Use of calcium lactate in preparation of pharmaceutical, food or nutritional compositions for treatment of disease

Abstract

The present invention relates to the use of calcium lactate in the preparation of pharmaceutical, food or nutritional compositions for the treatment of disease. The present invention provides the use of calcium lactate in the preparation of a pharmaceutical, food or nutritional composition for the treatment of a disease selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), inflammatory bowel disease (IBD), spinal cord injury, liver fibrosis and pulmonary fibrosis, wherein the pharmaceutical, food or nutritional composition comprises a therapeutically effective amount of calcium lactate as an active agent.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of January 11, 2019, an application number of "2019800080948", and an invention title of "Methods for Treating Chronic Inflammatory Diseases". Technical Field

[0002] The present invention belongs to the field of pharmaceuticals. Specifically, the present invention relates to the use of calcium lactate in the preparation of pharmaceutical compositions, foods or nutritional compositions for treating diseases. Background Art

[0003] Chronic inflammatory diseases (CID) underlie pathological conditions characterized by chronic inflammation, which has been defined as a long-term and persistent pro-inflammatory state (1). CID includes many common and rare CIDs, such as respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, pneumonia, and pulmonary fibrosis) (2), eye diseases (e.g., keratitis and age-related macular degeneration) (3), autoimmune diseases (4), kidney diseases (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), digestive diseases (e.g., non-alcoholic fatty liver disease, gastroenteritis, and chronic inflammatory bowel disease) (12), and skin disorders (e.g., psoriasis and atopic dermatitis) (13, 14).

[0004] An epidemiological study has shown that CID is a major cause of death worldwide and has been increasing over the past three decades. It is estimated that by 2030, 171 million people in the United States will be affected by CID (15). There are various types of anti-inflammatory drugs that can treat inflammation, such as aspirin, antihistamines, COX-2 inhibitors, corticosteroids, and non-steroidal anti-inflammatory drugs (NSAIDs). However, due to side effects and temporary effectiveness, there are limitations to the long-term administration of such existing drugs. Summary of the Invention

[0005] Methods for treating chronic inflammatory diseases are disclosed herein, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0006] Methods for treating Alzheimer's disease, Parkinson's disease, atherosclerosis, and / or stroke are disclosed herein, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0007] Methods for treating multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease (NAFLD) and / or rheumatoid arthritis are also disclosed, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate. In some embodiments, the method is for treating NAFLD, and the NAFLD is non-alcoholic steatohepatitis (NASH).

[0008] Methods for treating pulpitis and / or periodontitis are also disclosed, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0009] Methods for treating inflammatory bowel disease (IBD) are also disclosed, which methods comprise 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.

[0010] Methods for treating spinal cord injury, cerebral hemorrhage, myocardial infarction, keratitis and / or diabetic retinopathy are also disclosed, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0011] Methods for treating asthma, pulmonary fibrosis, obesity, gastroenteritis, chronic inflammatory bowel disease and / or atopic dermatitis are disclosed herein, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate. Methods for treating chronic obstructive pulmonary disease, pneumonia, keratitis, atherosclerosis, arteriosclerosis, myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis and / or psoriasis are also disclosed herein, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate. Methods for treating Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease, sepsis and / or osteoporosis are also disclosed herein, which methods comprise administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0012] In some embodiments, calcium lactate is administered in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient and / or diluent. In some embodiments, the pharmaceutical composition is formulated as a liquid, powder, aerosol, injection, infusion, patch, capsule, pill, tablet, depot (drug reservoir) or suppository.

[0013] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of calcium lactate as an active agent and a pharmaceutically 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 the polysaccharide is 1:<0.2 to 1:5. In some embodiments, the weight ratio of calcium lactate to the polysaccharide is 1:<0.2. In some embodiments, the weight ratio of calcium lactate to the polysaccharide is 1:0.2 to 1:5.

[0014] In some embodiments, the pharmaceutical composition further comprises a polymer and / or a lipid. In some embodiments, the pharmaceutical composition further comprises a polymer and a lipid, wherein the weight ratio of the polymer to the lipid is from 1:0.1 to 1:50. In some embodiments, the weight ratio of calcium lactate to the polymer and / or the lipid is at least 1:5. In some embodiments, the weight ratio of calcium lactate to the polymer and / or the lipid is from 1:5 to 1:30.

[0015] In some embodiments, the pharmaceutical composition comprises calcium lactate and a polymer and / or a lipid. In some embodiments, the pharmaceutical composition comprises a polymer and a lipid, wherein the weight ratio of the polymer to the lipid is from 1:0.1 to 1:50. In some embodiments, the weight ratio of calcium lactate to the polymer and / or the lipid is at least 1:5. In some embodiments, the weight ratio of calcium lactate to the polymer and / or the lipid is from 1:5 to 1:30.

[0016] 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.

[0017] 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.

[0018] In some embodiments, the polymer is poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), or a combination thereof.

[0019] In some embodiments, the lipid is a monoglyceride or a triglyceride 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 butter, cottonseed oil, sunflower oil, safflower oil, almond oil, olive oil, hydrogenated oil, 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, or a combination thereof.

[0020] In some embodiments, at least about 40% of the active agent is released after 6 hours when the pharmaceutical composition is placed in an in vitro dissolution test including an elution test method at 37 °C in 200 ml of an aqueous medium having a pH of 6.8 using a nylon filter with a pore size of 45 μm at 300 rpm.

[0021] In some embodiments, at least about 60% of the active agent is released after 12 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0022] In some embodiments, at least about 80% of the active agent is released after 24 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0023] In some embodiments, at least about 90% of the active agent is released after 48 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0024] In some embodiments, less than about 40% of the active agent is released after 24 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0025] In some embodiments, less than about 60% of the active agent is released after 48 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0026] In some embodiments, less than about 80% of the active agent is released after 72 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0027] In some embodiments, less than about 90% of the active agent is released after 144 hours in an in vitro dissolution test that includes an elution test method at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, after placing the pharmaceutical composition.

[0028] In some embodiments, the pharmaceutical composition is contained in a sterile glass or polyolefin container.

[0029] In some embodiments, calcium lactate is coated with a pharmaceutically acceptable enteric coating. In some embodiments, the enteric coating comprises hydroxypropyl methylcellulose 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 from 10:0.5 to 1:1.5. In some embodiments, in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than about 20% of the active agent is released after 30 minutes. In some embodiments, in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than 30% of the active agent is released after 60 minutes. In some embodiments, in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than 50% of the active agent is released after 120 minutes. In some embodiments, in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than 10% of the active agent is released after 120 minutes.

[0030] In some embodiments, calcium lactate is provided in a food or nutritional composition comprising calcium lactate. In some embodiments, the food or nutritional composition is an injectable nutritional supplement. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 . In vitro experiment on human leukemia monocytes. Western blot results showed that under hypoxic conditions, treatment with 2.5 mM calcium lactate reduced the inflammatory factor (NF-κb) in THP-1 cells (human leukemia monocytes).

[0032] Figure 2 . In vitro experiment on the differentiation of human leukemia monocytes into macrophages. Western blot results showed that under normoxic and hypoxic conditions, treatment with 2.5 mM calcium lactate reduced the inflammatory factor (NF-κb) in THP-1 cells (M0 macrophages) differentiated with PMA (phorbol 12-myristate 13-acetate).

[0033] Figure 3. Western blot results showed that under normoxia and hypoxia conditions, treatment with 2.5 mM calcium lactate reduced the inflammatory factor (NF-κb) in THP-1 cells (M1 macrophages) differentiated with interferon γ (IFN-γ) and lipopolysaccharide (LPS).

[0034] Figure 4 . Western blot results showed that under normoxia and hypoxia conditions, treatment with 2.5 mM calcium lactate reduced the inflammatory factor (NF-κb) in THP-1 cells (M2 macrophages) differentiated with interleukin 4 (IL-4) and interleukin 10 (IL-10).

[0035] Figure 5 . In vitro experiment on liver fibrosis. Western blot results showed that under hypoxia conditions, treatment with 2.5 mM calcium lactate reduced hypoxia-inducible factor-1α (HIF-1α) in LX-2 cells (human hepatic stellate cells).

[0036] Figure 6 . In vitro experiment on human endothelial cells. Western blot results showed that under hypoxia conditions, treatment with 2.5 mM calcium lactate reduced hypoxia-mediated factor (lactate dehydrogenase A) in human umbilical vein endothelial cells.

[0037] Figure 7 . In vitro experiment on human fibroblasts. Western blot results showed that under hypoxia conditions, treatment with 2.5 mM calcium lactate increased lactate dehydrogenase B in CCD-18Co cells (human colon fibroblasts).

[0038] Figure 8 . In vitro experiment on brain CID. Western blot results showed that under normoxia and hypoxia conditions, treatment with 2.5 mM calcium lactate reduced hypoxia-mediated factor in SK-N-SH cells (cerebral epithelial cells).

[0039] Figure 9 . In vitro experiment on liver CID. Western blot results showed that under hypoxia conditions, treatment with 2.5 mM calcium lactate altered the hypoxia-mediated factor in HepG2 cells (liver epithelial cells).

[0040] Figure 10 . Western blot results showed that under hypoxia conditions, treatment with 2.5 mM calcium lactate reduced the inflammatory factor (TLR-4) in HepG2 cells (liver epithelial cells).

[0041] Figure 11 . In vitro experiment on eye CID. Western blot results showed that under normoxia and hypoxia conditions, treatment with 2.5 mM calcium lactate reduced hypoxia-mediated factor in ARPE-19 cells (retinal pigment epithelial cells).

[0042] Figure 12 . Immunocytochemical results showed that under hypoxic conditions, hypoxia-inducible factor-1α (HIF-1α) in ARPE-19 cells (retinal pigment epithelial cells) decreased after treatment with 2.5 mM calcium lactate.

[0043] Figure 13 . Immunocytochemical results showed that under normoxic conditions, lactate dehydrogenase A in ARPE-19 cells (retinal pigment epithelial cells) decreased after treatment with 2.5 mM calcium lactate.

[0044] Figure 14 . Immunocytochemical results showed that under hypoxic conditions, inflammatory factor (nuclear factor-κB) in ARPE-19 cells (retinal pigment epithelial cells) decreased.

[0045] Figure 15 . Immunocytochemical results showed that under hypoxic conditions, inflammatory factor (toll-like receptor 4) in ARPE-19 cells (retinal pigment epithelial cells) decreased after treatment with 2.5 mM calcium lactate.

[0046] Figure 16 . Immunocytochemical results showed that under hypoxic conditions, lactate dehydrogenase B in ARPE-19 cells (retinal pigment epithelial cells) increased after treatment with 2.5 mM calcium lactate.

[0047] Figure 17 . Immunocytochemical results showed that under hypoxic conditions, drusen biomarker (apolipoprotein E) in ARPE-19 cells (retinal pigment epithelial cells) decreased after treatment with 2.5 mM calcium lactate.

[0048] Figure 18 . Western blot results showed that under hypoxic conditions, inflammatory factor (NF-κb) in ARPE-19 cells (retinal pigment epithelial cells) decreased.

[0049] Figure 19 . The photograph showed that there was no toxicity in ARPE-19 cells (retinal pigment epithelial cells) after treatment with 2.5 mM calcium lactate. Calcium lactate only played an important role in the metabolic changes of injured epithelial cells.

[0050] Figure 20 . In vivo experiment on liver CID (NASH). Blood chemistry results showed that after treatment with 2 mg / kg calcium lactate, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) decreased significantly. **P < 0.001 compared with methionine-choline deficient (MCD). Results are mean ± SD.

[0051] Figure 21 . Immunohistochemical results showed that treatment with 2 mg / kg calcium lactate could prevent hepatic steatosis.

[0052] Figure 22 . Immunohistochemical results showed that in the liver, similar to the control group, lipid droplets were absent in the 2 mg / kg calcium lactate treatment group, while they were found in the methionine-choline-deficient (MCD) group.

[0053] Figure 23 . Immunohistochemical results showed that in the liver, similar to the control group, immune cell infiltration was reduced in the 2 mg / kg calcium lactate treatment group, while it was increased in the methionine-choline-deficient (MCD) group.

[0054] Figure 24 . Immunohistochemical results showed that in the liver, similar to the control group, fibrosis was absent in the 2 mg / kg calcium lactate treatment group, but it was present in the methionine-choline-deficient (MCD) group.

[0055] Figure 25 . Western blot results showed that treatment with 2.5 mM calcium lactate reduced the inflammatory factor (NF-κb) in retinal pigment epithelial cell tissue.

[0056] Figure 26 . Immunohistochemical results showed that treatment with 2.5 mM calcium lactate restored retinal pigment epithelial cells to normal control epithelial cells with similar morphology.

[0057] Figure 27 . Fluorescent images of flat-mounted choroidal inner layer lesions in laser-induced age-related macular degeneration (AMD).

[0058] Figure 28 . Fluorescent images of flat-mounted choroidal inner layer lesions and neovascularization (new blood vessel formation) in laser-induced age-related macular degeneration (AMD).

[0059] Figure 29 . Experimental protocols for Alzheimer's disease and stroke were established.

[0060] Figure 30 . Representative images of brain injury by lipopolysaccharide (LPS). Contralateral: normal area. Ipsilateral: LPS injection.

[0061] Figure 31 . Recovery of damaged brain tissue and reduction of microglial cell infiltration recruited after brain injury.

[0062] Figure 32. Establish an experimental protocol for Parkinson's disease.

[0063] Figure 33 . Recovery through dopaminergic neurons treated with calcium lactate.

[0064] Figure 34 . Establish an experimental protocol for multiple sclerosis.

[0065] Figure 35 . Recovery through demyelinated spinal cord treated with calcium lactate and reduction of microglial cell infiltration.

[0066] Figure 36 . Representative histological features of aortic tissue. The left and middle tissues are stained with hematoxylin and eosin (H&E). The right tissue is stained with Oil Red O.

[0067] Figure 37 . Masson's trichrome staining of periodontal tissue.

[0068] Figure 38 . Masson's trichrome staining of the upper jaw.

[0069] Figure 39 . Masson's trichrome staining of foot and knee joint tissues.

[0070] Figure 40 . Establish an experimental protocol for inducing Crohn's disease.

[0071] Figure 41 . Representative images of Crohn's disease stained with hematoxylin and eosin (H&E).

[0072] Figure 42 . Establish an experimental protocol for inducing colitis.

[0073] Figure 43 . Representative images of colitis stained with hematoxylin and eosin (H&E). Detailed implementation

[0074] Recently, many studies have shown that CID is associated with aging and suggest that chronic inflammation is associated with age-related immunosenescence diseases (1-29). Chronic inflammation has been elucidated to occur through complex interactions between parenchymal cells and stromal cells such as immune cells, endothelial cells, and fibroblasts. Among them, bone marrow-derived cells such as monocytes and activated macrophages have been identified as the main effector cells in CID. After activation by bacterial lipopolysaccharide, IFN-γ, and damaged cells, macrophages produce many inflammatory cytokines and clear endogenous and exogenous inflammatory agents through transcriptional control of nuclear factor-κB (NF-κB). Macrophages are also further polarized into more specialized macrophage subtypes. However, in chronic inflammation, the finely controlled differentiation and activation of myeloid cells are abnormally regulated. Local tissue hypoxia causes metabolic changes in cells such as parenchymal cells and stromal cells by inducing hypoxia-inducible factors (HIFs). HIF, especially HIF-1, contributes to the abnormal activation of macrophages and is a major contributor to the chronic inflammatory response.

[0075] Most CIDs are characterized by inflammation. Injured tissues become resistant to blood flow and oxygen delivery, thus becoming a hypoxic environment. Pro-inflammatory mediators and hypoxic conditions can induce an angiogenic response by inducing HIF-1. Additionally, local inflammatory responses are present in most CIDs, including respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, pneumonia, and pulmonary fibrosis), eye diseases (e.g., keratitis and age-related macular degeneration), autoimmune diseases, kidney diseases, 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., non-alcoholic fatty liver disease, gastroenteritis, and chronic inflammatory bowel disease), and skin diseases (e.g., psoriasis and atopic dermatitis). During inflammation, the permeability of blood vessels increases, and monocytes, macrophages, platelets, mast cells, and other white blood cells are recruited under the attraction of chemokines. Accordingly, the present disclosure provides a therapeutic method for targeting macrophage activation and regulating extracellular matrix degradation by targeting HIF-1, NF-κB, and lactate dehydrogenase (LD) A and B.

[0076] Therapeutic Methods

[0077] Disclosed herein is a method for treating a chronic inflammatory disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of calcium lactate.

[0078] Methods for treating Alzheimer's disease, Parkinson's disease, atherosclerosis, and / or stroke are disclosed herein, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0079] Methods for treating multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease (NAFLD), and / or rheumatoid arthritis are also disclosed, the methods 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 the NAFLD is non-alcoholic steatohepatitis (NASH).

[0080] Methods for treating pulpitis and / or periodontitis are also disclosed, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0081] Methods for treating inflammatory bowel disease (IBD) are also disclosed, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof. In some embodiments, the IBD is Crohn's disease or ulcerative colitis.

[0082] Methods for treating spinal cord injury, cerebral hemorrhage, myocardial infarction, keratitis, and / or diabetic retinopathy are also disclosed, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0083] Methods for treating asthma, pulmonary fibrosis, obesity, gastroenteritis, chronic inflammatory bowel disease, and / or atopic dermatitis are disclosed herein, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof. Methods for treating chronic obstructive pulmonary disease, pneumonia, keratitis, atherosclerosis, arteriosclerosis, myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis, and / or psoriasis are also disclosed herein, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof. Methods for treating Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease, and / or sepsis are also disclosed herein, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0084] Methods for treating osteoporosis are also disclosed herein, the methods comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0085] The terms "subject" or "patient" include 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. Examples of non-mammals include, but are not limited to, birds, fish, etc. In some embodiments of the methods and compositions provided herein, the mammal is human or non-human.

[0086] As used herein, "treat", "treating", or "treatment" of one or more of the diseases disclosed herein by administering a pharmaceutical composition means any reduction in severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary, persistent or transient, attributable to or associated with the administration of the composition.

[0087] As used herein, the term "co-administer" and the like means including administering two or more selected active agents to a single patient, and is intended to include treatment regimens in which the agents are administered by the same or different routes of administration or at the same or different times.

[0088] The pharmaceutical compositions of the present disclosure may be administered in a therapeutically effective amount, and as used herein, the term "effective amount" or "therapeutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to any medical or prophylactic use. The effective dose level may be determined based on: the severity of the disease, the activity of the drug or active agent; the age, weight, health status, and gender of the patient, the sensitivity to the drug; the time of administration, route of administration, and excretion rate of the composition of the present disclosure; the duration of treatment, drugs used concomitantly or in combination with the composition of the present disclosure, and other factors known in the medical field. The pharmaceutical compositions of the present disclosure may be administered alone or in combination with other known drugs or components known to be used for treating one or more of the diseases disclosed herein. Taking all of the above factors into account, it is important to administer the composition in the smallest amount that can exhibit the greatest utility without producing side effects.

[0089] The pharmaceutical compositions of the present disclosure can be administered such that the daily dose of calcium lactate is, for example, from about 10 mg / kg to about 1,000 mg / kg, from about 10 mg / kg to about 500 mg / kg, from about 10 mg / kg to about 250 mg / kg, from about 10 mg / kg to about 200 mg / kg, from about 10 mg / kg to about 100 mg / kg, from about 1 mg / kg to about 100 mg / kg, from about 1 mg / kg to about 75 mg / kg, from about 1 mg / kg to about 50 mg / kg, from about 1 mg / kg to about 25 mg / kg, or from about 1 mg / kg to about 10 mg / kg. The dosing frequency of the composition can be, but is not particularly limited to, once, twice, three times, four times, etc. divided doses per day.

[0090] Compositions containing one or more of the compounds described herein can be administered for prophylactic and / or therapeutic treatment. In therapeutic applications, the composition is administered to a patient who already has a disease or disorder in an amount sufficient to cure or at least partially arrest (halt) the symptoms of the disease or disorder. The effective amount for such an application will depend on the severity and course of the disease or disorder, previous treatment, the health status, weight, and response of the patient to the drug, as well as the judgment of the treating physician. Determining such a therapeutically effective amount by routine experimentation (including, but not limited to, dose escalation clinical trials) is considered to be within the skill of the art.

[0091] In prophylactic applications, a composition containing the active agent described herein is administered to a patient who is predisposed to one or more of the diseases disclosed herein or otherwise at risk of developing one or more of the 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 health status, weight, etc. of the patient. Determining such a prophylactically effective amount by routine experimentation (such as dose escalation clinical trials) is considered to be within the skill of the art. When used in a patient, the effective amount for this use will depend on the severity and course of the disease, affliction, or disorder, previous treatment, the health status of the patient and response to the drug, as well as the judgment of the treating physician.

[0092] In cases where the patient's condition does not improve, the administration of the active agent described herein can, in the judgment of the physician, be a long-term administration, i.e., over a prolonged period of time, including over the entire life duration of the patient, in order to improve or otherwise control or limit the symptoms of the patient's disease or disorder.

[0093] The amount corresponding to such a dose of the given agent will vary depending on a variety of factors, such as the specific compound, the disease condition and its severity, the identity of the subject or host to be treated (e.g., age, weight, gender, etc.), but can still be routinely determined in a manner known in the art according to the specific circumstances surrounding the case, including, for example, the specific dose administered, the route of administration, the condition being treated, and the subject or host being treated.

[0094] Drug composition

[0095] The term "calcium lactate" refers to a class of metal lactate salts, which may exist, for example, in the form of hydrates, represented by C6H 10 O6Ca·5H2O, in which the calcium ion is bonded to the lactate radical. Calcium lactate can be in the form of a white powder or granules at room temperature, or anhydrous when heated at 120 °C, and has a solubility of 5% (w / v).

[0096] Calcium lactate can be formulated into a drug composition for the treatment of one or more diseases disclosed herein.

[0097] In different embodiments, the present invention provides a drug composition comprising a therapeutically effective amount of calcium lactate as an active agent for the treatment of one or more diseases disclosed herein and a pharmaceutically acceptable polysaccharide, polymer, lipid, or a combination of the above. In some embodiments, the drug composition comprises calcium lactate and a polysaccharide.

[0098] In some embodiments, the present invention provides an enteric coating for calcium lactate such that when the active agent is administered orally, the active agent can be protected from the acidic environment of the stomach and absorbed in the small intestine before it reaches the large intestine.

[0099] The present invention also provides short-acting and long-acting drug compositions comprising calcium lactate. In some embodiments, the long-acting composition comprises calcium lactate coated with at least one enteric coating substance, such as hydroxypropyl methylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, and polymers of methacrylic acid and its esters.

[0100] The drug composition of the present invention can be formulated into a pharmaceutical preparation for oral administration. Examples of the preparation include powders, tablets, capsules, granules, or syrups, tablets and capsules, but are not limited thereto.

[0101] The present invention also provides formulations of hydrogels, in particular formulations of methylcellulose, poloxamer, pectin and alginate hydrogels, which can be stored in vitro in solution or nanoparticle form and can form a gel and allow for sustained release of calcium lactate when injected into the body. The relatively short drug release time, which is a disadvantage of hydrogels, has been improved by increasing the interaction between the drug and the hydrogel or by delaying the diffusion of the drug in the hydrogel.

[0102] The weight ratio of calcium lactate to the 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 the polysaccharide can be 1:<0.2. The weight ratio of calcium lactate to the polysaccharide can be 1:0.2 to 1:5.

[0103] In some embodiments, the pharmaceutical composition further comprises a polymer or a lipid. The weight ratio of calcium lactate to the polymer or lipid can be at least 1:5. The weight ratio of calcium lactate to the 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.

[0104] In some embodiments, the pharmaceutical composition further comprises a polymer and a lipid. The weight ratio of calcium lactate to the polymer and lipid can be at least 1:5.

[0105] The weight ratio of calcium lactate to the 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.

[0106] In some embodiments, the pharmaceutical composition comprises calcium lactate and a polymer or a lipid. The weight ratio of calcium lactate to the polymer or lipid can be at least 1:5. The weight ratio of calcium lactate to the polymer or lipid can be 1:5 to 1:30. The weight ratio of calcium lactate to the 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.

[0107] In some embodiments, the pharmaceutical composition comprises calcium lactate and a polymer and a lipid. The weight ratio of calcium lactate to the polymer and lipid can be at least 1:5. The weight ratio of calcium lactate to the polymer and lipid can be 1:5 to 1:30. The weight ratio of calcium lactate to the 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.

[0108] In some embodiments, the weight ratio of the polymer to the lipid can be from 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.

[0109] Polysaccharides suitable for use in the composition can be cellulose derivatives (e.g., carboxymethyl cellulose (CMC), ethyl cellulose (EC), hydroxypropyl methyl cellulose (HPMC), methyl cellulose (MC)), pectin, hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen, alginate, alginic acid, or a combination of the above.

[0110] Polymers suitable for use in the composition can be poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), or a combination of the above.

[0111] Lipids suitable for use in the composition can be monoglycerides or triglycerides or polyethylene glycol, polyethylene glycol esters of vegetable oils, propylene glycol fatty acid esters, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa butter, cottonseed oil, sunflower 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, acetyl palmitate, lauryl alcohol, oleyl alcohol, cetyl alcohol, stearyl alcohol, or a combination of the above.

[0112] The present invention also relates 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 pharmaceutically acceptable enteric coating. In some embodiments, the enteric coating comprises hydroxypropyl methyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or a combination of the above. In some embodiments, the weight ratio of calcium lactate to the enteric coating is from 10:0.5 to 10:15, 10:0.5 to 1:1, 10:0.5 to 10:5, 10:0.5 to 10:3, 10:0.5 to 10:2, 10:0.5 to 10:1, 10:0.5 to 1:0.8.

[0113] As used herein, "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, i.e., the substance can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained.

[0114] As used herein, a pharmaceutical composition refers to a mixture of calcium lactate and other pharmaceutically 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 buffering agents. The pharmaceutical composition facilitates the administration of calcium lactate to a subject.

[0115] As used herein, the term "carrier" refers to a relatively non-toxic compound or agent that facilitates the incorporation of a compound into cells or tissues. The term "diluent" refers to a compound used to dilute a target compound 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 formers, permeants, gas formers, lubricants, and fluidizing agents well known in the art.

[0116] Diluents can include lactose, fructose, dextrose, sucrose, maltose, microcrystalline cellulose, starch, calcium hydrogen phosphate, mannitol, or a mixture thereof, but are not limited thereto. Other diluents include microcrystalline cellulose, lactose, mannitol, calcium phosphate, etc.

[0117] Examples of binders can include povidone, hydroxypropyl cellulose, polyvinyl alcohol, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and the like.

[0118] Solubilizers include surfactants, cyclodextrins and their derivatives, lipophilic substances, or a mixture thereof, but are not limited thereto.

[0119] Surfactants include water-soluble or water-dispersible nonionic, nonpolar nonionic, anionic, cationic, amphoteric, or ionic surfactants, or a mixture thereof, but are not limited thereto.

[0120] 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.

[0121] The pharmaceutical composition of the present invention may also include antimicrobial agents, such as benzyl alcohol, chlorobutanol, phenethyl alcohol, phenylmercuric acetate, potassium sorbate, and sorbic acid. Antifungal agents include compounds such as benzoic acid, butyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, and sodium benzoate.

[0122] An antimicrobial preservative can be added to the pharmaceutical compositions of the present invention to protect them from the growth of potentially harmful microorganisms which typically invade the aqueous phase, but which in some cases may also grow in the oil phase of the composition. Thus, preservatives having both water solubility and lipid solubility are desirable. Suitable antimicrobial preservatives include, for example, alkyl esters of p-hydroxybenzoic acid, propionates, phenoxyethanol, sodium methyl p-hydroxybenzoate, sodium propyl p-hydroxybenzoate, sodium dehydroacetate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, hydantoin derivatives, quaternary ammonium compounds and cationic polymers, imidazolidinyl urea, diazolidinyl urea, and trisodium ethylenediaminetetraacetate (EDTA).

[0123] Antioxidants can be added to protect all the components of the pharmaceutical composition from damage or degradation by oxidants present in the composition itself or in the environment of use, for example, anoxomer, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, potassium metabisulfite, propyl octyl and dodecyl gallates, sodium metabisulfite, sulfur dioxide, and tocopherols.

[0124] Once established, buffers can be used to maintain the pH required for the pharmaceutical composition, protecting it from the effects of external agents and shifts in the balance of the composition components.

[0125] The pharmaceutical compositions described herein can be prepared by techniques known in the art, for example, as described in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E. Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999), which are hereby incorporated by reference in their entirety.

[0126] In some embodiments, the pharmaceutical composition of the present invention is short-acting. The term "short-acting" means that when tested in the in vitro dissolution test described herein, the composition releases substantially all of the active agent within 48 hours, e.g., from delivery time zero until about 1 hour to about 48 hours, until about 3 hours to about 24 hours, until about 6 hours to about 24 hours, or until about 12 hours to about 24 hours.

[0127] For example, in an in vitro dissolution test including an elution test method (e.g., from Labfine Co., Mumbai, India) at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter with a pore size of 45 μm, after placing the composition, 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, in an in vitro dissolution test including an elution test method (e.g., from Labfine Co., Mumbai, India) at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter with a pore size of 45 μm, after placing the composition, 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.

[0128] 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 the active agent after an initial dose, e.g., from delivery time zero until about 48 hours to about 192 hours, until about 72 hours to about 192 hours, until about 96 hours to about 192 hours, until about 120 hours to about 192 hours or until about 144 hours to about 192 hours.

[0129] In some embodiments, after placing the composition in an in vitro dissolution test including an elution test method (e.g., from Labfine Co., Mumbai, India) at 300 rpm in 200 ml of an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, 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, after placing the composition in an in vitro dissolution test including an elution test method (e.g., from Labfine Co., Mumbai, India) at 300 rpm in 200 ml of a culture medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm, 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.

[0130] In some embodiments, after placing the composition in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then 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.

[0131] In some embodiments, the pharmaceutical composition of the present invention is in an injectable dosage form. For parenteral injections, suitable formulations may include aqueous or non-aqueous solutions, preferably with a physiologically compatible carrier.

[0132] Pharmaceutical preparations for parenteral administration include aqueous solutions of one or more active compounds in water-soluble form. Additionally, suspensions of the active agent can be prepared as suitable oily injection suspensions. Suitable lipid 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 monoglycerides or triglycerides or polyethylene glycol, polyethylene glycol esters of vegetable oils, propylene glycol esters of fatty acids, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa butter, cottonseed oil, sunflower 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 or combinations thereof.

[0133] Since the medical application of hydrogels, many 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 drug delivery, and various efforts have been made to solve these problems. Hydrogels are three-dimensional structures composed of hydrophilic polymer networks. More than 90% of the components are composed of water. Due to the similarity of hydrogels to biological tissues, such as high water content, porous structure, relatively soft properties and biocompatibility, they have been actively studied in the biomedical field. Hydrogels can exhibit various properties, depending on the type of polymer used as the backbone or the cross-linking method employed. When using polymers of the polyacrylic acid series or synthetic compounds such as polyvinyl alcohol, the biocompatibility is low, but chemical modification is easy, making engineering applications very easy. On the other hand, when using natural compounds, especially pectin, alginate, collagen, fibrin and hyaluronic acid as the backbone, chemical modification is difficult. However, using these substances as components of biological origin has many advantages, because it is suitable for clinical applications and has almost no side effects, such as immune inflammatory reactions during transplantation.

[0134] The crosslinking method affects the properties of the hydrogel and the type of polymer used. Even if the same polymer is used as the main chain, hydrogels with completely different properties can be obtained if the crosslinking methods are different. The methods of crosslinking hydrogels can be roughly divided into physical methods and chemical methods. Physical crosslinking methods include ionic interactions, hydrophobic interactions, hydrogen bonds, and reversible crosslinking through structural entanglement. These crosslinking methods can easily induce the formation of a three-dimensional network structure without the need for separate chemical additives or complex processes. On the other hand, compared with physical crosslinking methods, chemical crosslinking methods usually form covalent bonds, which result in the formation of an irreversible and stable network. Hydrogels with excellent biocompatibility and various physicochemical properties have been widely studied in biomedical fields such as drug delivery and tissue engineering. Most hydrogels show a shorter drug release time than other drug delivery systems due to their high water content, and there is a need to develop systems with a longer drug release time.

[0135] In some embodiments, the pharmaceutical composition of the present invention is an oral dosage form. For oral administration, the compounds described herein can be easily formulated by combining the active agent with pharmaceutically acceptable carriers or excipients well known in the art. Such carriers enable the formulation of the active agents described herein into tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, etc., for oral administration to the patient to be treated.

[0136] A pharmaceutical preparation for oral administration can be obtained by mixing one or more solid carriers with the compounds described herein, optionally grinding the resulting mixture, and, if necessary, processing the particulate mixture with the addition of suitable auxiliaries to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrants such as cross-linked sodium carboxymethylcellulose, polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, can be added.

[0137] The dragee cores have suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablet or dragee coatings to identify or characterize different combinations of the active compound doses.

[0138] The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, which include excipients and auxiliaries that facilitate processing the active agent into a pharmaceutically acceptable preparation. The appropriate preparation depends on the chosen route of administration. Any well-known techniques can be used appropriately and as understood in the art. The pharmaceutical compositions described herein can be manufactured in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, dragee-making, grinding, emulsifying, encapsulating, entrapping, or compression processes.

[0139] Aqueous suspensions may also contain one or more polymers as suspending agents. The polymers can be poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), or a combination thereof. Other useful polymers include water-soluble polymers such as cellulose polymers, for example hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Useful compositions may also include mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0140] In some embodiments, there are provided pharmaceutical dosage forms comprising calcium lactate and a pharmaceutically acceptable enteric coating to control the release of the active agent. In some embodiments, the coating is a film, and in other embodiments, it is a membrane. The enteric coating, such as a film or a membrane, can be used to delay release until after the stomach and to protect the active agent from gastric juice. The enteric coating can comprise one or more substances preferably having polymeric nature (e.g., methacrylates, etc.; polysaccharides, etc., as described in more detail below) or a combination of more than one such substance, optionally including other excipients such as, for example, plasticizers. 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 a combination thereof. In some embodiments of the present invention, the composition comprises a polymer that forms a hydrogel and other polymers capable of achieving a desired delay (or other variation) in the release of the active agent.

[0141] In the case of tablets for oral administration, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are usually also added. For oral administration in capsule form, useful diluents include lactose and dried corn starch.

[0142] Sterile injectable preparations can also be sterile injectable suspensions in a non-toxic parenterally acceptable carrier such as a lipid.

[0143] The amount of calcium lactate included in the pharmaceutical composition can be, but is not limited to, 1 wt% to 50 wt%, 1 wt% to 40 wt%, 1 wt% to 35 wt%, 1 wt% to 30 wt%, 1 wt% to 20 wt%, 1 wt% to 15 wt%, or 1 wt% to 10 wt% based on the total weight of the final composition. The concentration of calcium lactate included in a single dose of the pharmaceutical composition can be, but is not limited to, 2.5 mM to 100 mM, 2.5 mM to 50 mM, 2.5 mM to 25 mM, 5 mM to 100 mM, 5 mM to 50 mM, 5 mM to 25 mM, 10 mM to 100 mM, 10 mM to 50 mM, or 10 mM to 25 mM.

[0144] Solid oral pharmaceutical compositions can be prepared by conventional techniques such as dry granulation, direct compression, wet granulation, extrusion spheronization, melt granulation, or compression coating, but are not limited thereto. The coating can be applied as described below and can vary with respect to thickness and density. The amount of the coating is determined by the additional weight (obtained) added to the dry composition of the present invention (e.g., beads or cores containing calcium lactate). The weight increase can be in the range of 0.1% to 50%, 1% to 20%, 1% to 15%, 3% to 10%, 5% to 12%, or 8% to 12%.

[0145] The coating process can be carried out in any suitable manner, such as, for example, by using a coating machine that applies a solution of the polymer coating (especially as described above) to the composition. The polymer for coating can be provided by the manufacturer as a ready-to-use solution for direct use, or can be manufactured before use according to the manufacturer's instructions.

[0146] Unit Doses and Kits

[0147] The term "unit dosage form" refers to a physically discrete unit suitable as a single dose, each unit containing a predetermined amount of the active ingredient in association with a suitable pharmaceutical excipient, whereby it is used one or more times throughout the dosing regimen to produce the desired therapeutic effect, such as treating one or more of the diseases disclosed herein.

[0148] The pharmaceutical compositions described herein can be in unit dosage forms suitable for precise dosing in a single administration. In a unit dosage form, the formulation is divided into unit doses containing an appropriate amount of one or more active agents. A unit dose can be a packaged form containing a discrete amount of the formulation. Non-limiting examples are packaged tablets or capsules, and powders contained in vials or ampoules. An aqueous suspension composition can be packaged in a single-dose non-resealable container. Alternatively, a multi-dose resealable container can be used, in which case a preservative is usually included in the composition. By way of example only, the formulation of a parenteral injection can be in unit dosage form, which includes but is not limited to ampoules or in multi-dose containers.

[0149] Suitable daily doses for calcium lactate can be from 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 500 mg / kg of body weight. In larger mammals, including but not limited to humans, the daily dose ranges shown are from about 5 mg to about 100,000 mg, conveniently administered in divided doses, including but not limited to up to 4 times a day or in a long-acting form. Suitable unit dosage forms for administration include from 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 the active agent. The frequency of administration of the compositions of the present disclosure can be, but is not particularly limited to, once, twice, three times, four times, etc. in divided doses per day. In any individual case, techniques such as dose escalation studies can be used to determine the appropriate "effective" amount.

[0150] The foregoing ranges are merely suggestive, since the number of variables regarding individual treatment regimens is large, and fairly large deviations from these recommended values are not uncommon. Such doses can be varied according to many variables, not limited to the activity of the compound used, the disease or disorder to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or disorder being treated, and the judgment of the practitioner.

[0151] The toxicity and efficacy of such treatment regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including but not limited to determining the LD 50 (lethal dose for 50% of the population) and the ED 50 (therapeutically effective dose in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the ratio between LD 50 and ED 50 . Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies can be used to formulate a dose range for humans. The dose of the active agent is preferably within the range of circulating concentrations, which includes the ED 50 with minimal toxicity. The dose can vary within this range depending on the dosage form employed and the route of administration utilized.

[0152] 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 oily suspension. The pharmaceutical composition of the present invention can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.

[0153] The present invention also relates to sterile glass or polyolefin containers containing the pharmaceutical compositions 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).

[0154] The kit includes a suitable container such as a box, a single bottle, a bag or an ampoule. The suspension composition can be packaged in a single-dose non-reclosable container or a multi-dose reclosable container.

[0155] US2017 / 0360727A1, published on December 21, 2017, and PCT / IB2017 / 054091, with an international filing date of July 7, 2017, are incorporated herein by reference in their entirety. Also incorporated herein by reference in its entirety is US Application No. 62 / 616,923, filed on January 12, 2018.

[0156] Examples

[0157] Example 1

[0158] Cell culture of age-related macular disease

[0159] The human retinal pigment epithelial cell line ARPE-19 was purchased from the American Type Culture Collection (Manassas, VA, USA). The 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 (Welgene, Daegu, South Korea).

[0160] Example 2

[0161] Cell culture of Alzheimer's disease

[0162] The human brain cell line SK-N-SH was purchased from the American Type Culture Collection (Manassas, VA, USA). The 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).

[0163] Example 3

[0164] Cell culture of fibroblasts

[0165] The human colon fibroblast cell line CCD-18-Co was purchased from the American Type Culture Collection (Manassas, VA, USA). The 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).

[0166] Example 4

[0167] Cell culture of non-alcoholic steatohepatitis (NASH)

[0168] The human hepatocellular carcinoma cell line HepG2 was purchased from the American Type Culture Collection (Manassas, VA, USA). The 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).

[0169] Example 5

[0170] Cell culture of vascular diseases

[0171] The human umbilical vein endothelial cell line HUVEC was purchased from the American Type Culture Collection (Manassas, VA, USA). The HUVEC cells were maintained in endothelial cell growth medium 2 supplemented with MIX (PromoCell, Heidelberg, Germany).

[0172] Example 6

[0173] Macrophage culture

[0174] The human hepatic stellate cell line LX-2 was purchased from Sigma-Aldrich (St Louis, MO, USA). The 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).

[0175] 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. Cells were cultured under two conditions, one under a humid atmosphere at 37 °C with 5% CO2, and the other under hypoxic culture conditions maintained at 1% oxygen, 5%, and 94%.

[0176] Example 7

[0177] Reagents

[0178] 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 Life Sciences (Enzo Diagnostic, NY, USA). Human interferon-γ, human interleukin-4 (IL4), 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).

[0179] Example 8

[0180] Cell viability assay

[0181] The toxicity of CaLa was confirmed in ARPE-19 cells using the live cell imaging analyzer JuLI TM Br (NanoEnTek Inc., Seoul, Korea). Cells were seeded at a density of 2.5 x 10 5 cells / well in a 6-well culture plate in culture medium. After incubation for 24 h, the cells were treated with 2.5 mM CaLa for 12 h and 24 h. During the CaLa treatment period, the cells were continuously monitored for 24 h. (30, 31)

[0182] Example 9

[0183] Macrophage differentiation

[0184] 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 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 mg / ml streptomycin (Welgene, Daegu, South Korea), and 0.55 μM 2-mercaptoethanol (Gibco, Grand Island, NY, USA). THP-1 monocytes were maintained in a humidified atmosphere of 5% CO2 at 37 °C. THP-1 monocytes were differentiated into macrophages by incubating with phorbol 12-myristate 13-acetate (PMA) and 100 μg / ml (Enzo Diagnostic, NY, USA) in RPMI 1640 medium for 12 h. Macrophages were polarized into M1 macrophages by incubating with 20 ng / ml of IFN-γ (BPS Bioscience, San Diego, CA, USA) and 100 μg / ml of LPS (Sigma-Aldrich, St Louis, MO, USA) in RPMI 1640 medium for 24 h. Macrophage M2 polarization was obtained by incubating 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)

[0185] Example 10

[0186] Optical observation of cell viability

[0187] Using the live cell imaging analyzer JuLI TM Br (NanoEnTek Inc., Seoul, Korea), the toxicity of CaLa was confirmed in ARPE-19 cells. Cells were seeded into 6-well culture plates at a density of 2.5 x 10 5 cells / well. After incubation for 24 h, the cells were treated with 2.5 mM CaLa for 12 h and 24 h. During the CaLa treatment period, the cells were continuously monitored for 24 h.

[0188] Example 11

[0189] Protein immunoblot analysis

[0190] To prepare whole cell lysates, cells were lysed with 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 protease inhibitor mixture (Roche, Basel, Switzerland) and phosphatase inhibitors (NA3VO4, 1 mM, NaF 100 mM). Then, protein concentration was measured using a bicinchoninic acid (BCA) assay kit (Thermo Scientific, Waltham, MA). Whole cell or nuclear lysates were transferred onto polyvinylidene difluoride membranes (Merek Milliporc, Billerica, MA, USA). After blocking in 5% non-fat milk (Bio-Rad, Hercules, CA, USA) for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies diluted in TBST obtained from Sigma-Aldrich containing 100 mM Tris-HCl (pH 7.5), 1.5 M NaCl and 0.5% Tween-20 and supplemented with 5% BSA and 0.1% sodium azide (Sigma-Aldrich). Specific primary antibodies 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, the membranes were washed with TBST and incubated with anti-rabbit secondary antibody (1:10000) for 2 h. Immunoblots were developed using a protein immunoblot detection reagent (Abclone, Seoul, Korea) according to the manufacturer's protocol and exposed to x-ray film (Agfa, Leverkusen, Germany).

[0191] Example 12

[0192] Immunocytochemistry

[0193] ARPE-19 and SK-N-SH cells were fixed on a biocoated coverslip (BD bioscience, San Jose, CA, USA) with 4% PFA for 20 min and then incubated with primary antibodies for 15 h. The primary antibodies were as follows: 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); apoE (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with PBS, the cells were incubated with an anti-mouse secondary biotinylated antibody (1:2000, Vector Laboratorie, Burlingame, CA, USA) and visualized with streptavidin conjugated to fluorescein (Vector Laboratorie, Burlingame, CA, USA). The coverslip was mounted on a microscope slide using a Hard Set TM mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Confocal fluorescence images were obtained using a confocal laser scanning microscope (LSCM, Nikon A1+, Tokyo, Japan) under a 60X oil immersion lens.

[0194] Example 13

[0195] Animal model of NASH

[0196] All experiments were conducted according to 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-deficient diet (MCD) for 4 weeks. When the mice weighed 15 g each, the mice were randomly dissected to identify non-alcoholic steatohepatitis (NASH). The mice were injected subcutaneously with calcium lactate (2 mg / kg) twice a day (B.I.D) for 4 weeks.

[0197] Example 14

[0198] Animal model of age-related macular degeneration

[0199] All experiments were conducted according to 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 on a 12 h light / dark cycle (lights on, 08:00) and had free access to food and water. The mice were placed individually in an induction chamber and anesthetized with 2% isoflurane (HANA PHARM CO., Seoul, Korea). Injury (lesion) was 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 a 100-μm spot size, 700 mW power, and 1-s exposure time. A total of 15 mice were randomly divided into three groups: (i) no laser induction (n = 3); (ii) laser burn and injection of vehicle buffer (n = 5); and (iii) CaLa, 2 mg / kg / day (B.I.D) (n = 7). CaLa treatment was started several days after laser photocoagulation and was administered subcutaneously for 14 days. (33, 34)

[0200] Example 15

[0201] Histological analysis of the NASH model

[0202] The liver tissues of mice (n = 5) were dissected and fixed in 4% paraformaldehyde PBS solution at 4 °C for 15 h. The fixed liver tissues were embedded in paraffin and cut into 10-μm sections. The slides were incubated at 55 °C for 2 h, then dewaxed in xylene, rehydrated in a graded ethanol series, and used for hematoxylin (Merck, Darmstadt, Germany) and eosin (H&E) staining (35). The paraffin sections (10 μm thick) stained with H&E were imaged using a Leica DM 1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0203] Example 16

[0204] Histopathological analysis of age-related macular degeneration

[0205] Two weeks after subcutaneous administration of 2 mg / kg of CaLa, histopathological examination of retinopathy was performed. The enucleated eyes were fixed in 4% paraformaldehyde (PFA) at 4 °C for 24 h, and the eyes obtained by removing the anterior segment were washed three times in phosphate-buffered saline. The fixed flat mounts were dehydrated in a series of graded ethanol and embedded in paraffin. The 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 1000LED microscope (Leica Microsystems, Wetelar, Germany). Choroidal lesions and neovascularization on the flat mounts were observed by confocal microscopy (Carl Zeiss, Oberkochcn, Germany).

[0206] Example 17

[0207] Masson's trichrome staining

[0208] The liver tissues of mice (n = 5) were dissected and fixed in 4% paraformaldehyde PBS solution at 4 °C for 15 h. The fixed tissues were embedded in paraffin and cut into 5-mm sections. The slides were incubated at 55 °C for 2 h, then dewaxed in xylene, rehydrated in a graded ethanol series, and refixed in Bouin's solution at 56 °C for 1 h. The yellow color was removed by rinsing with running tap water for 5 - 10 min, and stained in Biebrich scarlet - acid fuchsin solution for 5 min, then stained in phosphomolybdic acid - phosphotungstic acid solution (1:1 ratio) for 30 min, and aniline blue solution and stained for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 2 - 5 min. Dehydrated with 95% ethanol and absolute ethanol and clarified in xylene. Fixed with a mounting medium. Images were taken using a Leica DM 1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0209] Example 18

[0210] Results

[0211] Figure 1 It was an in vitro experiment on human leukemia monocytes. The Western blot results showed that under hypoxic conditions in THP-1 cells (human leukemia monocytes), the inflammatory factor (NF-κb) decreased after treatment with 2.5 mM calcium lactate.

[0212] Figure 2 It was an in vitro experiment on human leukemia monocytes differentiated into macrophages. The Western blot results showed that under normoxic and hypoxic conditions, the inflammatory factor (NF-κb) decreased in THP-1 cells (M0 macrophages) differentiated with PMA (phorbol 12-myristate 13-acetate) after treatment with 2.5 mM calcium lactate.

[0213] Figure 3 The provided Western blot results showed that under normoxic and hypoxic conditions, the inflammatory factor (NF-κb) decreased in THP-1 cells (M1 macrophages) differentiated with interferon γ (IFN-γ) and lipopolysaccharide (LPS) after treatment with 2.5 mM calcium lactate.

[0214] Figure 4 The provided Western blot results showed that under normoxic and hypoxic conditions, the inflammatory factor (NF-κb) decreased in THP-1 cells (M2 macrophages) differentiated with interleukin - 4 (IL-4) and interleukin - 10 (IL-10) after treatment with 2.5 mM calcium lactate.

[0215] Figure 5It is an in vitro experiment on liver fibrosis. The Western blot results show that under hypoxic conditions, after treatment with 2.5 mM calcium lactate, the hypoxia-inducible factor-1α (HIF-1α) in LX-2 cells (human hepatic stellate cells) decreases.

[0216] Figure 6 It is an in vitro experiment on human endothelial cells. The Western blot results show that under hypoxic conditions, after treatment with 2.5 mM calcium lactate, the hypoxia-mediated factor (lactate dehydrogenase A) in human umbilical vein endothelial cells decreases.

[0217] Figure 7 It is an in vitro experiment on human fibroblasts. The Western blot results indicate that under hypoxic conditions, after treatment with 2.5 mM calcium lactate, the lactate dehydrogenase B in CCD-18Co cells (human colon fibroblasts) increases.

[0218] Figure 8 It is an in vitro experiment on brain CID. The Western blot results show that under normoxic and hypoxic conditions, after treatment with 2.5 mM calcium lactate, the hypoxia-mediated factor in SK-N-SH cells (cerebral epithelial cells) decreases.

[0219] Figure 9 It is an in vitro experiment on liver CID. The immunoblot results show that under hypoxic conditions, after treatment with 2.5 mM calcium lactate, the hypoxia-mediated factor in HepG2 cells (liver epithelial cells) changes.

[0220] Figure 10 The provided Western blot results show that under hypoxic conditions, after treatment with 2.5 mM calcium lactate, the inflammatory factor (TLR-4) in HepG2 cells (liver epithelial cells) decreases.

[0221] Figure 11 It is an in vitro experiment on eye CID. The Western blot results show that under normoxic and hypoxic conditions, after treatment with 2.5 mM calcium lactate, the hypoxia-mediated factor in ARPE-19 cells (retinal pigment epithelial cells) decreases.

[0222] Figure 12 The provided immunocytochemical results show that under hypoxic conditions, after treatment with 2.5 mM calcium lactate, the hypoxia-inducible factor-1α (HIF-1α) in ARPE-19 cells (retinal pigment epithelial cells) decreases.

[0223] Figure 13 The provided immunocytochemical results show that under normoxic conditions, after treatment with 2.5 mM calcium lactate, the lactate dehydrogenase A in ARPE-19 cells (retinal pigment epithelial cells) decreases.

[0224] Figure 14The provided immunocytochemical results show that under hypoxic conditions, treatment with 2.5 mM calcium lactate reduces the inflammatory factor (nuclear factor-κB) in ARPE-19 cells (retinal pigment epithelial cells).

[0225] Figure 15 The provided immunocytochemical results show that under hypoxic conditions, treatment with 2.5 mM calcium lactate reduces the inflammatory factor (toll-like receptor 4) in ARPE-19 cells (retinal pigment epithelial cells).

[0226] Figure 16 The provided immunocytochemical results show that under hypoxic conditions, treatment with 2.5 mM calcium lactate increases lactate dehydrogenase B in ARPE-19 cells (retinal pigment epithelial cells).

[0227] Figure 17 The provided immunocytochemical results show that under hypoxic conditions, treatment with 2.5 mM calcium lactate reduces the drusen marker (apolipoprotein E) in ARPE-19 cells (retinal pigment epithelial cells).

[0228] Figure 18 The provided western blot results show that under hypoxic conditions, treatment with 2.5 mM calcium lactate reduces the inflammatory factor (NF-κB) in ARPE-19 cells (retinal pigment epithelial cells).

[0229] Figure 19 It shows that treatment with 2.5 mM calcium lactate has no toxicity in ARPE-19 cells (retinal pigment epithelial cells). Calcium lactate only plays an important role in the metabolic changes of damaged epithelial cells.

[0230] Figure 25 The provided western blot results show that treatment with 2.5 mM calcium lactate reduces the inflammatory factor (NF-κb) in retinal pigment epithelial cell tissue.

[0231] Example 19

[0232] Targeted digestive diseases (NFALD, NASH)

[0233] Materials and methods

[0234] Animal model of liver disease. All experiments were conducted according to 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-deficient (MCD) diet for 4 weeks. Mice were injected subcutaneously with calcium lactate (2 mg / kg) twice a day (B.I.D.) for 4 weeks. Histological analysis. Mouse liver tissues (n = 5) were dissected and fixed in 4% paraformaldehyde in PBS solution at 4 °C for 15 h. The fixed liver tissues were embedded in paraffin and cut into 10-μm sections. The slides were incubated at 55 °C for 2 h and then deparaffinized in xylene and rehydrated in a 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 1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0235] 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. The fixed tissues were embedded in paraffin and cut into 5-mm sections. The slides were incubated at 55 °C for 2 h and then deparaffinized in xylene and rehydrated in a graded ethanol series and refixed in Bouin's solution at 56 °C for 1 h, decolorized by rinsing with running tap water for 5-10 min, and stained in Ponceau Scarlet-Acid Fuchsin solution for 5 min, then stained in Phosphomolybdic-Phosphotungstic acid solution (1:1 ratio) for 30 min, and Aniline Blue solution and stained for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 2-5 min. Dehydrated with 95% ethanol, absolute ethanol and cleared in xylene. Mounted with mounting medium. Images were taken using a Leica DM 1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0236] Figure 20 This is an in vivo experiment of liver CID (NASH). To induce NASH, mice were fed a methionine-choline-deficient (MCD) diet with polyunsaturated fat for 8 weeks. Blood chemistry results showed that serum AST and ALT were significantly decreased by 2 mg / kg calcium lactate treatment. **P < 0.001 vs. methionine-choline-deficient (MCD). Results are mean ± SD.

[0237] Figure 21The provided immunohistochemical results showed that no lipid droplets (white holes in liver tissue) were observed in the calcium lactate treatment group, while lipid droplets were observed in the methionine-choline deficient (MCD) group. Fatty degeneration could be prevented by treatment with 2 mg / kg calcium lactate. The results showed that fatty degeneration was prevented by calcium lactate treatment.

[0238] Figure 22 The provided immunohistochemical results showed that, similar to the control group, no hepatic lipid droplets (white holes in liver tissue) were present in the 2 mg / kg calcium lactate treatment group, while they were found in the methionine-choline deficient (MCD) group.

[0239] Figure 23 The provided immunohistochemical results showed that in the liver, similar to the control group, there was a reduction in immune cell infiltration in the 2 mg / kg calcium lactate treatment group, while there was an increase in immune cell infiltration in the methionine-choline deficient (MCD) group.

[0240] Figure 24 The provided immunohistochemical results showed that in the liver, similar to the control group, fibrosis (site of collagen accumulation; white arrow) was absent in the 2 mg / kg calcium lactate treatment group, but it was present in the methionine-choline deficient (MCD) group.

[0241] Example 20

[0242] Targeting eye diseases (age-related macular degeneration (AMD), keratitis, and diabetic retinopathy)

[0243] Materials and methods

[0244] Animals. All experiments were conducted according to 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 at 22 - 25 °C on a 12-hour light / dark cycle (lights on, 08:00) with free access to food and water.

[0245] Preparation of calcium lactate. Calcium lactate was purchased from Sigma Aldrich (St. Louis, MO, USA), and 2 mg / kg of calcium lactate was dissolved in saline for daily subcutaneous injection (for 21 days).

[0246] Animal model of eye diseases. Mice were individually placed in an induction chamber and anesthetized with 2% isoflurane (HANA PHARMCO., Seoul, Korea). Laser-induced Bruch's membrane rupture was induced in both eyes of the mice by laser photocoagulation (SDL405-700, SD Laser, China). The laser pulses were from a green laser (wavelength 532 nm; Visulas532; SD Laser). The laser parameters were a spot size of 100 μm, a power of 700 mw, and an exposure time of 1 s.

[0247] Preparation of choroidal flat mounts. Twenty-one days after laser injury, the mice in each group were anesthetized and perfused through the left ventricle with 1.0 mL of PBS containing 25 mg of fluorescein isothiocyanate-dextran (Sigma-Aldrich, St Louis, MO, USA). The eyes were enucleated and fixed in 4% paraformaldehyde for 1 hour. Retinal pigment epithelium-choroid-sclera eye cups were prepared by cutting the eyes in half, completely removing the lens, vitreous, and retina. The retinal pigment epithelium-choroid-sclera eye cups were flattened by making four or five radial incisions from the edge to the equator (mid-meridian) and flat-fixed in aqua-mount.

[0248] Histological analysis. The fixed flat mounts were dehydrated in a series of graded ethanols 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 1000LED microscope (Leica Microsystems, Wetzlar, Germany). Choroidal lesions and neovascularization on the flat mounts were visualized by confocal microscopy (Carl Zeiss, Oberkochen, Germany).

[0249] Results

[0250] Figure 26 Representative images of H&E staining of the inner layer lesions of the choroidal flat mounts are provided. Abnormal morphology of retinal pigment epithelial cells was observed after AMD induction (middle panel). Treatment with 2.5 mM calcium lactate restored the morphology of retinal pigment epithelial cells in a manner similar to that of normal control epithelial cells (left panel) (right panel).

[0251] Figure 27 Fluorescence images of the inner choroidal lesions on the flat mounts of laser-induced AMD are provided. Choroidal lesions were observed after AMD induction (middle panel). The choroidal lesions were restored after treatment with calcium lactate (right panel).

[0252] Figure 28 Fluorescent images of inner choroidal lesions and neovascularization on a flat scaffold of laser-induced AMD were provided. Choroidal lesions and neovascularization were observed after AMD induction (middle figure). After treatment with calcium lactate, choroidal lesions and neovascularization were restored (right figure), which was the same as the normal control (left figure).

[0253] Example 21

[0254] Targeting Alzheimer's disease & stroke

[0255] Materials and methods

[0256] Animals. All experiments were conducted in accordance with the animal care guidelines according to Korean law (11737, revised on April 5, 2013) and the guidelines established by the Gachon University Institutional Animal Care and Use Committee (LCDI-2018-0073). Eight-week-old male Sprague-Dawley (SD) rats were obtained from Samtako Co. (Osan, Gyeonggi-do, Republic of Korea) and bred (cultured) under specific pathogen-free (SPF) conditions. All animals were maintained at 22 - 25 °C on a 12-hour light / dark cycle with free access to food and water.

[0257] Preparation of lipopolysaccharide (LPS) and calcium lactate. 1 mg of LPS powder was dissolved in 1 ml of saline. If LPS was to be used freshly, the tube was placed on ice. For long-term storage, screw-cap vials were preferably used. Aliquots were stored at -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 injection (for 21 days).

[0258] Intracranial injection of LPS. The animals were placed in small acrylic cages equipped with an anesthesia machine, allowing them to breathe freely under gaseous anesthesia (2% isoflurane, Spartanburg, SC, USA). After achieving complete anesthesia of the animals, they were placed on the operating table (while maintaining gaseous anesthesia), with the upper side of the neck hyperextended to expose the posterior region. The animal fur on the head was shaved off with electric scissors, and the skin was wiped with iodine tincture. LPS was placed on ice before use. All surgical materials were disinfected with 70% ethanol. The internal space of the Hamilton syringe barrel was cleaned with distilled water and 70% ethanol. The Hamilton syringe was placed in the syringe holder. The animals in the stereotaxic device were placed in a horizontal position. A midline incision (about 1 cm) was made on the scalp with a scalpel. The needle of the Hamilton syringe was placed on the bregma. The three coordinates on the manipulator (AP: -3.6, LM: 2.0, and DV: -3.8) (in mm) were read and written down; this was used as the starting point. The DV coordinate of the injection point was slowly set; this would insert the tip of the needle into the brain until the injection point. The LPS solution (2 μl) was released at a flow rate of about 0.5 μl / min. After injection, the needle was withdrawn very slowly. The wound area was closed by suturing, and then a disinfectant was applied. The rats were marked for further identification.

[0259] Immunohistochemistry. The brain tissues were cut into 5 μm on a cryostat and stained for histological examination. Microscopic images were obtained at a magnification of 200X. For immunohistochemistry, the sections were blocked with 1% BSA and 10% NGS in 0.05 M PBS at room temperature for 1 h, and then reacted with rabbit anti-Iba-1 (1:1000, Bioss, MA, USA) at 4 °C for two overnights. The microglia (Iba-1) in the sections were visualized using the ABC staining system (Vector Laboratories, CA, USA).

[0260] Results

[0261] Figure 29 An experimental protocol for establishing Alzheimer's disease and stroke diseases is provided. OMT-110 is calcium lactate.

[0262] Figure 30 Representative images are provided, showing the damage to the brain by LPS. Contralateral: Normal area. Ipsilateral: LPS injection.

[0263] Figure 31 The recovery of the damaged brain tissue and the reduction in the infiltration of microglia recruited due to brain injury are shown. The results show that the inflammatory response exacerbating Alzheimer's disease and stroke is greatly alleviated.

[0264] Example 22

[0265] Targeting Parkinson's disease

[0266] Materials and methods

[0267] Animals. All experiments were conducted in accordance with the Korean legal guidelines for animal care (11737, revised on April 5, 2013) and the guidelines established by the Gachon University Institutional Animal Care and Use Committee (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 at 22 - 25 °C on a 12-hour light / dark cycle with free access to food and water.

[0268] Preparation of desipramine, 6-OHDA, and calcium lactate. Desipramine (12.5 mg / kg; norepinephrine 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. Calcium lactate (2 mg / kg, Sigma Aldrich, St Louis, USA) was dissolved in saline for daily subcutaneous injection (for 21 days).

[0269] Injections of desipramine and 6-OHDA. Thirty minutes before the 6-OHDA injection, desipramine was injected into the animals intraperitoneally (intraperitoneal injection). Thirty minutes after the desipramine injection, the animals were placed in small acrylic cages equipped with an anesthesia machine, allowing them to breathe freely under gaseous anesthesia (2% isoflurane, Spartanburg, SC, USA). After achieving complete anesthesia of the animals, they were placed on the operating table (while maintaining gaseous anesthesia), with the upper side of the neck hyperextended to expose the posterior region. The head fur of the animals was shaved with an electric scissor, and the skin was wiped with iodine tincture. All surgical materials were sterilized with 70% ethanol. The internal space of the Hamilton syringe barrel was cleaned with distilled water and 70% ethanol. The Hamilton syringe was placed in the syringe holder. The animals in the stereotaxic device were placed in a horizontal position. A midline incision (about 1 cm) was made on the scalp with a scalpel. The needle of the Hamilton syringe was placed on the bregma. Three coordinates on the manipulator were read (AP: -5.6, LM: 2.0, and DV: -7.6) (in mm) and used as the starting point. The DV coordinate of the injection point was slowly set; this inserted the tip of the needle into the brain until the injection point. The 6-OHDA solution (20 μg / 4 μl) was released at a flow rate of about 1 μl / min. After the injection, the needle was withdrawn very slowly. The wound area was closed by suturing, and then a disinfectant was applied. The rats were marked for further identification.

[0270] Immunohistochemistry. The brain tissues were cut into 5 μm on a cryostat and stained for histological examination. Microscopic images were obtained at a magnification of 200X. For immunohistochemistry, the sections were blocked with 1% BSA and 10% NGS in 0.05 M PBS for 1 h at room temperature, and then reacted with rabbit anti-tyrosine hydroxylase (1:500, Chemicon, Tokyo, Japan) for two overnights at 4 °C. The ABC staining system was used to visualize dopaminergic neurons in the sections (Vector Laboratories, CA, USA).

[0271] Results

[0272] Figure 32 An experimental protocol for establishing Parkinson's disease is provided. OMT-110 is calcium lactate.

[0273] Figure 33 It is shown that dopaminergic neurons are restored 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 (substantia nigra).

[0274] Example 23

[0275] Targeting multiple sclerosis and spinal cord injury

[0276] Materials and Methods

[0277] Animals. All experiments were conducted in accordance with the Korean legal animal care guidelines (11737, revised on April 5, 2013) and the guidelines established by the Gachon University Institutional Animal Care and Use Committee (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 at 22 - 25 °C on a 12-hour light / dark cycle and had free access to food and water.

[0278] Preparation of lipopolysaccharide (LPS) and calcium lactate. 1 mg of LPS powder was dissolved in 1 ml of saline. If LPS was to be used fresh, the tube was placed on ice. For long-term storage, screw-cap vials were preferably used. Aliquots were stored in a deep freezer (-20 °C). Calcium lactate was purchased from Sigma Aldrich (St. Louis, USA). 2 mg / kg of calcium lactate was dissolved in saline for daily subcutaneous injection (for 21 days).

[0279] Intrathecal injection of LPS. The animals were placed in a small acrylic cage equipped with an anesthesia machine so that they could breathe freely under gas anesthesia (2% isoflurane, Spartanburg, SC, USA). After the animals were fully anesthetized, they were placed on the operating table (while maintaining gas anesthesia). For intrathecal injection of LPS, the laminectomy site of the rats was exposed. Two small holes 1 mm apart were made in the dura mater above the left spine. 1 μl volume 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. Then, the injection site was sealed with surgical clips.

[0280] Immunohistochemistry. Spinal cord tissues were cut into 5-μm sections on a cryostat and stained for histological examination. Microscopic images were obtained at a magnification of 200X. For immunohistochemistry, the sections were blocked with 1% BSA and 10% NGS in 0.05 M PBS for 1 h at room temperature and then reacted with rabbit anti-Iba-1 (1:1000, Bioss, MA, USA) overnight at 4 °C. Microglia (Iba-1) in the sections were visualized using the ABC staining system (Vector Laboratories, CA, USA).

[0281] Results

[0282] Figure 34An experimental protocol for establishing multiple sclerosis is provided. OMT-110 is calcium lactate.

[0283] Figure 35 Recovery of demyelinated spinal cord and reduction of microglial infiltration treated with calcium lactate are shown. Demyelination of the central nervous system and the occurrence of immune infiltration are associated with a variety of disorders, including inflammation in multiple sclerosis and spinal cord injury.

[0284] Example 24

[0285] Targeting vascular diseases (atherosclerosis, intracerebral hemorrhage, and myocardial infarction)

[0286] Materials and Methods

[0287] Animals. All experiments were conducted in accordance with the Korean legal animal care guidelines (11737, revised on April 5, 2013) and the guidelines established by the Institutional Animal Care and Use Committee of the Gyeonggi bio center (2017-11-0008, South Korea). Male BALB / c ApoE mice at 8 weeks of age were obtained from SLC Inc. (Shizuoka, Japan) and bred under specific pathogen-free (SPF) conditions. All animals were maintained at 22-25 °C on a 12-hour light / dark cycle with free access to food and water. shi

[0288] Preparation of calcium lactate. Calcium lactate was purchased from Sigma Aldrich (St. Louis, MO, USA). 2 mg / kg of calcium lactate was dissolved in saline for daily subcutaneous injection (for 21 days).

[0289] Animal models of vascular diseases. BALB / c ApoE mice were used for spontaneously induced vascular diseases. In this study, a high-fat (40%) diet was provided for 4 weeks to induce severe atherosclerosis. shi

[0290]

[0291] Histological analysis. The thoracic aorta was incised, and half of the aorta was fixed in 10% neutral buffered formalin fixative. Then, histological analysis was performed. Histological analysis was performed using H&E and Oil Red O staining. The aortic wall thickness, atherosclerotic plaque area, number of foam cells, and lipidized area were analyzed.

[0291] Statistical analysis. Statistical analysis was performed using SPSS statistics, and the results were analyzed using the Student t-test. The Mann-Whitney method was used for histological analysis. P < 0.05 was considered statistically significant.

[0292] Results

[0293] Table 1. Quantitative analysis of histological analysis. After inducing the disease, all clinical indicators of arteriosclerosis increased significantly. However, after treatment with calcium lactate, these indicators decreased significantly. *: There was a significant difference between G2 and G1, P < 0.05; ++: There was a significant difference between G2 and G3, P < 0.01; +: There was a significant difference between G2 and G3, P < 0.05.

[0294] Histopathological analysis Male

[0295] Data are expressed as mean ± S.E.M. Statistical analysis of the results was performed by the Mann-Whitney method.

[0296] G1: Normal control (saline); G2: Vehicle control (saline); G3: Calcium lactate; G2-G3: ApoE-deficient mice fed a high-cholesterol diet (atherosclerosis)

[0297] Evidence of direct targeting of atherosclerosis

[0298] Table 2. Clinical blood chemistry

[0299] Clinical blood biochemistry (mg / dL) Male

[0300] Data are expressed as mean ± S.E.M. Statistical analysis was performed by the student t-test method.

[0301] **: There was a statistical difference between G2 and G1, P < 0.01

[0302] *: There was a statistical difference between G2 and G1, P < 0.05

[0303] G1: Normal control (saline, n = 3)

[0304] G2: Vehicle control (saline, n = 8)

[0305] G3: Test article (calcium lactate pentahydrate, n = 8)

[0306] G2-G3: ApoE-deficient mice fed a high-cholesterol diet

[0307] Pathogenic factors such as cholesterol (TCHO), LDL cholesterol (LDL), triglycerides (TG) and HDL cholesterol (HDL) were not controlled by calcium lactate. The combination form of calcium and lactate acts directly as a single component at the disease site (see Figure 36 ).

[0308] Figure 36Representative histological features of aortic tissue are provided. The tissues on the left and in the middle of the picture are H&E stained. The tissue on the right of the picture is Oil Red stained to distinguish atherosclerotic lesions. Atherosclerotic lesions were reduced by treatment with calcium lactate.

[0309] Example 25

[0310] Targeting periodontal diseases (pulpitis and periodontitis)

[0311] Materials and methods

[0312] Animals. All experiments were conducted in accordance with the Korean legal animal care guidelines (11737, revised on April 5, 2013) and the guidelines established by the Gachon University Institutional Animal Care and Use Committee (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 at 22 - 25 °C on a 12-hour light / dark cycle with free access to food and water.

[0313] Preparation of lipopolysaccharide (LPS) and calcium lactate. 1 mg of LPS powder was dissolved in 100 μl of saline. If LPS was to be used fresh, the tube was placed on ice. For long-term storage, screw-cap vials were preferably used. Aliquots were stored in a deep 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 injection (for 21 days).

[0314] Animal model of periodontal disease. Periodontal disease was induced by intradental injection of LPS (1 mg / 100 μl) in sterile saline. A fine subcutaneous injection needle was inserted into the mesiobuccal aspect of the first right mandibular molar, and the tip was moved distally to inject at the interdental papilla between the first and second molars. The injection was performed slowly, and the needle was kept in place for 10 seconds after injection to ensure that LPS was not lost through the needle track.

[0315] 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 embedded in paraffin and sectioned at 10 μm. The slides were incubated at 55 °C for 2 h and then dewaxed in xylene, rehydrated in a graded ethanol series, and refixed in Bouin's solution for 1 h, decolorized to remove the yellow color and rinsed with running tap water for 5 - 10 min, and stained with Ponceau - acid fuchsin solution for 5 min, then stained with phosphomolybdic - phosphotungstic acid solution (1:1 ratio) for 30 min, and stained with aniline blue solution for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 5 min. Dehydrated with 95% ethanol, absolute ethanol and clarified in xylene. Mounted with mounting medium. The stained tissues were imaged using a Leica DM1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0316] Results

[0317] Figure 37 Shown is the Masson's trichrome staining of the periodontal tissue. LPS treatment can induce periodontal disease, which results in the loss of the ligament and gingiva around the tooth root (middle panel). After treatment with calcium lactate, the ligament and gingiva were restored (right panel), which was the same as the normal control (left panel).

[0318] Figure 38 Shown is the Masson's trichrome staining of the upper gingiva. The upper gingiva around the periodontal tissue treated with LPS showed abnormal morphology due to severe inflammation (middle panel). After treatment with calcium lactate, the morphology of the upper gingiva was restored (right panel), which was the same as the normal control (left panel).

[0319] Example 26

[0320] Targeting musculoskeletal diseases (rheumatoid arthritis)

[0321] Materials and methods

[0322] Animals. All experiments were conducted in accordance with the Korean legal animal care guidelines (11737, revised on April 5, 2013) and the guidelines established by the Institutional Animal Care and Use Committee of Kyunggi BioCenter (2017 - 11 - 0003, South Korea). Six - week - old male DBA1 / J mice were obtained from SLC Inc. (Shizuoka, Japan) and bred under specific pathogen - free (SPF) conditions. All animals were maintained at 22 - 25 °C on a 12 - hour light / dark cycle and had free access to food and water.

[0323] Preparation of agents. Collagen type II at 2 mg / mL was mixed with an equal volume of complete Freund's adjuvant (CFA) for primary induction of rheumatoid arthritis. Collagen type II at 2 mg / mL was mixed with an equal volume of incomplete Freund's adjuvant (IFA) for secondary induction of rheumatoid arthritis. Calcium lactate at 2 mg / kg was dissolved in saline for daily subcutaneous injection (for 21 days).

[0324] Animal model of rheumatoid arthritis

[0325] Primary induction: 0.1 mL of the mixed solution (collagen type II + CFA) was subcutaneously injected into the area 1.5 cm from the base of the mouse tail. In all mice, the administration site and depth were the same.

[0326] Secondary induction: 21 days after primary induction, 0.1 mL of the mixed solution (collagen type II + IFA) was subcutaneously injected into the area 1.5 cm from the base of the mouse tail. In all mice, the administration site and depth were the same.

[0327] Histological analysis. The feet and knee joints of the mice were dissected and fixed (4% paraformaldehyde), and decalcified at 4°C for 2 days. The tissues were embedded in paraffin and cut into 15-μm sections. The slides were incubated at 55°C for 2 h, then dewaxed in xylene, rehydrated in a graded ethanol series, stained with Biebrich scarlet-acid fuchsin solution for 5 min, then stained with phosphomolybdic-phosphotungstic acid solution (1:1 ratio) for 30 min, and stained with aniline blue solution for 15 min. Briefly rinsed in distilled water and 1% acetic acid solution for 5 min. Dehydrated with 95% ethanol, absolute ethanol and clarified in xylene. Mounted with mounting medium. The stained tissues were imaged using a Leica DM 1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0328] Results

[0329] Figure 39 Shown is the Masson's trichrome staining of the foot and knee joint tissues. Inflammatory edema caused by rheumatoid arthritis was observed in the toes and knee joints (middle figure). 21 days after treatment with CaLa, the inflammatory edema in the toes and knee joints was significantly reduced (lower figure), and its morphology recovered, being the same as that of the normal control (upper figure).

[0330] Example 27

[0331] Targeting digestive diseases (inflammatory bowel disease: Crohn's disease and colitis)

[0332] Materials and methods

[0333] Animals. All experiments were conducted in accordance with the Korean legal guidelines for animal care (11737, revised on April 5, 2013) and the guidelines established by the Gachon University Institutional Animal Care and Use Committee (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 at 22 - 25 °C on a 12-hour light / dark cycle with free access to food and water.

[0334] Reagents 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 (2 mg / kg) was dissolved in saline for daily subcutaneous injection (for 21 days). Two inflammatory bowel disease models were used. Crohn's disease was induced by oral administration of indomethacin (7 mg / kg), and colitis was induced by daily subcutaneous injection of indomethacin (7 mg / kg) at 24-hour intervals ( Figure 40 and Figure 42 ).

[0335] Histological analysis. The fixed flat scaffolds were dehydrated in a 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 1000LED microscope (Leica Microsystems, Wetzlar, Germany).

[0336] Results

[0337] Figure 40 An experimental protocol for inducing Crohn's disease was provided.

[0338] Figure 41 Representative images of H&E staining of Crohn's disease were provided. Transmural inflammation and linear ulcers were observed in the intestine after induction of Crohn's disease (middle panel). After four weeks of treatment with CaLa, the transmural inflammation and linear ulcers in the intestinal tissue were significantly reduced (lower panel), and its morphology was restored to the same as that of the normal control (upper panel).

[0339] Figure 42 An experimental protocol for inducing colitis was provided.

[0340] Figure 43Representative images of H&E staining of colitis are provided. Superficial ulcers, polyps, and inflammation were observed in the intestine after induction of colitis (middle panel). After four weeks of treatment with CaLa, the superficial ulcers, polyps, and inflammation in the intestinal tissue were significantly reduced (lower panel), and its morphology was restored to the same as that of the normal control (upper panel).

[0341] Some additional embodiments disclosed herein include, but are not limited to, the following embodiments:

[0342] A. A method of treating asthma, pulmonary fibrosis, obesity, gastroenteritis, chronic inflammatory bowel disease, and / or atopic dermatitis, the method comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0343] B. A method of treating chronic obstructive pulmonary disease, pneumonia, keratitis, atherosclerosis, arteriosclerosis, myocarditis, diabetes, rheumatoid arthritis, pulpitis, periodontitis, and / or psoriasis, the method comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0344] C. A method of treating Alzheimer's disease, stroke, Parkinson's disease, multiple sclerosis, age-related macular degeneration, non-alcoholic fatty liver disease, sepsis, and / or osteoporosis, the method comprising administering a therapeutically effective amount of calcium lactate to a subject in need thereof.

[0345] D. The method according to any one of embodiments A-C, wherein the calcium lactate is administered in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[0346] E. The method according to any one of embodiments A-D, wherein the pharmaceutical composition is formulated as a liquid, powder, aerosol, injection, infusion, patch, capsule, pill, tablet, depot, or suppository.

[0347] F. The method according to any one of embodiments A-D, wherein the pharmaceutical composition comprises a therapeutically effective amount of calcium lactate as an active agent and a pharmaceutically acceptable polysaccharide, polymer, lipid, or combination thereof.

[0348] G. The method according to embodiment F, wherein the pharmaceutical composition comprises calcium lactate and a polysaccharide.

[0349] H. The method according to embodiment G, wherein the weight ratio of calcium lactate to the polysaccharide is 1:<0.2 to 1:5.

[0350] I. The method according to embodiment G, wherein the weight ratio of calcium lactate to the polysaccharide is 1:<0.2.

[0351] J. The method according to embodiment G, wherein the weight ratio of calcium lactate to the polysaccharide is 1:0.2 to 1:5.

[0352] K. The method according to any one of embodiments F-J further comprises a polymer and / or a lipid.

[0353] L. The method according to embodiment K further comprises a polymer and a lipid, wherein the weight ratio of the polymer to the lipid is from 1:0.1 to 1:50.

[0354] M. The method according to any one of embodiments K-L, wherein the weight ratio of calcium lactate to the polymer and / or lipid is at least 1:5.

[0355] N. The method according to embodiment M, wherein the weight ratio of calcium lactate to the polymer and / or lipid is from 1:5 to 1:30.

[0356] O. The method according to embodiment F comprises calcium lactate and a polymer and / or a lipid.

[0357] P. The method according to embodiment O comprises a polymer and a lipid, wherein the weight ratio of the polymer to the lipid is from 1:0.1 to 1:50.

[0358] Q. The method according to any one of embodiments O-P, wherein the weight ratio of calcium lactate to the polymer and / or lipid is at least 1:5.

[0359] R. The method according to any one of embodiments O-Q, wherein the weight ratio of calcium lactate to the polymer and / or lipid is from 1:5 to 1:30.

[0360] S. The method according to any one of embodiments O-R, wherein the composition is short-acting.

[0361] T. The method according to any one of embodiments O-R, wherein the composition is long-acting.

[0362] U. The method according to any one of embodiments F-T, wherein the composition is an injectable composition.

[0363] V. The method according to any one of embodiments F-U comprises a polysaccharide, which is a cellulose derivative, pectin, hyaluronic acid, starch, guar gum, chitosan, gelatin, collagen, alginate, alginic acid or a combination thereof.

[0364] W. The method according to any one of embodiments F-V comprises a polymer, which is poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA) or a combination thereof.

[0365] X. The method according to any one of embodiments F-W, comprising a lipid which is a monoglyceride or triglyceride or polyethylene glycol, polyethylene glycol ester of vegetable oil, propylene glycol fatty acid ester, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa butter, cottonseed oil, sunflower oil, safflower oil, almond oil, olive oil, hydrogenated oil, 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 or a combination thereof.

[0366] Y. The method according to any one of embodiments F-S and U-X, wherein at least about 40% of the active agent is released after 6 hours when the composition is placed in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter having a pore size of 45 μm.

[0367] Z. The method according to any one of embodiments F-S and U-Y, wherein at least about 60% of the active agent is released after 12 hours when the composition is placed in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter having a pore size of 45 μm.

[0368] AA. The method according to any one of embodiments F-S and U-Z, wherein at least about 80% of the active agent is released after 24 hours when the composition is placed in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter having a pore size of 45 μm.

[0369] BB. The method according to any one of embodiments F-S and U-AA, wherein at least about 90% of the active agent is released after 48 hours when the composition is placed in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter having a pore size of 45 μm.

[0370] CC. The method according to any one of embodiments F-H, J-R and T-X, wherein less than about 40% of the active agent is released after 24 hours when the composition is placed in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37 °C using a nylon filter having a pore size of 45 μm.

[0371] DD. The method according to any one of embodiments F-H, J-R, and T-CC, wherein less than about 60% of the active agent is released after 48 hours in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm after placing the composition.

[0372] EE. The method according to any one of embodiments F-H, J-R, and CC-DD, wherein less than about 80% of the active agent is released after 72 hours in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm after placing the composition.

[0373] FF. The method according to any one of embodiments F-H, J-R, and CC-EE, wherein less than about 90% of the active agent is released after 144 hours in an in vitro dissolution test comprising an elution test method at 300 rpm in an aqueous medium having a pH of 6.8 at 37°C using a nylon filter with a pore size of 45 μm after placing the composition.

[0374] GG. The method according to any one of embodiments F-FF, wherein the composition is contained in a sterile glass or polyolefin container.

[0375] HH. The method according to any one of embodiments A-D, wherein the calcium lactate is coated with a pharmaceutically acceptable enteric coating.

[0376] II. The method according to embodiment HH, wherein the enteric coating comprises hydroxypropyl methylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or a combination thereof.

[0377] JJ. The method according to any one of embodiments HH-II, wherein the weight ratio of calcium lactate to the enteric coating is from 10:0.5 to 1:1.5.

[0378] KK. The method according to any one of embodiments HH-JJ, wherein in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than about 20% of the active agent is released after 30 minutes.

[0379] LL. The method according to any one of embodiments HH-KK, wherein in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than 30% of the active agent is released after 60 minutes.

[0380] MM. The method according to any one of embodiments HH-LL, wherein in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than 50% of the active agent is released after 120 minutes.

[0381] NN. The method according to any one of embodiments HH-MM, wherein in an in vitro dissolution test using the USP paddle method at a paddle speed of 50 rpm at 37°C, when the composition is placed in 0.1 N HCl for 120 minutes and then adjusted to pH 6.8 with phosphate buffer, less than 10% of the active agent is released after 120 minutes.

[0382] OO. The method according to any one of embodiments A-B, wherein the calcium lactate is provided in a food or nutritional composition comprising calcium lactate.

[0383] PP. The method according to embodiment OO, wherein the composition is an injectable nutritional supplement.

[0384] The entire contents of U.S. Application No. 62 / 616,923, filed on January 12, 2018, are incorporated herein by reference.

[0385] The description of the foregoing specific embodiments will so fully disclose the general nature of the invention that others can, by applying knowledge within the scope of the art, readily modify and / or adapt various applications without departing from the general concept of the invention. Therefore, such adaptations and modifications are intended to fall within the meaning and scope of the equivalent forms of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that the words or terms herein are for the purpose of description and not limitation, so that the terms or phrases of this specification are to be interpreted by those skilled in the art in accordance with the teachings and guidance.

[0386] 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.

[0387] All aspects, embodiments, and options described herein can be combined in any and all variations.

[0388] References

[0389] 1. Shaw AC, Goldstein DR, Montgomery RR, “Age - dependent dysregulation of innate immunity,” Nature Reviews Immunol. 13:875 - 87(2013). 2. Sevenoaks MJ, Stockley RA, “Chronic Obstructive Pulmonary Disease, inflammation and co - morbidity--a common inflammatory phenotype", Respiratory Res. 7:70(2006).

[0390] 3. Hessen M, Akpek EK, “Dry eye: an inflammatory ocular disease,” J. Ophthalmic & Vision Res. 9:240 - 50(2014).

[0391] 4. Bianco P, Palucka AK, Pascuai V, Banchereau J, “Dendritic cells and cytokines in human inflammatory and autoimmune diseases,” Cytokine & Growth Factor Rev. 19:41 - 52(2008).

[0392] 5. Fomoni A, Ijaz A, Tejada T, Lenz O, "Role of inflammation in diabetic nephropathy,” Current Diabetes Rev. 4:10 - 7(2008).

[0393] 6. Minghetti L, “Role of inflammation in neurodegenerative diseases,” Current Opinion Neurology 18:315 - 21(2005).

[0394] 7. Kofier S, Nickel T, Weis M, “Role of cytokines in cardiovascular diseases: a focus on endothelial responses to inflammation,” Clinical Set. 108:205 - 13 (2005).

[0395] 8. Hotamisligil GS, “Inflammation and metabolic disorders,” Nature 444:860 - 7 (2006).

[0396] 9. Choy EH, Panayi GS, “Cytokine pathways and joint inflammation in rheumatoid arthritis,” New England J. Medicine 344:907 - 16 (2001).

[0397] 10. Mundy GR, “Osteoporosis and inflammation,” Nutrition Rev. 65: S 147 - 51 (2007).

[0398] 11. Page RC, Schroeder HE, “Pathogenesis of inflammatory periodontal disease, A summary of current work," Laboratory Investigation 34:235 - 49 (1976).

[0399] 12. Tilg H, Moschen AR, "Insulin resistance, inflammation, and non - alcoholic fatty liver disease,” Trends in Endocrinology and Metabolism: TEM 19:371 - 9 (2008).

[0400] 13. Hamid Q, Boguniewicz M, Leung DY, “Differential in situ cytokine gene expression in acute versus chronic atopic dermatitis,” J. Clinical Investigation 94:870 - 6(1994).

[0401] 14. Arican O, Aral M, Sasmaz S, Ciragil P, “Serum levels of TNF-alpha, IFN-gamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis and correlation with disease severity,” Mediators of Inflammation 2005:273 - 9(2005).

[0402] 15. Gilroy D, De Maeyer R, “New insights into the resolution of inflammation,” Seminars in Immunology 27:161 - 8(2015).

[0403] 16. Manabe I, “Chronic inflammation links cardiovascular, metabolic and renal diseases,” Circulation J. 75:2739 - 48(2011).

[0404] 17. Fujiwara N, Kobayashi K, “Macrophages in inflammation. Current drug targets,” Inflammation and Allergy 4:281 - 6(2005).

[0405] 18. Gilmore TD, “Introduction to NF-kappaB: players, pathways, perspectives,” Oncogene 25:6680 - 4(2006).

[0406] 19. Karin M, “How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex,” Oncogene 18:6867-74 (1999).

[0407] 20. Taylor CT, “Interdependent roles for hypoxia inducible factor and nuclear factor-kappaB in hypoxic inflammation,” J. Physiology 586:4055-9 (2008).

[0408] 21. Eltzschig HK, “Carmeliet P. Hypoxia and inflammation,” New England J. Medicine 364:656-65 (2011).

[0409] 22. Taylor CT, McElwain JC, “Ancient atmospheres and the evolution of oxygen sensing via the hypoxia-inducible factor in metazoans,” Physiology 25:272-9 (2010).

[0410] 23. Benizri E, Ginouves A, Berra E, “The magic of the hypoxia-signaling cascade. Cellular and molecular life sciences,” CMLS 65:1133-49 (2008).

[0411] 24. Wang GL, Jiang BH, Rue EA, Semenza GL, “Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension,” PNAS USA 92:5510-4 (1995).

[0412] 25. Barnes PJ, Karin M, “Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases," New England J. Medicine 336:1066-71(1997).

[0413] 26. Whyte MK, Walmsiey SR, "The regulation of pulmonary inflammation by the hypoxia-inducible factor hydroxylase oxygen-sensing pathway," Annals of the American Thoracic Society 11:Supp.5:8271-6(2014).

[0414] 27. Vadlapatla RK, Vadlapudi AD, Mitra AK, “Hypoxia-inducible factor-1(HIF-1): a potential target for intervention in ocular neovascular diseases," Current Drag Targets 14:919-35(2013).

[0415] 28. Mojsilovic-Petrovic J, Callaghan D, Cui H, Dean C, Stanimirovic DB, Zhang W, “Hypoxia-inducible factor-1(HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1(MCP-1 / CCL2) and MCP-5(Cell 2) in astrocytes," J. Neuroinflammation 4:12(2007).

[0416] 29. Medina J, Arroyo AG, Sanchez-Madrid F, Moreno-Otero R, “Angiogenesis in chronic inflammatory liver disease," Hepatology 39:1185-95 (2004).

[0417] 30. Jang JH, Cho YC, Kim KH, Lee KS, Lee J, Kim DE, et al., “BAI, a novel Cdk inhibitor, enhances famesyltransferase inhibitor LB42708-mediated apoptosis in renal carcinoma cells through the downregulation of Bcl-2 and c-FLIP(L),” Int. J. Oncology 45:1680-90 (2014).

[0418] 31. Narrima P, Paydar M, Looi CY, Wong YL, Taha H, Wong WF, et al, “Persea declinata (Bl.) kosterm bark crude extract induces apoptosis in MCF-7 cells via G0 / G1 cell cycle arrest, Bcl-2 / Bax / Bcl-xl signaling pathways, and ROS generation,” Evidence-Based Complementary and Alternative Medicine 2014: (2014).

[0419] 32. Genin M, Clement F, Fattaccioli A, Raes M, Michiels C, “M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide,” BMC Cancer 15:577 (2015).

[0420] 33. Kang S, Park KC, Yang KJ, Choi HS, Kim SH, Roh YJ, “Effect of cediranib, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, in a mouse model of choroidal neovascularization,” Clin. Exp. Ophthalmol. 41: 63 - 72 (2013).

[0421] 34. Roh YJ, Rho CR, Cho WK, Kang S, "The Amiangiogenic Effects of GoldNanoparticles on Experimental Choroidal Neovascularization in Mice,” Investig. Ophthalmol. Vis. Sci. 57: 6561 - 67 (2016).

[0422] 35. Jiao J, Mo B, Wei H, Jiang YR, “Comparative study of laser-inducedchoroidal neovascularization in rats by paraffin sections, frozen sections andhigh-resolution optical coherence tomography” Graefes Arch. Clin. Expt. Ophthalmol. 251: 301 - 7 (2013).

[0423] All publications, patents, and patent applications mentioned in this specification are hereby 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. Use of calcium lactate in the preparation of a pharmaceutical composition, food or nutritional composition for treating a disease selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), inflammatory bowel disease (IBD), spinal cord injury, liver fibrosis and pulmonary fibrosis, wherein the pharmaceutical composition, food or nutritional composition comprises a therapeutically effective amount of calcium lactate as an active agent.

2. The use according to claim 1, wherein, The pharmaceutical composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient or diluent.

3. The use according to claim 1, wherein, The pharmaceutical composition is formulated as a liquid, powder, aerosol, injection, infusion, patch, capsule, pill, tablet, depot or suppository.

4. The use according to claim 1, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable polysaccharide, polymer, lipid or a combination thereof.

5. The use according to claim 4, wherein, The pharmaceutical composition comprises the polysaccharide, and the weight ratio of calcium lactate to the polysaccharide is from 1:0.2 to 1:

5.

6. The use according to claim 4, wherein, The pharmaceutical composition comprises the polymer and the lipid, and the weight ratio of the polymer to the lipid is from 1:0.1 to 1:

50.

7. The use according to claim 5, wherein the polysaccharide is selected from the group consisting of cellulose derivatives, pectin, hyaluronic acid, starch, guar gum, chitosan, alginate and alginic acid or a combination thereof.

8. The use according to claim 6, wherein the polymer is selected from the group consisting of poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG) and poly(lactic-co-glycolic acid) (PLGA) or a combination thereof.

9. The use according to claim 6, wherein the lipid is selected from the group consisting of monoglyceride or triglyceride or polyethylene glycol, polyethylene glycol esters of vegetable oils, propylene glycol esters of fatty acids, sesame oil, soybean oil, castor oil, corn oil, palm oil, peanut oil, cocoa butter, cottonseed oil, sunflower oil, safflower oil, almond oil, olive oil, hydrogenated oil, 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 or a combination thereof.

10. The use according to claim 1, wherein, The calcium lactate is coated with a pharmaceutically acceptable enteric coating.

11. The use according to claim 10, wherein, The enteric coating comprises hydroxypropyl methylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), shellac, polymers of methacrylic acid and its esters, or a combination thereof.

12. The use according to claim 10, wherein The weight ratio of calcium lactate to the enteric coating is from 10:0.5 to 1:1.5.

Images