Pharmaceutical composition for preventing or treating peripheral arterial disease, including aspartic acid

Abstract

An aspect of the present invention provides a pharmaceutical composition for preventing or treating peripheral arterial disease (PAD), including aspartic acid or a pharmaceutically acceptable salt thereof. The composition increases perfusion, enhances the density of CD31-positive capillary lumens, and overexpresses VEGF to promote angiogenesis, thereby exhibiting excellent effects in preventing or treating peripheral arterial disease. In addition, the composition may further comprise ornithine or a pharmaceutically acceptable salt thereof to exhibit greater prophylactic and therapeutic effects on peripheral arterial disease through better angiogenesis than single administration of aspartic acid or a pharmaceutically acceptable salt thereof.

Description

Pharmaceutical composition for preventing or treating peripheral arterial disease containing aspartic acid

[0001] It relates to a pharmaceutical composition for preventing or treating peripheral arterial disease containing aspartic acid.

[0002]

[0003] As life expectancy continues to increase worldwide, the number of older adults with risk factors for atherosclerosis, such as type 2 diabetes, obesity, and hypertension, is increasing. The incidence of peripheral arterial disease (PAD), a condition characterized by the progression of atherosclerotic plaque, is rapidly increasing, affecting 230 million adults worldwide. Critical limb ischemia (CLI), a progressive stage of PAD, carries a significant risk of amputation, stroke, myocardial infarction, and death, and diminishes quality of life.

[0004] In real-world clinical settings, physicians struggle to select the optimal treatment for patients with critical limb ischemia (CLI). Currently, approximately 60,000 major amputations are performed annually in the United States alone, but the use of endovascular procedures is decreasing worldwide. Endovascular revascularization is preferred over open surgery for CLI due to its lower morbidity and mortality. Endovascular intervention remains the standard treatment for patients with PAD, particularly high-risk patients who are not suitable for open revascularization. Furthermore, the need for reintervention in endovascular procedures is less cost-effective.

[0005] However, despite the increase in endovascular interventions, the number of major amputations has decreased, while the number of minor amputations has increased, resulting in a relatively stable overall number of amputations. Surgical or interventional revascularization is currently not suitable for a significant portion of the population. Despite improvements in limb salvage rates and mortality rates due to advances in medical therapy, including antiplatelet agents and statins, and improved management of comorbidities, the overall survival rate for patients with CLI remains lower than that of patients with serious conditions such as heart failure and certain cancers (average 3.5 years).

[0006] Given the limitations of current CLI therapies, therapeutic angiogenesis has emerged as a promising option for overcoming ischemia, but it remains a clinically challenging task. Furthermore, various genetic (e.g., hepatocyte growth factor gene) and cellular (e.g., bone marrow-derived mononuclear cells, mesenchymal stromal cells, human pluripotent stem cell-derived endothelial cells) therapies are being developed for the treatment of PAD. However, their application to humans remains challenging due to the difficulty.

[0007] Meanwhile, nitric oxide (NO) is a well-known regulator of endothelial function and plays a crucial role in endothelial vasorelaxation. NO also upregulates Vascular Endothelial Growth Factor (VEGF), a key stimulator of angiogenesis. Endothelial nitric oxide synthase (eNOS) promotes collateral arterial adaptation and blood flow recovery in a rat hind limb ischemia (HLI) model. However, clinically applicable therapies for the complex regulation of eNOS expression and activity, which involves numerous interrelated mechanisms, are lacking.

[0008] Meanwhile, L-ornithine-L-aspartate (LOLA), which exists as a crystalline salt, is currently used in the clinical management of hepatic encephalopathy. That is, the pharmacokinetic and pharmacological properties of LOLA are well established in the treatment of hepatic encephalopathy, and it is already commercially available.

[0009] The present inventors directly confirmed that LOLA induced increased perfusion, increased CD31-positive capillary luminal density, and overexpression of VEGF in ischemic limbs, and thus propose LOLA as a therapeutic agent for PAD, particularly CLI patients.

[0010]

[0011] One aspect is to provide a pharmaceutical composition for preventing or treating peripheral arterial disease (PAD), comprising aspartic acid or a pharmaceutically acceptable salt thereof.

[0012] Another aspect is to provide a health functional food for preventing or improving peripheral arterial disease (PAD) containing aspartic acid or a food-based acceptable salt thereof.

[0013] Another aspect provides a method for preventing or treating peripheral arterial disease (PAD), comprising administering aspartic acid or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0014] Another aspect provides the use of aspartic acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of peripheral arterial disease (PAD).

[0015]

[0016] One aspect provides a pharmaceutical composition for preventing or treating peripheral arterial disease (PAD), comprising aspartic acid or a pharmaceutically acceptable salt thereof.

[0017] According to one aspect, the aspartic acid may be L-aspartic acid, D-aspartic acid, or a mixture thereof, specifically, it may be L-aspartic acid, and more specifically, it may be a compound represented by the following chemical formula 1:

[0018] [Chemical Formula 1]

[0019] .

[0020] In one aspect, the aspartic acid or a pharmaceutically acceptable salt thereof may be derived from natural sources or synthesized using known organic synthesis methods. Furthermore, the aspartic acid or a pharmaceutically acceptable salt thereof may be a non-protein compound, a peptide, an extract of plant-derived tissue or cells, or a product obtained by culturing microorganisms (e.g., bacteria or fungi, and particularly yeast).

[0021] The above aspartic acid or a pharmaceutically acceptable salt thereof increases perfusion, increases the density of CD31-positive capillary lumen, and promotes angiogenesis by overexpressing VEGF, thereby exhibiting an excellent effect in preventing or treating peripheral arterial disease.

[0022] More specifically, the aspartic acid or a pharmaceutically acceptable salt thereof promotes the conversion of citrulline or a pharmaceutically acceptable salt thereof into arginine or a pharmaceutically acceptable salt thereof within the administered subject. The converted arginine produces nitric oxide (NO) together with endothelial nitric oxide synthase (eNOS), and the nitric oxide upregulates the expression of VEGF, thereby exhibiting an excellent angiogenic effect and an excellent preventive or therapeutic effect on peripheral arterial disease.

[0023] In one aspect, the composition may further comprise ornithine or a pharmaceutically acceptable salt thereof.

[0024] According to one aspect, the ornithine may be L-ornithine, D-ornithine or a mixture thereof, specifically, it may be L-ornithine, and more specifically, it may be a compound represented by the following chemical formula 2:

[0025] [Chemical Formula 2]

[0026] .

[0027] In one aspect, the ornithine or a pharmaceutically acceptable salt thereof may be derived from natural sources or may be synthesized using known organic synthesis methods. Furthermore, the ornithine or a pharmaceutically acceptable salt thereof may be a non-protein compound, a peptide, an extract of plant-derived tissue or cells, or a product obtained by culturing microorganisms (e.g., bacteria or fungi, and particularly yeast).

[0028] When the above composition further comprises ornithine or a pharmaceutically acceptable salt thereof, angiogenesis is further promoted, thereby exhibiting a better effect in preventing or treating peripheral arterial disease.

[0029] More specifically, the ornithine or a pharmaceutically acceptable salt thereof is converted into citrulline or a pharmaceutically acceptable salt thereof within the mitochondria. As a result, the aspartic acid or a pharmaceutically acceptable salt thereof contained in the composition produces more arginine, ultimately demonstrating a superior angiogenic effect and an excellent preventive or therapeutic effect against peripheral arterial disease.

[0030] The term "salt" means a salt prepared using a specific compound and a relatively non-toxic acid or base, depending on the nature of the compound.

[0031] Additionally, the “salt” in the present specification may be a “pharmaceutically acceptable salt.”

[0032] The term "pharmaceutically acceptable" means that a compound exhibits the property of being non-toxic to cells or humans when exposed to it under certain conditions.

[0033] The term "pharmaceutically acceptable salt" refers to a salt prepared using a compound according to one aspect and a relatively non-toxic acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of a base, either in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include salts of sodium, potassium, calcium, ammonium, organic amines, or magnesium, or similar salts. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of an acid, either in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate ion, phosphoric acid, monohydrogen phosphate ion, dihydrogen phosphate ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphorous acid, and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and further include salts of amino acids (e.g., arginine) and salts of organic acids such as glucuronic acid.

[0034] The above pharmaceutically acceptable salts can be synthesized by conventional chemical methods from parent compounds containing acidic or basic moieties. Typically, these salts are prepared by reacting the free acid or base form of these compounds with a stoichiometrically appropriate amount of base or acid in water, an organic solvent, or a mixture of the two. Non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0035] Additionally, in one aspect, the composition may increase at least one selected from the group consisting of perfusion, CD31-positive capillary lumen density, and nitric oxide, specifically, may increase at least two selected from the group consisting of perfusion, CD31-positive capillary lumen density, and nitric oxide, and more specifically, may increase all of perfusion, CD31-positive capillary lumen density, and nitric oxide.

[0036] In addition, in one aspect, the composition may be one that promotes the expression of one or more proteins selected from the group consisting of VEGF (Vascular Endothelial Growth Factor), VEGFR (Vascular Endothelial Growth Factor Receptor), FGF (Fibroblast Growth Factor), FGFR (Fibroblast Growth Factor Receptor), CD31 and endothelial nitric oxide synthase (eNOS), and specifically, one or more proteins selected from the group consisting of VEGFR (Vascular Endothelial Growth Factor Receptor), FGF (Fibroblast Growth Factor), FGFR (Fibroblast Growth Factor Receptor) and endothelial nitric oxide synthase (eNOS), VEGF (Vascular Endothelial Growth Factor) and CD31, and more specifically, VEGF (Vascular Endothelial Growth Factor), VEGFR (Vascular It may enhance the expression of all of the endothelial growth factor receptors (EGFR), fibroblast growth factor receptor (FGF), fibroblast growth factor receptor (FGFR), CD31, and endothelial nitric oxide synthase (eNOS).

[0037] In addition, in one aspect, the composition may promote the expression of at least one selected from the group consisting of a gene encoding VEGF (Vascular Endothelial Growth Factor), a gene encoding VEGFR (Vascular Endothelial Growth Factor Receptor), a gene encoding FGF (Fibroblast Growth Factor), a gene encoding FGFR (Fibroblast Growth Factor Receptor), a gene encoding CD31, and a gene encoding endothelial nitric oxide synthase (eNOS), and specifically, at least one selected from the group consisting of a gene encoding VEGFR (Vascular Endothelial Growth Factor Receptor), a gene encoding FGF (Fibroblast Growth Factor), a gene encoding FGFR (Fibroblast Growth Factor Receptor), and a gene encoding endothelial nitric oxide synthase (eNOS), VEGF (Vascular Endothelial Growth Factor) It may be to promote the expression of a gene encoding a growth factor (Growth Factor) and a gene encoding CD31, and more specifically, a gene encoding a vascular endothelial growth factor (VEGF), a gene encoding a vascular endothelial growth factor receptor (VEGFR), a gene encoding a fibroblast growth factor (FGF), a gene encoding a fibroblast growth factor receptor (FGFR),It may be by enhancing the expression of both the gene encoding CD31 and the gene encoding endothelial nitric oxide synthase (eNOS).

[0038] In addition, in one aspect, the peripheral arterial disease may be one or more diseases selected from the group consisting of atherosclerosis, diabetic foot disease, limb ischemia, ischemic cerebrovascular disease, ischemic cardiovascular disease, thromboangiitis obliterans (Berger's disease), and polyarteritis nodosa.

[0039] The above limb ischemia may be critical limb ischemia (CLI).

[0040] In addition, according to one aspect, the ischemic cerebrovascular disease may be one or more diseases selected from the group consisting of stroke, cerebral embolism, cerebral thrombosis, transient ischemic attack (TIA), cerebral hemorrhage, and cerebral infarction.

[0041] Additionally, in one aspect, the ischemic cardiovascular disease may be one or more diseases selected from the group consisting of myocardial infarction and angina pectoris.

[0042] The above term “prevention” may mean any act of inhibiting or delaying peripheral arterial disease in an individual by administering a pharmaceutical composition according to one aspect.

[0043] The above term “treatment” may mean any action that improves or beneficially changes the symptoms of peripheral arterial disease in an individual by administering a pharmaceutical composition according to one aspect.

[0044] The term "administration" refers to introducing a given substance into a subject in an appropriate manner, and "subject" refers to any living organism, including rats, mice, and livestock, including humans, that may have peripheral arterial disease. A specific example may be a mammal, including humans.

[0045] Additionally, the pharmaceutical composition may be provided as a pharmaceutical composition containing only the active ingredient, or including one or more pharmaceutically acceptable carriers, excipients or diluents.

[0046] Specifically, the carrier may be, for example, a colloidal suspension, a powder, a saline solution, a lipid, a liposome, microspheres, or nano-spheres. These may be complexed or associated with a carrier vehicle and may be transported in vivo using carrier systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.

[0047] When the above pharmaceutical composition is formulated, it can be prepared using diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. When manufacturing in the form of eye drops, known diluents or excipients may be used.

[0048] In one aspect, the pharmaceutical composition may further comprise a peripheral arterial disease treatment agent in addition to aspartic acid or a pharmaceutically acceptable salt thereof and / or ornithine or a pharmaceutically acceptable salt thereof.

[0049] The above pharmaceutical composition may be provided in combination with another peripheral artery disease treatment agent, and the other peripheral artery disease treatment agent may be a conventionally known peripheral artery disease treatment agent or a newly developed peripheral artery disease treatment agent.

[0050] When the above pharmaceutical composition further includes another peripheral arterial disease treatment agent, it is important to mix the agent in an amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

[0051] Furthermore, in one aspect, the pharmaceutical composition may be administered alone or in combination with another peripheral arterial disease treatment. Specifically, the pharmaceutical composition may be administered in combination with a known composition having a preventive or therapeutic effect on peripheral arterial disease or another peripheral arterial disease treatment, and may be administered simultaneously, separately, or sequentially, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves maximum effect with the minimum amount without adverse effects, and this can be readily determined by those skilled in the art.

[0052] The above pharmaceutical composition can be administered orally or parenterally, and when administered parenterally, the method of injection can be selected from external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, rectal injection, or intrathoracic injection.

[0053] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. Specifically, the pharmaceutical composition can be administered at 0.001 to 4,700 mg / kg / day, and more specifically, at 0.1 to 4,000 mg / kg / day. The administration may be administered once a day or in several divided doses. For example, it may be administered every other day or once a week.

[0054]

[0055] Another aspect provides a health functional food for preventing or improving peripheral arterial disease (PAD) containing aspartic acid or a food-wise acceptable salt thereof.

[0056] The above “aspartic acid”, “salt”, “peripheral arterial disease”, “prevention”, etc. may be within the aforementioned range.

[0057] The above aspartic acid or a food-based acceptable salt thereof increases perfusion, increases the density of CD31-positive capillary lumen, and promotes angiogenesis by overexpressing VEGF, thereby exhibiting an excellent effect in preventing or improving peripheral arterial disease.

[0058] More specifically, the aspartic acid or a food-acceptable salt thereof promotes the conversion of citrulline or a food-acceptable salt thereof into arginine or a food-acceptable salt thereof within the administered subject. The converted arginine produces nitric oxide (NO) together with endothelial nitric oxide synthase (eNOS), and the nitric oxide upregulates the expression of VEGF, thereby exhibiting an excellent angiogenic effect and an excellent preventive or ameliorating effect on peripheral arterial disease.

[0059] In one aspect, the health functional food may further contain ornithine or a food-wise acceptable salt thereof.

[0060] When the above health functional food further contains ornithine or a food-related acceptable salt thereof, it further promotes angiogenesis, thereby exhibiting a better effect in preventing or improving peripheral arterial disease.

[0061] More specifically, the above-mentioned ornithine or a food-acceptable salt thereof is converted into citrulline or a food-acceptable salt thereof within the mitochondria. As a result, the aspartic acid or a food-acceptable salt thereof contained in the health functional food produces more arginine, ultimately demonstrating a superior angiogenic effect and an excellent preventive or ameliorating effect on peripheral arterial disease.

[0062] The term "improvement" can refer to any action that at least reduces a parameter associated with the condition being treated, such as the severity of symptoms. In this case, the health functional food can be used to prevent or improve peripheral arterial disease, either before or after the onset of the disease, or simultaneously with or separately from a treatment medication.

[0063] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0064] The above health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further comprising one or more of a carrier, diluent, excipient, and additive. Foods to which compounds according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0065] Specific examples of the carrier, excipient, diluent and additive may include at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

[0066] In addition to containing the above-mentioned effective ingredient, the above-mentioned health functional food may contain other ingredients as essential ingredients without special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0067] In addition to the above, health functional foods according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0068] In one aspect, the health functional food may further include a health functional food for preventing or improving peripheral arterial disease in addition to aspartic acid or a food-scientifically acceptable salt thereof and / or ornithine or a food-scientifically acceptable salt thereof.

[0069] The above health functional food may be provided in combination with a health functional food for preventing or improving peripheral arterial disease known in the past or a health functional food for preventing or improving peripheral arterial disease that is newly developed.

[0070] When the above health functional food further includes a health functional food for preventing or improving peripheral arterial disease, it is important to mix the amount that can achieve the maximum effect with the minimum amount without causing side effects, and this can be easily determined by a person skilled in the art.

[0071] In addition, in one aspect, the health functional food may be consumed alone or in combination with a health functional food for preventing or improving peripheral arterial disease.

[0072] The above health functional food can be taken in conjunction with known compositions or newly developed health functional foods for the prevention or improvement of peripheral arterial disease. These can be taken simultaneously, separately, or sequentially, and can be taken singly or in multiple doses. It is important to take all of the above factors into consideration and consume the amount that achieves maximum effect with the minimum amount possible without causing side effects, which can be readily determined by those skilled in the art.

[0073]

[0074] Another aspect provides a method for preventing or treating peripheral arterial disease (PAD), comprising administering aspartic acid or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0075] The above “aspartic acid”, “pharmaceutically acceptable salt”, “substance”, “administration”, “peripheral arterial disease”, “prevention”, “treatment”, etc. may be within the aforementioned scope.

[0076] In one aspect, the method may further comprise the step of administering ornithine or a pharmaceutically acceptable salt thereof to the subject.

[0077] Additionally, in one aspect, the aspartic acid or a pharmaceutically acceptable salt thereof may be administered simultaneously with ornithine or a pharmaceutically acceptable salt thereof.

[0078]

[0079] Another aspect provides the use of aspartic acid or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of peripheral arterial disease (PAD).

[0080] The above “aspartic acid”, “pharmaceutically acceptable salt”, “substance”, “administration”, “peripheral arterial disease”, “prevention”, “treatment”, etc. may be within the aforementioned scope.

[0081] In one aspect, the aspartic acid or a pharmaceutically acceptable salt thereof may include ornithine or a pharmaceutically acceptable salt thereof.

[0082]

[0083] According to one aspect, a pharmaceutical composition comprises aspartic acid or a pharmaceutically acceptable salt thereof, thereby increasing perfusion, increasing the density of CD31-positive capillary lumen, and promoting angiogenesis by overexpressing VEGF, thereby exhibiting an excellent effect in preventing or treating peripheral arterial disease. In addition, the composition further comprises ornithine or a pharmaceutically acceptable salt thereof, thereby exhibiting an excellent effect in preventing or treating peripheral arterial disease through superior angiogenesis compared to administration of aspartic acid or a pharmaceutically acceptable salt thereof alone.

[0084]

[0085] Figures 1 to 4 illustrate changes in perfusion after single ligation of the proximal femoral artery in the HLI model. Specifically, 1) Figure 1 illustrates the surgical procedure for unilateral femoral artery ligation distal to the deep branch of the right leg of a rat. 2) Figure 2 illustrates laser Doppler perfusion imaging, with ROIs drawn around the foot and ankle to calculate the perfusion ratio of the ischemic limb after surgery and before surgery. 3) Figure 3 illustrates a point on a linear plot of the experiment representing the perfusion recovery rate (n = 5), with error bars representing the standard error of the mean. 4) Figure 4 illustrates a schematic protocol. More specifically, the protocol is as follows: Perfusion imaging was performed before surgery. Substances corresponding to each group were injected intraperitoneally five times over two weeks after surgery. Perfusion imaging was scanned on POD 7 and 14. The gastrocnemius muscles of the ischemic limbs were sampled for immunohistochemical analysis and Western blotting immediately after final perfusion.

[0086] Figure 5 shows the perfusion recovery of the three groups on POD 7 and 14. Specifically, the bar graph of the experiment represents the perfusion recovery rate, and the error bars represent the standard error of the mean. * indicates a significant difference at p < 0.05. The p value was calculated using a two-sample t-test. Group 1 (control group; n = 8); Group 2 (sorbitol; n = 8); Group 3 (LOLA; n = 8).

[0087] Figures 6 and 7 are diagrams showing angiogenesis in three groups after LOLA administration. Specifically, 1) Figure 6 shows the expression of CD31 protein detected by immunohistochemistry in the gastrocnemius muscle of HLI mice. The scale bar represents 100 μm. 2) Figure 7 shows the density of CD31-positive cells. The error bars represent the standard error of the mean. *** indicates a significant difference at p < 0.001.

[0088] Figures 8 to 10 are diagrams showing angiogenesis in three groups after LOLA administration. Specifically, 1) Figure 8 shows the expression of VEGF protein detected by immunohistochemistry in the gastrocnemius muscle of HLI mice. The scale bar represents 100 μm. 2) Figure 9 shows Western blotting of VEGF. The relative expression of the protein was normalized to the endogenous control GAPDH. 3) Figure 10 is a diagram showing the expression level of VEGF. * and *** indicate significant differences at p < 0.05 and p < 0.001, respectively.

[0089]

[0090] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0091]

[0092] Example

[0093] 1. Experimental methods and materials

[0094] (1) Animals

[0095] This experiment was approved by the institutional animal care and use committee of the inventors' institution in accordance with the guidelines of the National Institutes of Health (IACUC-230034). The experiment was conducted in compliance with ARRIVE guidelines. Male Sprague Dawley rats (Seongnam, South Korea), weighing 280–320 g (8 weeks old), were used. The rats were housed in a controlled environment (room temperature 20–24°C, humidity 40–60%) with ad libitum access to standard food and water throughout the experiment.

[0096]

[0097] (2) Hind Limb Ischemia

[0098] Anesthesia was induced using 5% isoflurane dissolved in O2. After anesthesia, analgesia, sedation, and muscle relaxation were achieved by intramuscular injection of tiletamine and zolazepam (Zoletil, Virbac, Carros, France) mixed with xylazine (Rompun, Elanco, Indianapolis, IN, US) (1:1). Each concentration was 0.7 mg / kg. Unilateral femoral artery ligation was then performed distal to the origin of the deep branch of the right leg of the rats. An oblique incision was made along the inguinal region using Metzenbaum scissors. The femoral nerve, artery, and vein were separated, and the femoral artery was tied around the inguinal region with a single ligature (4-0 Vicryl). The skin was sutured with 4-0 nylon.

[0099]

[0100] (3) Protocol

[0101] Perfusion imaging was performed before surgery. Immediately after surgery, postoperative perfusion images were taken (postoperative day 0). The rats were then randomly divided into three groups, with eight rats per group: Group 1 (control group; saline injection), Group 2 (sorbitol group; sorbitol injection), and Group 3 (LOLA treatment group). Group 1 received saline (1.7 ml / kg), Group 2 distilled water, sorbitol, and saline (20:2:30, 1.7 ml / kg), and Group 3 received intraperitoneal injections of LOLA (333 mg / kg) (Hepa-Merz infusion, Hanwha Pharma, Chuncheon, Republic of Korea) five times at 3, 5, 7, and 10 ALC 12 POD. Because Herpa-Merz infusion consists of LOLA, sorbitol, and distilled water, group 2 was administered the same volume of sorbitol and distilled water along with saline instead of LOLA. To create a normal control group, group 2 was also assigned the same volume of saline. Perfusion imaging was performed on POD 7 and 14. Immunohistochemical analysis and Western blotting of the gastrocnemius muscle of the ischemic leg were performed after the final perfusion imaging.

[0102]

[0103] (4) Perfusion Imaging

[0104] Rats were imaged for approximately 3 minutes using a laser Doppler imaging device (Moor LDI, Axminster, UK). Images were analyzed using Moor LDI Imaging Review. Regions of interest (ROIs) were drawn around the paw and ankle, and the perfusion ratio was calculated comparing the ischemic limb postoperatively to the ischemic limb preoperatively.

[0105]

[0106] (5) Immunohistochemical analysis

[0107] The mice were euthanized and fixed by perfusion with 4% paraformaldehyde 14 days after surgery. Muscles were removed, cut into blocks, embedded in paraffin, sectioned at 5 μm using a microtome, and mounted on glass slides. The sections were blocked with Proteinase K (S3020; Agilent DAKO, Santa Clara, CA, USA) and then incubated with antibodies to VEGF (1:100; MA1-16629; Thermo Scientific, Waltham, MA, USA) and CD31 (1:100; MA1-80069; Thermo Scientific, Waltham, MA, USA) for 10 minutes. The samples were then incubated with secondary antibodies (Envision + system-HRP Labeled Polymer - Antimouse, K4001; Agilent DAKO, Santa Clara, CA, USA) for 20 minutes at room temperature. Digital images of five microscopic fields obtained from four sections of each animal were saved and observed using a Zeiss Slide scanner (ZENblue3.1_ZENblack_3-OSR-lite, Carl Zeiss Microscopy, LLC, White Plains, NY, USA; magnification, x400). Capillary density was expressed in mm 2 It was expressed as the number of CD31-positive cells per case and was written after consensus review by two doctors for each case.

[0108]

[0109] (6) Western blotting

[0110] Frozen muscle samples were homogenized in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Walthan, MA, USA) with 1% Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Walthan, MA, USA) and centrifuged at 13,000 g for 15 min at 4°C. Protein concentrations were determined using the BCA assay kit (cat. no. 23250; Pierce; Thermo Fisher Scientific, Walthan, MA, USA) according to the manufacturer's instructions. Equal amounts of proteins were loaded onto 12% Tris-glycine SDS-polyacrylamide gels and separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA). After electrophoresis, the gels were transferred to polyvinylidene difluoride (PVDF) membranes (GenDEPOT, Baker, TX, USA). Membranes were blocked in Tween-20 buffer (Biosoulution, Suwon, Korea) containing 5% bovine serum albumin (BSA) (bioWORLD, Dublin, OH, USA) for 1 h at room temperature in Tris-buffered saline (TBS). The membranes were incubated overnight at 4°C with primary antibodies against VEGF (MA1-16629; Thermo Fisher Scientific, Walthan, MA, USA) and GAPDH diluted 1:1,000 in 5% BSA-TBS-T. The membranes were washed five times for 5 min in TBS-T buffer, incubated with goat anti-mouse IgG antibody (HRP, GeneTex, Irvine, CA, USA) diluted 1:10,000 in 5% BSA-TBS-T at room temperature, and washed five times for 5 min in TBS-T buffer.Bands were detected using Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Relative protein expression was normalized to the endogenous control GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

[0111]

[0112] (7) Statistical analysis

[0113] Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's post-hoc test. All p-values ​​< 0.05 were considered statistically significant. Data were analyzed using SPSS software (version 29.0, SPSS, Inc.).

[0114]

[0115] 2. Experimental Results

[0116] To induce hind limb ischemia (HLI) and stimulate angiogenesis, particularly in the gastrocnemius muscle, the femoral artery of five 8-week-old rats was unilaterally ligated (Fig. 1). Immediately after surgery, perfusion decreased by approximately 40% compared to normal preoperative levels. Perfusion then increased over 14 days, reaching a plateau with approximately 60% recovery between weeks 3 and 4 (Figs. 2 and 3). The period leading to this plateau was defined as the perfusion recovery period. To evaluate the effects of L-ornithine-L-aspartate (LOLA) on angiogenesis during the subacute phase of HLI, changes in perfusion and the microenvironment were examined using this model. LOLA was administered five times, starting on postoperative day (POD) three, over the next 2 weeks (Fig. 4).

[0117]

[0118] (1) Perfusion changes

[0119] On POD 7, the LOLA-administered group (group 3) showed a significantly higher perfusion recovery rate than the control group (group 1) and sorbitol-administered group (group 2) (group 1 vs group 2 vs group 3: 51.72 ± 13.81 vs 58.42 ± 15.95 vs 71.90 ± 18.66; p = 0.022) (Fig. 5). Although not statistically significant, the perfusion recovery rate on POD 14 in group 3 showed a tendency to increase compared to the other groups (group 1 vs group 2 vs group 3: 62.97 ± 11.02 vs 60.69 ± 16.11 vs 72.76 ± 18.66; p = 0.204) (Fig. 5).

[0120]

[0121] (2) Histological evaluation of capillary density

[0122] To further evaluate the differences in perfusion between groups, immunohistochemistry targeting the endothelial cell marker CD31 was performed to quantify the vascular lumen density in muscle samples on POD 14. The CD31-positive lumen density, indicative of capillary angiogenesis, was significantly higher in LOLA-treated rats compared with the other groups (Group 1 vs Group 2 vs Group 3: 157.25 ± 34.49 vs 162.50 ± 34.75 vs 352.25 ± 44.3; p < 0.001) (Figs. 6 and 7).

[0123]

[0124] (3) Expression of VEGF

[0125] The expression of VEGF in the ischemic limb determined by Western blotting on the post-operative day (POD) was significantly higher in LOLA-treated rats (group 3) than in the other groups (group 1 vs group 2 vs group 3: 0.76 ± 0.12 vs 0.63 ± 0.14 vs 1.00 ± 0.19; p < 0.001). In addition, immunohistochemical staining for VEGF was enhanced in group 3 compared with the other groups (Figs. 8 to 10).

[0126]

[0127] Reference example

[0128] The murine model experiments on LOLA administration described in the above examples demonstrated significantly increased perfusion in the ischemic limb, high CD31-positive capillary lumen density and significant overexpression of VEGF during the subacute phase of HLI.

[0129] L-Ornithine can enter the mitochondria and be converted to L-citrulline, whereas L-aspartate promotes the conversion of L-citrulline to L-arginine, the only physiologically important substrate for NO synthesis. Thus, LOLA increases L-arginine, which is important for nitric oxide (NO) synthesis, through the conversion of L-ornithine. NO plays an important role in angiogenesis by upregulating VEGF-induced angiogenesis and coordinating with FGF-induced angiogenesis. Meanwhile, endothelial nitric oxide synthase (NOS) is important for ischemic remodeling and collateral arterial adaptation, and induces flow recovery in a hind limb ischemia (HLI) rat model. Aspartate regulates the endothelial translational machinery for VEGFR2 and FGFR1 synthesis via mTORC1. Accordingly, LOLA administration increased VEGF expression and angiogenesis in our CLI model.

[0130] The experiments described in the examples above were designed to allow sufficient reperfusion on POD 7 to 14 via unilateral proximal artery ligation without gross incision or coagulation to be suitable for assessing angiogenesis in the calf muscle. Perfusion imaging was performed only in the ischemic hindlimb, without comparison with the non-ischemic contralateral limb, consistent with the need for quantitative perfusion imaging of the affected limb in patients with CLI before and after revascularization.

[0131] The pre- and postoperative perfusion volumes and recovery patterns observed in the experimental results of the above examples were similar to those observed in a conventional rat HLI model. Considering that PAD patients often do not receive immediate treatment, LOLA was administered starting on POD 3 and continued throughout the perfusion recovery period prior to POD 14, which represents the subacute phase of arterial occlusion. The LOLA dose was determined based on a previous toxicity report in Sprague-Dawley rats (50% lethal dose: 4.7 g / kg, with no systemic toxicity observed at 1 to 4 g / kg).

Claims

1. A pharmaceutical composition for preventing or treating peripheral arterial disease (PAD), comprising aspartic acid or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition according to claim 1, wherein the aspartic acid is a compound represented by the following chemical formula 1: [Chemical Formula 1] .

3. A pharmaceutical composition according to claim 1, further comprising ornithine or a pharmaceutically acceptable salt thereof.

4. In claim 3, the pharmaceutical composition wherein the ornithine is a compound represented by the following chemical formula 2: [Chemical formula 2] .

5. A pharmaceutical composition according to claim 1, wherein the composition promotes angiogenesis.

6. A pharmaceutical composition according to claim 1, wherein the composition increases at least one selected from the group consisting of perfusion, CD31-positive capillary lumen density, and nitric oxide.

7. A pharmaceutical composition according to claim 1, wherein the composition promotes the expression of at least one protein selected from the group consisting of VEGF (Vascular Endothelial Growth Factor), VEGFR (Vascular Endothelial Growth Factor Receptor), FGF (Fibroblast Growth Factor), FGFR (Fibroblast Growth Factor Receptor), CD31, and endothelial Nitric Oxide Synthase (eNOS).

8. A pharmaceutical composition according to claim 1, wherein the composition promotes the expression of at least one gene selected from the group consisting of a gene encoding VEGF (Vascular Endothelial Growth Factor), a gene encoding VEGFR (Vascular Endothelial Growth Factor Receptor), a gene encoding FGF (Fibroblast Growth Factor), a gene encoding FGFR (Fibroblast Growth Factor Receptor), a gene encoding CD31, and a gene encoding endothelial nitric oxide synthase (eNOS).

9. A pharmaceutical composition according to claim 1, wherein the aspartic acid or a pharmaceutically acceptable salt thereof promotes conversion of citrulline or a pharmaceutically acceptable salt thereof into arginine or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition according to claim 3, wherein the ornithine or a pharmaceutically acceptable salt thereof is converted into citrulline or a pharmaceutically acceptable salt thereof within the mitochondria.

11. A pharmaceutical composition according to claim 1, wherein the peripheral arterial disease is at least one disease selected from the group consisting of atherosclerosis, diabetic foot disease, limb ischemia, ischemic cerebrovascular disease, ischemic cardiovascular disease, thromboangiitis obliterans, and polyarteritis nodosa.

12. A pharmaceutical composition according to claim 11, wherein the limb ischemia is critical limb ischemia (CLI).

13. A pharmaceutical composition according to claim 11, wherein the ischemic cerebrovascular disease is at least one disease selected from the group consisting of stroke, cerebral embolism, cerebral thrombosis, transient ischemic attack (TIA), cerebral hemorrhage, and cerebral infarction.

14. A pharmaceutical composition according to claim 11, wherein the ischemic cardiovascular disease is at least one disease selected from the group consisting of myocardial infarction and angina pectoris.

15. Health functional food containing aspartic acid or a food-wise acceptable salt thereof for preventing or improving peripheral arterial disease (PAD).

16. A health functional food further comprising ornithine or a food scientifically acceptable salt thereof according to claim 15.

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