Use of CHP (Cyclo-Hispro) for lowering blood pressure

Abstract

The present invention relates to the use of CHP (cyclo-hispro) for lowering blood pressure, and more specifically to a pharmaceutical composition for lowering blood pressure containing CHP and a functional health food composition containing the same; a method for lowering blood pressure using CHP; the use of CHP in the production of an antihypertensive agent; a pharmaceutical composition for preventing or treating hypertension or its complications containing CHP and a functional health food composition for preventing or ameliorating hypertension or its complications containing the same; a method for preventing or treating hypertension or its complications using CHP; and the use of CHP in the production of a drug for preventing or treating hypertension or its complications.

Description

[Technical Field]

[0001] The present invention relates to the use of CHP (cyclo-hispro) for lowering blood pressure, and more specifically to a pharmaceutical composition for lowering blood pressure containing CHP and a functional health food composition containing the same; a method for lowering blood pressure using CHP; the use of CHP in the production of an antihypertensive agent; a pharmaceutical composition for preventing or treating hypertension or its complications containing CHP and a functional health food composition for preventing or ameliorating hypertension or its complications containing the same; a method for preventing or treating hypertension or its complications using CHP; and the use of CHP in the production of a drug for preventing or treating hypertension or its complications. [Background technology]

[0002] Recently, nutritional status has improved dramatically due to economic growth, improved educational standards, and scientific advances, and diseases caused by changes in various lifestyles, including dietary habits, and an increase in average life expectancy are also showing a tendency to diversify.

[0003] Cerebrovascular disease, the second leading cause of death in Korea after cancer, is caused by the high incidence of hyperlipidemia and high blood pressure. High blood pressure, in particular, is a major cause of complications such as cerebrovascular disease and cardiovascular disease, and is showing an increasing trend as dietary habits become more Westernized and the population ages.

[0004] The number of patients with kidney disorders is gradually increasing. This trend is due to factors such as changes in lifestyle, aging, and the rise in diabetic nephropathy caused by the increasing prevalence of diabetes. The number of patients who develop renal failure or are forced to undergo dialysis due to declining kidney function is increasing every year. Dialysis therapy is associated with side effects such as impaired red blood cell production, complications due to microglubulin accumulation, and increased cardiovascular disease. Patients with renal failure are treated with antihypertensive therapy, as well as medications to suppress the rise in blood electrolytes and a low-protein diet. In cases of renal anemia, erythropoietin is administered, but these are not sufficient to prevent the progression of the condition. Renal diseases such as nephritis, diabetic nephropathy, and renal failure are often accompanied by hypertension. Because hypertension is considered a contributing factor to the progression of kidney disease, antihypertensive drugs are also used to slow the progression of kidney disease.

[0005] There are drugs for treating high blood pressure, such as calcium channel blockers, diuretics, and angiotensin converting enzyme inhibitors, but drugs for treating high blood pressure manufactured by chemical synthesis have side effects such as headache, liver disease, facial flushing, atrioventricular block, and gout. To solve these problems, research has been ongoing recently to find ways to prevent and treat high blood pressure using natural substances.

[0006] The most well-known cellular model of hypertension is the angiotensin II (Ang II) model. Ang II binds to angiotensin receptors present in blood vessels and exerts various effects. The type 1 receptor, AT1 (AT1R), induces vasoconstriction, while the type 2 receptor, AT2 (AT2R), relaxes blood vessels, acting in the opposite direction to AT1 (Elena Kaschina & Thomas Unger, Blood Press, 12(2), 70-88, 2003). In fact, hypertensive patients develop hypertension because their blood Ang II levels remain high or AT1 is more strongly expressed than AT2, leading to blood vessels remaining constricted rather than relaxed. Therefore, drugs that reduce AT1 expression levels or blockers that prevent Ang II from binding to the AT1 receptor can be used as hypertension treatments (Le TH, et al., Hypertension, 42(4), 507-514, 2003).

[0007] High blood pressure, arteriosclerosis, peripheral circulatory disorders, vascular stenosis, cerebral infarction, angina pectoris, myocardial infarction, and ischemic cerebral disease are all diseases caused by vasoconstriction. Vasoconstriction is the narrowing of blood vessels, such as arteries (including the aorta) or veins, due to the contraction of the vascular wall. Smooth blood circulation requires increased blood flow, which in turn requires vasodilation. Vasodilation involves the production of nitric oxide (NO) through the action of endothelial nitric oxide synthase (eNOS) in vascular endothelial cells (Ulrich Forstermann & Thomas Munzel, Circulation, 113, 1708-1714, 2006). In fact, NO production is reduced in hypertension. In general, vascular smooth muscle is affected by various factors, and the cGMP signaling pathway in vascular smooth muscle cells via NO produced and secreted by vascular endothelial cells is a very important pathway in vascular relaxation (Zhu J, et al., Mol Brain, 9, 20, 2016). NO is produced from L-arginine by eNOS in vascular endothelial cells. NO secreted from vascular endothelial cells flows into vascular smooth muscle cells, activates soluble guanylyl cyclase (sGC), and increases cGMP production through a signaling system that relaxes vascular smooth muscle and regulates blood pressure. Therefore, NO production promoters can also be used as antihypertensive drugs.

[0008] Meanwhile, "Cyclo-HisPro (CHP)" is a naturally occurring cyclic dipeptide composed of histidine-proline, a metabolic product of thyrotropin-releasing hormone (TRH), or a physiologically active dipeptide that can be synthesized de novo in the body during the TRH metabolic process. Its blood sugar regulating effects (Korean Patent Publication No. 10-2013-0006170), fibrosis treatment effects (Korean Patent Registration No. 10-2140910), and peritoneal fibrosis treatment effects (Korean Patent Registration No. 10-2133151) are known, but the blood pressure lowering effect of CHP is unknown. Summary of the Invention [Problem to be solved by the invention]

[0009] Under these circumstances, the present inventors have confirmed that cyclo-HisPro (CHP) exerts its antihypertensive effect in vascular endothelial cells by reducing the expression of the Angiotensin II type I receptor gene or protein; increasing the expression of the Angiotensin II type II receptor gene or protein; increasing the expression of the VE-cadherin gene or protein; increasing the expression of the eNOS gene or protein; and / or increasing the production of NO, thereby completing the present invention.

[0010] Therefore, an object of the present invention is to provide a pharmaceutical composition for lowering blood pressure, which comprises cyclo-hispro or a pharmaceutically acceptable salt thereof.

[0011] Another object of the present invention is to provide a health functional food composition for lowering blood pressure, which contains cyclo-hispro or a nutrient-acceptable salt thereof.

[0012] It is still another object of the present invention to provide a method for lowering blood pressure using cyclo-hispro or a pharmaceutically acceptable salt thereof.

[0013] Yet another object of the present invention is to provide a use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of an antihypertensive agent.

[0014] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating hypertension or its complications, comprising cyclo-hispro or a pharmaceutically acceptable salt thereof.

[0015] Another object of the present invention is to provide a health functional food composition for preventing or ameliorating hypertension or its complications, which comprises cyclo-hispro or a nutrient-acceptable salt thereof.

[0016] It is still another object of the present invention to provide a method for preventing or treating hypertension or its complications using cyclo-hispro or a pharmaceutically acceptable salt thereof.

[0017] It is still another object of the present invention to provide the use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of hypertension or its complications. [Means for solving the problem]

[0018] In order to solve the above-mentioned problems, in a first aspect, the present invention provides a pharmaceutical composition for lowering blood pressure, which contains cyclo-hispro or a pharmaceutically acceptable salt thereof; and a functional health food composition for lowering blood pressure, which contains cyclo-hispro or a nutritively acceptable salt thereof.

[0019] The present invention also provides a method for lowering blood pressure, comprising administering cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need thereof; and the use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of an antihypertensive agent.

[0020] According to a preferred embodiment of the present invention, the cyclo-hispro or a pharmaceutically or food-based acceptable salt thereof can exert a blood pressure lowering effect in vascular endothelial cells through one or more activities selected from the group consisting of the following a) to e): a) Decreased expression of the Angiotensin II type I receptor gene or protein; b) Increased angiotensin II type II receptor gene or protein expression; c) increased VE-cadherin gene or protein expression; d) increased eNOS gene or protein expression; and e) Increased production of NO.

[0021] In a second aspect, the present invention provides a pharmaceutical composition for preventing or treating hypertension or complications thereof, comprising cyclo-hispro or a pharmaceutically acceptable salt thereof, and a functional health food composition for preventing or ameliorating hypertension or complications thereof, comprising cyclo-hispro or a nutritively acceptable salt thereof.

[0022] The present invention also provides a method for preventing or treating hypertension or a complication thereof, comprising administering cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need thereof; and the use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of hypertension or a complication thereof.

[0023] According to a preferred embodiment of the present invention, the complication may be selected from the group consisting of arteriosclerosis, coronary artery disease, myocardial infarction, heart failure, angina pectoris, peripheral blood circulation disorder, vascular stenosis, stroke, cerebral infarction, cerebral hemorrhage, ischemic encephalopathy, renal failure, glomerulitis, diabetes, diabetic nephropathy, diabetic retinopathy, proteinuria, uremia, edema, visual impairment due to ocular vascular stenosis, and glaucoma. [Effects of the Invention]

[0024] The composition containing CHP of the present invention exerts a blood pressure lowering effect by reducing the expression of the Angiotensin II type I receptor gene or protein; increasing the expression of the Angiotensin II type II receptor gene or protein; increasing the expression of the VE-cadherin gene or protein; increasing the expression of the eNOS gene or protein; and / or increasing NO production in vascular endothelial cells, and is therefore useful for preventing, ameliorating, or treating hypertension and its complications.Since it is a naturally occurring ingredient and is safe for the human body, it can be used in therapeutic agents and food compositions that can replace conventional synthetic ACE inhibitors, which exhibit some side effects. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is a graph showing the results of administering cyclo-Hispro (pre-treatment) 3 days before preparing 5 / 6 nephrectomized rats, and measuring systolic blood pressure at 2, 6, and 8 weeks. [Figure 2] FIG. 2 is a graph showing the results of administering cyclo-hispro to 5 / 6 nephrectomized rats starting 2 weeks after preparation (post-treatment), and measuring systolic blood pressure at 2, 6, and 8 weeks. [Figure 3] FIG. 3 shows changes in the expression levels of AT1R (Angiotensin II type I receptor), AT2R (Angiotensin II type 2 receptor), and VE-cadherin proteins in HUVEC cells following treatment with angiotensin II and CHP. [Figure 4] FIG. 4 is a graph showing that eNOS gene expression in SVEC4-10 cells increases in a CHP concentration-dependent manner. [Figure 5]FIG. 5 shows an image confirming the increase in NO production by CHP treatment in HMVEC-L cells using DAF-FM DA (scale bar 50 μm). DETAILED DESCRIPTION OF THE INVENTION

[0026] As mentioned above, conventional antihypertensive agents produced by chemical synthesis methods, etc., exhibit side effects such as headache, liver disease, facial flushing, atrioventricular block, and gout. Therefore, efforts are ongoing to search for natural substances with fewer side effects that have a blood pressure lowering effect and to develop them as pharmaceutical and food ingredients. As a result, the present inventors have confirmed that cyclo-HisPro (CHP), a naturally occurring cyclic dipeptide composed of histidine and proline, which is a metabolite of thyrotropin-releasing hormone (TRH), has a blood pressure lowering effect without side effects, and have sought a solution to the above problems.

[0027] Therefore, a first aspect of the present invention relates to a pharmaceutical composition for lowering blood pressure, which contains cyclo-hispro or a pharmaceutically acceptable salt thereof, and a functional health food composition for lowering blood pressure, which contains cyclo-hispro or a nutritively acceptable salt thereof.

[0028] In the present invention, "cyclo-HisPro (CHP)" refers to a naturally occurring cyclic dipeptide composed of histidine-proline, which is a metabolic product of thyrotropin-releasing hormone (TRH), or a physiologically active dipeptide that can be synthesized de novo in the body during the TRH metabolic process, and is widely distributed throughout the brain, spinal cord, gastrointestinal tract, etc.

[0029] In the composition of the present invention, the CHP may be synthesized or commercially available, or may be purified from a CHP-containing substance, such as a prostate extract or soybean hydrolysate.

[0030] By use of the term "purified," it is intended to mean that the CHP is in a concentrated form relative to the form obtained from a naturally occurring source, such as a prostate extract. Purified components can be enriched from their natural sources or obtained by chemical synthesis methods.

[0031] A first aspect of the present invention relates to a method for lowering blood pressure, further comprising the step of administering an effective amount of cyclo-hispro or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0032] Furthermore, the first aspect of the present invention relates to the use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of an antihypertensive agent.

[0033] In a first aspect of the present invention, the CHP or a pharmaceutically or food-based acceptable salt thereof can exert a blood pressure lowering effect in vascular endothelial cells through one or more activities selected from the group consisting of the following a) to e): a) Decreased expression of the Angiotensin II type I receptor gene or protein; b) Increased angiotensin II type II receptor gene or protein expression; c) increased VE-cadherin gene or protein expression; d) increased eNOS gene or protein expression; and e) Increased production of NO.

[0034] Therefore, the CHP of the present invention or a pharmaceutically or food-based acceptable salt thereof can be utilized to prevent, improve, or treat hypertension and / or its complications.

[0035] A second aspect of the present invention relates to a pharmaceutical composition containing cyclo-hispro or a pharmaceutically acceptable salt thereof for preventing or treating hypertension or complications thereof, and a functional health food composition containing cyclo-hispro or a nutritively acceptable salt thereof for preventing or ameliorating hypertension or complications thereof.

[0036] Regarding the term "hypertension" in this invention, the World Health Organization (WHO) defines hypertension as a systolic blood pressure of 160 mmHg or higher and a diastolic blood pressure of 95 mmHg or higher. Hypertension is broadly classified into essential hypertension, which has an unknown cause, and secondary hypertension caused by an underlying disease, and it is known that more than 90% of cases of these fall into the category of essential hypertension.

[0037] The term "hypertensive complication" as used herein refers to a symptom or disease that causes hypertension or a symptom or disease that occurs due to hypertension. Specifically, the hypertensive complication may be a symptom or disease selected from the group consisting of arteriosclerosis, coronary artery disease, myocardial infarction, heart failure, angina pectoris, peripheral blood circulation disorder, vascular stenosis, stroke, cerebral infarction, cerebral hemorrhage, ischemic encephalopathy, renal failure, glomerulitis, diabetes, diabetic nephropathy, diabetic retinopathy, proteinuria, uremia, edema, visual impairment due to ocular vascular stenosis, and glaucoma.

[0038] In the compositions of the present invention for preventing, ameliorating, or treating hypertension or its complications, the terms "prevention," "amelioration," and / or "treatment" refer to any action that inhibits and delays the onset of a disease or condition, any action that improves or favorably alters the state of a disease or condition, and any action that delays, halts, or reverses the progression of a disease or condition.

[0039] Because the kidney is involved in water and sodium metabolism and is closely related to the renin-angiotensin system, kidney disease itself, i.e., glomerulitis and renal failure, can cause hypertension and also play an important role in the development of primary hypertension. Therefore, in one embodiment of the present invention, the blood pressure lowering effect of CHP was confirmed in a nephrectomy animal model, and as shown in Figures 1 and 2, it was confirmed that administration of CHP before and / or after nephrectomy significantly reduced systolic blood pressure.

[0040] Therefore, the CHP of the present invention is useful for preventing, ameliorating or treating complications associated with kidney diseases, such as renal failure, glomerulitis, diabetic nephropathy, proteinuria or uremia.

[0041] The most well-known cellular model of hypertension is the angiotensin II (Ang II) model. Ang II binds to angiotensin receptors present in blood vessels and exerts various effects. The type 1 receptor, AT1 (AT1R), induces vasoconstriction, while the type 2 receptor, AT2 (AT2R), relaxes blood vessels, acting in the opposite direction to AT1 (Elena Kaschina & Thomas Unger, Blood Press, 12(2), 70-88, 2003). In fact, hypertensive patients develop hypertension because their blood Ang II levels remain high or AT1 is more strongly expressed than AT2, leading to blood vessels remaining constricted rather than relaxed. Therefore, the development of drugs that reduce AT1 expression levels or blockers that prevent Ang II from binding to the AT1 receptor is essential for the development of therapeutic agents for hypertension (Le TH, et al., Hypertension, 42(4), 507-514, 2003). Therefore, in another example of the present invention, the expression levels of AT1R, AT2R, and VE-cadherin proteins in HUVEC cells were examined following treatment with AngII and CHP. As shown in Figure 3, CHP treatment reduced the expression level of AT1R protein, which was increased by AngII, increased the expression level of AT2R protein, which was decreased by AngII, and increased the expression level of VE-cadherin protein, which was decreased by AngII. Thus, CHP increased the expression level of AT1R protein, which induces vascular constriction. decrease This increases the expression level of the AT2R protein, which relaxes blood vessels. increase It was confirmed that this has a blood pressure lowering effect.

[0042] Typical diseases caused by vasoconstriction include high blood pressure, arteriosclerosis, peripheral circulatory disorders, vascular stenosis, cerebral infarction, angina pectoris, myocardial infarction, and ischemic cerebral disease. Vasoconstriction is the narrowing of blood vessels, such as arteries (including the aorta) or veins, due to the contraction of the vascular wall. Smooth blood circulation requires increased blood flow, which in turn requires vasodilation. Vasodilation involves the production of nitric oxide (NO) through the action of eNOS (Endothelial Nitric Oxide Synthase), an enzyme that synthesizes nitric oxide in vascular endothelial cells (Ulrich Forstermann & Thomas Munzel, Circulation, 113, 1708-1714, 2006). In fact, NO production is reduced in hypertension. Vascular smooth muscle is generally affected by various factors, and the cGMP signaling pathway in vascular smooth muscle cells via NO produced and secreted by vascular endothelial cells is a crucial pathway for vasorelaxation (Zhu J, et al., Mol Brain, 9, 20, 2016). NO is produced from L-arginine by eNOS in vascular endothelial cells. NO secreted from vascular endothelial cells enters vascular smooth muscle cells, activates soluble guanylyl cyclase (sGC), and increases cGMP production via a signaling pathway that relaxes vascular smooth muscle and regulates blood pressure. Therefore, the development of drugs that can promote NO production is necessary to treat blood pressure disorders caused by vascular diseases. Accordingly, in another embodiment of the present invention, we investigated the eNOS gene expression level following CHP treatment in SVEC4-10 cells and the NO production following CHP treatment in HMVEC-L cells. As shown in Figure 4, CHP treatment increased eNOS gene expression levels, and as shown in Figure 5, CHP treatment increased NO production. This confirmed that CHP relaxes blood vessels by increasing NO production, thereby lowering blood pressure.

[0043] A second aspect of the present invention relates to a method for preventing or treating hypertension or complications thereof, further comprising the step of administering to an individual in need thereof an effective amount of cyclo-hispro or a pharmaceutically acceptable salt thereof.

[0044] Furthermore, a second aspect of the present invention relates to the use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of hypertension or its complications.

[0045] In this application, the term "pharmaceutically acceptable" means that a substance is physiologically acceptable and does not generally cause an allergic reaction or a similar reaction when administered to humans, and the salt is preferably an acid addition salt formed from a pharmaceutically acceptable free acid.

[0046] The pharmaceutically acceptable salt may be an acid addition salt formed with an organic or inorganic acid, such as formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid, and methanesulfonic acid. The inorganic acid may be, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, and boric acid. The acid addition salt may preferably be in the form of a hydrochloride or acetate, more preferably a hydrochloride.

[0047] Further possible salt forms include gaba salts, gabapentin salts, pregabalin salts, nicotinate salts, adipate salts, hemimalonate salts, cysteine ​​salts, acetylcysteine ​​salts, methionine salts, arginine salts, lysine salts, ornithine salts or aspartate salts.

[0048] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include carriers for oral administration and carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration may include water, a suitable oil, saline, aqueous glucose, glycol, etc. Stabilizers and preservatives may also be included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be found in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0049] The pharmaceutical compositions of the present invention may be administered to mammals, including humans, by any method, for example, orally or parenterally, including, but not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0050] The pharmaceutical compositions of the present invention may be formulated for oral or parenteral administration by the administration routes described above, and may be formulated using one or more buffers (e.g., saline or PBS), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants, surfactants, diluents, or excipients.

[0051] Formulations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions, and capsules, and such solid formulations can be prepared by mixing the pharmaceutical composition of the present invention with at least one or more excipients, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose, or gelatin. For example, tablets or sugar-coated tablets can be obtained by blending the active ingredient with a solid excipient, pulverizing the mixture, adding suitable excipients, and then processing the granule mixture.

[0052] In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives in addition to water or liquid paraffin, which are commonly used simple diluents.

[0053] In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, sodium alginate, or the like may be added as a disintegrant depending on the case, and further additives such as an anti-coagulating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, and a preservative may be included.

[0054] For parenteral administration, the pharmaceutical compositions of the present invention may be formulated into injections, transdermal preparations, and nasal inhalants with suitable parenteral carriers by methods known in the art. Injections must be sterilized and protected from microbial contamination, such as bacteria and fungi. Suitable carriers for injections include, but are not limited to, solvents or dispersion media such as water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferred carriers include Hank's solution, Ringer's solution, triethanolamine-containing phosphate buffered saline (PBS), or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injections from microbial contamination, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal, may be used. In most cases, the injections may further contain isotonic agents such as sugars or sodium chloride.

[0055] Transdermal administration includes ointments, creams, lotions, gels, topical solutions, pastes, liniments, aerosols, etc. As used above, "transdermal administration" means that a pharmaceutical composition is topically administered to the skin, and an effective amount of an active ingredient contained in the pharmaceutical composition is delivered into the skin.

[0056] For inhalation administration, the compounds used according to the present invention can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch. Dosage forms for parenteral administration are described in the literature (Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour), a formulary generally known to all pharmaceutical scientists.

[0057] When the pharmaceutical composition of the present invention contains an effective amount of cyclo-hispro, it can provide a desirable blood pressure lowering effect and prevent, ameliorate, or treat hypertension or its complications. As used herein, the term "effective amount" refers to an amount that shows a response greater than or equal to that of a negative control group, and preferably refers to an amount sufficient to prevent, ameliorate, or treat hypertension or its complications. The pharmaceutical composition of the present invention may contain 0.01 to 99.9% cyclo-hispro, with the remainder being a pharmaceutically acceptable carrier. The effective amount of cyclo-hispro contained in the pharmaceutical composition of the present invention varies depending on the form in which the composition is manufactured.

[0058] The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. For example, the amount of cyclo-hispro may be preferably 0.001 to 100 mg, more preferably 0.01 to 70 mg, per kg of body weight per day, administered once or several times in divided doses. However, the effective dose of cyclo-hispro for a patient is determined by taking into account various factors, such as the patient's age, body weight, health condition, sex, severity of the disease, diet, and excretion rate, as well as the route of administration and frequency of treatment. Taking these factors into consideration, a person skilled in the art would be able to determine an appropriate effective dose of cyclo-hispro for a specific purpose, such as for lowering blood pressure or preventing, ameliorating, or treating hypertension or its complications. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, route of administration and method of administration, as long as it exhibits the effects of the present invention.

[0059] The pharmaceutical compositions of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy or methods using biological response modifiers.

[0060] The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing cyclo-hispro. In this respect, the composition of the present invention can be a quasi-drug composition for lowering blood pressure or preventing or ameliorating hypertension or its complications, and a quasi-drug containing the composition.

[0061] The topical preparation can be applied directly to the skin. When used as an external preparation, the pharmaceutical composition of the present invention may further contain any other ingredient commonly used in dermatological preparations, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, moisturizing agents, essential oils, dyes, pigments, hydrophilic active agents, lipophilic active agents, or lipid vesicles. The ingredients may be added in amounts commonly used in dermatological preparations.

[0062] When the pharmaceutical composition of the present invention is provided as an external preparation, it may be in the form of, but is not limited to, a liquid, ointment, patch, gel, cream, spray, etc. According to one embodiment of the present invention, the external preparation of the present invention may include an ointment, a mask, a plaster, a patch, a transdermal absorption agent, etc.

[0063] When the pharmaceutical composition of the present invention is used as a quasi-drug composition, cyclo-hispro can be added directly or can be used appropriately with other quasi-drug ingredients by a conventional method. The amount of the active ingredient to be mixed can be suitably determined depending on the purpose of use (prevention, health, or therapeutic treatment).

[0064] The contents of the pharmaceutical composition and functional health food composition of the present invention can be applied mutatis mutandis to the quasi-pharmaceutical composition and quasi-pharmaceutical product of the present invention.

[0065] In the present invention, the term "health functional food" includes both the meanings of "functional food" and "health food."

[0066] In the present invention, the term "functional food" is the same as food for special health use (FoSHU), and refers to food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrients.

[0067] In the present invention, the term "health food" refers to a food that has a more active effect on maintaining or improving health than general foods, and "health supplement food" refers to a food intended for health supplementation. Depending on the circumstances, the terms "functional food," "health food," and "health supplement food" may be used interchangeably. The food may be prepared in various forms, such as tablets, capsules, powders, granules, liquids, and pills.

[0068] As a specific example of such functional foods, processed foods can be produced by using the composition to modify agricultural, livestock or marine products to take advantage of their properties and to improve their shelf life.

[0069] The health functional food composition of the present invention may also be produced in the form of a nutritional supplement, food additive, feed, etc., and is intended for consumption by humans or animals including livestock.

[0070] The food compositions of the above types can be prepared in various forms by conventional methods known in the art. Common foods that can be prepared by adding cyclo-hispro include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meat and processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candies, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., miso, soy sauce, sauces, etc.).

[0071] Additionally, nutritional supplements can be prepared by adding cyclo-hispro to, but are not limited to, capsules, tablets, pills, and the like.

[0072] Furthermore, functional health foods include, but are not limited to, cyclo-hispro, which can be liquefied, granulated, encapsulated, or powdered to be consumed in the form of tea, juice, or drink (health drink). Furthermore, cyclo-hispro can be prepared in the form of powder or concentrated liquid for use as a food additive. Furthermore, cyclo-hispro can be mixed with well-known active ingredients known to be effective in preventing or ameliorating hypertension or its complications to prepare a composition.

[0073] When the food composition of the present invention is used as a health drink composition, the health drink composition can contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates per 100 mL of the composition of the present invention is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g.

[0074] Cyclo-hispro can be contained as an active ingredient in a food composition for lowering blood pressure or preventing or ameliorating hypertension or its complications in an amount effective for achieving the preventive or ameliorating effect, for example, preferably 0.01 to 100 wt% based on the total weight of the entire composition, but is not particularly limited thereto. The food composition of the present invention can be produced by mixing cyclo-hispro with other active ingredients known to be effective in lowering blood pressure or preventing or ameliorating hypertension or its complications.

[0075] In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloids, thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. The health food of the present invention may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

[0076] In the methods of the present invention, the term "individual" includes, but is not limited to, any animal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent). Such term does not denote a particular age or sex. Thus, it is intended to include fetuses, as well as adult and newborn subjects, whether female or male. A patient refers to a subject suffering from a disease or disorder. The term patient includes human and veterinary subjects.

[0077] In the method of the present invention, the effects of CHP and the configuration including the administration route, number of administrations, dosage, etc. have been explained above, and therefore further description thereof will be omitted.

[0078] The present invention will be described in more detail below with reference to examples. However, since the present invention can be modified in various ways and can have various forms, the specific examples and explanations described below are only for the purpose of understanding the present invention and are not intended to limit the present invention to the specific disclosed forms. The scope of the present invention should be understood to include all modifications, equivalents, and alternatives that fall within the spirit and technical scope of the present invention. [Example]

[0079] [Preparation example] Cyclo-Hispro (CHP) used in the following examples was purchased from BACHEM. [Example 1] Determination of the preventive effect of cyclo-Hispro treatment on blood pressure regulation in rats 1-1. Creation of a 5 / 6 nephrectomy model and administration of cyclo-Hispro The kidney is involved in water and sodium metabolism and is closely related to the renin-angiotensin system, so kidney disease itself, i.e., glomerulitis and renal failure, can cause hypertension and also play an important role in the development of primary hypertension. Chronic renal failure caused by nephrectomy in rats leads to the continued progression of hypertension, uremia, proteinuria, and myocardial sclerosis. Seven-week-old SD rats purchased from Coretec Co., Ltd. were bred for over a week, and then the experimental group underwent surgery in two stages. After general anesthesia, an abdominal midline incision was made to separate the left kidney from the adrenal gland and surrounding tissue. The renal blood vessels were then clamped with forceps to prevent bleeding, and the upper and lower thirds of the left kidney were removed. One week later, the rats were again subjected to general anesthesia, and the entire right kidney was removed to complete the 5 / 6 nephrectomy model.

[0080] The animals were divided into three groups of eight each. The first group was a normal control group (SHAM) that had not undergone nephrectomy and was administered phosphate-buffered saline. The second group was a disease control group that had undergone 5 / 6 nephrectomy (5 / 6 Nx) and was administered phosphate-buffered saline without cyclo-Hispro. The third group was orally administered cyclo-Hispro at 35 mg / kg every other day for 8 weeks starting three days before nephrectomy.

[0081] 1-2. Blood pressure measurement in rats To reduce the error range of blood pressure measurements, the experimental animals were placed in a holder for a blood pressure measuring device (BP-2000 blood pressure analysis system, rat platform 2-channel, Visitech Systems, Apex, NC, USA) and then adapted for 30 minutes in a 37°C heating chamber. Systolic blood pressure was measured at least five times in the tail artery and averaged. A group-by-group t-test was performed to test the significance of the above experimental results between the disease control group (Nx), the normal control group, and the cyclo-Hispro group, and showed statistically significant differences (**p<0.005, ***p<0.0005, ****p<0.00005).

[0082] As shown in Figure 1, a significant time-dependent increase in systolic blood pressure was observed in the disease control group (Nx) compared to the normal control group (SHAM). Meanwhile, the cyclo-Hispro administration group demonstrated a significant decrease in systolic blood pressure compared to the disease control group (Nx). Specifically, the systolic blood pressure in the normal control group (SHAM) was 127.8 mmHg, while that in the disease control group (Nx) was 196.2 mmHg. The systolic blood pressure in the cyclo-Hispro administration group was 156.7 mmHg. Therefore, it was confirmed that administration of cyclo-Hispro has a preventive effect against hypotension.

[0083] [Example 2] Assessment of the therapeutic effect of cyclo-Hispro on blood pressure regulation in rats 2-1. Creation of a 5 / 6 nephrectomy model and administration of cyclo-Hispro A 5 / 6 nephrectomy model was prepared using the same method as in Example 1-1 and divided into three groups of eight animals each. Groups 1 and 2 were treated in the same manner, but group 3 was orally administered cyclo-hispro at 35 mg / kg every other day for six weeks starting two weeks after nephrectomy.

[0084] 2-2. Blood pressure measurement in rats The blood pressure of each group of rats was measured using the same method as in Examples 1-2. As shown in Figure 2, the systolic blood pressure of the diseased control group (Nx) increased significantly in a time-dependent manner compared to the normal control group (SHAM). Meanwhile, the systolic blood pressure of the cyclo-Hispro administered group was significantly decreased compared to the normal control group (Nx). Specifically, the systolic blood pressure of the normal control group (SHAM) was 127.8 mmHg, the systolic blood pressure of the diseased control group (Nx) was 196.2 mmHg, and the systolic blood pressure of the cyclo-Hispro administered group was 171.6 mmHg. This confirms that administration of cyclo-Hispro is effective in blood pressure regulation therapy.

[0085] [Example 3] Confirmation of AT1R, AT2R, or VE-cadherin protein expression in HUVEC cells treated with angiotensin II and CHP 3-1. HUVEC cell culture Human umbilical vein endothelial cells (HUVEC, CRL-1730) were obtained from ATCC (American Type Culture Collection), and EBM-2 basal medium and EGM-2 MV microvascular endothelial cell growth medium SingleQuots supplement were purchased from Lonza.

[0086] HUVEC cells were cultured in EGM-2 medium supplemented with EGM-2 MV SingleQuots at 37°C and 5% CO. The medium was changed every 2–3 days, and subculture was performed at 70–80% confluency.

[0087] 3-2. Confirmation of AT1R, AT2R, and VE-cadherin protein expression levels HUVEC cells were treated in three groups as shown in Table 1.

[0088] [Table 1]

[0089] Angiotensin II- and CHP-treated HUVEC cells were placed in 500 μl of RIPA buffer supplemented with protease and phosphatase inhibitors and disrupted using an IKA T10 homogenizer. The cells were then placed on ice for 15 minutes and centrifuged at 15,000 rpm at 4°C. The supernatants were collected and their protein concentrations were measured using the BCA assay. Equal amounts of the samples were separated using a Bolt™ protein gel electrophoresis system and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies against AT1R (Angiotensin II type I receptor), AT2R (Angiotensin II type 2 receptor), VE-cadherin, and β-actin. After washing three times with TBST for 10 minutes each, the cells were incubated with secondary antibodies at room temperature for 1 hour. After washing three times with TBST for 10 minutes each, the cells were incubated with ECL and the expression level was measured. The band size was quantified using ImageJ and corrected by dividing each band size by the size of the β-actin band. Statistical significance was analyzed using Student's t-test (*p<0.05, **p<0.05, ***p<0.0005) compared with the angiotensin II-treated control group (Ang II).

[0090] As shown in Figure 3, angiotensin II (Ang II) treatment of HUVEC cells increased the expression of AT1 (AT1R) protein, which induces vascular contraction, while decreasing the expression of AT2 (AT2R) protein, which relaxes blood vessels and has the opposite effect to AT1. In the CHP-treated group, a statistically significant decrease in AT1R was observed, while a tendency toward an increase in AT2 was confirmed. Furthermore, VE-cadherin, which plays an important role in blood vessel formation, was decreased by angiotensin II (Ang II), but a statistically significant increase in VE-cadherin protein expression was confirmed in the group treated with CHP.

[0091] The results of this example demonstrate that CHP induces vascular contraction by suppressing the expression of angiotensin II receptor 1 (AT1R). Decline This increases the expression of angiotensin II receptor 2 (AT2R), which relaxes blood vessels. increase This confirms that CHP has a blood pressure lowering effect, which fully demonstrates that it can be used to treat hypertension.

[0092] [Example 4] Confirmation of eNOS gene expression by CHP treatment in SVEC4-10 cells 4-1. SVEC4-10 cell culture The mouse endothelial cell line SVEC4-10 (CRL-2181) was obtained from ATCC (American Type Culture Collection), and RPMI-1640 medium and FBS were purchased from Hyclone.

[0093] SVEC4-10 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2, and subcultured every two days.

[0094] 4-2. Confirmation of eNOS gene expression levels 5 x 10 SVEC4-10 cells in a 6-well plate 5 The cells were cultured for 24 hours in serum-free medium. The cells were treated with CHP at concentrations of 0, 10, 100, and 250 μM, and then cultured for 2 hours at 37°C in 5% CO2. The medium was then removed, the cells were washed once with cold PBS, and then immediately dissolved using NucleoZOL.

[0095] RNA was extracted using NucleoZOL (Macherey-Nagel) according to the manufacturer's total RNA isolation protocol. 1 μg of RNA was used to synthesize cDNA by reverse transcription polymerase chain reaction (RTC) using the iScript cDNA synthesis kit (Bio-Rad). The synthesized cDNA was analyzed by real-time PCR using the eNOS gene primer set in iQ SYBR Green Supermix (Bio-Rad). The primer set used was synthesized by Bioneer with the base sequences shown in Table 2.

[0096] [Table 2]

[0097] The expression value of each gene was corrected by dividing it by the expression value of the housekeeping gene β-actin. Statistical significance was analyzed using Student's t-test (*p<0.05).

[0098] As a result of the experiment, it was confirmed that CHP treatment significantly increased the expression of the eNOS gene, which produces nitric oxide, as shown in Figure 4. This suggests that the promotion of nitric oxide production would relax blood vessels and effectively regulate blood pressure.

[0099] [Example 5] Confirmation of NO production by CHP treatment in HMVEC-L cells 5-1. HMVEC-L cell culture Human lung microvascular endothelial cells (HMVEC-L, CC2527) were obtained from ATCC (American Type Culture Collection), and EBM-2 basal medium and EGM-2 MV microvascular endothelial cell growth medium SingleQuots supplement were purchased from Lonza.

[0100] HUVEC-L cells were cultured in EGM-2 medium supplemented with EGM-2 MV SingleQuots at 37°C and 5% CO2. The medium was changed every 2–3 days, and subculture was performed at 70–80% confluency.

[0101] 5-2. Confirmation of NO generation 5 x 10 in a black 96-well plate 4 After 24 hours of incubation, cells were plated in serum-free medium at concentrations of 0, 1, 10, 100, and 500 μM CHP and cultured for 2 hours at 37°C and 5% CO2. 5 μM DAF-FM DA (DAF-FM Diacetate; Invitrogen) was added and the cells were further cultured for 30 minutes. After washing once with PBS, the cells were observed under a fluorescence microscope at the FITC wavelength. Photographs were taken with identical exposure times, and the fluorescence intensity of each cell was measured using ImageJ. Statistical significance was determined using one-way ANOVA, and comparisons with the untreated control group were analyzed using Dunnett's test (****p<0.0001).

[0102] As a result of the experiment, it was confirmed that CHP treatment increased the DAF-FM DA fluorescence intensity in HMVEC-L cells, as shown in Figure 5. Since fluorescence intensity reflects the amount of NO in the cells, this indicates that CHP treatment significantly increased NO production in vascular endothelial cells.

[0103] The production of NO by vascular endothelial cells promotes the relaxation of vascular smooth muscle cells, which in turn lowers blood pressure. Therefore, the results of this example confirmed that CHP can improve blood pressure regulation by increasing NO production.

[0104] Statistical analysis of results Statistical significance was analyzed using Student's t-test and one-way ANOVA statistical methods, and outliers were analyzed using Grubb's Outlier calculator in GraphPad. Significant outliers were excluded from the pooled results.

Claims

1. A pharmaceutical composition for lowering blood pressure by relaxing blood vessels in a hypertensive patient, comprising cyclo-HisPro or a pharmaceutically acceptable salt thereof, the composition consisting of i) cyclo-HisPro or a pharmaceutically acceptable salt thereof, or ii) cyclo-HisPro or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition for lowering blood pressure according to claim 1, wherein the cyclo-hispro or a pharmaceutically acceptable salt thereof exerts a blood pressure lowering effect in vascular endothelial cells by one or more activities selected from the group consisting of the following a) to e): a) decreased angiotensin II type I receptor gene or protein expression; b) increased angiotensin II type II receptor gene or protein expression; c) increased VE-cadherin gene or protein expression; d) increased eNOS gene or protein expression; and e) Increased production of NO.

3. A functional health food composition for lowering blood pressure by relaxing the blood vessels of hypertensive patients, comprising cyclo-hispro or a nutmologically acceptable salt thereof, said composition consisting of i) cyclo-hispro or a nutmologically acceptable salt thereof, or ii) cyclo-hispro or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

4. The health functional food composition for lowering blood pressure according to claim 3, wherein the cyclo-hispro or a food-based acceptable salt thereof exerts a blood pressure lowering effect in vascular endothelial cells by one or more activities selected from the group consisting of the following a) to e): a) decreased angiotensin II type I receptor gene or protein expression; b) increased angiotensin II type II receptor gene or protein expression; c) increased VE-cadherin gene or protein expression; d) increased eNOS gene or protein expression; and e) Increased production of NO.

5. Use of cyclo-hispro or a pharmaceutically acceptable salt thereof, which has a vasorelaxant effect, in the manufacture of an antihypertensive agent.

6. In claim 5, the cyclo-hispro or a pharmaceutically acceptable salt thereof exerts a blood pressure lowering effect in vascular endothelial cells by one or more activities selected from the group consisting of the following a) to e): a) decreased angiotensin II type I receptor gene or protein expression; b) increased angiotensin II type II receptor gene or protein expression; c) increased VE-cadherin gene or protein expression; d) increased eNOS gene or protein expression; and e) Increased production of NO.

7. A pharmaceutical composition for preventing or treating hypertension by relaxing blood vessels, comprising cyclo-hispro or a pharmaceutically acceptable salt thereof.

8. A functional health food composition for preventing or ameliorating high blood pressure by relaxing blood vessels, comprising cyclo-hispro or a nutritively acceptable salt thereof.

9. Use of cyclo-hispro or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of hypertension by relaxing blood vessels.

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