [Diagram 31] Regeneration of damaged brain tissue and reduced microglial infiltration recruited during brain injury. [Diagram 32] Experimental scheme for establishing Parkinson's disease. [Diagram 33] Regeneration of dopaminergic neurons by treatment with calcium lactate. [Diagram 34] Experimental scheme for establishing multiple sclerosis. [Diagram 35] Regeneration of spinal cord demyelination and reduction of microglial infiltration by treatment with calcium lactate. [Diagram 36] Representative histological profiles of aortic tissue. Tissues on the left and center are hematoxylin and eosin (H&E) stained. Tissues on the right are Oil Red O stained. [Figure 37] Masson's trichrome staining for periodontal tissues. [Figure 38] Masson's trichrome stain for the upper gingiva. [Figure 39] Masson's trichrome staining of leg and knee joint tissues. [Diagram 40]Experimental scheme for inducing Crohn's disease. [Diagram 41] Representative images of hematoxylin and eosin (H&E) staining of Crohn's disease. [Diagram 42] Experimental scheme for inducing colitis. [Diagram 43] Representative images of hematoxylin and eosin (H&E) staining of colitis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description of the Invention Recently, many studies have shown that CID is related to aging and suggested that chronic inflammation is associated with age-related immunosenescence diseases (1-29). Chronic inflammation was elucidated to occur through complex interactions between parenchymal and stromal cells (such as immune cells, endothelial cells, and fibroblasts). Among them, cells originating from the bone marrow, such as monocytes and activated macrophages, were identified as the main effector cells in CID. Upon activation by bacterial lipopolysaccharide, IFN-gamma, and damaged cells, macrophages produced many inflammatory cytokines and cleared endogenous and exogenous inflammatory substances through transcriptional regulation by nuclear factor kappa B (NF-κB). Macrophages were also further polarized into more specialized macrophage subtypes. However, the exquisitely regulated differentiation of macrophages and activation of myeloid cells were dysregulated in chronic inflammation. Local tissue hypoxia caused metabolic changes in cells, such as parenchymal and interstitial cells, through the induction of hypoxia-inducible factors (HIFs). HIFs, especially HIF-1, contribute to the abnormal activation of macrophages and are major contributors to chronic inflammatory responses.

[0031] Most CIDs are characterized by inflammation. Injured tissues become resistant to blood flow and oxygen transport, resulting in a hypoxic environment. Proinflammatory mediators and hypoxic conditions can induce an angiogenic response through induction of HIF-1. In addition, local inflammatory responses exist in most CIDs, including respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, pneumonitis, and pulmonary fibrosis), eye diseases (e.g., keratitis and age-related macular degeneration), autoimmune diseases, nephropathy, neurodegenerative diseases (e.g., Alzheimer's disease, stroke, Parkinson's disease, and multiple sclerosis), cardiovascular diseases (e.g., atherosclerosis, arteriosclerosis, and myocarditis), metabolic disorders (e.g., diabetes and obesity), musculoskeletal diseases (e.g., rheumatoid arthritis and osteoporosis), periodontal diseases (e.g., pulpitis and periodontitis), digestive diseases (e.g., nonalcoholic fatty liver disease, gastroenteritis, and chronic inflammatory bowel disease), and skin disorders (e.g., psoriasis and atopic dermatitis). During inflammation, vascular permeability increases, and monocytes, macrophages, platelets, mast cells, and other leukocytes are recruited under the attraction of chemokines. Therefore, disclosed herein are therapeutic approaches targeted at regulating macrophage activation and extracellular matrix degradation by targeting HIF-1, NF-κB and lactate dehydrogenase (LAD) A and B.

[0032] Treatment Method Disclosed herein is a method of treating a chronic inflammatory disease, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0033] Disclosed herein is a method for treating Alzheimer's disease, Parkinson's disease, atherosclerosis, and / or stroke, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0034] Also disclosed is a method for treating multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease (NAFLD) and / or rheumatoid arthritis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.In some embodiments, the method is for treating NAFLD, and NAFLD is non-alcoholic steatohepatitis (NASH).

[0035] Also disclosed is a method of treating pulpitis and / or periodontitis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0036] Also disclosed is a method of treating inflammatory bowel disease (IBD), comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. In some embodiments, the IBD is Crohn's disease or ulcerative colitis.

[0037] Further disclosed is a method for treating spinal cord injury, cerebral hemorrhage, myocardial infarction, keratitis, and / or diabetic retinopathy, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0038] Disclosed herein is a method for treating asthma, pulmonary fibrosis, obesity, gastroenteritis, chronic inflammatory bowel disease, and / or atopic dermatitis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.Disclosed herein is a method for treating chronic obstructive pulmonary disease, pneumonitis, keratitis, atherosclerosis, arteriosclerosis, myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis, and / or psoriasis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.Disclosed herein is a method for treating Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease, and / or sepsis, comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0039] Also disclosed herein is a method of treating osteoporosis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0040] The term "subject" or "patient" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In some embodiments of the methods and compositions provided herein, the mammal is a human or a non-human.

[0041] As used herein, "treat," "treating," or "treatment" of one or more diseases disclosed herein by administration of a pharmaceutical composition refers to either a reduction in severity, a delay in onset, a slowing in progression, or a shortening in duration, whether permanent or temporary, persistent or interim, of one or more diseases disclosed herein that may result from or be associated with the administration of the composition.

[0042] The term "co-administration" and similar terms, as used herein, is meant to encompass the administration of two or more selected active agents to one patient, and is intended to include treatment regimens in which the active agents are administered by the same or different routes of administration or at the same or different times.

[0043] The pharmaceutical compositions of the present disclosure can be administered in a therapeutically effective amount, and as used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to any medical treatment or prevention. The effective dosage level can be determined according to the severity of the disease, the activity of the drug or active agent, the patient's age, weight, health and sex, sensitivity to drugs, administration time, administration route; as well as the excretion rate of the compositions of the present disclosure, the duration of treatment, drugs used simultaneously or in combination with the compositions of the present disclosure, and other factors known in the medical field. The pharmaceutical compositions of the present disclosure can be administered alone or in combination with other known drugs or ingredients known for the treatment of one or more diseases disclosed herein. Taking all of the above factors into consideration, it is important to administer the composition in the smallest amount that can show maximum effect without causing side effects.

[0044] The pharmaceutical composition of the present disclosure can be administered so that the daily dose of calcium lactate is, for example, about 10 mg / kg to about 1,000 mg / kg, about 10 mg / kg to about 500 mg / kg, about 10 mg / kg to about 250 mg / kg, about 10 mg / kg to about 200 mg / kg, about 10 mg / kg to about 100 mg / kg, about 1 mg / kg to about 100 mg / kg, about 1 mg / kg to about 75 mg / kg, about 1 mg / kg to about 50 mg / kg, about 1 mg / kg to about 25 mg / kg, or about 1 mg / kg to about 10 mg / kg. The frequency of administration of the composition can be divided into divided doses, such as once, twice, three times, or four times a day, but is not particularly limited thereto.

[0045] The compositions containing the compounds described herein can be administered for preventive and / or therapeutic treatment. In therapeutic applications, the compositions are administered to patients already suffering from disease or pathology in an amount sufficient to cure or at least partially prevent the symptoms of disease or pathology. The amount effective for this use depends on the severity and course of disease or pathology, treatment history, the patient's health condition, weight and response to drugs, and the judgment of the attending physician. It is considered well within the skill of one of ordinary skill in the art to determine such therapeutically effective amounts by routine experimentation (including but not limited to dose escalation clinical trials).

[0046] In prophylactic applications, compositions containing the active agents described herein are administered to patients susceptible to or otherwise at risk of one or more diseases disclosed herein. Such an amount is defined as a "prophylactically effective amount or dose." In this use, the exact amount also depends on the patient's health condition, weight, etc. It is considered well within the skill of those skilled in the art to determine such prophylactically effective amounts by routine experimentation (e.g., dose escalation clinical trial). When used in patients, the effective amount for this use depends on the severity and course of the disease, disorder or condition, previous treatment, the patient's health condition and response to drugs, and the judgment of the attending physician.

[0047] If the patient's condition does not improve, administration of the active agents described herein can be carried out chronically, i.e., for an extended period of time (including the entire life of the patient), at the physician's discretion, to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.

[0048] The amount of a given agent that corresponds to such an amount will vary depending on factors such as the particular compound, the disease state and its severity, the identity of the subject or host requiring treatment (e.g., age, weight, sex, etc.), but regardless can be routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, for example, the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0049] Pharmaceutical Compositions The term "calcium lactate" means C 6 H 10 O 6 Ca 5H 2 It refers to a type of metal lactate, represented by O, which can exist as a hydrate, for example, where calcium ions are bound to lactic acid. Calcium lactate can be in the form of a white powder or granules at room temperature and anhydrous at heating conditions of 120°C, and has a solubility of 5% (w / v).

[0050] Calcium lactate can be formulated into a pharmaceutical composition for treating one or more of the diseases disclosed herein.

[0051] In various embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of calcium lactate as an active agent for treating one or more diseases disclosed herein, and a pharma- ceutically acceptable polysaccharide, polymer, lipid, or combination thereof. In some embodiments, the pharmaceutical composition comprises calcium lactate and a polysaccharide.

[0052] In some embodiments, the present invention provides an enteric coating of calcium lactate such that the active agent is protected from the acidic environment of the stomach and is absorbed in the small intestine before reaching the large intestine when the active agent is orally administered.

[0053] The present invention also provides short-acting and long-acting pharmaceutical compositions comprising calcium lactate. In some embodiments, the long-acting compositions comprise calcium lactate coated with at least one enteric coating material, such as hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, and polymers of methacrylic acid and its esters.

[0054] The pharmaceutical composition of the present invention can be formulated into pharmaceutical preparations for oral administration.Examples of preparations include, but are not limited to, powders, tablets, capsules, granules or syrups, tablets and capsules.

[0055] Also provided herein are formulations of hydrogels, specifically methylcellulose, poloxamer, pectin, and alginate hydrogels, that can remain in the form of a solution or nanoparticles in vitro and form gels when injected into the body, allowing sustained release of calcium lactate. The drawback of hydrogels, the relatively short drug release time, has been improved by increasing the interaction between the drug and the hydrogel or by slowing down the diffusion of the drug in the hydrogel.

[0056] The weight ratio of calcium lactate to polysaccharide can be, for example, 1:<0.2 to 1:5, 1:0.01 to 1:5, 1:0.05 to 1:5, or 1:0.1 to 1:5. The weight ratio of calcium lactate to polysaccharide can be 1:<0.2. The weight ratio of calcium lactate to polysaccharide can be 1:0.2 to 1:5.

[0057] In some embodiments, the pharmaceutical composition further comprises a polymer or lipid. The weight ratio of calcium lactate to polymer or lipid can be at least 1:5. The weight ratio of calcium lactate to polymer or lipid can be 1:5 to 1:30, for example, 1:5 to 1:30, 1:5 to 1:20, 1:5 to 1:10, 1:10 to 1:30, 1:10 to 1:20, or 1:20 to 1:30.

[0058] In some embodiments, the pharmaceutical composition further comprises a polymer and lipid. The weight ratio of calcium lactate to polymer and lipid can be at least 1:5. The weight ratio of calcium lactate to polymer and lipid can be 1:5 to 1:30, for example, 1:5 to 1:30, 1:5 to 1:20, 1:5 to 1:10, 1:10 to 1:30, 1:10 to 1:20, or 1:20 to 1:30.

[0059] In some embodiments, the pharmaceutical composition comprises calcium lactate and a polymer or lipid. The weight ratio of calcium lactate to polymer or lipid can be at least 1:5. The weight ratio of calcium lactate to polymer or lipid can be 1:5 to 1:30. The weight ratio of calcium lactate to polymer or lipid can be 1:5 to 1:30, for example, 1:5 to 1:30, 1:5 to 1:20, 1:5 to 1:10, 1:10 to 1:30, 1:10 to 1:20, or 1:20 to 1:30.

[0060] In some embodiments, the pharmaceutical composition comprises calcium lactate, a polymer and a lipid. The weight ratio of calcium lactate to polymer and lipid can be at least 1:5. The weight ratio of calcium lactate to polymer and lipid can be 1:5 to 1:30. The weight ratio of calcium lactate to polymer and lipid can be 1:5 to 1:30, for example, 1:5 to 1:30, 1:5 to 1:20, 1:5 to 1:10, 1:10 to 1:30, 1:10 to 1:20, or 1:20 to 1:30.

[0061] In some embodiments, the weight ratio of polymer to lipid can be 1:0.1 to 1:50, 1:0.1 to 1:20, 1:0.1 to 1:10, 1:0.1 to 1:5, 1:0.1 to 1:2, 1:0.1 to 1:1, 1:0.1 to 0.5, or 1:0.1 to 1:0.2.

[0062] Polysaccharides suitable for use in the composition can be cellulose derivatives (e.g., carboxymethylcellulose (CMC), ethylcellulose (EC), hydroxypropylmethylcellulose (HPMC), methylcellulose (MC)), pectin, hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen, alginates, alginic acid, or combinations thereof.

[0063] Polymers suitable for use in the composition can be poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic acid-lactic acid (PLGA), or combinations thereof.

[0064] Lipids suitable for use in the composition can be mono- or tri-fatty acid glycerol esters or polyethylene glycols, polyethylene glycol esters of vegetable oils, fatty acid propylene glycol esters, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa butter, cottonseed oil, sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenated oils, oleic acid, linoleic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, or combinations thereof.

[0065] The present invention is further directed to a pharmaceutical composition comprising a therapeutically effective amount of calcium lactate as an active agent for treating one or more diseases disclosed herein, wherein the calcium lactate is coated with a pharma- ceutically acceptable enteric coating. In some embodiments, the enteric coating comprises hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or combinations thereof. In some embodiments, the weight ratio of calcium lactate to the enteric coating is 10:0.5-10:15, 10:0.5-1:1, 10:0.5-10:5, 10:0.5-10:3, 10:0.5-10:2, 10:0.5-10:1, 10:0.5-1:0.8.

[0066] "Pharmaceutically acceptable," as used herein, refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic, i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0067] Pharmaceutical composition, as used herein, refers to a mixture of calcium lactate with other pharma- ceutically acceptable chemical components, such as, but not limited to, carriers, stabilizers, diluents, disintegrants, suspending agents, thickening agents, binders, antimicrobial agents, antimicrobial preservatives, antioxidants, and / or buffers. Pharmaceutical compositions facilitate administration of calcium lactate to a subject.

[0068] The term "carrier" as used herein refers to a relatively non-toxic compound or agent that facilitates the uptake of a compound into cells or tissues.The term "diluent" refers to a compound that is used to dilute the compound of interest prior to delivery.Diluents can also be used to stabilize compounds, as they can provide a more stable environment.Pharmaceutically acceptable additives include, but are not limited to, diluents, binders, solubilizers, solubility enhancers, pore-forming agents, osmotic agents, gas-forming agents, lubricants and flow agents that are well known in the art.

[0069] Diluents may include, but are not limited to, lactose, fructose, dextrose, sucrose, maltose, microcrystalline cellulose, starch, calcium hydrogen phosphate, mannitol, or mixtures thereof. These diluents include microcrystalline cellulose, lactose, mannitol, calcium phosphate, and the like.

[0070] Examples of binders can include povidone, hydroxypropyl cellulose, polyvinyl alcohol, hydroxypropyl methylcellulose, sodium carboxymethyl-cellulose and the like.

[0071] Solubilizing agents include, but are not limited to, surfactants, cyclodextrin and its derivatives, lipophilic substances or mixtures thereof.

[0072] Surfactants include, but are not limited to, water-soluble or water-dispersible nonionic, nonpolar nonionic, anionic, cationic, amphoteric or ionic surface active agents or mixtures thereof.

[0073] Examples of disintegrants include crospovidone, croscarmellose sodium, sodium starch glycolate, and examples of lubricants include magnesium stearate, calcium stearate, sodium stearyl fumarate, and the like.

[0074] The pharmaceutical compositions of the present invention may further include antibacterial agents such as benzyl alcohol, chlorobutanol, phenylethyl alcohol, phenyl-mercuric acetate, potassium sorbate, and sorbic acid. Antifungal agents include compounds such as benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, and sodium benzoate.

[0075] Antimicrobial preservatives can be added to the pharmaceutical composition of the present invention to protect it from the growth of potentially harmful microorganisms that usually invade the aqueous phase but can also grow in the oil phase of the composition.Therefore, preservatives that have both water solubility and lipid solubility are desirable.Suitable antimicrobial preservatives include, for example, alkyl esters of p-hydroxybenzoic acid, propionates, phenoxyethanol, sodium methylparaben, sodium propylparaben, sodium dehydroacetate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, hydantoin derivatives, quaternary ammonium compounds and cationic polymers, imidazolidinyl urea, diazolidinyl urea, and trisodium ethylenediaminetetraacetate (EDTA).

[0076] Antioxidants, such as anoxomer, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, potassium metabisulfite, propyloctyl and dodecyl gallate, sodium metabisulfite, sulfur dioxide, and tocopherols, can be added to protect all of the components of the pharmaceutical composition from damage or degradation by oxidants present in the composition itself or in its environment of use.

[0077] Buffers can be used to maintain the desired pH of a pharmaceutical composition once established against the action of external agents and shifts in the equilibrium of the components of the composition.

[0078] The pharmaceutical compositions described herein can be prepared according to techniques known in the art, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference in their entireties.

[0079] In some embodiments, the pharmaceutical compositions of the present invention are short-acting. The term "short-acting" refers to a composition that releases substantially all of the active agent within 48 hours, e.g., from the time of delivery (i.e., 0 hours), from about 1 hour to about 48 hours, from about 3 hours to about 24 hours, from about 6 hours to about 24 hours, or from about 12 hours to about 24 hours, when tested in the in vitro dissolution test described herein.

[0080] For example, when the composition is placed in an in vitro dissolution test including a dissolution test method (e.g., provided by Labfine Co., Mumbai, India) using a nylon filter having a pore size of 45 μm, in 200 ml aqueous medium having a pH of 6.8 at 37° C., at 300 rpm, at least about 40% of the active agent is released after 6 hours, at least about 60% of the active agent is released after 12 hours, at least about 80% of the active agent is released after 24 hours, and / or at least about 90% of the active agent is released after 48 hours. In some embodiments, when the composition is placed in an in vitro dissolution test comprising a dissolution test method (e.g., from Labfine Co., Mumbai, India) using a nylon filter having a pore size of 45 μm, in 200 ml aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, at least about 40% to about 60% of the active agent is released after 6 hours, at least about 60% to about 80% of the active agent is released after 12 hours, at least about 80% to about 90% of the active agent is released after 24 hours, and / or at least about 90% to about 100% of the active agent is released after 48 hours.

[0081] In some embodiments, the pharmaceutical composition of the present invention is long-acting. The term "long-acting" is intended to mean a composition that slowly releases an active agent after initial administration, for example, from the time of delivery (i.e., 0 hours) for about 48 hours to about 192 hours, about 72 hours to about 192 hours, about 96 hours to about 192 hours, about 120 hours to about 192 hours, or about 144 hours to about 192 hours.

[0082] In some embodiments, when the composition is placed in an in vitro dissolution test involving a dissolution test method (e.g., from Labfine Co., Mumbai, India) using a nylon filter having a pore size of 45 μm, in 200 ml aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, less than about 40% of the active agent is released after 24 hours, less than about 60% of the active agent is released after 48 hours, less than about 80% of the active agent is released after 72 hours, and less than about 90% of the active agent is released after 144 hours. In some embodiments, when the composition is placed in an in vitro dissolution test involving a dissolution test method (e.g., provided by Labfine Co., Mumbai, India) using a nylon filter having a pore size of 45 μm, at 37° C. in 200 ml medium having a pH of 6.8, at 300 rpm, about 20% to about 50% of the active agent is released after 24 hours, about 20% to about 40% of the active agent is released after 24 hours, about 40% to about 70% of the active agent is released after 48 hours, about 40% to about 60% of the active agent is released after 48 hours, about 40% to about 80% of the active agent is released after 72 hours, about 50% to about 80% of the active agent is released after 72 hours, about 60% to about 90% of the active agent is released after 144 hours, or about 70% to about 90% of the active agent is released after 144 hours.

[0083] In some embodiments, when the composition is placed in an in vitro dissolution test involving the USP Paddle Method at 37° C. with a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than about 20% of the active agent is released after 30 minutes, less than 30% of the active agent is released after 60 minutes, less than 50% of the active agent is released after 120 minutes, and / or less than 10% of the active agent is released after 120 minutes.

[0084] In some embodiments, the pharmaceutical composition of the present invention is in an injectable form. For parenteral injection, suitable formulations can include aqueous or non-aqueous solutions, preferably with a physiologically compatible carrier.

[0085] Pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds in water-soluble form.In addition, suspensions of active agents can be prepared as suitable oily injection suspensions.Suitable lipids or lipophilic carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. The lipids can be mono- or tri-fatty acid glycerol esters or polyethylene glycols, polyethylene glycol esters of vegetable oils, fatty acid propylene glycol esters, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa butter, cottonseed oil, sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenated oils, oleic acid, linoleic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, or combinations thereof.

[0086] Since hydrogels were applied in medicine, numerous hydrogels have been developed and studied in many fields, including the medical, pharmaceutical, and cosmetic industries. Although hydrogels are generally biocompatible, they have various problems that limit the delivery of drugs, and various efforts have been made to solve these problems. Hydrogels are three-dimensional structures composed of a network of hydrophilic polymers. More than 90% of their components are composed of water. Hydrogels have been actively studied in the field of biomedicine due to their similarity to living tissues, such as high water content, porous structure, relatively soft properties, and biocompatibility. Hydrogels can exhibit various properties depending on the type of polymer used as the backbone or the crosslinking method employed. When synthetic compounds such as polyacrylic acid series polymers or polyvinyl alcohol are used, they have low biocompatibility, but their chemical modification is easy, and therefore engineering applications are very easy. On the other hand, when natural compounds, especially pectin, alginic acid, collagen, fibrin, and hyaluronic acid, are used as the backbone, chemical modification is difficult. Nevertheless, there are advantages to using these materials, which are biologically derived components, since they are suitable for clinical applications and cause almost no side effects such as immune inflammatory reactions upon implantation.

[0087] The crosslinking method affects not only the type of polymer used but also the properties of hydrogels. Even if the same polymer is used as the backbone, different crosslinking methods can result in hydrogels with completely different characteristics. Methods for crosslinking hydrogels can be broadly divided into physical and chemical methods. Physical crosslinking methods include reversible crosslinking through ionic interactions, hydrophobic interactions, hydrogen bonds, and structural entanglements. These crosslinking methods can easily cause the formation of a three-dimensional network structure without the need for separate chemical additives or complicated processes. On the other hand, chemical crosslinking methods typically form covalent bonds that are irreversible and result in a stable network structure compared to physical crosslinking methods. Hydrogels with excellent biocompatibility and various physicochemical properties have been widely studied in the fields of biomedicine, such as drug delivery and tissue engineering. Most hydrogels exhibit shorter drug release times than other drug delivery systems due to their high water content, and there is a need to develop systems with longer drug release times.

[0088] In some embodiments, the pharmaceutical composition of the present invention is in oral dosage form.For oral administration, the compound described herein can be easily formulated by combining active agent with pharma-ceutically acceptable carrier or excipient well known in the art.Such carrier allows the active agent described herein to be formulated as tablet, powder, pill, dragee, capsule, liquid, gel, syrup, elixir, slurry, suspension, etc., for oral ingestion by the patient to be treated.

[0089] The pharmaceutical preparation for oral use can be obtained by mixing one or more solid carriers with the compound described herein, optionally grinding the mixture obtained, and then processing the mixture of granules after adding suitable auxiliary agents.Suitable excipients include, but are not limited to, fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations: such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.; or others: such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.Optionally, disintegrants can be added, such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts (e.g., sodium alginate).

[0090] Dragee core is provided with suitable coating.For this purpose, concentrated sugar solution can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solution, and suitable organic solvent or solvent mixture.Dyes or pigments can be added to tablet or dragee coating for identification or to characterize different combinations of active compound doses.

[0091] Pharmaceutical compositions can be formulated in a conventional manner using one or more pharma- ceutically acceptable carriers, including excipients and auxiliaries that facilitate the processing of active agents into pharma- ceutically usable preparations.Appropriate formulations depend on the route of administration selected.Any well-known technique can be used as appropriate and as understood in the art.The pharmaceutical compositions described herein can be prepared in a conventional manner, for example, by way of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, encapsulating or compressing process, for example, by way of example only.

[0092] Aqueous suspensions can also contain one or more polymers as suspending agents. The polymer can be poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic acid-lactic acid (PLGA), or a combination thereof. Other useful polymers include water-soluble polymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Useful compositions can also include a mucoadhesive polymer selected from, e.g., carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0093] In some embodiments, a pharmaceutical dosage form is provided that includes calcium lactate and a pharma- ceutically acceptable enteric coating for controlling the release of the active agent. In some embodiments, the coating is a film, and in other embodiments, a membrane. The enteric coating, e.g., a film or membrane, can serve to delay release until after the stomach and to protect the active agent from gastric juices. The enteric coating can include one or more preferably polymeric substances (e.g., methacrylates, etc.; polysaccharides, etc., as described in detail below), or a combination of two or more such substances, optionally including other excipients, e.g., plasticizers, etc. In some embodiments, the enteric coating includes hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or combinations thereof. In some embodiments of the present invention, the composition includes a hydrogel-forming polymer and additional polymers that can achieve a desired delay (or other change) in the release of the active agent.

[0094] In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Typically, lubricants such as magnesium stearate are also added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch.

[0095] The sterile injectable preparation may also be a sterile injectable suspension in a non-toxic parenterally acceptable carrier such as a lipid.

[0096] The amount of calcium lactate contained in the pharmaceutical composition can be, but is not limited to, 1 wt%-50 wt%, 1 wt%-40 wt%, 1 wt%-35 wt%, 1 wt%-30 wt%, 1 wt%-20 wt%, 1 wt%-15 wt%, or 1 wt%-10 wt% based on the total weight of the final composition. The concentration of calcium lactate contained in a single dose of the pharmaceutical composition can be, but is not limited to, 2.5 mM-100 mM, 2.5 mM-50 mM, 2.5 mM-25 mM, 5 mM-100 mM, 5 mM-50 mM, 5 mM-25 mM, 10 mM-100 mM, 10 mM-50 mM, or 10 mM-25 mM.

[0097] The solid oral pharmaceutical compositions can be prepared by conventional techniques, such as, but not limited to, dry granulation, direct compression, wet granulation, extrusion spheronization, melt granulation, or compression coating. The coating can be applied as described below and can vary in terms of thickness and density. The amount of coating is defined by the additional weight added to (and thereby obtained by) the dry composition of the invention (e.g., beads or cores containing calcium lactate). The weight gain can range from 0.1% to 50%, 1% to 20%, 1% to 15%, 3% to 10%, 5% to 12%, or 8% to 12%.

[0098] The coating process can be carried out by any suitable means, such as by using a coating device that applies a solution of the polymer coat (particularly as described above) to the composition. The polymers for coating are either provided by the manufacturer in a ready-made solution for direct use or can be constituted before use according to the manufacturer's instructions.

[0099] Unit Dose and Kits The term "unit dosage form" refers to physically discrete units suitable as single dosages, each unit containing a predetermined amount of active ingredient in combination with a suitable pharmaceutical excipient, one or more of which can be used throughout a dosing regimen to produce a desired therapeutic effect, e.g., to treat one or more of the diseases disclosed herein.

[0100] The pharmaceutical compositions described herein can be in unit dosage form suitable for single administration of precise dosage. In unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more active agents. The unit dosage can be in packaged form containing discrete amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection can be presented in unit dosage form (including but not limited to ampoules) or in multi-dose containers.

[0101] Suitable daily dosages for calcium lactate can be about 1 mg / kg to about 1000 mg / kg, about 10 mg / kg to about 750 mg / kg, about 10 mg / kg to about 500 mg / kg, or about 100 mg / kg to about 500 mg / kg (body weight). Indicated daily dosages in large mammals (including but not limited to humans) range from about 5 mg to about 100,000 mg, but are conveniently administered in divided doses (including but not limited to up to four times a day) or in long-acting form. Unit dosage forms suitable for administration contain about 10 mg to about 1000 mg, about 100 mg to about 1000 mg, about 500 mg to about 750 mg, about 25 mg to about 250 mg, about 50 mg to about 100 mg, about 10 mg to about 200 mg, or about 10 mg to about 250 mg of active agent. The frequency of administration of the compositions of the present disclosure can be, but is not limited to, divided doses once, twice, three times, four times, etc. per day. An appropriate "effective" amount in any individual case can be determined using techniques such as a dose escalation study.

[0102] The foregoing ranges are merely suggestions, due to the large number of variables involved in each individual treatment regimen, and significant deviations from these recommendations are not uncommon. Such dosages can vary depending on a number of variables, including, but not limited to, the activity of the compound used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0103] The toxicity and therapeutic efficacy of such treatment regimens may be determined using, but not limited to, LD 50 (a dose lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 and ED 50The therapeutic index can be expressed as a ratio between the ED and the dose at which toxicity is minimal. Compounds that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dose of the active agent is determined to be the ED at which toxicity is minimal. 50 The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0104] According to the present invention, the pharmaceutical composition can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.The pharmaceutical composition of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.

[0105] The present invention is also directed to a sterile glass or polyolefin container containing a pharmaceutical composition disclosed herein. In some embodiments, the container is non-DEHP (bis(2-ethylhexyl) phthalate (di-2-ethylhexyl phthalate, diethylhexyl phthalate, DEHP; dioctyl phthalate, DOP) or non-PVP (polyvinylpyrrolidone).

[0106] The kit comprises suitable containers such as boxes, individual bottles, bags or ampoules. The suspension compositions can be packaged in single-dose non-reclosable containers or multi-dose reclosable containers.

[0107] US2017 / 0360727A1, published December 21, 2017, and PCT / IB2017 / 054091 (International Filing Date: July 7, 2017), are hereby incorporated by reference in their entireties. Also, U.S. Patent Application No. 62 / 616,923, filed January 12, 2018, is hereby incorporated by reference in its entirety. EXAMPLES

[0108] Example 1 Cell Culture for Age-Related Macular Disease The human retinal pigment epithelial cell line ARPE-19 was purchased from the American Type Culture Collection (Manassas, VA, USA). ARPE-19 cells were maintained in RPMI 1640 containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Welgenc, Daegu, South Korea).

[0109] Example 2 Cell culture for Alzheimer's disease The human brain cell line SK-N-SH was purchased from the American Type Culture Collection (Manassas, VA, USA). SK-N-SH cells were maintained in RPMI 1640 containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Welgene, Daegu, South Korea).

[0110] Example 3 Cell Culture for Fibroblasts The human colonic fibroblast cell line CCD-18-Co was purchased from the American Type Culture Collection (Manassas, VA, USA). CCD-18Co cells were maintained in DMEM containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Welgene, Daegu, South Korea).

[0111] Example 4 Cell Culture for Nonalcoholic Steatohepatitis (NASH) The human hepatocellular carcinoma cell line HepG2 was purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2 cells were maintained in RPMI 1640 containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Welgene, Daegu, South Korea).

[0112] Example 5 Cell Culture for Vascular Disease Human umbilical vein endothelial cell line HUVEC was purchased from the American Type Culture Collection (Manassas, VA, USA). HUVEC cells were maintained in Endothelial Cell Growth Medium 2 (PromoCell, Heidelberg, Germany) containing Endothelial Cell Growth Medium 2 Supplement Mix.

[0113] Example 6 Macrophage culture Human hepatic stellate cell line LX-2 was purchased from Sigma-Aldrich (St. Louis, MO, USA). LX-2 cells were maintained in DMEM containing 2% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA), 100 IU / ml penicillin, and 100 μg / ml streptomycin (Welgene, Daegu, South Korea).

[0114] The human monocytic cell line THP-1 was purchased from the American Type Culture Collection (Manassas, VA, USA). THP-1 cells were maintained in RPMI 1640 containing 10% fetal bovine serum (Life Technologies, Gaithersburg, MD, USA), 100 IU / ml penicillin, 100 μg / ml streptomycin (Welgene, Daegu, South Korea), and 0.55 μM 2-mercaptoethanol. The cells were incubated at 4°C for 1 h at 20°C for 2 h at 20°C for 1 h at 20°C. 2Cells were cultured in both conditions: a humidified atmosphere at 37° C. containing 0.1% CO and hypoxic culture conditions maintained at 1%, 5% and 94% oxygen.

[0115] Example 7 reagent Calcium lactate (CaLa) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 2-mercaptoethanol was purchased from Gibco (Grand Island, NY, USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Enzo Lifesciences (Enzo Diagnostic, NY, USA). Human interferon-gamma, human interleukin-4 (IL-4), and human interleukin-10 (IL-10) were purchased from BPS Bioscience (San Diego, CA, USA). Paraformaldehyde (PFA) solution (4%) was purchased from Biosesang (Biosesang Inc., Gyeonggi, Korea).

[0116] Example 8 Cell viability assay The toxicity of CaLa was confirmed in ARPE-19 cells using a live cell movie analyzer JuLI™Br (NanoEnTek Inc., Seoul, Korea). 5 Cells were seeded at a density of 1000 cells / well. After 24 h of incubation, cells were treated with 2.5 mM CaLa for 12 and 24 h. During the CaLa treatment period, cells were continuously monitored for 24 h (30, 31).

[0117] Example 9 Macrophage differentiation The human mononuclear cell line THP-1 was purchased from the American Type Culture Collection (Manassas, VA, USA). THP-1 cells were maintained in RPMI 1640 medium (Hyclone Laboratories, Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT, USA), 100 IU / ml penicillin (Welgene, Daegu, South Korea), 100 μg / ml streptomycin (Welgene, Daegu, South Korea), and 0.55 μM 2-mercaptoethanol (Gibco, Grand Island, NY, USA). THP-1 cells were maintained at 37°C in a humidified atmosphere of 5% C02. THP-1 monocytes were differentiated into macrophages by incubation with 100 μg / ml phorbol 12-myristate 13-acetate (PMA) (Enzo Diagnostic, NY, USA) in RPMI 1640 medium for 12 h. Macrophages were polarized into Ml macrophages by incubation with 20 ng / ml IFN-γ (BPS Bioscience, San Diego, CA, USA) and 100 μg / ml LPS (Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640 medium for 24 h. Macrophage M2 polarization was obtained by incubation with 20 ng / ml IL-4 (BPS Bioscience, San Diego, CA, USA) and 20 ng / ml IL-10 (BPS Bioscience, San Diego, CA, USA) in RPMI 1640 medium for 24 h (32).

[0118] Example 10 Optical observation of cell viability The toxicity of CaLa was confirmed in ARPE-19 cells using a live cell movie analyzer JuLI™Br (NanoEnTek Inc., Seoul, Korea). 5Cells were seeded at a density of 1000 cells / well. After 24 hours of incubation, cells were treated with 2.5 mM CaLa for 12 and 24 hours. During the CaLa treatment period, cells were continuously monitored for 24 hours.

[0119] Example 11 Western blot analysis To prepare whole cell lysates, a protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitors (NA 3 VO 4Cells were lysed in lysis buffer (1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 10% glycerol) containing 1 mM NaF, 100 mM NaCl. Protein concentrations were then measured using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, MA). Total cell or nuclear lysates were transferred to polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk (Bio-Rad, Hercules, CA, USA) for 1 h, the membranes were incubated overnight at 4°C with primary antibodies diluted in TBST containing 100 mM Tris-HCI (pH 7.5), 1.5 M NaCl, and 0.5% Tween-20 (obtained from Sigma-Aldrich) supplemented with 5% BSA and 0.1% sodium azide (Sigma-Aldrich). Specific primary antibodies were against HIF-1α (1:1000), HIF-2α (1:1000), LDH-A (1:1000), LDH-B (1:1000), TLR-4 (1:1000), NF-κB (1:1000), VEGF (1:1000), α-tubulin (1:1000), actin (1:1000), and GAPDH (1:5000). The next day, membranes were washed with TBST and incubated with anti-rabbit secondary antibody (1:10000) for 2 h. Immunoblots were developed using Western blot detection reagent (Abclone, Seoul, Korea) and exposed to X-ray film (Agfa, Leverkusen, Germany) according to the manufacturer's protocol.

[0120] Example 12 immunocytochemistry ARPE-19 and SK-N-SH cells were fixed on Biocoat coverslips (BD bioscience, San Jose, CA, USA) with 4% PFA for 20 min and then incubated with primary antibodies for 15 h: HIF-1α (1:300, BD Biosciences, San Jose, CA, USA), LDH-A (1:300, Santa Cruz Biotechnology, Santa Cruz, CA USA), LDH-B (1:300, Santa Cruz Biotechnology, Santa Cruz, CA USA), NF-κB (1:300, Santa Cruz Biotechnology, Santa Cruz, CA USA), TLR-4 (1:300, Santa Cruz Biotechnology, Santa Cruz, CA USA), and apoE (1:200, Santa Cruz Biotechnology, Santa Cruz, CA USA). After washing with PBS, cells were incubated with anti-mouse secondary biotinylated antibody (1:2000, Vector Laboratories, Burlingame, CA USA) and visualized with streptavidin conjugated to fluorescein (Vector Laboratories, Burlingame, CA USA). Coverslips were mounted on microscope slides with VECTASHIELD® Hard Set™ mounting medium containing DAPI (Vector Laboratories, Burlingame, CA USA). Confocal fluorescent images were acquired under a 60× oil immersion lens by confocal laser scanning microscopy (LSCM, Nikon A1+, Tokyo, Japan).

[0121] Example 13 Animal Models for NASH All experiments were performed under the institutional guidelines established by the Institutional Animal Care and Use Committee of Gachon University (IACUC-2017-0008). Female 5-week-old C57BL / 6 mice were purchased from KOATEC (Pyeongtaek, South Korea) and fed a methionine-choline diet (MCD) for 4 weeks. When the mice weighed 15 g each, they were randomly dissected to identify nonalcoholic steatohepatitis (NASH). Mice were subcutaneously injected with calcium lactate (2 mg / kg) twice daily (BID) for 4 weeks.

[0122] Example 14 Animal Models for Age-Related Macular Degeneration All experiments were performed under the institutional guidelines (IACUC-2017-0008) established by the Institutional Animal Care and Use Committee of Gachon University. Six-week-old female C57BL / 6 mice (20–25 g) were purchased from Orient (Charles River Korea, Seoul, Korea). All animals were maintained at 22°C–25°C with food and water available ad libitum and in a 12-h light / dark cycle (lights on at 08:00). Mice were individually placed on an induction chamber and anesthesia was induced with 2% isoflurane (HANA PHARM CO., Seoul, Korea). Lesions were induced in each eye of the mice by laser photocoagulation (SDL405-700, SD Laser, China). The laser pulses were from a blue laser (wavelength, 405 nm; SD Laser). The laser parameters were spot size 100 μm, power 700 mw, and exposure time 1 s. A total of 15 mice were randomized into three groups: (i) no laser induction (n=3), (ii) laser burn and vehicle buffer injection (n=5), and (iii) CaLa, 2 mg / kg / day (BID) (n=7). CaLa treatment was subcutaneous for 14 days starting the day after laser photocoagulation (33, 34).

[0123] Example 15 Histological analysis of NASH models Mouse liver tissues (n=5) were dissected and fixed in 4% paraformaldehyde in PBS for 15 h at 4°C. Fixed liver tissues were embedded in paraffin and sectioned at 10 μm. Slides were incubated at 55°C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol series, and used for hematoxylin (Merck, Darmstadt, Germany) and eosin (H&E) staining (35). H&E stained paraffin sections (10 μm thick) were imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0124] Example 16 Histological analysis of age-related macular degeneration Two weeks after subcutaneous administration of 2 mg / kg CaLa, histopathological examination of retinal lesions was performed. Enucleated eyes were fixed with 4% paraformaldehyde (PFA) for 24 h at 4 °C, and the eyes obtained by removing the anterior segment were washed three times in phosphate-buffered saline. Fixed flat mounts were dehydrated in graded series of ethanol and embedded in paraffin. Paraffin sections (10 μm thick) were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma-Aldrich, St. Louis, MO, USA) (H&E) and imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetelar, Germany). Choroidal lesions and neovascularization on the flat mounts were observed by confocal microscopy (Carl Zeiss, Oberkochn, Germany).

[0125] Example 17 Masson's trichrome staining Mouse liver tissues (n=5) were dissected and fixed in 4% paraformaldehyde in PBS solution at 4°C for 15 h. Fixed tissues were embedded in paraffin and sectioned at 5 mm. Slides were incubated at 55°C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol series, refixed in Bouin's solution at 56°C for 1 h, rinsed in tap water for 5-10 min to remove yellow color, stained in Biebrich scarlet-acid fuchsin solution for 5 min, then in phosphomolybdic acid-phosphotungstic acid solution (1:1 ratio) for 30 min, and aniline blue solution for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 2-5 min. Dehydrated in 95% ethyl alcohol, absolute ethyl alcohol, and cleared in xylene. Mounted with mounting medium. Imaging was performed using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0126] Example 18 result Figure 1 is an in vitro experiment of human leukemia mononuclear cells. Western blot results show that inflammatory factors (NF-κb) were reduced by treatment with 2.5 mM calcium lactate in THP-1 cells (human leukemia mononuclear cells) under hypoxic conditions.

[0127] Figure 2 is an in vitro experiment of human leukemic monocytes differentiated into macrophages. Western blot results show that inflammatory factors (NF-κb) were reduced by 2.5 mM calcium lactate treatment in THP-1 cells (M0 macrophages) differentiated with PMA (phorbol-12-myristate-13-acetate) under normoxic and hypoxic conditions.

[0128] Figure 3 provides Western blot results showing that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in interferon gamma (IFN-γ) and lipopolysaccharide (LPS)-differentiated THP-1 cells (M1 macrophages) under normoxic and hypoxic conditions.

[0129] FIG. 4 provides Western blot results showing that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in interleukin-4 (IL-4) and interleukin-10 (IL-10)-differentiated THP-1 cells (M2 macrophages) under normoxic and hypoxic conditions.

[0130] Figure 5 is an in vitro experiment of liver fibrosis. Western blot results show that hypoxia-inducible factor-1α (HIF-1α) was decreased by 2.5 mM calcium lactate treatment in LX-2 cells (human hepatic stellate cells) under hypoxic conditions.

[0131] Figure 6 is an in vitro experiment of human endothelial cells. Western blot results show that 2.5 mM calcium lactate treatment reduced hypoxia mediator (lactate dehydrogenase A) in human umbilical vein endothelial cells under hypoxic conditions.

[0132] Figure 7 is a human fibroblast in vitro experiment. Western blot results show that 2.5 mM calcium lactate treatment increased lactate dehydrogenase B in CCD-18Co cells (human colon fibroblasts) under hypoxic conditions.

[0133] Figure 8: Brain CID in vitro experiment. Western blot results show that 2.5 mM calcium lactate treatment reduced hypoxia mediators in SK-N-SH cells (brain epithelium) under normoxic and hypoxic conditions.

[0134] Figure 9 is an in vitro liver CID experiment. Western blot results show that 2.5 mM calcium lactate treatment altered hypoxia-mediated factors in HepG2 cells (liver epithelium) under hypoxic conditions.

[0135] FIG. 10 provides Western blot results showing that inflammatory factors (TLR-4) were reduced by 2.5 mM calcium lactate treatment in HepG2 cells (hepatic epithelium) under hypoxic conditions.

[0136] Figure 11: Ocular CID in vitro experiment. Western blot results show that 2.5 mM calcium lactate treatment reduced hypoxia mediators in ARPE-19 cells (retinal pigment epithelium) under normoxic and hypoxic conditions.

[0137] FIG. 12 provides immunocytochemistry results showing that 2.5 mM calcium lactate treatment reduced hypoxia-inducible factor-1α (HIF-1α) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions.

[0138] FIG. 13 provides immunocytochemistry results showing that 2.5 mM calcium lactate treatment reduced lactate dehydrogenase A in ARPE-19 cells (retinal pigment epithelium) under normoxic conditions.

[0139] FIG. 14 provides immunocytochemistry results showing that 2.5 mM calcium lactate treatment reduced inflammatory factors (nuclear factor kappa B) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions.

[0140] FIG. 15 provides immunocytochemistry results showing that 2.5 mM calcium lactate treatment reduced inflammatory factors (Toll-like receptor 4) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions.

[0141] FIG. 16 provides immunocytochemistry results showing that 2.5 mM calcium lactate treatment increased lactate dehydrogenase B in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions.

[0142] FIG. 17 provides immunocytochemistry results showing that 2.5 mM calcium lactate treatment reduced drusen marker (apolipoprotein E) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions.

[0143] FIG. 18 provides Western blot results showing that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in ARPE-19 cells (retinal pigment epithelium) under hypoxic conditions.

[0144] Figure 19 shows the lack of toxicity of 2.5 mM calcium lactate treatment in ARPE-19 cells (retinal pigment epithelium). Calcium lactate plays an important role only in the metabolic changes of injured epithelial cells.

[0145] FIG. 25 provides Western blot results showing that 2.5 mM calcium lactate treatment reduced inflammatory factors (NF-κb) in retinal pigment epithelium tissue.

[0146] Example 19 Targeting gastrointestinal diseases (NFALD, NASH) material and method Animal models for liver disease. All experiments were performed under the institutional guidelines (IACUC-2017-0008) established by the Institutional Animal Care and Use Committee of Gachon University. Female 5-week-old C57BL / 6 mice were purchased from KOATEC (Pyeongtaek, South Korea) and fed a methionine-choline (MCD) diet for 4 weeks. Mice were subcutaneously injected with calcium lactate (2 mg / kg) twice daily (BID) for 4 weeks. Histological analysis. Mouse liver tissues (n=5) were dissected and fixed in 4% paraformaldehyde in PBS solution for 15 h at 4 °C. Fixed liver tissues were embedded in paraffin and sectioned at 10 μm. Slides were incubated at 55°C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol series, and used for hematoxylin (Merck, Darmstadt, Germany) and eosin (H&E) staining. H&E stained paraffin sections (10 μm thick) were imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0147] Masson's trichrome staining. Mouse liver tissues (n=5) were dissected and fixed in 4% paraformaldehyde in PBS at 4°C for 15 h. Fixed tissues were embedded in paraffin and sectioned at 5 mm. Slides were incubated at 55°C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol series, refixed in Bouin's solution at 56°C for 1 h, rinsed in tap water for 5-10 min to remove yellow color, stained in Biebrich scarlet-acid fuchsin solution for 5 min, then in phosphomolybdic acid-phosphotungstic acid solution (1:1 ratio) for 30 min, and aniline blue solution for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 2-5 min. Dehydrated in 95% ethyl alcohol, absolute ethyl alcohol, and cleared in xylene. Mounted with mounting medium. Imaging was performed using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0148] Figure 20 is an in vivo experiment of liver CID (NASH). To induce NASH, mice were fed a polyunsaturated fat-containing methionine-choline deficient (MCD) diet for 8 weeks. Blood chemistry results show that 2mg / kg calcium lactate treatment significantly reduced serum AST and ALT. **P<0.001 vs. methionine-choline deficient (MCD). Results are mean±SD.

[0149] Figure 21 provides immunohistochemistry results showing that no lipid droplets (white holes in liver tissue) were observed in the calcium lactate-treated group, while lipid droplets were frequently observed in the methionine-choline deficient (MCD) group. Calcium lactate treatment at 2 mg / kg prevented fatty degeneration. The results show that calcium lactate treatment prevented fatty degeneration.

[0150] FIG. 22 provides immunohistochemistry results showing that the 2 mg / kg calcium lactate treatment group, like the control group, had no lipid droplets (white holes in the liver tissue) in the liver, while the methionine-choline deficient (MCD) group did.

[0151] FIG. 23 provides immunohistochemistry results showing that the 2 mg / kg calcium lactate treatment group had reduced immune cell infiltration in the liver, similar to the control group, while the methionine-choline deficient (MCD) group had increased immune cell infiltration.

[0152] FIG. 24 provides immunohistochemistry results showing that the 2 mg / kg calcium lactate treatment group, like the control group, had no fibrosis (areas of collagen accumulation; white arrows) in the liver, whereas the methionine-choline deficient (MCD) group did.

[0153] Example 20 Targeting eye diseases (AMD, keratitis, and diabetic retinopathy) material and method Animals. All experiments were performed under the institutional guidelines established by the Institutional Animal Care and Use Committee of Gachon University (IACUC-2017-0008). Six-week-old C57BL mice were purchased from Orient (Charles River Korea, Seoul, Korea). All animals were maintained on a 12-h light / dark cycle (lights on at 08:00) at 22–25 °C with food and water available ad libitum.

[0154] Preparation of calcium lactate. Calcium lactate was purchased from Sigma Aldrich (St. Louis, MO, USA) and 2 mg / kg calcium lactate was dissolved in saline for daily subcutaneous injection (21 days).

[0155] Animal model for eye disease. Mice were individually placed on an induction chamber and anesthesia was induced with 2% isoflurane (HANA PHARM CO., Seoul, Korea). Laser-induced rupture of Bruch's membrane was induced in both eyes of mice by laser photocoagulation (SDL405-700, SD Laser, China). The laser pulse was from a green laser (wavelength, 532 nm; Visulas, 532; SD Laser). The laser parameters were spot size 100 μm, power 700 mw, and exposure time 1 s.

[0156] Preparation of choroidal flat mounts. Twenty-one days after laser injury, mice from each group were anesthetized and perfused into the left ventricle with 1.0 mL of PBS containing 25 mg fluorescein isothiocyanate-dextran (Sigma-Aldrich, St. Louis, MO, USA). Eyes were enucleated and fixed in 4% paraformaldehyde for 1 h. RPE-choroid-scleral eye cups were prepared after hemisection of the eyes and removal of the entire lens, vitreous, and retina. RPE-choroid-scleral eye cups were flattened by making four or five radial incisions from the edge to the equator and flat mounted in aquamount.

[0157] Histological analysis. Fixed flat mounts were dehydrated in graded ethanol series and embedded in paraffin. Paraffin sections (10 μm thick) were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma-Aldrich, St. Louis, MO, USA) (H&E) and imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany). Choroidal lesions and neovascularization on flat mounts were observed by confocal microscopy (Carl Zeiss, Oberkochen, Germany).

[0158] result Figure 26 provides representative images of H&E staining of the lesions in the inner layer of the choroidal flat mount. Abnormal morphology of the retinal pigment epithelium was observed after AMD induction (middle image). Treatment with 2.5 mM calcium lactate (right image) regenerated the morphology of the retinal pigment epithelium similar to that of normal control epithelium (left image).

[0159] Figure 27 provides fluorescence images of lesions in the inner layer of the choroid on flat mounts due to laser-induced AMD. Choroidal lesions were observed after AMD induction (middle image). Choroidal lesions were restored after treatment with calcium lactate (right image).

[0160] Figure 28 provides fluorescence images of the lesions and neovascularization of the inner layer of the choroid on flat mounts due to laser-induced AMD. After AMD induction, choroidal lesions and neovascularization are observed (middle image). After treatment with calcium lactate, choroidal lesions and neovascularization are restored (right image), similar to normal control (left image).

[0161] Example 21 Targeting Alzheimer's Disease and Stroke material and method Animals. All experiments were performed in accordance with the Animal Care Guidelines under the Korean Law (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of Gachon University (LCDI-2018-0073). Eight-week-old male Sprague-Dawley (SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea) and bred under specific pathogen-free (SPF) conditions. All animals were maintained under a 12-h light / dark cycle at 22–25 °C with food and water available ad libitum.

[0162] Preparation of Lipopolysaccharide (LPS) and Calcium Lactate. Dissolve 1 mg of LPS powder in 1 ml of saline. Keep the tube on ice if the LPS is going to be used immediately. For long-term storage, screw-cap vials are preferred. Aliquots were stored at -20°C. Calcium lactate was purchased from Sigma Aldrich (St. Louis, MO, USA). 2 mg / kg calcium lactate was dissolved in saline for daily subcutaneous injections (21 days).

[0163] Intracranial injection of LPS. The animal was placed in a small acrylic cage connected to an anesthesia machine under gas anesthesia (2% isoflurane, Spartanburg, SC, USA) to allow easy breathing. After sufficient anesthesia was achieved, the animal was placed on the operating table (while maintaining gas anesthesia) with the upper neck extended upwards to expose the posterior neck. The hair on the animal's head was shaved with electric clippers and the skin was smeared with iodine. LPS was kept on ice until use. All surgical instruments were disinfected with 70% ethanol. The inner space of the Hamilton syringe barrel was cleaned with distilled water and 70% ethanol. The Hamilton syringe was placed in the syringe holder. The animal was placed in a lateral position in the stereotaxic apparatus. A midline incision (approximately 1 cm) was made on the skin of the head with a scalpel. The needle of the Hamilton syringe was attached above the bregma. The three coordinates (AP: -3.6, LM: 2.0 and DV: -3.8) on the manipulator were read (in mm) and recorded; this was used as the starting point. Gradually, the DV coordinate of the injection point was determined; the needle tip was inserted into the brain to the injection point. The LPS solution (2 μl) was released at a constant flow rate of 0.5 μl / min. After injection, the needle was very slowly withdrawn. The wound was closed by sutures and then painted with disinfectant. The rat was marked for further identification.

[0164] Immunohistochemistry. Brain tissues were sectioned at 5 μm on a cryostat and stained for histological examination. Microscopic images were acquired at 200X magnification. For immunohistochemistry, sections were blocked with 1% BSA and 10% NGS in 0.05M PBS for 1 h at room temperature and then reacted with rabbit anti-Iba-1 (1:1000, Bioss, MA, USA) at 4°C for two nights. Microglia (Iba-1) were visualized in the sections using the ABC staining system (Vector Laboratories, CA, USA).

[0165] result Figure 29 provides an experimental scheme for establishing Alzheimer's disease and stroke. OMT-110 is calcium lactate. Figure 30 provides representative images showing brain damage by LPS. Contralateral: normal area. Ipsilateral: LPS injection.

[0166] Figure 31 shows the regeneration of damaged brain tissue and the reduction of microglial infiltration recruited upon brain injury. The results show that the inflammatory response that exacerbates Alzheimer's disease and stroke is greatly reduced.

[0167] Example 22 Targeting Parkinson's Disease material and method Animals. All experiments were performed in accordance with the Animal Care Guidelines under the Korean Law (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of Gachon University (LCDI-2018-0073). Eight-week-old male Sprague-Dawley (SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea) and bred under specific pathogen-free (SPF) conditions. All animals were maintained under a 12-h light / dark cycle at 22–25 °C with food and water available ad libitum.

[0168] Preparation of desipramine, 6-OHDA, and calcium lactate. Desipramine (12.5 mg / kg; noradrenaline transporter blocker; Sigma Aldrich, St. Louis, USA) was dissolved in saline for intraperitoneal injection. 6-OHDA (20 μg / rat, Sigma Aldrich, St. Louis, USA) was dissolved in saline for intracranial injection. 2 mg / kg calcium lactate (Sigma Aldrich, St. Louis, USA) was dissolved in saline for daily subcutaneous injection (21 days).

[0169] Injection of desipramine and 6-OHDA. Thirty minutes before the injection of 6-OHDA, the animals were injected with desipramine into the abdominal cavity (intraperitoneal injection). Thirty minutes after the injection of desipramine, the animals were placed in a small acrylic cage connected to an anesthesia machine that allowed them to breathe comfortably under gas anesthesia (2% isoflurane, Spartanburg, SC, USA). After sufficient anesthesia of the animals was achieved, the animals were placed on the operating table (while maintaining gas anesthesia) with the upper neck extended upwards to expose the posterior neck. The hair on the animal's head was shaved with electric clippers, and the skin was painted with iodine. All surgical instruments were disinfected with 70% ethanol. The inner space of the barrel of the Hamilton syringe was cleaned with distilled water and 70% ethanol. The Hamilton syringe was placed in the syringe holder. The animals were placed in a lateral position in the stereotaxic apparatus. A midline incision (approximately 1 cm) was made on the skin of the head with a scalpel. The needle of the Hamilton syringe was attached on the bregma. Three coordinates (AP:-5.6, LM:2.0 and DV:-7.6) were read (in mm) on the manipulator and used as the starting point. The DV coordinate of the injection point was gradually determined; the needle tip was inserted into the brain to the injection point. The 6-OHDA solution (20μg / 4μl) was released at a constant flow rate of 1μl / min. After injection, the needle was very slowly withdrawn. The wound was closed by suture and then painted with disinfectant. The rat was marked for further identification.

[0170] Immunohistochemistry. Brain tissues were sectioned at 5 μm on a cryostat and stained for histological examination. Microscopic images were acquired at a magnification of 200X. For immunohistochemistry, sections were blocked with 1% BSA and 10% NGS in 0.05M PBS for 1 h at room temperature, and then reacted with rabbit anti-tyrosine hydroxylase (1:500, Chemicon, Tokyo, Japan) at 4°C for two nights. Dopaminergic neurons in the sections were visualized using the ABC staining system (Vector Laboratories, CA, USA).

[0171] result Figure 32 provides an experimental scheme for establishing Parkinson's disease. OMT-110 is calcium lactate.

[0172] Figure 33 shows regeneration of dopaminergic neurons by treatment with calcium lactate. Parkinson's disease is a neurodegenerative disorder that affects dopamine production by destroying dopaminergic neurons in a specific region of the brain (the substantia nigra).

[0173] Example 23 Targeting Multiple Sclerosis and Spinal Cord Injury material and method Animals. All experiments were performed in accordance with the Animal Care Guidelines under the Korean Law (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of Gachon University (LCDI-2018-0073). Eight-week-old male Sprague-Dawley (SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea) and bred under specific pathogen-free (SPF) conditions. All animals were maintained under a 12-h light / dark cycle at 22–25 °C with food and water available ad libitum.

[0174] Preparation of Lipopolysaccharide (LPS) and Calcium Lactate. Dissolve 1 mg of LPS powder in 1 ml of saline. Keep the tube on ice if the LPS is going to be used immediately. For long-term storage, it is preferable to use screw-cap vials. Aliquots were stored in an ultra-low temperature freezer (-20°C). Calcium lactate was purchased from Sigma Aldrich (St. Louis, USA). Dissolve 2 mg / kg of calcium lactate in saline for daily subcutaneous injections (21 days).

[0175] Intrathecal injection of LPS. The animals were placed in a small acrylic cage connected to an anesthesia machine under gas anesthesia (2% isoflurane, Spartanburg, SC, USA) to allow easy breathing. After sufficient anesthesia of the animals was achieved, the animals were placed on the operating table (while maintaining gas anesthesia). For intrathecal injection of LPS, the rat laminectomy site was exposed. Two small holes were made at 1 mm intervals in the dura covering the left spinal column. A volume of 1 μl of LPS solution was carefully aspirated into a sterile Hamilton syringe, and the LPS solution was slowly injected into the spinal cord. The syringe was kept in place for an additional 2 min to prevent backflow from the injection site. The injection site was then closed by a hemostat.

[0176] Immunohistochemistry. Spinal cord tissues were sectioned at 5 μm on a cryostat and stained for histological examination. Microscopic images were acquired at 200X magnification. For immunohistochemistry, sections were blocked with 1% BSA and 10% NGS in 0.05M PBS for 1 h at room temperature and then reacted with rabbit anti-lba-1 (1:1000, Bioss, MA, USA) at 4°C for two nights. Microglia (Iba-l) were visualized in the sections using the ABC staining system (Vector Laboratories, CA, USA).

[0177] result Figure 34 provides an experimental scheme for establishing multiple sclerosis. OMT-110 is calcium lactate.

[0178] Figure 35 shows the regeneration of spinal cord demyelination and reduction of microglial infiltration by treatment with calcium lactate. Demyelination and immune infiltration of the central nervous system occur in association with inflammation in many disorders, including multiple sclerosis and spinal cord injury.

[0179] Example 24 Targeting vascular diseases (atherosclerosis, cerebral hemorrhage, and myocardial infarction) material and method Animals. All experiments were performed in accordance with the animal care guidelines under the Korean Act (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of the Gyeonggi Biocenter (2017-11-0008, South Korea). Five-week-old male BALB / c ApoE shi Mice were obtained from Japan SLC Inc. (Shizuoka, Japan) and bred under specific pathogen-free (SPF) conditions. All animals were maintained at 22–25°C with a 12-h light / dark cycle and free access to food and water.

[0180] Preparation of calcium lactate. Calcium lactate was purchased from Sigma Aldrich (St. Louis, MO, USA). 2 mg / kg calcium lactate was dissolved in saline for daily subcutaneous injection (21 days).

[0181] Animal model for vascular disease. BALB / c ApoE for spontaneous induction of vascular disease. shi In this study, mice were fed a high fat (40%) diet for 4 weeks to induce extensive atherosclerosis.

[0182] Histological analysis. The thoracic aorta was sectioned and half of the aorta was fixed in 10% neutral buffered formalin fixative. Histological analysis was then performed. Histological analysis was performed with H&E and Oil Red O staining. Aortic wall thickness, atherosclerotic plaque area, foam cell number, and lipidated area were analyzed.

[0183] Statistical analysis. Statistical analysis was performed using SPSS statistics and the results were analyzed using Student's t-test. Histological analysis was performed using the Mann-Whitney method. P<0.05 was considered statistically significant.

[0184] result Table 1: Quantitative analysis for histological analysis. After disease induction, all clinical indices of arteriosclerosis were significantly increased. However, the indices were significantly decreased after treatment with calcium lactate. *: significant difference between G2 and G1, P<0.05; ++: significant difference between G2 and G3, P<0.01; +: significant difference between G2 and G3, P<0.05. Data are expressed as mean ± SEM. Results were statistically analyzed by the Mann-Whitney method. G1: normal control (saline); G2: vehicle control (saline); G3: calcium lactate; G2-G3: high cholesterol diet-fed ApoE-deficient mice (atherosclerosis)

[0185] Evidence for Direct Targeting of Atherosclerosis (Table 2) Clinical blood chemistry Data are expressed as mean ± SEM. Statistical analysis was performed by Student's t test. ** : There is a significant difference between G2 and G1, P<0.01 * : There is a significant difference between G2 and G1, P<0.05 G1: Normal control (saline, n=3) G2: Vehicle control (saline, n=8) G3: Test article (calcium lactate pentahydrate, n=8) G2-G3: ApoE-deficient mice fed a high-cholesterol diet

[0186] Pathogenic factors such as cholesterol (TCHO), LDL cholesterol (LDL), triglycerides (TG), and HDL-cholesterol (HDL) were not regulated by calcium lactate. The combined form of calcium and lactate acts directly on the disease site as a single component (see Figure 36).

[0187] Figure 36 provides a representative histological profile of aortic tissue. The tissues on the left and center of the photograph are H&E stained. The tissues on the right of the photograph are oil red stained to distinguish atherosclerotic lesions. The atherosclerotic lesions were reduced by treatment with calcium lactate.

[0188] Example 25 Targeting Periodontal Disease (Pulpitis and Periodontitis) material and method Animals. All experiments were performed in accordance with the Animal Care Guidelines under the Korean Law (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of Gachon University (LCDI-2018-0073). Eight-week-old male Sprague-Dawley (SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea) and bred under specific pathogen-free (SPF) conditions. All animals were maintained under a 12-h light / dark cycle at 22–25 °C with food and water available ad libitum.

[0189] Preparation of Lipopolysaccharide (LPS) and Calcium Lactate. Dissolve 1 mg of LPS powder in 100 μl of saline. Keep the tube on ice if the LPS is going to be used immediately. For long-term storage, it is preferable to use screw-cap vials. Aliquots were stored in an ultra-low temperature freezer (-20°C). Calcium lactate was purchased from Sigma Aldrich (St. Louis, MO, USA). 2 mg / kg of calcium lactate was dissolved in saline for daily subcutaneous injections (21 days).

[0190] Animal model for periodontal disease. Periodontal disease was induced by intragingival injection of LPS (1 mg / 100 μl) in sterile saline. A fine hypodermic needle was inserted into the mesial aspect of the right first mandibular molar and the tip was moved distally so that the injection was at the interdental papilla between the first and second molars. The injection was made slowly and the needle was held in place for 10 seconds after injection to prevent LPS from being lost through the needle track.

[0191] Histological analysis. The rat gingiva, including the teeth, was dissected and fixed in 4% paraformaldehyde at 4°C for 2 days. The fixed tissues were paraffin-embedded and sectioned at 10 μm. The slides were incubated at 55°C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol series, refixed in Bouin's solution for 1 h, rinsed in tap water for 5–10 min to remove the yellow color, stained with Biebrich scarlet-acid fuchsin solution for 5 min, then in phosphomolybdic-phosphotungstic acid solution (1:1 ratio) for 30 min, and aniline blue solution for 15 min. They were briefly rinsed in distilled water and 1% acetic acid solution for 5 min. They were dehydrated in 95% ethyl alcohol, absolute ethyl alcohol, and cleared in xylene. They were mounted with mounting medium. Stained tissues were imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0192] result Figure 37 shows Masson's trichrome staining of periodontal tissue. LPS treatment induces periodontal disease, which causes loss of ligaments and gums around the tooth root (middle image). After treatment with calcium lactate (right image), the ligaments and gums regenerated, similar to the normal control (left image).

[0193] Figure 38 shows Masson's trichrome staining of the upper gingiva. The upper gingiva surrounding the periodontium treated with LPS shows abnormal morphology due to severe inflammation (middle image). After treatment with calcium lactate (right image), the morphology of the upper gingiva was restored to the same as that of the normal control (left image).

[0194] Example 26 Targeting musculoskeletal disorders (rheumatoid arthritis) material and method Animals. All experiments were performed in accordance with the Animal Care Guidelines under the Korean Act (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of Gyeonggi Biocenter (2017-11-0003, South Korea). Six-week-old male DBA1 / J mice were obtained from Japan SLC Inc. (Shizuoka, Japan) and bred under specific pathogen-free (SPF) conditions. All animals were maintained under a 12-h light / dark cycle at 22–25 °C with food and water available ad libitum.

[0195] Preparation of agents. For primary induction of rheumatoid arthritis, 2 mg / mL type II collagen was mixed with an equal volume of complete Freund's adjuvant (CFA). For secondary induction of rheumatoid arthritis, 2 mg / mL type II collagen was mixed with an equal volume of incomplete Freund's adjuvant (IFA). 2 mg / kg calcium lactate was dissolved in saline for daily subcutaneous injection (21 days).

[0196] Animal Models for Rheumatoid Arthritis Primary induction: 0.1 mL of the mixed solution (type II collagen + CFA) was injected subcutaneously into the area 1.5 cm from the base of the tail of the mice. The injection site and depth were the same for all mice.

[0197] Secondary induction: 21 days after primary induction, 0.1 mL of the mixed solution (type II collagen + IFA) was injected subcutaneously into the area 1.5 cm from the base of the tail of the mice. The injection site and depth were the same for all mice.

[0198] Histological analysis. Mouse legs and knee joints were dissected, fixed (4% paraformaldehyde), and decalcified at 4°C for 2 days. Tissues were paraffin embedded and sectioned at 15 μm. Slides were incubated at 55°C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol series, stained with Biebrich scarlet-acid fuchsin solution for 5 min, then phosphomolybdic-phosphotungstic acid solution (1:1 ratio) for 30 min, and aniline blue solution for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 5 min. Dehydrated in 95% ethyl alcohol, absolute ethyl alcohol, and cleared in xylene. Mounted with mounting medium. Stained tissues were imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0199] result Figure 39 shows Masson's trichrome staining of foot and knee joint tissues. Rheumatoid arthritis inflammatory edema was observed in the toes and knee joints (middle image). After 21 days of treatment with CaLa, inflammatory edema was obviously reduced in the toes and knee joints (lower image), showing the same morphological recovery as normal control (upper image).

[0200] Example 27 Targeting gastrointestinal diseases (inflammatory bowel disease: Crohn's disease and colitis) material and method Animals. All experiments were performed in accordance with the Animal Care Guidelines under the Korean Law (11737, revised April 5, 2013) and the guidelines set forth by the Institutional Animal Care and Use Committee of Gachon University (LCDI-2018-0073). Six-week-old male Sprague-Dawley (SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea) and bred under specific pathogen-free (SPF) conditions. All animals were maintained under a 12-h light / dark cycle at 22–25 °C with food and water available ad libitum.

[0201] Active agents and animal models. To induce Crohn's disease and colitis, indomethacin was purchased from Sigma Chemical (St. Louis, Missouri, USA) and dissolved in saline. Calcium lactate was dissolved in saline at 2 mg / kg for daily subcutaneous injection (for 21 days). Two models of inflammatory bowel disease were used. Crohn's disease was induced by oral administration of indomethacin (7 mg / kg) and colitis was induced by subcutaneous injection of indomethacin (7 mg / kg) daily at 24-hour intervals (Figures 40 and 42).

[0202] Histological analysis. Fixed flat mounts were dehydrated in graded ethanol series and embedded in paraffin. Paraffin sections (5 μm thick) were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma-Aldrich, St. Louis, MO, USA) (H&E) and imaged using a Leica DM 1000 LED microscope (Leica Microsystems, Wetzlar, Germany).

[0203] result FIG. 40 provides an experimental scheme for inducing Crohn's disease.

[0204] Figure 41 provides representative images of H&E staining of Crohn's disease. After induction of Crohn's disease, transmural inflammation and linear ulceration in the intestine were observed (middle image). After 4 weeks of treatment with CaLa, transmural inflammation and linear ulceration were obviously reduced in intestinal tissue (lower image), showing the same morphological recovery as normal control (upper image).

[0205] FIG. 42 provides an experimental scheme for inducing colitis.

[0206] Figure 43 provides representative images of H&E staining of colitis. After induction of colitis, superficial ulcers, polyps, and inflammation in the intestine were observed (middle image). After 4 weeks of treatment with CaLa, superficial ulcers, polyps, and inflammation were obviously reduced in the intestinal tissue (lower image), showing the same morphological recovery as the normal control (upper image).

[0207] Some of the additional aspects disclosed herein include, but are not limited to, the following: A. A method of treating asthma, pulmonary fibrosis, obesity, gastroenteritis, chronic inflammatory bowel disease, and / or atopic dermatitis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. B. A method for treating chronic obstructive pulmonary disease, pneumonitis, keratitis, atherosclerosis, arteriosclerosis, myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis, and / or psoriasis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. C. A method for treating Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease, sepsis, and / or osteoporosis, comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate. D. The method of any one of aspects A-C, wherein the calcium lactate is administered in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, excipient, or diluent. E. The method of any one of embodiments A-D, wherein the pharmaceutical composition is formulated into a liquid, powder, aerosol, injection, infusion, patch, capsule, pill, tablet, depot, or suppository. F. The method of any one of embodiments A-D, wherein the pharmaceutical composition comprises a therapeutically effective amount of calcium lactate as an active agent, and a pharma- ceutically acceptable polysaccharide, polymer, lipid, or combination thereof. G. The method of embodiment F, wherein the pharmaceutical composition comprises calcium lactate and a polysaccharide. H. The method of embodiment G, wherein the weight ratio of calcium lactate to polysaccharide is 1:<0.2 to 1:5. I. The method of embodiment G, wherein the weight ratio of calcium lactate to polysaccharide is 1:<0.2. J. The method of embodiment G, wherein the weight ratio of calcium lactate to polysaccharide is from 1:0.2 to 1:5. K. The method of any one of embodiments F-J, further comprising a polymer and / or a lipid. L. The method of embodiment K, further comprising a polymer and a lipid, wherein the weight ratio of polymer to lipid is from 1:0.1 to 1:50. M. The method of any one of embodiments KL, wherein the weight ratio of calcium lactate to polymer and / or lipid is at least 1:5. N. The method of embodiment M, wherein the weight ratio of calcium lactate to polymer and / or lipid is from 1:5 to 1:30. O. The method of embodiment F, comprising calcium lactate and a polymer and / or lipid. P. The method of embodiment O, comprising a polymer and a lipid, the weight ratio of polymer to lipid being from 1:0.1 to 1:50. Q. The method of any one of embodiments O-P, wherein the weight ratio of calcium lactate to polymer and / or lipid is at least 1:5. R. The method of any one of embodiments O-Q, wherein the weight ratio of calcium lactate to polymer and / or lipid is from 1:5 to 1:30. S. The method of any one of embodiments O-R, wherein the composition is short-acting. T. The method of any one of embodiments O-R, wherein the composition is long-acting. U. The method of any one of embodiments F through T, wherein the composition is an injectable composition. V. The method of any one of aspects F-U, comprising a polysaccharide that is a cellulose derivative, pectin, hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen, alginate, alginic acid, or a combination thereof. W. The method of any one of embodiments F-V, comprising a polymer that is a poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic-lactic acid (PLGA), or a combination thereof. X. The method of any one of embodiments F-W, comprising a lipid that is a mono- or tri-fatty acid glycerol ester or polyethylene glycol, a polyethylene glycol ester of a vegetable oil, a propylene glycol fatty acid ester, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa oil, cottonseed oil, sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenated oil, oleic acid, linoleic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, or a combination thereof. Y. The method of any one of embodiments F-S and U-X, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 40% of the active agent is released after 6 hours. Z. The method of any one of embodiments F-S and U-Y, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 60% of the active agent is released after 12 hours. AA. The method of any one of embodiments F-S and U-Z, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 80% of the active agent is released after 24 hours. BB. The method of any one of embodiments F-S and U-AA, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and at least about 90% of the active agent is released after 48 hours. CC. The method of any one of embodiments FH, JR, and TX, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and wherein less than about 40% of the active agent is released after 24 hours. DD. The method of any one of embodiments FH, JR, and TCC, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and wherein less than about 60% of the active agent is released after 48 hours. EE. The method of any one of embodiments F-H, J-R, and CC-DD, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and wherein less than about 80% of the active agent is released after 72 hours. FF. The method of any one of embodiments F-H, J-R, and CC-EE, wherein the composition is placed in an in vitro dissolution test comprising a dissolution test method using a nylon filter having a pore size of 45 μm in 200 ml of aqueous medium having a pH of 6.8 at 37° C. at 300 rpm, and less than about 90% of the active agent is released after 144 hours. GG. The method of any one of embodiments F-FF, wherein the composition is contained in a sterile glass or polyolefin container. HH. The method of any one of embodiments AD, wherein the calcium lactate is coated with a pharma-ceutically acceptable enteric coating. II. The method of embodiment HH, wherein the enteric coating comprises hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and esters thereof, or combinations thereof. JJ. The method of any one of embodiments HH-II, wherein the weight ratio of calcium lactate to enteric coating is 10:0.5 to 1:1.5. KK. The method of any one of embodiments HH-JJ, wherein in an in vitro dissolution test involving the USP Paddle Method at 37° C. with a paddle speed of 50 rpm, when the composition is placed in 0.1 HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than about 20% of the active agent is released after 30 minutes. LL. The method of any one of embodiments HH-KK, wherein in an in vitro dissolution test involving the USP Paddle Method at 37° C. with a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than 30% of the active agent is released after 60 minutes. MM. The method of any one of embodiments HH-LL, wherein in an in vitro dissolution test involving the USP Paddle Method at 37° C. with a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than 50% of the active agent is released after 120 minutes. NN. The method of any one of embodiments HH through MM, wherein in an in vitro dissolution test including the USP Paddle Method at 37° C. with a paddle speed of 50 rpm, when the composition is placed in 0.1 N HCl for 120 minutes and subsequently adjusted to pH 6.8 with phosphate buffer, less than 10% of the active agent is released after 120 minutes. OO. The method of any one of aspects A-B, wherein the calcium lactate is provided in a food or nutritional composition comprising calcium lactate. PP. The method of embodiment OO, wherein the composition is an injectable nutritional supplement.

[0208] U.S. Patent Application No. 62 / 616,923, filed January 12, 2018, is hereby incorporated by reference in its entirety.

[0209] Although the description of the specific embodiments above will fully reveal the general nature of the present invention, others can easily modify and / or adapt such specific embodiments for various applications by applying knowledge within the skill of the art without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that the expressions or terms in this specification are intended to be descriptive and not limiting, as the terms or terms in this specification are interpreted by those skilled in the art in light of the teachings and guidance.

[0210] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0211] All of the various aspects, embodiments, and options described herein can be combined in any and all variations.

[0212] References TIFF0007665220000003.tif25150TIFF0007665220000004.tif231151TIFF0007665220000005.tif218153TIFF0007665220000006.tif80151

[0213] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A pharmaceutical composition for treating one or more diseases selected from the group consisting of multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease (NAFLD), rheumatoid arthritis, pulpitis, periodontitis, inflammatory bowel disease (IBD), spinal cord injury, and fibrosis in a subject in need thereof, comprising a therapeutically effective amount of calcium lactate as an active agent, and a pharma- ceutical acceptable polymer and lipid.

2. 2. The pharmaceutical composition of claim 1, wherein the weight ratio of polymer to lipid is 1:0.1 to 1:

50.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the polymer is a polymer selected from the group consisting of poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), polyglycolic acid-lactic acid (PLGA), and combinations thereof.

4. 3. The pharmaceutical composition of claim 1 or 2, wherein the lipid is selected from the group consisting of mono- or tri-fatty acid glycerol esters or polyethylene glycols, polyethylene glycol esters of vegetable oils, fatty acid propylene glycol esters, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa oil, cottonseed oil, sunflower seed oil, safflower oil, almond oil, olive oil, hydrogenated oils, oleic acid, linolenic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, myristic acid, capric acid, caprylic acid, lauric acid, stearic acid, ethyl oleate, isopropyl palmitate, octyldodecyl myristate, cetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, and combinations thereof.

5. 3. The pharmaceutical composition according to claim 1 or 2, further comprising a pharma- ceutically acceptable polysaccharide, gelatin and / or collagen.

6. 6. The pharmaceutical composition of claim 5, wherein the polysaccharide is selected from the group consisting of cellulose derivatives, pectin, hyaluronic acid, starch, guar gum, chitosan, alginates, alginic acid, and combinations thereof.

7. 3. The pharmaceutical composition of claim 1 or 2, wherein the calcium lactate is coated with a pharma- ceutically acceptable enteric coating.

8. 8. The pharmaceutical composition of claim 7, wherein the enteric coating comprises hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or combinations thereof.

9. 8. The pharmaceutical composition of claim 7, wherein the weight ratio of calcium lactate to enteric coating is from 10:0.5 to 1:1.5.

Images