Pharmaceutical composition for preventing or treating cardiovascular aging diseases containing enavogliflozin

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cardiovascular aging diseases, comprising enavogliflozin as an active ingredient. The enavogliflozin of the present invention is highly effective in preventing and treating endothelial dysfunction caused by vascular endothelial cell senescence and related cardiovascular aging diseases.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cardiovascular aging diseases, which contains enavogliflozin as an active ingredient. [Background technology]

[0002] Senescence is a complex process that occurs during the course of human life, where physiological, psychological, environmental, and behavioral changes interact with one another. Vascular senescence is a typical example of human aging. In healthy blood vessels, vascular endothelium plays the most important role in regulating vascular homeostasis. Cardiovascular aging diseases such as atherosclerosis rapidly increase due to endothelial dysfunction caused by endothelial cell senescence.

[0003] Therefore, in order to derive innovative targets for the prevention and treatment of diseases caused by cardiovascular aging, a strategy is needed to suppress the occurrence of cardiovascular diseases by inhibiting the occurrence of vascular aging and the premature aging process caused by specific factors.

[0004] The recent increase in environmental pollutants, such as particulate matter and microplastics, has emerged as a major social issue, prompting research into various aspects of pollution, including its causes and countermeasures. According to a WHO report, 72% of particulate matter-related deaths are related to cardiovascular aging diseases, further exacerbating the condition of elderly people and those with existing cardiovascular aging diseases. Microplastics are formed when plastic fragments are released into the environment and gradually break down into fine (≦5 mm) or nano (≦100 nm) particles. They are reported to be ingested and accumulated by other marine and freshwater organisms, fish, and birds and mammals, including humans. Exposure to microplastics and their accumulation in the body may induce oxidative stress, potentially affecting growth, reproduction, movement, behavior, and aging (lifespan). However, the effects and mechanisms of long-term microplastic exposure are currently unknown.

[0005] Sodium-glucose cotransporters (SGLTs) are glucose transport proteins. SGLT-1 is expressed in the small intestine, liver, kidney, and heart, while SGLT-2 is primarily expressed in the kidney. SGLT-2 inhibitors are a new class of antidiabetic drugs that reduce glucose reabsorption in the proximal nephron and increase glucose excretion through an insulin-independent mechanism. SGLT-2 inhibitors have also been reported to reduce mortality and hospitalization rates associated with cardiovascular aging independently of blood glucose regulation. Nonclinical studies have observed increased expression in endothelial cells in response to high glucose levels and angiotensin II (angiotensin II, a key factor involved in vascular aging and endothelial dysfunction).

[0006] Therefore, there is growing interest in the use of SGLT-2 inhibitors for the prevention or treatment of cardiovascular aging diseases. Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors have made extensive efforts to develop a composition for preventing or treating cardiovascular aging diseases, and as a result have found that enavogliflozin has excellent effects in preventing and treating endothelial dysfunction caused by vascular endothelial cell senescence and related cardiovascular aging diseases, thereby completing the present invention.

[0008] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cardiovascular aging diseases, which contains enavogliflozin as an active ingredient.

[0009] Another object of the present invention is to provide a method for preventing or treating cardiovascular aging diseases, comprising administering to a subject in need thereof a pharmaceutical composition for preventing or treating cardiovascular aging diseases, the pharmaceutical composition comprising enavogliflozin as an active ingredient.

[0010] Another object of the present invention is to provide a use of enavogliflozin for the preparation of a pharmaceutical composition for the prevention or treatment of cardiovascular aging diseases. [Means for solving the problem]

[0011] The present invention relates to a pharmaceutical composition for preventing or treating cardiovascular aging diseases, comprising enavogliflozin as an active ingredient, and the prevention or treatment of cardiovascular aging diseases using the same, and it has been confirmed that the pharmaceutical composition according to the present invention is far superior in the prevention or treatment of cardiovascular aging diseases.

[0012] The present invention will now be described in more detail.

[0013] One aspect of the present invention is a pharmaceutical composition for preventing or treating cardiovascular aging diseases, comprising enavogliflozin as an active ingredient.

[0014] The term "enavogliflozin" as used herein refers to an SGLT-2 (Sodium-Glucose Cotransporter 2) inhibitor, which selectively inhibits SGLT-2, which is involved in the reabsorption of glucose from the kidney, thereby preventing glucose from being absorbed into the body and excreting it in the urine.

[0015] "Enavogliflozin" in the present invention may have a structure represented by the following chemical formula 1.

[0016] [ka]

[0017] The term "prevention" as used herein means any action of suppressing or delaying the onset of cardiovascular aging diseases by administering the pharmaceutical composition according to the present invention.

[0018] The term "treatment" as used herein means any action that improves or alters cardiovascular aging diseases by administering the pharmaceutical composition according to the present invention.

[0019] In the following examples, nanoplastics were used to induce vascular endothelial cell senescence, and it was confirmed that enavogliflozin can prevent or treat endothelial dysfunction caused by vascular endothelial cell senescence and related cardiovascular aging diseases.

[0020] Nanoplastics are tiny plastic particles (<100 nm) generated during the decomposition of plastic products. It has been reported that the accumulation of nanoplastics in the body induces oxidative stress, affecting growth, reproduction, movement, behavior, and aging (lifespan). In the following study, we confirmed that nanoplastics penetrated porcine coronary artery endothelial cells and increased the activity of β-galactosidase (SA-β-Gal), an immunohistochemical marker of cellular senescence, leading to decreased endothelial cell proliferation, increased the expression of cellular senescence markers p53 and p21, and increased the expression of NADPH oxidase, which is known to induce cellular senescence by producing peroxides in endothelial cells and impair endothelial cell function. These results indicate that the accumulation of nanoplastics induces vascular endothelial cell senescence and resulting endothelial dysfunction. It is well known that increased levels of p53 and p21 in endothelial cells lead to cellular senescence and impaired cardiac function. The accumulation of these senescent cells leads to a decline in vascular function, which has been reported to cause atherosclerosis (aortic aneurysms, coronary artery disease, peripheral artery disease, and carotid artery disease) (G. Katsuumi et al., Vascular Senescence in Cardiovascular and Metabolic Diseases Front Cardiovasc Med. 2018; 5: 18.). Cellular senescence impairs the essential role that the endothelium plays in maintaining vascular homeostasis, promoting endothelial dysfunction and the development of age-related vascular diseases.SA-β-Gal has been detected in aged retinal vessels, atherosclerotic plaques of the aorta and coronary arteries, and adipose tissue from obese subjects. Increased p53 expression has been reported in patients with congestive heart failure or hypertrophic cardiomyopathy (YE Han & SY Kim, Endothelial senescence in vascular diseases: current understanding and future opportunities in senotherapeutics. Experimental & Molecular Medicine volume 55, pages 1-12 (2023)). Strong SA-β-Gal staining was observed in atherosclerotic lesions of coronary arteries obtained by autopsy from patients with ischemic heart disease, and immunohistochemical analysis using an anti-factor VIII antibody confirmed that SA-β-Gal-stained cells were vascular endothelial cells. Endothelial cell senescence increased ICAM-1 expression and decreased eNOS activity, both of which are associated with atherosclerosis (T. Minamino et al., Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation. 2002 Apr 2;105(13):1541-4.). Furthermore, LDLR mice fed a high-fat diet for 2 to 12 weeks were used as a model of atherosclerosis. - / - (low-density lipoprotein receptor - / -SA-β-Gal staining of aortic endothelial cells from mice revealed increased SA-β-Gal activity and p53 expression. When comparing the outer curvature (undisturbed flow) and inner curvature (disturbed flow) of the aorta, senescent endothelial cells were found to accumulate in the inner curvature during the atherosclerosis process, demonstrating that endothelial cell senescence is induced through the p53-p21 signaling pathway. These results suggest that endothelial cell senescence may be involved in the initiation and progression of atherosclerosis (CM Warboys et al., Disturbed Flow Promotes Endothelial Senescence via a p53-Dependent Pathway. Arterioscler Thromb Vasc Biol. 2014;34:985-995). Senescent vascular cells have been reported to accumulate in human atherosclerotic tissue and exhibit various dysfunctional features, suggesting that cellular senescence may contribute to the development of atherosclerosis in humans. It has been shown that the interaction between monocytes and vascular endothelial cells becomes stronger with endothelial cell senescence and also promotes atherosclerosis (T. Minamino et al., Vascular Cell Senescence. Circulation Research. 2007;100:15-26).

[0021] In the present invention, the vascular endothelial cell aging experiment using nanoplastics was adopted as an experimental model to demonstrate that enavogliflozin is effective in preventing or treating endothelial dysfunction caused by aging of vascular endothelial cells, and the present invention is in no way limited to the prevention or treatment of cardiovascular aging diseases caused by nanoplastics.

[0022] In the present invention, cardiovascular aging diseases are associated with endothelial dysfunction caused by vascular endothelial cell aging, and may be, for example, one or more selected from the group consisting of hypertrophic obstructive cardiomyopathy, severe obstructive coronary artery disease, aortic valve stenosis, hemodynamically significant aortic or mitral valve stenosis, aortic stenosis, coronary artery disease, peripheral artery disease, and carotid artery disease, but are not limited thereto.

[0023] Another aspect of the present invention is a method for preventing or treating cardiovascular aging diseases, comprising administering a pharmaceutical composition for preventing or treating cardiovascular aging diseases, which comprises enavogliflozin as an active ingredient, to a subject in need thereof.

[0024] The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier in addition to the active ingredient, enavogliflozin of Chemical Formula 1 or a pharmaceutically acceptable salt thereof, and may be formulated together with the carrier.

[0025] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not stimulate living organisms and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated as liquid solutions include sterilized and biocompatible carriers such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, to which other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added, if necessary.

[0026] Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare the compound into injection forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0027] In the present invention, the pharmaceutical composition may have an oral dosage form such as a tablet, a capsule, etc. In one embodiment of the present invention, the pharmaceutical composition may have a purified dosage form.

[0028] In the present invention, the pharmaceutical composition may have a dosage form for parenteral administration. For example, but not limited to, a dosage form for parenteral administration containing the composition of the present invention as an active ingredient may be formulated into an injectable form such as a subcutaneous injection, an intravenous injection, or an intramuscular injection.

[0029] To prepare an injectable dosage form, the composition of the present invention is mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can be formulated into an ampule or vial for unit administration.

[0030] Alternatively, the compositions of the present invention may be formulated into various forms for parenteral administration, such as eye drops, microneedles, patches, depots, and the like.

[0031] The compositions of the present invention are administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease. The effective dose level is determined by factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. They can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. That is, the total effective amount of the compositions of the present invention can 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. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without causing side effects, which can be easily determined by one skilled in the art.

[0032] The optimal once-daily dosage of enavogliflozin determined during clinical trials is 0.1 mg to 0.5 mg.

[0033] When the pharmaceutical composition is formulated into a unit dosage form, the content of the active ingredient in the pharmaceutical composition may be 0.1 mg to 0.5 mg.

[0034] The pharmaceutical composition according to the present invention may be administered once to three times a day, for example, once a day, but is not limited thereto.

[0035] In the present invention, the dose of enavogliflozin that can be used for the prevention or treatment of cardiovascular aging diseases is not particularly limited and can be appropriately adjusted depending on the severity of the disease, body weight, age, sex, and presence or absence of other complications of the subject to be administered.

[0036] The method for administering the pharmaceutical composition for preventing or treating cardiovascular aging diseases uses a pharmaceutical composition for preventing or treating cardiovascular aging diseases, so overlapping content between the two will be omitted to avoid excessive description in the specification.

[0037] Enavgliflozin used as an active ingredient in the present invention can be synthesized according to published prior art. In the present invention, enavogliflozin may be in a crystalline or amorphous form. For example, enavogliflozin may be enavogliflozin crystalline form A, crystalline form B, crystalline form C, crystalline form D, crystalline form E, or amorphous enavogliflozin, which are reported to have the following X-ray diffraction spectra in Korean Patent Publication No. 2017-0142904 or Korean Patent Application No. 2022-0123673.

[0038] Crystalline form A: A crystalline form having an X-ray diffraction (XRD) spectrum including peaks at 2[θ] values ​​selected from 6.2°±0.2°, 7.2°±0.2°, 8.8°±0.2°, 17.6°±0.2°, 19.0°±0.2°, 22.5°±0.2°, and 25.1°±0.2°. Crystalline form B: A crystalline form having an X-ray diffraction (XRD) spectrum including peaks at 2[θ] values ​​selected from 7.0°±0.2°, 14.9°±0.2°, 17.7°±0.2°, 18.8°±0.2°, 20.6°±0.2°, 21.8°±0.2°, and 23.5°±0.2°. Crystalline form C: A crystalline form having an X-ray diffraction (XRD) spectrum including peaks at 2[θ] values ​​selected from 5.6°±0.2°, 7.3°±0.2°, 15.7°±0.2°, 17.2°±0.2°, 18.9°±0.2°, 21.2°±0.2°, and 21.9°±0.2°. Crystalline form D: A crystalline form having an X-ray diffraction (XRD) spectrum including peaks at 2[θ] values ​​selected from 5.5°±0.2°, 7.2°±0.2°, 15.3°±0.2°, 17.2°±0.2°, 17.6°±0.2°, 18.9°±0.2°, and 21.1°±0.2°. Crystalline form E: A crystalline form having an X-ray diffraction (XRD) spectrum including peaks at 2[θ] values ​​selected from 4.93°±0.2°, 6.12°±0.2°, 7.43°±0.2°, 8.89°±0.2°, 9.74°±0.2°, 14.79°±0.2°, 15.79°±0.2°, 16.11°±0.2°, 19.79°±0.2°, and 22.83°±0.2°. The crystalline forms A, B, C, D and E can each be identified by an X-ray diffraction spectrum having four or more, e.g., four, five, six, seven, eight or more, peaks from the 2[θ] values ​​listed above.

[0039] Without being limited thereto, the pharmaceutical composition containing enavogliflozin according to the present invention may have the configuration of the pharmaceutical composition of PCT / KR2022 / 014640.

[0040] For example, the pharmaceutical composition of the present invention may comprise a compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, an excipient, a disintegrant, and a binder, and the compound of Chemical Formula 1 may have an average particle size of 15 μm or less.

[0041] In an embodiment of the present invention, the average particle size of enavogliflozin may be 15 um or less, preferably 10 um or less.

[0042] When the average particle size of enavogliflozin exceeded 15 μm, the dissolution rate after 5 minutes was very low at less than 40% of the total enavogliflozin content, and the dissolution rate after 30 minutes was also less than 80%, indicating that the final dissolution rate was unsuitable.

[0043] If drug particle size refinement is required, it can be achieved using conventional mills capable of refining particles, such as a jet mill, hammer mill, ball mill, or fluid energy mill. Alternatively, drug particle size can be refined using size classification methods, such as sieving or air current classification. Methods for achieving the desired particle size are well known in the art. For example, see the following reference: Pharmaceutical Dosage Forms: Volume 2, 2nd Edition, Ed.: H. A. Lieberman, L. Lachman, and J. B. Schwartz (Chapter 3: Size Reduction).

[0044] In this specification, the particle size of a drug is expressed based on the particle size distribution, D(X) = Y (where X and Y are positive numbers). D(X) = Y means that when the particle size distribution of a drug obtained by measuring the particle size of a certain drug in a formulation is represented by a cumulative curve, the particle size at which the particle size accumulates from smallest to largest to reach X% (% is calculated based on number, volume, or weight) is Y. For example, D(10) represents the particle diameter at the 10% point when the particle sizes of the drug are accumulated from smallest to largest, D(50) represents the particle diameter at the 50% point when the particle sizes of the drug are accumulated from smallest to largest, and D(90) represents the particle diameter at the 90% point when the particle sizes of the drug are accumulated from smallest to largest.

[0045] Whether the particle size distribution D(X) represents the percentage of the total cumulative particles based on number, volume, or weight varies depending on the method used to measure the particle size distribution. Methods for measuring particle size distribution and the associated percentage types are well known in the art. For example, when particle size distribution is measured by the well-known laser diffraction method, the X value in D(X) represents the percentage calculated by volume average. Those skilled in the art are well aware that particle size distribution measurements obtained from a particular method can be empirically correlated with those obtained from other techniques through routine experimentation. For example, laser diffraction provides a volume-average particle size depending on the volume of the particles, which corresponds to the weight-average particle size when the density is constant.

[0046] In the present invention, the particle size distribution of drug particles can be measured using a commercially available laser diffraction / scattering method based on Mie scattering. For example, measurements can be made using commercially available devices such as the Mastersizer laser diffraction device from Malvern Instruments. This device irradiates particles with a helium-neon laser beam and a blue light-emitting diode, causing scattering and resulting in a light scattering pattern appearing on the detector. The particle size distribution can be determined by analyzing this light scattering pattern according to Mie scattering. Measurements can be made using either dry or wet methods.

[0047] In the examples of the present invention, the particle size of the drug was measured as the volume average particle size by laser diffraction method.

[0048] In an embodiment of the present invention, the compound of Chemical Formula 1 may be contained in an amount of less than 1 part by weight relative to 100 parts by weight of the total pharmaceutical composition.

[0049] The appropriate once-daily dose of enavogliflozin determined through clinical trials is 0.1 mg to 0.5 mg, and when the pharmaceutical composition is formulated into a unit dosage form, the content of the active ingredient in the pharmaceutical composition may be 0.1 mg to 0.5 mg.

[0050] The pharmaceutical composition according to the present invention contains the compound of Formula 1 as an active ingredient as well as pharmaceutically acceptable additives.

[0051] The pharmaceutical composition of the present invention contains excipients, disintegrants, binders, and the like as additives.

[0052] Examples of excipients include lactose (including hydrates), dextrin, mannitol, sorbitol, starch, microcrystalline cellulose (e.g., Celphere®), silicified microcrystalline cellulose (e.g., Prosolv®), calcium phosphate hydrate, calcium phosphate anhydrous, calcium carbonate, sugars, or mixtures thereof. In an embodiment of the present invention, the preferred excipient is microcrystalline cellulose.

[0053] Examples of disintegrants include crospovidone, croscarmellose sodium, sodium starch glycolate, and low-substituted hydroxypropyl cellulose. In an embodiment of the present invention, the preferred excipient is croscarmellose sodium.

[0054] Examples of binders include polyvinylpyrrolidone, povidone, gelatin, starch, sucrose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl alkylcellulose (e.g., hydroxypropylmethylcellulose), and mixtures thereof. In an embodiment of the present invention, the preferred binder is hydroxypropylcellulose.

[0055] Examples of other additives include lubricants and colorants.

[0056] The lubricants include stearic acid, stearates (e.g., magnesium stearate), hard anhydrous silicic acid, talc, corn starch, canauba wax, magnesium silicate, synthetic aluminum silicate, hydrogenated oil, white lead, titanium oxide, microcrystalline cellulose, macrogol 4000 and 6000, isopropyl myristate, calcium hydrogen phosphate, and mixtures thereof.

[0057] In an embodiment of the present invention, the excipient may be included in an amount of 80 to 95 parts by weight per 100 parts by weight of the total pharmaceutical composition.

[0058] In a specific example of the present invention, the disintegrant may be included in an amount of 2 to 8 parts by weight per 100 parts by weight of the total pharmaceutical composition. If the amount of disintegrant is less than 2 parts by weight per 100 parts by weight of the total pharmaceutical composition, the initial disintegrating power may be low, resulting in a delayed dissolution rate, which may affect the Cmax in the body. Also, if the amount of disintegrant is more than 8 parts by weight per 100 parts by weight of the total pharmaceutical composition, the amount of disintegrant in the post-mixing section may be increased, which may reduce the overall fluidity of the granules.

[0059] In a specific embodiment of the present invention, the binder may be present in an amount of 3 to 10 parts by weight per 100 parts by weight of the total pharmaceutical composition. If the binder is present in an amount less than 3 parts by weight per 100 parts by weight of the total pharmaceutical composition, it may be difficult to form and maintain a suitable dry granule, which may affect the maintenance of homogeneous dispersion of the active ingredient and the flowability of the granules due to the generation of fine powder. Furthermore, if the binder is present in an amount more than 10 parts by weight per 100 parts by weight of the total pharmaceutical composition, granules with strong binding strength may be formed, which may affect the solubility of the initially disintegrated granule particles during dissolution, and may also affect Cmax in the body.

[0060] The pharmaceutical composition according to the present invention may be an immediate release formulation.

[0061] In one embodiment of the present invention, the dissolution rate of the pharmaceutical composition after 5 minutes may be 50% or more, preferably 60% or more of the total content of the active ingredient.

[0062] In one embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 80% or more, preferably 85% or more of the total content of the active ingredient after 15 minutes.

[0063] In one embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 85% or more, preferably 90% or more of the total content of the active ingredient after 30 minutes.

[0064] The dissolution rate of the active ingredient in a pharmaceutical composition affects the maximum blood concentration (Cmax) and area under the blood concentration-time curve (AUC) upon drug administration. Conversely, adjusting the dissolution rate of the pharmaceutical composition is important to achieve an appropriate Cmax and AUC. Since enavogliflozin has a Tmax of 1-2 hours, the drug absorption rate in the stomach is considered important. The dissolution rate was measured under conditions of dissolution medium 1.2 according to Method 2 (paddle method) of the Korean Pharmacopoeia for dissolution testing. Specific conditions may be found in the following experimental examples.

[0065] The present invention also provides a pharmaceutical composition comprising a granule obtained by mixing a pre-blended granule containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof and a post-blended portion.

[0066] In the course of research into formulating the compound of Chemical Formula 1, the present inventors have confirmed that preparing granules and then purifying them into a dosage form is advantageous in terms of uniformity of the drug content and the formulation.

[0067] In the pharmaceutical composition, the granules are prepared by mixing pre-mixed granules with a post-mixing portion.

[0068] The pre-blended granules may include the compound of Formula 1 or a pharmaceutically acceptable salt thereof, an excipient, a binder, and a lubricant.

[0069] The post-mixing section may also contain an excipient, a disintegrant, and a lubricant.

[0070] The explanations for excipients, binders, disintegrants, lubricants, etc. are the same as those described above, so they will be omitted to avoid duplication.

[0071] In a specific example of the present invention, the pre-blended granules and the post-blended portion may each contain an excipient, more specifically, the pre-blended granules and the post-blended portion may each contain microcrystalline cellulose as an excipient.

[0072] According to the following examples, it was found that the microcrystalline cellulose contained in the pre-blended granules and post-blended portion affects the drug content uniformity depending on its particle size and volume density.

[0073] In a specific example of the present invention, the particle size of the microcrystalline cellulose in the pre-blended granules may be 130 μm or less, preferably 60 μm to 130 μm. The volume density of the microcrystalline cellulose in the pre-blended granules may be 0.26 to 0.33 μm. When the particle size and volume density of the microcrystalline cellulose in the pre-blended granules satisfy the above conditions, a formulation with low content uniformity deviation (SD) can be obtained. When the particle size of the microcrystalline cellulose in the pre-blended granules is 130 μm or less, the content uniformity of the total blended granules, the content uniformity of the final granules, and the formulation are all at good levels. The Carr's index value, which indicates the physical properties of the final granules, is also good, and the fluidity of the formulation is also excellent. On the other hand, when the particle size of the microcrystalline cellulose in the pre-blended granules exceeds 130 μm, the content uniformity of the total blended granules and the content uniformity of the final granules both increase in deviation, which is unsuitable, and the formulation uniformity is also poor.

[0074] Meanwhile, the particle size of the microcrystalline cellulose in the post-mixing section may be 130 μm or more, preferably 130 μm to 250 μm. The volume density of the excipient in the post-mixing section may be 0.28 to 0.37. It has been confirmed that when the particle size of the microcrystalline cellulose in the post-mixing section is less than 130 μm, the Carr's index value, which indicates the physical properties of the final granules, is inappropriate, and the fluidity of the granules is weakened.

[0075] When comparing the microcrystalline cellulose in the pre-mixed granules with the microcrystalline cellulose in the post-mixed portion, it was found that, unlike the fact that a small particle size of the microcrystalline cellulose contained in the pre-mixed granules is preferable, the particle size of the microcrystalline cellulose in the post-mixed portion is preferably relatively larger than the particle size of the microcrystalline cellulose contained in the pre-mixed granules.

[0076] According to the following examples, it was found that not only the particle size of the microcrystalline cellulose in the pre-blended granules and the microcrystalline cellulose in the post-blended portion, but also the weight ratio of the excipient in the pre-blended granules and the excipient in the post-blended portion affect the drug content uniformity.

[0077] In a specific example of the present invention, the weight ratio of the excipient in the pre-blended granules to the excipient in the post-blended portion may be 4: 1 to 1: 1. As the proportion of microcrystalline cellulose in the post-blended portion increases, the fluidity of the granules improves, but the content deviation increases, so it is considered preferable to maintain the weight ratio within the appropriate range.

[0078] Meanwhile, in the pharmaceutical composition according to the present invention, the binder may be at least one selected from the group consisting of hydroxypropyl cellulose, povidone, copovidone, and hypromellose.

[0079] In one embodiment of the present invention, the binder may be hydroxypropyl cellulose, and the weight-average molecular weight may be less than 200,000. If hydroxypropyl cellulose with a weight-average molecular weight of 200,000 or more is used, the dissolution rates after 5 minutes and 30 minutes are both low, which is undesirable in terms of bioavailability.

[0080] On the other hand, with regard to the Carr's index, which is used as a measure of flowability in formulation, the Carr's index of the granules is preferably 21-25.

[0081] The granules in the pharmaceutical composition of the present invention may be, but are not limited to, dry granules. In another embodiment, the granules may be wet granules.

[0082] In the present invention, the pharmaceutical composition may have an oral dosage form such as a tablet, capsule, etc. In one embodiment of the present invention, the pharmaceutical composition may have a purified dosage form.

[0083] In a preferred embodiment, the pharmaceutical composition may contain the compound of Formula 1 in a dosage of 0.3 mg.

[0084] The pharmaceutical composition according to the present invention may be orally administered once a day, but is not limited thereto. [Effects of the Invention]

[0085] The present invention relates to a pharmaceutical composition for preventing or treating cardiovascular aging diseases, comprising enavogliflozin as an active ingredient. The enavogliflozin of the present invention exhibits excellent effects in preventing and treating endothelial dysfunction caused by vascular endothelial cell senescence and related cardiovascular aging diseases. [Brief explanation of the drawings]

[0086] [Figure 1] FIG. 1 is a diagram showing the spherical morphology of nanoplastic according to an embodiment of the present invention. [Figure 2] 1 is a graph showing zeta potential and hydrodynamic diameter according to one embodiment of the present invention. [Figure 3] 1 is a graph showing the results of cytotoxicity of nanoplastics according to an embodiment of the present invention. [Figure 4] FIG. 1 shows the penetration of fluorescently tagged nanoplastics into cells according to one embodiment of the present invention. [Figure 5] 1 is a diagram and graph showing β-galactosidase (SA-β-Gal) activity in coronary artery rings exposed to nanoplastics according to one embodiment of the present invention. [Figure 6] 1 is a graph showing the degree of improvement in cell proliferation according to an embodiment of the present invention. [Figure 7] 1 is a diagram and graph showing the expression of cellular senescence markers p53 and p21 according to one embodiment of the present invention. [Figure 8] 1 is a diagram and graph showing ROS production index in a group exposed to nanoplastics according to one embodiment of the present invention. [Figure 9] 1 is a diagram and graph showing the expression of Nox2 and p22phox, which are membrane catalytic subunits of NADPH oxidase, upon exposure to nanoplastics according to one embodiment of the present invention. [Figure 10] 1 is a graph showing the degree of vasorelaxation due to exposure to nanoplastics according to an embodiment of the present invention. [Figure 11] 1 is a diagram and graph showing the expression of eNOS protein in a nanoplastics-exposed group according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0087] Hereinafter, one or more specific examples will be described in more detail through examples, but these examples are intended to illustrate one or more specific examples and are not intended to limit the scope of the present invention.

[0088] Example 1. Physicochemical properties of nanoplastics The polystyrene nanoplastics used in the experiment had an average particle size of 25 nm and a concentration of 10 mg / mL (dissolved in 10% DMSO (dimethyl sulfoxide)). The shape and structure of the nanoplastics were measured using an ultra-high-resolution field emission scanning electron microscope (Regulus 8230; Hitachi High-tech, Japan), and the zeta potential distribution and hydrodynamic diameter were measured using a dynamic light scattering (DLS) Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK).

[0089] Example 2. Evaluation of cytotoxicity and intracellular penetration of nanoplastics 2-1. Cytotoxicity of nanoplastics After isolating porcine coronary arteries, porcine coronary artery endothelial cells were isolated by collagenase treatment for 20 minutes and then centrifuged. They were then cultured in DMEM (high-glucose; GenDEPOT) medium containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μg / mL). 1 × 10 cells were cultured in a 96-well plate. 4 Cells were dispensed at 1000 cells / well to confirm the cytotoxicity of nanoplastics. Nanoplastics were treated at various concentrations (0.1, 0.3, 1, 3, 10, 30, and 100 μg / mL) and then cultured in a 96-well plate for 24 hours. Because serum (FBS) inhibits nanoplastic absorption, cells were treated with nanoplastics in serum-free medium and then cultured for 24 hours. After treatment with MTT (0.1 mg / mL) for 4 hours, 200 μL of DMSO was added to dissolve the formazan crystals. Absorbance was then measured at 540 nm (Enspire Multilabel Reader; Perkin Elmer Ltd.), and the results are shown in Figures 1–3.

[0090] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and Tukey's test was used for post hoc tests of each type. The significance level was set at p<0.05.

[0091] As can be seen in Figures 1-3, the nanoplastics were spherical (Figure 1), and the zeta potential and hydrodynamic diameter were confirmed to be -40.93 ± 0.58 mV and 21.97 ± 0.53 nm, respectively (Figure 2). This indicates that the nanoplastics did not aggregate in solution. Furthermore, the cytotoxicity of nanoplastics was not observed up to treatments of 0.1-10 μg / mL. However, treatments of 30 μg / mL and 100 μg / mL showed cytotoxicity, with cell viabilities of 73.33% and 45.15%, respectively (Figure 3).

[0092] 2-2. Penetration of nanoplastics into cells The physicochemical properties of the nanoplastics were examined to confirm their penetration into cells. After isolating porcine coronary arteries, they were treated with collagenase for 20 minutes and then centrifuged to obtain porcine coronary artery endothelial cells. Next, they were cultured in DMEM (high-glucose; GenDEPOT) culture medium containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μg / mL). 1 × 10 cells were placed in a 2-well Lab-Tek Chamber Side (Thermo Scientific, USA). 5 Cells were plated at 100 cells / well and divided into a control group and a fluorescently tagged nanoplastic (Life Technologies Corporation, Eugene, Oregon) group. The fluorescently tagged nanoplastic was treated at 10 μg / mL. After 24 hours of incubation, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 15 minutes. Then, the cells were stained with DAPI (4',6-Diamidino-2-phenylindole, 10 μM). The intracellular penetration of the nanoplastic was analyzed using a ZEISS LSN980 confocal microscope, and the results are shown in Figure 4.

[0093] As can be seen in Figure 4, we confirmed that the fluorescently tagged nanoplastics penetrated into the cells.

[0094] Example 3. Evaluation of aging The activity of β-galactosidase (SA-β-Gal), cell proliferation and cellular senescence markers were measured.

[0095] 3-1. Evaluation of β-galactosidase activity Senescence-associated β-galactosidase (SA-β-Gal) activity is a characteristic of senescent cells and is used as an immunohistochemical marker for senescence.

[0096] Specifically, porcine coronary arteries were cut into 2-3 mm pieces to create coronary artery rings. The coronary artery rings were then divided into three groups: a control group, a group treated with 10 μg / mL nanoplastics, and a group treated with 10 μg / mL nanoplastics plus enavogliflozin at different concentrations (0.01, 0.1, and 1 μM). After 24 hours of culture, the rings were fixed with 4% formaldehyde and stained with X-gal staining reagent for 1 day. The coronary artery rings were then immersed in optimal cutting temperature (OCT) solution (Leica Biosystems) at -25°C, and 10 μm sections were prepared using a cryotome. The sections were then observed under a microscope. The results are shown in Figure 5. Furthermore, β-galactosidase activity in porcine coronary artery-derived endothelial cells was evaluated as follows. 5 × 10 4Coronary artery endothelial cells were dispensed at 100 cells / well and divided into a control group, a group treated with 10 μg / mL of nanoplastics, and a group treated with nanoplastics plus enavogliflozin at different concentrations (0.01, 0.1, and 1 μM). After 24 hours of incubation, the cells were fixed with 4% formaldehyde solution (1 mL / well) for 15 minutes, and then treated with X-gal staining reagent (1 mg / mL) for 16 hours. X-gal was hydrolyzed by β-galactosidase activity, turning blue. The changes in each group were observed under a microscope, and the results are shown graphically in Figure 5.

[0097] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Test results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and post hoc tests for each type were performed using Tukey's test. The significance level was set at p<0.05.

[0098] As can be seen in Figure 5, β-galactosidase (SA-β-Gal) activity increased in coronary artery rings exposed to nanoplastics. On the other hand, β-galactosidase (SA-β-Gal) activity significantly decreased in the groups treated with different concentrations of enavogliflozin (0.01, 0.1, and 1 μM).

[0099] 3-2. Evaluation of cell proliferation efficacy Cell cycle arrest is one of the main characteristics of cellular senescence. We investigated whether enavogliflozin could improve the reduction in endothelial cell proliferation, i.e., cellular senescence, caused by exposure to nanoplastics.

[0100] Specifically, porcine coronary arteries were isolated, treated with collagenase for 20 minutes, and then centrifuged to obtain porcine coronary artery endothelial cells. 1 × 10 cells were then plated in a 96-well plate. 4Cells were plated at 100 cells / well. Cell culture medium was DMEM containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μM) and cultured at 37°C in a 5% CO2 incubator. Experiments were conducted using a control group, nanoplastics at 10 μg / mL, and nanoplastics + enavogliflozin at different concentrations (0.01, 0.1, and 1 μM). Cell proliferation was measured using the CellTiter 96 Aqueos One Solution Cell Proliferation Assay (Promega Corporation, USA). After culturing the treated cells for 24 hours, MTS tetrazolium solution (20 μL / well) was added and incubated for 3 hours. Absorbance was measured at 490 nm using an Enspire Multilabel Reader (Perkin Elmer Ltd.). The results are shown in Figure 6.

[0101] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Test results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and post-hoc tests for each type were performed using Tukey's test. The significance level was set at p<0.05.

[0102] As can be seen in Figure 6, cell proliferation was significantly reduced in the nanoplastic-exposed group, while the enavogliflozin-treated group confirmed that enavogliflozin improved cell proliferation.

[0103] 3-3. Evaluation of cellular senescence marker expression in endothelial cells The expression of cellular senescence markers p53 and p21 was confirmed in porcine coronary artery endothelial cells.

[0104] Specifically, 1 × 10 cells were placed in a 6-well plate. 5 Cells were plated at 100 cells / well. The cell culture medium was DMEM containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μg / mL), and the cells were cultured at 37°C in a CO2 incubator. The cells were divided into a control group, a nanoplastics group (10 μg / mL), and a nanoplastics + enavogliflozin group (0.01, 0.1, and 1 μM). After treatment, the cells were cultured for 24 hours.

[0105] To lyse porcine coronary artery endothelial cells, 1X RIPA buffer was added to each well and allowed to lyse for 10 minutes. The cells were scraped with a scraper and centrifuged at 14,000 rpm at 4°C for 15 minutes. Protein quantification of the resulting cell lysates was measured using Bio-Rad DC protein reagent. 15µg of protein was loaded equally for analysis.

[0106] The primary antibodies, p53 (1:1,000), p21 (1:1,000), and beta-actin (1:1,000), were incubated at 4°C for 24 hours, followed by 3-5 washes with 1X TBS-T buffer for 10 minutes. The secondary antibodies were incubated at 1:20,000 for 1 hour at room temperature, followed by 3-5 washes with 1X TBS-T buffer for 10 minutes. Protein bands were visualized using an Amersham Imager 680 after treatment with ECL solution. The results are shown in Figure 7.

[0107] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and Tukey's test was used for post hoc tests of each type. The significance level was set at p<0.05.

[0108] As can be seen in Figure 7, the nanoplastic-treated group showed a significant increase in the expression of p53 and p21 proteins, while the enavogliflozin-treated group showed a significant decrease in the proteins of cellular senescence markers in a concentration-dependent manner (0.01, 0.1, and 1 μM).

[0109] Example 4. Evaluation of cellular oxidative stress Oxidative stress was measured using dihyroethidium (DHE) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA).

[0110] Specifically, 1 x 10 cells were placed in a black 96-well plate. 4 Porcine coronary artery endothelial cells were seeded at 100 cells / well. Experiments were conducted by treating the cells with either a control group, 10 μg / mL nanoplastics, or 10 μg / mL nanoplastics plus enavogliflozin at different concentrations (0.01, 0.1, and 1 μM). The cell culture medium was DMEM containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μg / mL). The cells were cultured at 37°C in a CO2 incubator. After 24 hours of culture, the amount of reactive oxygen species (ROS) produced was measured by fluorescence absorbance (excitation / emission 485 / 535 nm).

[0111] To measure reactive oxygen species produced in the coronary artery rings, samples were processed into groups of coronary artery rings, immersed in molds containing optimal cutting temperature (OCT) solution (Leica Biosystems), and frozen in liquid nitrogen. 10 μm sections were cut using a cryotome, stained with DHE (10 μM), incubated at 37°C for 45 minutes, washed with PBS, and then observed under a fluorescence microscope. The results are shown in Figure 8.

[0112] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and Tukey's test was used for post hoc tests of each type. The significance level was set at p<0.05.

[0113] As can be seen in Figure 8, the fluorescence intensity of DCF-DA and DHE, which are indicators of ROS generation, increased significantly in the group exposed to nanoplastics, while the fluorescence intensity significantly decreased in the groups treated with enavogliflozin at different concentrations (0.01, 0.1, and 1 μM).

[0114] 4-2. Evaluation of marker expression related to cellular oxidative stress NADPH oxidase in porcine coronary artery endothelial cells is known to generate peroxides from endothelial cells, leading to cellular senescence and endothelial cell dysfunction. We assessed the oxidative stress of cells by measuring the expression of Nox2 and p22phox, which are membrane and catalytic subunits of NADPH oxidase.

[0115] Specifically, 1 × 10 cells were placed in a 6-well plate. 5 Cells were plated at 100 cells / well. The cell culture medium was DMEM containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μg / mL), and the cells were cultured at 37°C in a CO2 incubator. The cells were divided into a control group, a nanoplastics group (10 μg / mL), and a nanoplastics + enavogliflozin group (0.01, 0.1, and 1 μM). After treatment, the cells were cultured for 24 hours.

[0116] To lyse porcine coronary artery endothelial cells, 1X RIPA buffer was added to each well and allowed to lyse for 10 minutes. The cells were scraped with a scraper and centrifuged at 14,000 rpm at 4°C for 15 minutes. Protein quantification of the resulting cell lysates was measured using Bio-Rad DC protein reagent. 15µg of protein was loaded equally for analysis.

[0117] The primary antibodies were Nox2 (1:1,000), p22phox (1:1,000), and beta-actin (1:1,000) for 24 hours at 4°C, followed by 3-5 washes with 1X TBS-T buffer for 10 minutes. The secondary antibodies were incubated at a ratio of 1:20,000 for 1 hour at room temperature, followed by 3-5 washes with 1X TBS-T buffer for 10 minutes. Protein bands were visualized using an Amersham Imager 680 after treatment with ECL solution. The results are shown in Figure 9.

[0118] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and Tukey's test was used for post hoc tests of each type. The significance level was set at p<0.05.

[0119] As shown in Figure 9, Nox2 and p22phox, membrane catalytic subunits of NADPH oxidase, were significantly increased by nanoplastic exposure. Meanwhile, enavogliflozin treatment at different concentrations (0.01, 0.1, and 1 μM) significantly decreased the associated proteins in a concentration-dependent manner.

[0120] This confirmed that enavogliflozin inhibited the production of reactive oxygen species caused by nanoplastics and reduced oxidative stress.

[0121] Example 5. Evaluation of vascular reactivity 5-1.Evaluation of vascular function Vascular endothelial cells play an important role in maintaining vascular homeostasis, but aging is known to induce vascular dysfunction. Therefore, we evaluated the vascular reactivity of vascular endothelial cells to see whether enavogliflozin prevents vascular dysfunction caused by nanoplastic exposure. We also evaluated the effects of empagliflozin, a competitor, on the vascular endothelial cells.

[0122] Specifically, the anterior descending branch of the left coronary artery from pig hearts was excised and cut into 2-3 mm rings. Experiments were conducted in the following groups: a control group, a group treated with 10 μg / mL nanoplastics, a group treated with 10 μg / mL nanoplastics plus different concentrations of enavogliflozin (0.01, 0.1, and 1 μM), and a group treated with 10 μg / mL nanoplastics plus 1 μM empagliflozin. The coronary artery rings were placed in Krebs solution at 37°C in an organ chamber (95% O2, 5% CO2) and changes in isometric contraction were measured. After 90 minutes of isotonic contraction at 5 g, the viability of each coronary artery ring was confirmed by repeated contractions up to 80 mM KCl. After 30 minutes of washing, the coronary artery rings were contracted to approximately 80% (maximum contraction rate) using the thromboxane mimetic U46619, and then treated with bradykinin to calculate the rate of vascular relaxation relative to the bradykinin concentration. The results are shown in the graph in Figure 10.

[0123] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). Two-way ANOVA was performed to verify differences between groups, and Bonferroni's test was used for post hoc tests of each type. The significance level was set at p<0.05.

[0124] Bradykinin is a biological substance that, when the bradykinin receptor is activated, activates eNOS (endothelial nitric oxide synthase), releasing NO (nitric oxide), thereby relaxing blood vessels.

[0125] As shown in Figure 10, normal blood vessels without vascular dysfunction contracted in the presence of the thromboxane mimetic U46619 and relaxed in a concentration-dependent manner upon treatment with bradykinin, achieving nearly 100% relaxation at high concentrations of 100 nM bradykinin (control group). However, unlike the control group, the coronary artery rings in the nanoplastic-treated group showed reduced relaxation compared to the control group, even after treatment with bradykinin in a concentration-dependent manner. The maximum relaxation rate did not reach 100% and the rings remained contracted. This confirmed the occurrence of endothelial dysfunction due to nanoplastics. Meanwhile, the groups treated with enavogliflozin at different concentrations (0.01, 0.1, and 1 μM) showed significantly increased bradykinin-induced vascular relaxation compared to the nanoplastic-only group. These results indicate that enavogliflozin ameliorates endothelial dysfunction caused by nanoplastics (Figure 10).

[0126] The effects of bradykinin on vasorelaxation were compared between groups treated with nanoplastics and 1 μM enavogliflozin or 1 μM empagliflozin. In the enavogliflozin and nanoplastic treatment group, vasorelaxation was significantly increased at bradykinin concentrations of 0.3 nM or higher compared to the group treated with nanoplastics alone. In the group treated with the same concentrations of empagliflozin and nanoplastics, vasorelaxation was significantly increased at relatively high concentrations of 30 nM or higher compared to the group treated with nanoplastics alone (Figure 10).

[0127] This confirmed that enavogliflozin prevents endothelial dysfunction caused by exposure to nanoplastics, and was found to be more effective than empagliflozin in improving vascular dysfunction.

[0128] 5-2. Measurement of endothelial nitric oxide synthase expression Decreased expression of endothelial nitric oxide synthase (eNOS) is a common characteristic of endothelial cell senescence, which is known to induce endothelial dysfunction. Therefore, we measured the expression of endothelial nitric oxide synthase and confirmed that enavogliflozin improves endothelial function damage caused by nanoplastic exposure.

[0129] Specifically, 1 × 10 cells were placed in a 6-well plate. 5 Cells were plated at 100 cells / well. The cell culture medium was DMEM containing 10% FBS, penicillin (100 U / mL), streptomycin (100 U / mL), and fungizone (250 μg / mL), and were cultured at 37°C in a CO2 incubator. Cells were divided into a control group, nanoplastics 10 μg / mL, and nanoplastics + enavogliflozin concentration groups (0.01, 0.1, and 1 μM). After treatment, the cells were cultured for 24 hours.

[0130] To lyse porcine coronary artery endothelial cells, 1X RIPA buffer was added to each well and allowed to lyse for 10 minutes. The cells were scraped with a scraper and centrifuged at 14,000 rpm at 4°C for 15 minutes. Protein quantification of the resulting cell lysates was measured using Bio-Rad DC protein reagent. 15µg of protein was loaded equally for analysis.

[0131] The primary antibodies, eNOS (1:1,000) and beta-actin (1:1,000), were incubated at 4°C for 24 hours, followed by 10-minute washes three to five times with 1X TBS-T buffer. The secondary antibodies were incubated at 1:20,000 for 1 hour at room temperature, followed by 10-minute washes three to five times with 1X TBS-T buffer. Protein bands were visualized using ECL solution and then visualized using an Amersham Imager 680. The results are shown in Figure 11.

[0132] Statistical analysis of data was performed using Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA). Results are presented as mean ± standard error of the mean (SEM). One-way ANOVA was performed to verify differences between groups, and Tukey's test was used for post hoc tests of each type. The significance level was set at p<0.05.

[0133] As can be seen in Figure 11, eNOS protein expression was significantly reduced in the nanoplastics exposure group, while enavogliflozin significantly increased eNOS expression in a concentration-dependent manner (0.01, 0.1, and 1 μM).

Claims

1. A pharmaceutical composition for preventing or treating cardiovascular aging diseases, comprising enavogliflozin as an active ingredient.

2. 2. The pharmaceutical composition for preventing or treating cardiovascular aging diseases according to claim 1, wherein the cardiovascular aging disease is one or more selected from the group consisting of hypertrophic obstructive cardiomyopathy, severe obstructive coronary artery disease, aortic stenosis, hemodynamically significant aortic or mitral stenosis, aortic, coronary artery disease, peripheral artery disease, and carotid artery disease.

3. 2. The pharmaceutical composition for preventing or treating cardiovascular aging diseases according to claim 1, wherein the pharmaceutical composition is for oral or parenteral administration.

4. 2. The pharmaceutical composition for preventing or treating cardiovascular aging diseases according to claim 1, wherein the single dose of enavogliflozin is 0.1 mg to 0.5 mg.

5. 2. The pharmaceutical composition for preventing or treating cardiovascular aging diseases according to claim 1, which is administered once a day.

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