Amorphous calcium carbonate for the prevention and treatment of myocardial ischemia

Abstract

The present invention provides a method for the treatment and / or prevention of myocardial ischemia and myocardial infarction, comprising administering a composition containing stabilized amorphous calcium carbonate.

Description

Technical Field

[0001] The present invention provides a method for treating or preventing myocardial ischemia by administering stabilized amorphous calcium carbonate to a subject suffering from myocardial ischemia or having a predisposition to develop myocardial ischemia.

Background Art

[0002] Myocardial ischemia is a condition in which the blood perfusion of the heart decreases, resulting in a decrease in oxygen supply to the heart, irregular myocardial energy metabolism, and abnormal heart function. Clinical studies have revealed that coronary artery stenosis, the main cause of which is arteriosclerosis, is the main and most common cause of myocardial ischemia. Coronary artery disease, which people often refer to as heart disease, is caused by coronary atherosclerotic arteriosclerosis. Currently, there are many treatment methods for myocardial ischemia, a type of coronary artery disease, such as stent placement and balloon dilation, but drug therapy remains the mainstream. Nitrate esters (e.g., isosorbide mononitrate or its sustained release), beta blockers such as propranolol, and calcium channel blockers such as nifedipine can be selected to dilate the coronary arteries, increase myocardial oxygen supply, and decrease peripheral resistance, heart function, and myocardial oxygen consumption. Statins (e.g., atorvastatin, simvastatin) should also be taken to reduce plasma cholesterol and stable atherosclerotic plaques and prevent the formation of thrombosis that can cause stroke by plaque detachment.

[0003] Myocardial infarction is myocardial necrosis resulting from persistent ischemia of a portion of the myocardium, which arises from intravascular thrombus formation resulting from atherosclerosis of the coronary arteries and atherosclerosis of the coronary arteries, which can lead to rapid, permanent, and complete blockage of the lumen of a blood vessel and interruption of blood flow. It can be divided into three phases according to the clinical process and ECG findings, which are the acute, subacute, and chronic phases. Clinical symptoms mainly occur in the acute phase, and the highest mortality rate occurs in the first week of the acute phase. Acute myocardial infarction (AMI) is a common severe case that threatens human life and is currently one of the most harmful cardiac conditions.

[0004] International Publication No. 2008 / 041236 describes the treatment of musculoskeletal disorders with a composition comprising an organic substance essentially consisting of chitin and polypeptides (Pp) and finely mixed calcium carbonate (CaCO3), and specifies the particular ratio between CaCO3 and the organic substance or polypeptide.

[0005] International Publication No. 2013 / 088440 discloses that the bioavailability of amorphous calcium carbonate is significantly higher than that of crystalline calcium carbonate. Meiron (Journal of Bone and Mineral Research, Vol.26, No.2, 2011, pp 364-372) reached similar observations and further states that amorphous calcium carbonate is approximately 120 times more soluble than calcite (one of the crystalline forms of calcium carbonate).

[0006] Seung-Kwon Myung et al., (Nutrients. 2021 Feb;13(2):368), and many other publications have described the destructive role of calcium supplementation in people with cardiovascular disease.

[0007] There is a need to develop new and effective means to treat and prevent myocardial ischemia, but this has not yet been addressed. [Overview of the project]

[0008] This invention is based on the surprising finding that the administration of calcium carbonate, which is considered an undesirable supplement in cases of cardiovascular disease, brings about significant improvement in subjects suffering from myocardial ischemia, particularly myocardial infarction.

[0009] According to one embodiment, the present invention provides a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the treatment or prevention of myocardial ischemia. According to some embodiments, myocardial ischemia is caused by myocardial infarction. According to some embodiments, myocardial ischemia is acute. According to some embodiments, myocardial ischemia is chronic.

[0010] According to some embodiments, the composition is administered in a manner selected from sublingual, inhalation, IV, and oral administration. According to some embodiments, the composition is administered in a manner selected from oral and sublingual, inhalation and sublingual, oral and inhalation and oral, inhalation and sublingual administration, and inhalation and IV administration.

[0011] According to some embodiments, the composition is administered in doses of stabilized ACC ranging from about 5 to about 200 mg / kg / day.

[0012] According to some embodiments, the powder composition contains secondary particles of ACC having a size of less than 500 μm for sublingual and / or inhalation administration.

[0013] According to some embodiments, the stabilizer is selected from the group consisting of polyphosphates, organic acids, phosphorylated amino acids, phosphorylated, phosphorylated, sulfated or sulfonated organic compounds, phosphate or sulfate esters of hydroxycarboxylic acids, bisphosphonates, organic polyphosphates, polyphosphates, hydroxyl-containing organic compounds, derivatives thereof, proteins, and any combination thereof.

[0014] According to some embodiments, the stabilizer is selected from the group consisting of triphates or salts thereof, phosphoserine, citric acid, sodium triphosphate and citric acid, adenosine triphosphate, adenosine diphosphate, phytic acid, etidronic acid, pyrophosphate, polyphosphate, hexametaphosphate, ethanol, and any combination thereof.

[0015] In another aspect, the present invention provides a method for treating or preventing myocardial ischemia in a subject, comprising administering to the subject a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer.

[0016] According to any one of the embodiments described above, the composition may be formulated as a food supplement or as a pharmaceutical composition, which may be in the form of a powder, suspension, tablet, or capsule. [Brief explanation of the drawing]

[0017] [Figure 1] The echocardiographic results for the three treatment groups on days 0, 2, and 28 are shown (one-way ANOVA, P=0.0120). [Figure 2] This shows the percentage change in ejection fraction for each treatment group on day 28 compared to baseline (day 0). [Figure 3A] This shows the percentage of proliferation of cardiomyocytes cultured with 1 or 2 mM ACC or CaCl2. [Figure 3B] This shows the proliferation of cardiomyocytes in the presence of ACC compared to proliferation in the presence of CaCl2 (equal concentration of calcium). [Figure 3C] This shows cTNT mRNA expression in the presence of ACC, compared to expression in the presence of CaCl2 (at the same concentration of calcium). [Figure 4] This shows the mRNA levels of CD31 (endothelial marker) in cells cultured with 2 mM ACC (double repeat) or CaCl2. [Figure 5]The ratio of new blood vessels to the area of ​​injury in the two treatment groups (ACC and control) is shown (p<0.05). [Modes for carrying out the invention]

[0018] According to one embodiment, the present invention provides a method for treating or preventing myocardial ischemia in a subject, comprising administering to the subject a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer.

[0019] In another embodiment, the present invention provides a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the treatment or prevention of myocardial ischemia. In some embodiments, the present invention provides a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the treatment of myocardial ischemia. In some embodiments, the present invention provides a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the prevention of myocardial ischemia.

[0020] The terms “subject” and “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock (including cattle, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats). According to other embodiments, the subject is an animal such as livestock or domesticated animals. According to some embodiments, the subject is a human subject.

[0021] As used herein, the terms "myocardial ischemia" and "cardiac ischemia" are defined as insufficient blood supply to the myocardium caused by a reduction in the volume of the cardiovascular system. Myocardial ischemia can be caused by coronary artery disease. As used herein, the term "coronary artery disease" is defined as a disease / disorder of cardiac function resulting from an imbalance between myocardial function and the ability of the coronary vessels to supply sufficient blood flow for normal function. Specific coronary artery diseases / disorders associated with coronary artery disease that can be treated with the compositions and methods described herein include myocardial ischemia, angina pectoris, coronary aneurysm, coronary thrombosis, coronary vasospasm, coronary artery disease, coronary heart disease, coronary occlusion, and coronary stenosis. Another cause of myocardial ischemia is caused by cardiac trauma. According to some embodiments, myocardial ischemia is caused by atherosclerosis, blood clots, or coronary constriction.

[0022] As used herein, the term "obstructive peripheral vascular disease" (also known as peripheral arterial occlusive disease) is an occlusion involving vascular disorders in the carotid or femoral arteries, including the iliac arteries. Obstructive vascular diseases include, but are not limited to, conditions in patients with sickle cell anemia. Occlusion of the femoral artery causes pain and restricted movement. A specific disorder associated with obstructive peripheral vascular disease is diabetic foot, which affects diabetic patients and often results in amputation of the foot.

[0023] According to some embodiments, myocardial ischemia includes myocardial infarction. Thus, according to some embodiments, the present invention provides a composition comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer for use in the treatment or prevention of myocardial infarction.

[0024] As used herein, the terms “treating” and “treatment” are interchangeable and refer to taking measures to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, the alleviation or improvement of one or more symptoms associated with myocardial ischemia and myocardial infarction, the delay or slowing of the onset or progression of ischemia, the improvement, alleviation or stabilization of the disease, and other beneficial outcomes. In particular, according to one embodiment, treating ischemia includes at least one of the following: stopping, delaying, reversing, or preventing the progression of ischemia. According to some embodiments, treating or preventing myocardial ischemia or myocardial infarction includes enhancing angiogenesis, i.e., neovascularization. As used herein, the term “preventing,” when used in relation to a condition or disease, refers to the administration of a composition that reduces the frequency, probability, or delays the onset of symptoms of a medical condition in a subject compared to a subject not administered the composition. Therefore, preventing myocardial ischemia or infarction involves administering stabilized ACC to subjects who are predisposed to or at risk of developing myocardial ischemia or infarction. Such subjects are, for example, selected from subjects suffering from diabetes, hypertension, high blood cholesterol levels, high blood triglyceride levels, obesity, genetic predisposition, stress, a history of MI heart disease, or smoking. Accordingly, according to some embodiments, the present invention provides amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the treatment or prevention of myocardial ischemia in subjects suffering from diabetes. According to another embodiment, the present invention provides amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the treatment or prevention of myocardial ischemia in subjects suffering from hypertension. According to another embodiment, the present invention provides amorphous calcium carbonate (ACC) stabilized with at least one stabilizer for use in the treatment or prevention of myocardial ischemia in subjects suffering from high blood cholesterol levels.According to another embodiment, the present invention provides amorphous calcium carbonate (ACC) stabilized by at least one stabilizer for use in the treatment or prevention of myocardial ischemia in a subject suffering from high blood triglyceride levels.

[0025] The term "administration" or "administering" of a substance, compound or composition to a subject can be effected using one of the various methods known to those skilled in the art. For example, a compound or composition can be administered enterally or parenterally. Enteral refers to administration via the gastrointestinal tract, including oral or rectal. Parenteral administration includes intravenous, sublingual, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, intranasal, inhalation, intraspinal, intracerebral, and transdermal (e.g., by absorption through a skin duct). A compound or composition can also be suitably introduced by a refillable or biodegradable polymer device or other "drug delivery" and "controlled release" devices, such as patches and pumps, or formulations that provide extended, slow or controlled release of the compound or composition. Administration can also be effected, for example, once, multiple times, and / or over one or more extended periods. In some aspects, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug or a medical food.

[0026] According to some embodiments, administration is oral administration. According to other embodiments, administration is sublingual administration. According to further embodiments, administration is a combination of oral and sublingual administrations. According to other embodiments, administration is administration by inhalation. According to other embodiments, administration is a combination of administration by inhalation and oral and / or sublingual administrations. According to some embodiments, the composition is administered in a mode selected from sublingual, inhalation, IV, oral administration, or by a combination of administrations selected from oral and sublingual, inhalation and sublingual, oral and inhalation, and oral, inhalation and sublingual administrations.

[0027] According to certain embodiments, administration, for example, by oral administration, sublingual administration, or a combination thereof, involves administering less than 200 mg / kg of calcium per day as stabilized ACC. The dose according to any one aspect and embodiment of the present invention refers to the amount of elemental calcium in ACC. According to one embodiment, the method of the present invention involves administering less than 150 mg / kg / day or less than 100 mg / kg / day of calcium as stabilized ACC. According to another embodiment, the dose of ACC is less than 50 mg / kg / day, less than 30 mg / kg / day, or less than 20 mg / kg / day as calcium in stabilized ACC. According to some embodiments, administration according to the present invention, for example, by oral, sublingual, or combination administration, involves administering 5-150, 10-120, 15-100, 20-80, 30-70, or 40-60 mg / kg / day of calcium as stabilized ACC. According to certain embodiments, the administration according to the present invention, for example, by oral, sublingual, or combined administration, includes the administration of 5-80, 10-75, 15-70, 20-65, 25-60, 30-55, 35-50, or 40-45 mg / kg / day of calcium as stabilized ACC. According to some embodiments, the administration according to the present invention, for example, by oral, sublingual, or combined administration, includes the administration of about 10-45, about 15-40, or about 20-35 mg / kg / day of calcium as stabilized ACC. According to further embodiments, the administration according to the present invention, for example, by oral, sublingual, or combined administration, includes the administration of 0.1-30, 0.2-28, 0.3-26, 0.5-24, 1-22, 2-20, 3-18, 3-16, 4-15, 5-14, 6-12, or 8-10 mg / kg / day of calcium as stabilized ACC. According to several embodiments, the administration according to the present invention, for example, orally, sublingually, or in combination, includes the administration of 0.2-10, 0.5-8, 0.8-6, 1-5, 1.5-4, or 2-3 mg / kg / day of stabilized ACC. According to another embodiment, the administration includes the administration of 500-8000 mg / day, 800-6000, or 100-4000 mg / day of ACC. According to several embodiments, the administration includes the administration of 200-3000 mg / day, 400-2500, or 600-2000 mg / day of stabilized ACC.According to other embodiments, the administration includes the administration of 800-4000 mg / day, 1000-3000 mg / day, or 1500-2500 mg / day of stabilized ACC.

[0028] According to some embodiments, administration, for example, oral administration, sublingual administration, or combined administration, includes a daily dose of approximately 600 to approximately 23,500 mg of ACC. According to one embodiment, administration includes doses of 600 to 20,000, 800 to 18,000, 1,000 to 15,000, 1,200 to 12,000, 1,500 to 10,000, and 2,000 to 8,000 mg / day of ACC. According to some embodiments, administration includes doses of 1,000 to 12,000, 2,000 to 11,000, 3,000 to 10,000, 3,500 to 9,000, and 4,000 to 8,000 mg / day of ACC.

[0029] According to some embodiments, the administration includes the administration of stabilized ACC at a dose of 10-350 mg / kg / day. According to some embodiments, the administration includes the administration of ACC at a dose of 2-100, 3-90, 4-85, 5-80, 10-70, or 15-60 mg / kg / day. According to some embodiments, the administration includes the administration of ACC at a dose of 2-10, 3-9, 4-8.5, 5-8, or 6-8 mg / kg / day.

[0030] According to some embodiments, administration may include administration in a single dose or in multiple separate doses. For example, a daily dose may be divided into 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 separate doses.

[0031] According to some embodiments, the administration is short-term, for example, for at least 1, 2, 3, 5, or 7 days. According to other embodiments, the administration is for 1, 2, 3, or 4 weeks. According to further embodiments, the administration is long-term, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. According to other embodiments, the administration is for more than one year, such as 2, 3, 4, 5 years, or more.

[0032] According to some embodiments, use includes prevention. Therefore, according to some embodiments, administration is prophylactic. According to some embodiments, as described above, use for the prevention of myocardial ischemia or infarction is prophylactic for subjects predisposed to developing myocardial ischemia or infarction.

[0033] According to some embodiments, the composition for sublingual administration is in the form of ACC powder. The sublingual powder consists of ACC particles.

[0034] As used herein, the term “particles” refers to separate fine particles or nanoparticles of ACC stabilized by the stabilizers defined above, as well as aggregates or aggregates thereof. According to some embodiments, the particles are primary particles of stabilized ACC. Basic nanoparticles are in the range of 5–500 nm, 10–300 nm, or 20–100 nm. Often, these nanoparticles readily aggregate and agglomerate into much larger secondary particles. These aggregates and aggregates can then be broken down into smaller particles by grinding and dissolution techniques. According to other embodiments, the particles are aggregates or aggregates of primary particles, i.e., secondary particles. As used herein, the term “particle size” refers to a typical diameter measurement of secondary particles, such as aggregates or broken aggregates, in at least one dimension.

[0035] The particle size of the aggregate can be adjusted within a range by a combination of grinding and sieving techniques. In some embodiments, at least 70% of the treated particles of the composition have a particle size of 600 μm or less. In other embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles of the composition have a particle size of 600 μm or less. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles of the composition have a particle size of 500 μm or less. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles of the composition have a particle size of 400 μm or less. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles of the composition have a particle size of 300 μm or less. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles of the composition have a particle size of 200 μm or less. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles in the composition have a particle size of 100 μm or less. In some embodiments, at least 80%, at least 85%, at least 90%, or at least 95% of the particles in the composition have a particle size of 70, 50, or 30 μm or less. According to some embodiments, the particle size is about 20 to about 500 μm, about 30 to about 450 μm, or about 50 to about 400 μm.

[0036] According to any one of the embodiments described above, the ACC is a stabilized ACC, i.e., an ACC that maintains an amorphous state for a long period of time even under high humidity conditions or in an aqueous environment.

[0037] The terms “amorphous calcium carbonate,” “ACC (amorphous calcium carbonate),” “stable ACC,” “stabilized ACC,” and “ACC comprising a stabilizer” are used interchangeably herein and refer to the amorphous form of calcium carbonate. The term “stable” as used herein indicates that the calcium carbonate is maintained in an amorphous form for a long period, for example, at least about 7 days in a solid form having about 30% or less crystalline calcium carbonate. According to any one of the above embodiments, the composition is stable for at least 7 days. According to some embodiments, the composition is stable for at least 1 month. According to other embodiments, the composition is stable for at least 3 months. According to further embodiments, the composition is stable for 6 months. According to certain embodiments, the composition is stable for at least 1 year. According to certain embodiments, the composition is stable for at least 2 years.

[0038] According to any one of the embodiments described above, ACC is stabilized by at least one stabilizer. The term “stabilized ACC” means ACC containing a stabilizer as part of the ACC particles. The terms “stabilizing agent” and “stabilizer” are used interchangeably herein and refer to any molecule, ion, or substance that contributes to keeping calcium carbonate in an amorphous state during the manufacture, formulation, and / or storage of ACC. According to the teachings of the present invention, ACC acts as an activator that gives improvement in exercise and muscle performance. According to the teachings of the present invention, any ACC that maintains stability can be used. Any compound that can stabilize ACC in its amorphous form is suitable for carrying out the present invention. The terms “stable ACC” and “stabilized ACC” are used interchangeably herein and refer to calcium carbonate being maintained in an amorphous form for a long period of time with less than about 30% or about 30% conversion to a crystalline form. According to some embodiments, this term refers to solid stabilized ACC, as well as stabilized ACC dispersed in a liquid carrier such as an aqueous (e.g., water) or non-aqueous liquid carrier.

[0039] In certain embodiments, the stabilizer is a single component. In other embodiments, the use of several stabilizers is involved. In some cases, the stabilizer is located within the molecular matrix of the ACC particles. In some cases, the stabilizer is stored externally, and in other cases, they are located both internally and externally. The internal stabilizer or combination of stabilizers may be the same as or different from the external stabilizer.

[0040] ACC stabilizer The stabilizer may include, but is not limited to, molecules having one or more functional groups selected from hydroxyl, carboxyl, ester, amine, phosphino, phosphono, phosphate, sulfonyl, sulfate, or sulfino groups. Hydroxyl-containing compounds combined with hydroxides may optionally also have other functional groups such as carboxyl, but the hydroxyl is not esterified.

[0041] According to some embodiments, the stabilizer is low-toxicity or non-toxicity to mammalian cells or organisms, particularly humans. According to some embodiments, the stabilizer is food, dietary supplement, or pharmaceutical grade.

[0042] In certain embodiments, the ACC stabilizer is, independently in each appearance, an organic acid, a phosphorylated, phosphorylated, sulfonated or sulfonated organic compound, a phosphoric acid or sulfuric acid ester of a hydroxyl carboxylic acid, an organic amine compound, an organic compound containing hydroxyl, an organic phosphorus compound or its salt, a phosphorylated amino acid and its derivative, a bisphosphonate compound, an organic phosphate compound, an organic phosphonate compound, an inorganic phosphoric acid, an organic compound having the above-defined functional groups, an inorganic phosphate and a polyphosphate compound, a polyphosphate chain, an organic surfactant, a bioessential inorganic ion, or any combination thereof.

[0043] According to several embodiments, the stabilizer is an organic acid. According to a particular embodiment, the organic acid is selected from ascorbic acid, citric acid, lactic acid, acetic acid, oxalic acid, malonic acid, glutaconic acid, succinic acid, maleic acid, lactic acid, and aconitic acid, and optionally includes compounds with at least two carboxyl groups and a molecular weight of 250 g / mol or less, such as citric acid, tartaric acid, and malic acid. According to one particular embodiment, the stabilizer is citric acid.

[0044] In another embodiment, the phosphate ester of a hydroxyl carboxylic acid is phosphoenolpyruvic acid. In another embodiment, the phosphate or sulfate ester of a hydroxyl carboxylic acid includes an amino acid. Examples of such esters are phosphoserine, phosphothreonine, sulfoserine, sulfothreonine, and phosphocreatine.

[0045] Hydroxyl-containing compounds combined with hydroxides may include, for example, mono-, di-, tri-, oligo-, and polysaccharides such as sucrose, or other polyols such as glycerol. Hydroxyl-containing compounds may further include hydroxy acids such as citric acid, tartaric acid, and malic acid, or hydroxyl-containing amino acids such as serine or threonine. Each possibility represents a distinct embodiment of the present invention.

[0046] Some specific, unrestricted examples of such ACC stabilizers include phytic acid, citric acid, dibasic sodium pyrophosphate, sodium adenosine 5'-monophosphate (AMP) salt, sodium adenosine 5'-diphosphate (ADP) salt and disodium adenosine 5'-triphosphate (ATP) salt hydrate, phosphoserine, phosphorylated amino acids, food-grade surfactants, sodium stearoyl lactylate, and combinations thereof.

[0047] According to some embodiments, the stabilizer comprises at least one component selected from phosphate or sulfate esters of hydroxyl carboxylic acids such as phosphoenolpyruvic acid, phosphoserine, phosphothreonine, sulfoserine, or sulfothreonine, and a hydroxyl-containing organic compound selected from mono-, di-, tri-, oligo-, and -polysaccharides, such as sucrose, mannose, and glucose.

[0048] The hydroxyl-containing compound may further comprise at least one alkali hydroxide, such as sodium hydroxide or potassium hydroxide. Phosphorylated acids may be present in oligopeptides and polypeptides. In other embodiments of the present invention, the stabilizer is an organic acid selected from monocarboxylic acids or polycarboxylic acids, e.g., dicarboxylic acids or tricarboxylic acids. Each possibility represents a distinct embodiment of the present invention. The organic acid may be as defined above.

[0049] In some embodiments of the present invention, the ACC stabilizer is selected from phosphorylated amino acids, polyols, and combinations thereof. In some embodiments, the stable ACC comprises a phosphorylated compound as a stabilizer, in which phosphorylation occurs to the hydroxyl group of the organic compound. In some embodiments, the stable ACC comprises a stabilizer selected from the group consisting of citric acid, phosphoserine, phosphothreonine, and combinations thereof. Non-exclusive examples of stabilizers containing phosphates, phosphites, phosphonate groups and their salts or esters include phytic acid, dimethyl phosphate, trimethyl phosphate, sodium pyrophosphate, tetraethyl pyrophosphate, ribulose bisphosphate, etidronic acid and other medical bisphosphonates, 3-phosphoglycerates, glyceraldehyde 3-phosphate, 1-deoxy-D-xylulose-5-sodium phosphate, diethylenetriaminepentakis(methylphosphonic acid), nitrilotri(methylphosphonic acid), 5-phospho-D-ribose-1-bisphosphate pentasodium salt, adenosine 5'-bisphosphate sodium salt, and adenosine 5'-triphosphate disodium salt hydrate. This includes α-D-galactosamine 1-phosphate, 2-phospho-L-ascorbate trisodium salt, α-D-galactose 1-phosphate dipotassium salt pentahydrate, α-D-galactosamine 1-phosphate, O-phosphorylethanolamine disodium salt hydrate, 2,3-diphospho-D-glycerate pentasodium salt, phospho(enol)pyruvate monosodium salt hydrate, D-glyceraldehyde 3-phosphate, sn-glycerol 3-phosphate lithium salt, D-(-)-3-phosphoglycerate disodium salt, D-glucose 6-phosphate sodium salt, phosphatidic acid, ibandronate sodium salt, phosphonoacetic acid, DL-2-amino-3-phosphonopropionic acid, or combinations thereof.

[0050] In some embodiments, the stabilizer may be, in particular, bioessential inorganic ions including Na, K, Mg, Zn, Fe, P, S, N, P, or S in the oxide phase, or ammonia or N as a nitro group.

[0051] Stabilized ACC can be stabilized by two or more stabilizers, for example, two, three, or more stabilizers. Stabilizers can be added during the synthesis and precipitation of ACC primary particles and are defined as “internal stabilizers”. Stabilizers can be added after synthesis and bonded to the outer surface of the particles. These are defined as “external stabilizers”. In some embodiments in which both internal and external stabilizers are used, the internal and external stabilizers are similar. In other embodiments, the internal and external stabilizers are different stabilizers. The internal and external stabilizers may be independent of each other as defined above, and each may be a combination of multiple types of stabilizers.

[0052] Stable ACC can contain three or more stabilizers, and one or more stabilizers are added to ACC during its formation and precipitation.

[0053] According to some embodiments, at least one stabilizer is selected from the group consisting of polyphosphates, bisphosphonates, phosphorylated amino acids, citric acid, and any combination thereof. In some embodiments, two or more stabilizers, for example, two, three, or four stabilizers are added.

[0054] According to one embodiment, ACC is stabilized by a combination of phosphoserine and citrate. According to another embodiment, ACC is stabilized by a combination of triphosphate and citrate.

[0055] According to some embodiments, the stabilizer is a polyphosphate or a pharmaceutically acceptable salt thereof. According to some embodiments, the polypolyphosphate is a physiologically compatible, water-soluble polypolyphosphate selected from the group consisting of sodium, potassium, and any other essential cations of polyphosphate. In one embodiment, the polyphosphate is an organic or inorganic polyphosphate. As used herein, the term “polyphosphate” refers to a high molecular weight ester of PO4. According to some embodiments, the polypolyphosphate is a physiologically compatible, water-soluble polyphosphate selected from the group consisting of sodium polyphosphate and potassium polyphosphate. In some embodiments, the polyphosphate is an inorganic polyphosphate or a pharmaceutically acceptable salt thereof. Not limited examples of such salts are Na, K, Mg, Mn, and Zn. According to some embodiments, the inorganic (poly)phosphate contains 2 to 10 phosphate groups, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups. According to some embodiments, the inorganic polyphosphate is selected from pyrophosphates, triphosphates, and hexametaphosphates. According to one embodiment, the stabilizer is a pyrophosphate or a pharmaceutically acceptable salt thereof, such as sodium pyrophosphate. According to another embodiment, the stabilizer is an inorganic triphosphate or a pharmaceutically acceptable salt thereof, such as sodium triphosphate. The terms "triphosphate" and "tripolyphosphate" are used interchangeably herein. According to a further embodiment, the stabilizer is a pharmaceutically acceptable salt thereof, such as hexametaphosphate or sodium hexametaphosphate.

[0056] According to some embodiments, the stabilizer is a bisphosphonate or a pharmaceutically acceptable salt thereof. Non-limiting examples of salts include Na, K, Mg, Mn, and Zn.

[0057] As used herein, the term “bisphosphonate” refers to an organic compound having two phosphonate (PO(OH)2) groups. The term further relates to compounds having a PO3-organic-PO3 skeleton. Most typically, there is a range of bisphosphonates used as pharmaceuticals for the treatment of osteoporosis. According to some embodiments, the bisphosphonate is selected from the group consisting of etidronic acid, zoledronic acid, medronic acid, alendronic acid, and pharmaceutically acceptable salts thereof. According to some embodiments, the stabilizer is etidronic acid or a pharmaceutically acceptable salt thereof. According to another embodiment, the stabilizer is zoledronic acid or a pharmaceutically acceptable salt thereof. According to a further embodiment, the stabilizer is medronic acid or a pharmaceutically acceptable salt thereof. According to a particular embodiment, the stabilizer is alendronic acid or a pharmaceutically acceptable salt thereof.

[0058] According to certain embodiments, the stabilizer is a phosphorylated amino acid. According to one embodiment, the phosphorylated amino acid is phosphoserine. According to another embodiment, the phosphorylated amino acid is phosphothreonine.

[0059] According to some embodiments, the ACC composition comprises the combination of stabilizers disclosed above.

[0060] According to some embodiments, the stabilizer is an inorganic polyphosphate or bisphosphonate as defined above, and the molar ratio between the P atoms of the stabilizer and the Ca atoms of ACC (P:Ca molar ratio) is about 1:90 to 1:1. In one embodiment, the P:Ca molar ratio is about 1:40 to about 1:1. In further embodiments, the P:Ca molar ratio is about 1:35 to about 1:2. In certain embodiments, the P:Ca molar ratio is about 1:30 to about 1:3. In certain embodiments, the P:Ca molar ratio is about 1:28 to about 1:3. In other embodiments, the P:Ca molar ratio is about 1:25 to about 1:4. In further embodiments, the P:Ca molar ratio is about 1:20 to about 1:5. In another embodiment, the P:Ca molar ratio is about 1:20 to about 1:6. In certain embodiments, the P:Ca molar ratio is about 1:15 to about 1:5. In another specific embodiment, the P:Ca molar ratio is about 1:25 to about 1:5. According to some embodiments, such inorganic polyphosphates are pyrophosphates, triphophosphates, hexametaphosphates, or pharmaceutically acceptable salts thereof. According to another embodiment, the bisphosphonate is alendronate, etidronic acid, zoledronic acid, or medronate, and the P:Ca molar ratio is as defined above.

[0061] According to some embodiments, the calcium content (Ca content) of such compositions containing stabilizers is about 1% to about 39% by weight, about 5% to about 39% by weight, about 10% to about 39% by weight, about 15% to about 39% by weight, about 20% to about 38% by weight, about 25% to about 38% by weight, or about 30% to about 38% by weight of the dry ACC particles. The terms "Ca content" and "calcium content" are used interchangeably herein and refer to the calcium content of ACC in the final composition.

[0062] In certain embodiments, the molar ratio of P:Ca is about 1:40 to about 1:1, and the Ca content is about 20% to about 39% by weight. In some embodiments, the molar ratio is 1:28 to about 1:3, and the Ca content is about 30% to about 38% by weight of the dry ACC particles. In other embodiments, the molar ratio is 1:25 to about 1:5, and the Ca content is about 30% to about 36% by weight of the dry ACC particles.

[0063] According to some embodiments, the stabilized ACC powder contains about 1% to about 18% by weight, about 4% to about 15% by weight, and about 6% to about 10% by weight of absorbent and adsorbed water. According to some embodiments, the stabilizer is a polyphosphate or bisphosphonate, and the molar ratio of the P atoms of the stabilizer to the Ca atoms of ACC is about 1:90 to 1:1.

[0064] According to some embodiments, the stabilizer is selected from the group consisting of polyphosphates, phosphorylated amino acids, bisphosphonates, citric acid, tartaric acid, and any combination thereof. According to one embodiment, the polyphosphate is selected from the group consisting of triphates, pyrophosphates, and hexametaphosphates, the phosphorylated amino acid is phosphoserine or phosphothreonine, and the bisphosphonate is selected from the group consisting of alendronate, etidronic acid, zoledronic acid, and medronic acid. According to some embodiments, the polyphosphate is an inorganic polyphosphate.

[0065] According to one embodiment, the stabilizer is selected from the group consisting of organic acids, phosphorylated, phosphornated, sulfated or sulfonated organic compounds, phosphate or sulfate esters of hydroxycarboxylic acids, phosphorylated amino acids, bisphosphonates, organic polyphosphates, hydroxyl-containing organic compounds, derivatives thereof, proteins, and any combination thereof.

[0066] According to another embodiment, the stabilizer is selected from the group consisting of phosphoserine, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, polyphosphate, inorganic triphosphate, hexametaphosphate, ethanol, and any combination thereof.

[0067] In most cases, ACC contains 1 to 20% by weight, preferably 10% by weight or less of adsorbed water, maintains stability in the presence of a stabilizer, and is further stored under dry conditions. The ACC powder used in the examples contained about 6 to 10% by weight of water when formulated. Regarding calcium content, this means that the calcium content of ACC is in a practical range of 28 to 38% by weight of its composition. In practical calculations, the average calcium content is defined as 30% by weight in this application.

[0068] As used herein, the term “pharmaceutical composition” means any composition comprising at least a stabilized ACC and optionally at least one additional pharmaceutically acceptable carrier, stabilizer, and / or expander.

[0069] According to any of the above embodiments and models, the composition of the present invention is a food supplement. According to any of the above embodiments and models, the composition of the present invention is a pharmaceutical composition.

[0070] Formulations of the compositions of the present invention can be adjusted according to the required application. In particular, compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient after administration to a mammal. For example, formulations may be any one selected from plasters, granules, lotions, topical preparations, lemonades, aromatic waters, powders, syrups, eye ointments, liquids and solutions, aerosols, extracts, elixirs, ointments, liquid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injectables, injectable preparations, spirits, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules. According to some embodiments, the composition is a powder. According to certain embodiments, the composition is a powder for sublingual administration. According to other embodiments, the composition is in the form of tablets or capsules for oral administration. According to some embodiments, the composition for oral administration may be an enteric-coated composition or an enteric-coated capsule.

[0071] The formulation may also contain excipients to aid in the manufacture, storage, and efficiency of the medication. Examples include silicon dioxide and microcellulose as anticaking agents, magnesium stearate as a lubricant, and sucralose, mannitol, sorbitol, erythritol, menthol, and citric acid as flavorings.

[0072] According to some embodiments, the composition is a nutritional supplement composition. As used herein, the term “nutritional supplement composition” means a composition suitable for use in humans or animals, comprising one or more natural products that provide health benefits or have therapeutic effects related to the prevention or alleviation of disease.

[0073] The term "food supplement" is used to mean a product containing such a composition, intended to supplement food by providing nutrients beneficial to health in accordance with acceptable directives such as European directives. For example, a food supplement may be a capsule or tablet for swallowing, or a powder or small vial for mixing with food to provide beneficial health effects. Food supplements may also be formulated as sublingual compositions. Food supplements may contain, in addition to activators, food carriers and / or excipients. According to some embodiments, the food carriers and / or excipients are pharmaceutically acceptable carriers and / or excipients.

[0074] The term “food carrier” refers to a compound, material, composition, and / or dosage form suitable for use in contact with a target tissue. Each carrier must also be “acceptable” in the sense that it is compatible with other components of the formulation. As used herein, the term “food carrier” means a material that can be administered, consumed, digested, or passed through the digestive system of an animal or a human without toxic effects. These food carrier materials may exist as either a solid or a liquid at room temperature.

[0075] As used herein, the terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refer to all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption retardants, surfactants, etc., that are compatible with pharmaceutically active substances. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may also contain other active compounds that provide auxiliary, additional, or enhanced therapeutic functions.

[0076] The terms "pharmaceutically acceptable" and "pharmacologically acceptable" include molecular entities and compositions that, when administered to animals or humans as necessary, do not produce harmful, allergic, or other undesirable reactions.

[0077] According to any one of the embodiments described above, the composition of the present invention is formulated in any known form, such as a powder, suspension, tablet, or capsule.

[0078] While the present invention has been described in general terms, these will be more readily understood by referring to the following examples, which are provided for illustrative purposes and are not intended to limit the invention. [Examples]

[0079] Example 1 Model - A mouse model of myocardial infarction (MI).

[0080] C57 / b 7-week-old male mice were used.

[0081] The three treatment groups were treated as follows (the number of mice refers to the final quantity used in the calculation): 1. IP administration of a solution containing stabilized ACC immediately after induction of MI for 28 days (6 mice, labeled as post-treatment) 2. IP administration of a stabilized ACC solution 7 days before MI induction and for an additional 28 days after MI induction (5 mice, labeled as pre-treatment). 3. Immediate administration of saline solution as an intra-invasive therapy (IP) immediately after induction of myocardial infarction (MI), followed by 28 days of continued treatment with saline solution. (8 mice, labeled as control)

[0082] Stabilized ACC: ACC was stabilized using inorganic triphodes (8-12%, optionally containing 1% citrate). The solution contained 1% w / v stabilized ACC (0.45% (w / v) elemental calcium), and 0.2 mL (0.01 mL / gr) of stabilized ACC was injected into each mouse. Alternatively, ACC can be stabilized with phosphoserine, polyphosphates containing 2-10 phosphate groups, bisphosphonates, organic acids, phytic acid, citrate, and any combination thereof. Exemplary preparation methods for stabilized ACC are described below.

[0083] Preparation of 10% TP-1% citrate ACC (ACC stabilized with 10% inorganic triphosphate and 1% citrate) formulated as a cell culture medium supplement.

[0084] 36 mL of 3% calcium chloride solution was mixed with 4 mL of 0.27% citric acid solution and 10 mL of 0.5406% tripphosphate solution. Then, 40 mL of 1.9485% sodium carbonate solution was added to precipitate ACC. A stabilized ACC suspension was prepared by adding 10 mL of a stabilizing solution containing 0.5406% tripphosphate to the ACC suspension. The resulting suspension was used. Alternatively, the suspension was filtered using a Buchner funnel, the cake was washed with water, and the cake was further dried, for example, in an oven. In another method, ACC is stabilized using inorganic polyphosphates having 2 to 10 phosphates, phosphoserines, bisphosphonates, organic acids, and any combination thereof. Further examples of stabilized ACC and its preparation can be found in International Publications 2009 / 053967, 2014 / 024191, and 2016 / 193982.

[0085] MI Procedure The animals were acclimatized for 5 days and maintained with a 12-hour day / night regimen, with free access to food and water. On the day of MI induction, the mice were anesthetized with 4% isoflurane and connected to a ventilator. During surgery, the left anterior descending artery (LAD) was ligated using 8-0 surgical sutures. Ischemia could be observed immediately after LAD ligation. After the surgical procedure, the mice were returned to their cages.

[0086] At the end of the experiment (28 days after MI induction), the animals were euthanized, and their hearts were collected for histopathological evaluation using Masson's trichrome staining.

[0087] Echocardiographic evaluation was performed on all mice before surgery (day 0), and on days 2 and 28.

[0088] The results are shown in Figures 1 and 2. Figure 1 shows the ejection fraction (percentage of blood pumped out of the left ventricle in each cardiac bit) for different treatments. The results show that only the group treated before MI induction was able to recover ejection fraction levels similar to those seen before injury induction (p<0.05). Figure 2 shows the percentage change in ejection fraction for each treatment group at day 28 compared to baseline (day 0). This figure clearly shows that prophylactic treatment with stabilized ACC led to faster healing of mice compared to other treatments (p<0.05), and showed almost complete recovery with no change in ejection fraction at 28 days compared to baseline.

[0089] Example 2 - Effect of stabilized ACC compared to calcium chloride on cardiomyocytes and endothelial cells in vitro. In this study, cardiomyocytes and endothelial cells were obtained from 3-day-old ICR mice (male and female). Cardiac sections from 3-day-old mice were finely chopped, enzymatically isolated (using collagenase), and plated in culture dishes. A general protocol can be found in Ehler E, Moore-Morris T, Lange S. Isolation and culture of neonatal mouse cardiomyocytes. J Vis Exp. 2013;(79):50154. Cells were cultured in 24-well plates. Calcium-depleted DMEM / F-12 (Ca-depleted) medium was used. 1 mM and 2 mM ACC or CaCl2 were added to the Ca-depleted medium. Cells were cultured in triplicates under these different conditions for 4 days. At the end of the culture period, cells were stained with cTnT (staining cardiac troponin T), a marker for cardiomyocytes, and Ki67, which stains proliferating cells. Cells were examined by fluorescence microscopy. Cells showing both stains (green for cTnT and red for Ki67) were proliferating cardiomyocytes. Many photographs were analyzed and quantified.

[0090] The results are shown in Figures 3A to 3C, demonstrating that ACC induced increased cardiomyocyte proliferation compared to CaCl2 at both concentrations. As can be seen in Figure 3B, which shows cardiomyocyte proliferation in the presence of ACC compared to proliferation in the presence of CaCl2 (at the same concentration of calcium), there is a significant increase in cTnT, a marker of cardiomyocyte proliferation. This can also be observed at the mRNA level (Figure 3C).

[0091] Furthermore, ACC had a beneficial effect on cardiomyocyte contraction compared to CaCl2 (data not shown, as seen in the video), and cells cultured with ACC contracted much more spontaneously compared to those cultured with CaCl2.

[0092] Endothelial cells were also evaluated using the CD31 marker and quantified by assessing relative mRNA expression using RT-PCR. The results are shown in Figure 4. It can be seen that in the presence of cultured ACC (2 mM), endothelial cells (expressing CD31) were found to be more abundant than in the presence of the corresponding concentration of CaCl2 (referring to calcium).

[0093] These results suggest that ACC improves the functionality and performance of cardiomyocytes and endothelial cells, which may also indicate improved repair mechanisms. The interaction between endothelial cells and cardiomyocytes is well established to regulate early cardiac development and adult cardiomyocyte function, including its effects on systolic state. Cardiomyocytes depend on endothelial cells not only for oxygenated blood supply but also for local protective signals that promote cardiomyocyte organization and survival [Hsieh PC, Davis ME, Lisowski LK, Lee RT. Endothelial-cardiomyocyte interactions in cardiac development and repair. Annu Rev Physiol. 2006;68:51-66].

[0094] Example 3 - Effect of ACC on the regeneration of new blood vessels in mice treated with MI Cardiac tissue from the injured area (i.e., scar tissue) of mice in Experiment 1 described above was used for histopathological evaluation. This tissue was obtained from either the group treated with ACC one week before MI induction and continued for a further 28 days until the mice were euthanized, or from the control group that received saline for 28 days after MI induction until euthanasia. The following staining was performed: cTnT (for cardiac troponin T), α-SMA (staining smooth muscle actin alpha), and DAPI (staining dead cell nuclei) on slides obtained from these two treatment groups. The ratio of new blood vessels in the area of ​​scar tissue (as seen by α-SMA staining) compared to the area of ​​injured tissue (cTnT and DAPI staining) was analyzed. The results are shown in Figure 5.

[0095] The results in Figure 5 show that mice that received ACC one week before MI induction and continued to receive ACC for an additional 28 days had significantly more new blood vessels compared to control mice that received only saline / vehicle for 28 days after MI induction. These results demonstrate the protective effect of ACC in minimizing damage caused by MI, as well as the improved healing process seen in the newly generated blood vessels.

[0096] While the present invention has been described herein in preferred embodiments, it may be modified without departing from the spirit and nature of the invention as defined in the appended claims.

Claims

1. A composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer, for use in the treatment or prevention of myocardial ischemia.

2. The composition for use according to claim 1, wherein the myocardial ischemia is caused by myocardial infarction.

3. The composition for use according to claim 1 or 2, administered in a manner selected from sublingual, inhalation, IV, or oral administration, or by a combination of administrations selected from oral and sublingual, inhalation and sublingual, oral and inhalation, and oral, inhalation and sublingual administration.

4. A composition for use according to any one of claims 1 to 3, comprising administering stabilized ACC at a dose of approximately 5 to approximately 200 mg / kg / day.

5. The composition for use according to any one of claims 1 to 4, wherein the administration comprises administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 separate doses per day.

6. The composition for use according to claim 3, wherein the composition for sublingual administration is a powder composition comprising secondary particles of ACC having a size of less than 500 μm.

7. The treatment comprises enhanced angiogenesis, according to any one of claims 1 to 6.

8. The composition according to any one of claims 1 to 7, wherein the use comprises administering the composition to a subject predisposed to developing myocardial ischemia.

9. The composition according to claim 8, wherein the subject is selected from subjects suffering from diabetes, hypertension, high blood cholesterol levels, high blood triglyceride levels, or obesity, and is a subject with a genetic predisposition, stress, a history of MI heart disease, or is a smoker.

10. The composition according to any one of claims 1 to 9, wherein the stabilizer is selected from the group consisting of inorganic polyphosphates, organic acids, phosphorylated amino acids, phosphorylated, phosphornated, sulfated or sulfonated organic compounds, phosphate or sulfate esters of hydroxycarboxylic acids, bisphosphonates, organic polyphosphates, hydroxyl-containing organic compounds, derivatives thereof, proteins, and any combination thereof.

11. The composition according to claim 10, wherein the stabilizer is selected from the group consisting of inorganic triphosphate or its salt, phosphoserine, citric acid, sodium triphosphate and citric acid, adenosine triphosphate, adenosine diphosphate, phytic acid, etidronic acid, pyrophosphate, polyphosphate, hexametaphosphate, ethanol, and any combination thereof.

12. A method for treating or preventing myocardial ischemia in a subject requiring treatment or prevention of myocardial ischemia, comprising administering to the subject a composition comprising amorphous calcium carbonate (ACC) stabilized with at least one stabilizer.

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