Pharmaceutical composition for preventing or treating calcific valvular disease comprising enavogliflozin as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating calcific valvular disease, comprising enavogliflozin as an active ingredient. According to the present invention, enavogliflozin significantly reduces valvular calcification caused by calcium deposition via a mechanism that suppresses the expression of RUNX2 and BMP2, which are biomarkers of calcification. Accordingly, the pharmaceutical composition of the present invention comprising enavogliflozin as an active ingredient can provide a new clinical means of non-invasive drug therapy for calcific valvular disease, which has so far required reliance on surgical operations or invasive procedures due to the absence of effective drug therapeutic agents. In addition, the pharmaceutical composition provides an effect of suppressing progression of valvular calcification in addition to conventional disease management for patients with type 2 diabetes, which is a major high-risk group for calcific valvular disease, thereby having very high clinical usefulness.

Description

Pharmaceutical composition for the prevention or treatment of calcific valvular disease containing inabogliflozin as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of calcific valvular disease comprising inabogliflozin as an active ingredient.

[0002] Aortic valve stenosis, a representative disease of calcific valvular disease, is a condition in which inflammation and calcification progress in the valve, causing it to thicken and leading to adhesions that prevent the aortic valve from opening properly. It is one of the three major cardiovascular diseases, following coronary artery disease and hypertension. If stenosis persists, symptoms of heart failure, such as reduced cardiac output, and myocardial ischemia may occur; if patients do not receive treatment after experiencing symptoms, the average mortality rate reaches 50% after two years. It is emerging as a common disease and a major cause of death in an aging society, with an incidence rate of 2% in the population aged 65 to 74 and over 6% of the population aged 75 and older exhibiting severe aortic valve stenosis requiring treatment.

[0003] Known risk factors for aortic valve stenosis include obesity, diabetes, chronic kidney disease, hypertension, age, and smoking; however, it is particularly strongly associated with age, resulting in a high probability of occurrence in elderly patients. While the exact cause of the disease has not yet been clearly identified, it is gradually becoming clear that the calcification reaction of valve cells is a significant contributing factor. Although several studies are currently underway, target proteins or substances capable of effectively blocking valve calcification have yet to be developed, and drug treatment is currently unavailable. Aortic valve replacement surgery, which involves replacing the aortic valve with an artificial one, is the only treatment option; however, surgical or percutaneous replacement procedures are not feasible due to the high proportion of elderly patients. Furthermore, the procedure has disadvantages such as high costs (approximately 30 million KRW), an increased risk of stroke, the possibility of leakage around the valve, and durability issues associated with long-term use. Therefore, there is an urgent need for the development of drug therapies to replace aortic valve replacement surgery, and the development of biomarkers for easy diagnosis is also required because early diagnosis is difficult. Consequently, there is an urgent need to discover target proteins that can diagnose and treat aortic valve stenosis.

[0004] The surface of the aortic valve leaflet is covered with valvular endothelial cells (VECs), and valvular interstitial cells (VICs) are spread inward. Because heart valves are subjected to strong stress from blood flow, they produce various biochemical signals, matrix proteins, and matrix remodeling enzymes to maintain the homeostasis of valve cell tissue. It is known that when continuous stress occurs, such as aging and calcification, excessive collagen infiltration occurs and matrix proteins are destroyed; consequently, calcification increases and valvular interstitial cells lose their ability to maintain homeostasis.

[0005] Recent studies provide diverse information on the calcification response induced by valve cells from a molecular biological perspective, with one of the most significant areas of focus being the mechanism of osteogenic formation by valve interstitial cells. The calcification process of valve interstitial cells proposed to date is divided into an initiation phase and a propagation phase. The initiation of valve stenosis is believed to be caused by damage to the valve endothelium due to mechanical stress or other risk factors. It is suggested that low-density lipoprotein (LDL) and lipoprotein(a) [Lp(a)] infiltrate the subendothelial layer, accumulate, and oxidize, inducing chronic inflammatory responses by macrophages, T cells, and mast cells, which can lead to the activation of valve interstitial cells (VICs). In the propagation phase, activated VICs produce unorganized collagen, causing fibrosis and progressive valve thickening. Osteoblast-like cells generate calcification vesicles, inducing valve calcification. It has been suggested that as the valve calcifies, it increases mechanical stress, injury, apoptosis, and osteoblast activation, leading to further calcification and causing a vicious cycle.

[0006] Research began in earnest in the 2010s and is currently being conducted primarily abroad; however, there are still no therapeutic drugs available because a definitive target protein capable of effectively preventing valve calcification has not yet been identified. Most existing studies have focused on the indirect involvement of interstitial cells in calcification—specifically, hyperlipidemia, inflammation, and the angiotensin system—rather than on direct changes in proteins within interstitial cells during the calcification process, which are similar to targets and directions in atherosclerosis. Based on this hypothesis, clinical trials to develop drugs for treating valve stenosis are being conducted using lipid-lowering and osteoporosis treatments such as PCSK9 inhibitors, niacin, and alendronic acid; however, these attempts are presumed to be aimed at expanding the indications of existing drugs rather than developing specific treatments for valve stenosis, and clinical trials using some lipid-lowering agents have reported no efficacy.

[0007] Meanwhile, SGLT-2 (sodium glucose cotransporter 2) is a transporter responsible for the excessive reabsorption of glucose in the kidneys, along with SGLT-1 (sodium glucose cotransporter 1), with SGLT-2 playing the majority of the role. Therefore, when SGLT-2 inhibitors suppress the SGLT-2 transporter, the amount of glucose excreted in the urine increases, which ultimately lowers blood glucose levels and further excretes the calories contained in the glucose, resulting in a weight loss effect.

[0008] One of the drugs developed as an SGLT-2 inhibitor that can be usefully used as a treatment for type 2 diabetes due to such effects is Enavogliflozin, represented by the following structural formula (Chemical Formula 1), and has been disclosed in Korean Patent Publication No. 2014-0022086 (Patent Document 1).

[0009] [Chemical Formula 1]

[0010]

[0011] Compound Name: (2S,3R,4R,5S,6R)-2-(7-chloro-6(4-cyclopropylbenzyl)-2,3-dihydrobenzofuran-4-yl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0012] However, it has not yet been reported that inabogliflozin is useful for preventing or treating calcific valvular disease.

[0013] The inventors have completed the present invention by confirming that inabogliflozin can prevent or treat calcific valve disease.

[0014] Therefore, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of calcific valvular disease comprising inabogliflozin as an active ingredient.

[0015] The present invention will be described in more detail below.

[0016] The present invention provides the use of inabogliflozin for the prevention or treatment of calcific valvular disease, a pharmaceutical composition for the prevention or treatment of calcific valvular disease comprising inabogliflozin as an active ingredient, and a method for the prevention or treatment of calcific valvular disease comprising administering an effective amount of inabogliflozin to a subject who requires it.

[0017] In the present invention, the term 'Calcific Valvular Disease' comprehensively refers to a chronic and progressive pathological condition characterized by the abnormal deposition and accumulation of calcium in the leaflets or annulus of one or more heart valves, particularly the aortic valve and / or mitral valve. This calcification refers to a group of clinical diseases in which the normal opening and closing function of the valve is impaired and hemodynamic disorders are caused by stiffening, thickening, and reduced mobility of the valve tissue.

[0018] The pathophysiology of the aforementioned 'calcific valvular disease' is understood not as a previously known passive degenerative process associated with aging, but as an active, cell-mediated process. Calcific valvular disease is initiated by complex stimuli, including damage to the valve endothelium, lipid deposition (such as Lp(a)) and oxidation, chronic inflammatory responses, and mechanical stress. These stimuli activate Valvular Interstitial Cells (VICs), which are the cells native to the valve. Activated VICs undergo a phenotypic transition into osteoblast-like cells via myofibroblasts. These osteoblast-like cells express Rund-associated transcription factor 2 (Runx2) and other factors similar to the process of osteoogenesis, increase the activity of alkaline phosphatase (ALP), and secrete bone matrix proteins (e.g., osteocalcin, osteopontin). Ultimately, this induces the deposition of calcium and phosphorus in the extracellular matrix, forming calcific nodules and causing ectopic ossification of the valve.

[0019] In the present invention, the diagnosis of 'calcified valvular disease' can be confirmed through non-invasive imaging examinations, in addition to the patient's clinical symptoms (e.g., dyspnea during exercise, chest pain, syncope) and the identification of murmurs through cardiac auscultation. The most critical diagnostic method is echocardiography, through which the severity of the disease can be determined by quantitatively evaluating (a) the degree of thickening, sclerosis, and calcification of the valve leaflets, (b) the limitation of valve movement and whether the valve is open or closed, (c) a decrease in the valve orifice area, and (d) an increase in blood flow velocity and pressure gradient passing through the valve. Additionally, computed tomography (CT) can be used to quantify the severity of 'calcified valvular disease' as defined in the present invention. The 'Valve Calcium Score' can be utilized as such a quantitative indicator, and the aforementioned 'Valve Calcium Score' can be used in a comprehensive sense including the Aortic Valve Calcium Score (AVC score) and the Mitral Annular Calcification (MAC score).

[0020] In one embodiment of the present invention, the calcific valve disease may be calcific aortic valve disease (CAVD), for example, aortic valve sclerosis or aortic valve stenosis.

[0021] Calcific valvular disease exhibits a progressive disease spectrum depending on the severity of the disease. Specifically, Calcific Aortic Valve Disease (CAVD), the most important and common form, can begin with Aortic Valve Sclerosis, an early stage of the disease.

[0022] The above 'aortic valve sclerosis' refers to a condition in which thickening of the valve leaflets and localized calcification have begun, but the reduction in valve mobility is still mild and does not cause significant hemodynamic disturbances (e.g., high blood flow velocity or pressure gradient). If this 'aortic valve sclerosis' continues to progress, and the calcification worsens and the opening and closing of the valve becomes severely restricted, it progresses to 'aortic valve stenosis (AVS),' which results in a significant reduction in the valve opening area and impaired blood flow to the left ventricular outflow tract.

[0023] Therefore, aortic valve sclerosis is a precursor disease of aortic valve stenosis and the most common early clinical manifestation of calcific valve disease.

[0024] In another embodiment of the present invention, the 'calcified valve disease' includes mitral anterior calcification (MAC), which can cause mitral valve stenosis or mitral valve regurgitation.

[0025] These valvular dysfunctions place a chronic pressure overload on the left ventricle, causing left ventricular hypertrophy (LVH) and resulting in myocardial fibrosis and remodeling. As the disease progresses further, it can lead to left ventricular systolic or diastolic dysfunction, causing heart failure, particularly heart failure with preserved ejection fraction (HFpEF), which is a major cause of rapidly increasing morbidity and mortality in patients.

[0026] Furthermore, 'calcified valvular disease' shares many pathophysiological mechanisms (e.g., lipid deposition, inflammation) and risk factors with atherosclerosis. Therefore, chronic kidney disease (CKD), end-stage renal disease (ESRD), diabetes mellitus, hypertension, dyslipidemia, metabolic syndrome, obesity, and smoking are major associated diseases or risk factors that accelerate the development and progression of calcified valvular disease. Congenitally, a bicuspid aortic valve is a strong risk factor that causes severe calcified valvular disease early on due to abnormal hemodynamic stress.

[0027] In the following examples, the efficacy of inabogliflozin in inhibiting calcification was evaluated in a human valvular interstitial cell model in which calcification was induced with NaH2PO4. As a result, it was confirmed through Alizarin red staining and CPC analysis that valvular calcification was significantly inhibited in the group treated with inabogliflozin (see Fig. 1). Furthermore, upon investigating the mechanism of this phenotypic inhibition, it was observed that the expression of RUNX2 and BMP2, key biomarkers of osteoblast differentiation that were significantly increased in the calcification-induced group, was significantly reduced in the inabogliflozin-treated group (see Fig. 2). These results demonstrate that inabogliflozin effectively inhibits the phenotypic conversion of valvular interstitial cells into osteoblast-like cells and the resulting ectopic ossification process, indicating that it can be usefully employed for the prevention or treatment of calcific valvular disease.

[0028] In one embodiment of the present invention, the present invention may be administered to a patient with Type 2 Diabetes Mellitus to prevent or treat calcified valve disease.

[0029] Specifically, type 2 diabetes is well known as a major independent risk factor that promotes the development and progression of calcific valvular disease, particularly calcific aortic valve disease (CAVD). The pathophysiological environment of patients with type 2 diabetes, particularly chronic hyperglycemia, acts as a direct harmful stimulus to valve tissue.

[0030] A hyperglycemic environment amplifies oxidative stress and chronic inflammation within valve tissue, and this inflammatory and oxidative stress environment strongly promotes the phenotype conversion of valvular interstitial cells (VICs) into osteoblast-like cells. In other words, diabetes directly accelerates the process of ectopic ossification, which is a key mechanism of calcific valvular disease.

[0031] Consequently, patients with type 2 diabetes have a significantly higher risk of developing severe aortic valve stenosis due to the progression of valve calcification at an earlier age and at a faster rate compared to patients without diabetes.

[0032] Therefore, the pharmaceutical composition of the present invention, comprising the SGLT2 inhibitor inabogliflozin as an active ingredient, may be particularly useful for a group of patients with calcific valvular disease accompanied by type 2 diabetes.

[0033] A pharmaceutical composition containing inabogliflozin as an active ingredient can be used in the form of single administration or combination administration.

[0034] The above 'calcific valvular disease' is a multifactorial disease with a very complex pathophysiology, in which multiple pathways, such as inflammatory responses and metabolic abnormalities, in addition to the osteoblast differentiation pathway, are simultaneously involved in the progression of the disease. Therefore, the inabogliflozin of the present invention may be administered in combination with one or more other drugs to obtain a complementary or synergistic effect by simultaneously blocking different pathological pathways of calcific valvular disease.

[0035] In one embodiment, a pharmaceutical composition containing inabogliflozin may be intended for co-administration with metformin.

[0036] In another embodiment, a pharmaceutical composition containing inabogliflozin may be intended for co-administration with a DPP4 inhibitor.

[0037] Although not limited thereto, for example, the above DPP4 inhibitor may be gemigliptin.

[0038] In another embodiment, the above co-administration may be co-administration with two or more different drugs. For example, a pharmaceutical composition containing inabogliflozin may be intended for co-administration with metformin and a DPP4 inhibitor.

[0039] Alternatively, a pharmaceutical composition containing inabogliflozin may be administered in combination with a sulfonylurea insulin secretagogue.

[0040] In another embodiment, a pharmaceutical composition containing inabogliflozin may be administered in combination with insulin.

[0041] In another embodiment, the pharmaceutical composition of the present invention may be administered in combination with a treatment for major cardiovascular risk factors associated with patients with calcific valvular disease, and such a drug may be a hyperlipidemia treatment (e.g., statin class drugs, ezetimibe, PCSK9 inhibitors, etc.) or an antihypertensive agent (e.g., ARB, ACE inhibitor, calcium channel blocker, diuretic, etc.).

[0042] The above co-administration may be a method of co-administering a first formulation containing inabogliflozin and a second formulation containing another drug simultaneously or sequentially, or may include administration in the form of a fixed-dose combination (FDC) containing inabogliflozin and one or more other drugs as a single formulation.

[0043] In one embodiment, for convenience of co-administration, a pharmaceutical composition containing inabogliflozin may be formulated together with metformin and / or a DPP4 inhibitor.

[0044] As used herein, the term "prevention" refers to any act of suppressing or delaying the onset of calcific valve disease by administering a pharmaceutical composition according to the present invention.

[0045] Additionally, the term "treatment" as used herein refers to any act in which calcific valve disease is improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0046] The dosage of inabogliflozin that may be used for the prevention or treatment of calcific valvular disease is not specifically limited and can be appropriately adjusted based on the severity of the patient's disease, weight, age, gender, presence or absence of other complications, etc.

[0047] The duration of administration of inabogliflozin can be appropriately adjusted by the clinician based on the preventive or therapeutic effect of inabogliflozin administration on calcific valvular disease.

[0048] Since the above-mentioned methods for the prevention or treatment of calcific valve disease use the pharmaceutical composition of the present invention, any overlap between the two is omitted to avoid excessive description in the specification.

[0049] Inabogliflozin used as an active ingredient in the present invention can be synthesized through known prior art. In the present invention, inabogliflozin may be in a crystalline or amorphous form. For example, inabogliflozin may be inabogliflozin crystalline form A, crystalline form B, crystalline form C, crystalline form D, crystalline form E, or inabogliflozin amorphous form, which are reported to have the following X-ray diffraction spectra through Korean Patent Publication No. 2017-0142904 or Korean Patent Application No. 2022-0123673.

[0050] Crystalline Form A: A crystalline form having an X-ray diffraction (XRD) spectrum including peaks at a 2[θ] value 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°.

[0051] Crystalline Form B: Crystalline form having an X-ray diffraction (XRD) spectrum including peaks at a 2[θ] value 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°.

[0052] Crystalline form C: Crystalline form having an X-ray diffraction (XRD) spectrum including peaks at a 2[θ] value 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°.

[0053] Crystalline form D: Crystalline form having an X-ray diffraction (XRD) spectrum including peaks at a 2[θ] value 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°.

[0054] Crystalline form E: Crystalline form having an X-ray diffraction (XRD) spectrum including peaks at a 2[θ] value 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°.

[0055] The above crystal forms A, B, C, D, and E can each be identified by an X-ray diffraction spectrum having four or more, for example, four, five, six, seven, eight or more peaks at the 2[θ] value presented above.

[0056] Although not limited thereto, a pharmaceutical composition containing inabogliflozin according to the present invention may have the composition of a pharmaceutical composition of PCT / KR2022 / 014640.

[0057] For example, the pharmaceutical composition of the present invention may be a pharmaceutical composition comprising inabogliflozin or its pharmaceutically acceptable salt, excipients, disintegrants, and binders as active ingredients, wherein the average particle size of inabogliflozin is 15 μm or less.

[0058] In a specific embodiment of the present invention, the average particle size of inabogliflozin may be 15 µm or less, preferably 10 µm or less.

[0059] When the average particle size of inabogliflozin exceeds 15 µm, the 5-minute dissolution rate is very low at less than 40% of the total content of inabogliflozin, and the 30-minute dissolution rate is also less than 80%, so the final dissolution rate is found to be unsuitable.

[0060] If finer particle size reduction of the drug is required, the drug can be ground using conventional mills capable of fine particle reduction, such as Z-mills, hammer mills, ball mills, and fluid energy mills. Additionally, the particle size of the drug can be further refined using size classification methods, such as sieving or air current classification. Methods for controlling the desired particle size are well known in the art. For example, refer to the following literature: [Pharmaceutical dosage forms: volume 2, 2nd edition, Ed.: HALieberman, L. Lachman, JBSchwartz (Chapter 3: SIZE REDUCTION)].

[0061] In this specification, the particle size of a drug is expressed based on a particle size distribution such as 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 diameter of a drug within a formulation is represented by a cumulative curve, the particle diameter at the point where the particle size accumulates in order of smallest to X% (where % is calculated based on number, volume, or weight) is Y. For example, D(10) represents the particle diameter at the point where the particle size of the drug accumulates in order of smallest to 10%, D(50) represents the particle diameter at the point where the particle size of the drug accumulates in order of smallest to 50%, and D(90) represents the particle diameter at the point where the particle size of the drug accumulates in order of smallest to 90%.

[0062] Whether the particle size distribution D(X) represents a percentage of the total accumulated particles based on number, volume, or weight depends on the method used to measure the particle size distribution. Methods for measuring particle size distribution and the types of percentages associated therewith are known in the art. For example, when measuring particle size distribution by the well-known laser diffraction method, the X value in D(X) represents the percentage calculated by the volume average. Those skilled in the art are well aware that the results of particle size distribution measurements obtained by a particular method may correlate with those obtained from other techniques based on experience from conventional experiments. For example, the laser diffraction method provides a volume-averaged particle size in response to the volume of the particles, which corresponds to the weight-averaged particle size when the density is constant.

[0063] In the present invention, the measurement of the particle size distribution of drug particles can be performed using a commercially available device based on the laser diffraction and scattering method according to Mie theory. For example, the measurement is performed using a commercially available device such as the Mastersizer laser diffraction device from Malvern Instruments. This device obtains the particle diameter distribution by irradiating particles with a helium-neon laser beam and a blue light-emitting diode, causing scattering to occur and a light scattering pattern to appear on a detector, and interpreting this light scattering pattern according to Mie theory. Either a dry or wet method may be used for the measurement.

[0064] For reference, in a specific embodiment of the present invention, the particle size of the drug was measured by the volume average particle size by laser diffraction.

[0065] In a specific embodiment of the present invention, the inabogliflozin may be included in an amount of less than 1 part by weight per 100 parts by weight of the total pharmaceutical composition.

[0066] The appropriate once-daily dosage of inabogliflozin identified during clinical trials is 0.1 mg to 0.5 mg, and when the above pharmaceutical composition is formulated into a unit dosage form, the content of the active ingredient in the pharmaceutical composition may be 0.1 to 0.5 mg.

[0067] The pharmaceutical composition according to the present invention includes pharmaceutically acceptable additives in addition to the active ingredient inabogliflozin.

[0068] The pharmaceutical composition of the present invention includes excipients, disintegrants, and binders as additives.

[0069] 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, anhydrous calcium phosphate, calcium carbonate, sugars, or mixtures thereof. In embodiments of the present invention, a preferred excipient is microcrystalline cellulose.

[0070] Examples of disintegrants include crospovidone, sodium croscarmellose, sodium starch glycolate, and low-substituted hydroxypropylcellulose. In a specific embodiment of the present invention, a preferred excipient is sodium croscarmellose.

[0071] Examples of binders include polyvinylpyrrolidone, povidone, gelatin, starch, sucrose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylalkylcellulose (e.g., hydroxypropylmethylcellulose), and mixtures thereof. In a specific embodiment of the present invention, a preferred binder is hydroxypropylcellulose.

[0072] Examples of other additives include lubricants and coloring agents.

[0073] The above lubricant comprises stearic acid, stearate (e.g., magnesium stearate), hard anhydrous silica, talc, corn starch, carnauba wax, magnesium silicate, synthetic aluminum silicate, hardened oil, white wax, titanium oxide, microcrystalline cellulose, macrogol 4000 and 6000, isopropyl myristate, calcium hydrogen phosphate, and mixtures thereof.

[0074] In a specific embodiment of the present invention, the excipient may be included in an amount of 80 to 95 parts by weight relative to 100 parts by weight of the total pharmaceutical composition.

[0075] In a specific embodiment of the present invention, the disintegrant may be included in an amount of 2 to 8 parts by weight relative to 100 parts by weight of the total pharmaceutical composition. If the amount of disintegrant is less than 2 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, the initial disintegration power is low and the dissolution rate may be delayed, which may affect the Cmax in the body. In addition, if the amount exceeds 8 parts by weight relative to 100 parts by weight of the total pharmaceutical composition, the amount of disintegrant in the post-mixing portion increases, so the overall flowability of the granules may decrease.

[0076] In a specific embodiment of the present invention, the binder may be included in an amount of 3 to 10 parts by weight per 100 parts by weight of the total pharmaceutical composition. If the binder is less than 3 parts by weight per 100 parts by weight of the total pharmaceutical composition, it may be difficult to form and maintain suitable dry granules, which may affect the maintenance of homogeneous dispersion of the main ingredient and the granule flowability due to the generation of fine particles. In addition, if it exceeds 10 parts by weight per 100 parts by weight of the total pharmaceutical composition, granules with strong binding force are formed, which affects the solubility of the granule particles that disintegrate initially upon dissolution, and this may also affect the Cmax in the body.

[0077] The pharmaceutical composition according to the present invention may be an immediate-release formulation.

[0078] In one embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 50% or more, preferably 60% or more, of the total content of the active ingredient after 5 minutes.

[0079] In one embodiment of the present invention, the pharmaceutical composition may have a dissolution rate of 80% or more of the total content of the active ingredient after 15 minutes, preferably 80% or more.

[0080] 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.

[0081] Since the dissolution rate of the active ingredient in a pharmaceutical composition affects the peak blood concentration (Cmax) and the area under the blood concentration-time curve (AUC) upon drug administration, conversely, it is important to adjust the dissolution rate of the pharmaceutical composition to achieve appropriate Cmax and AUC. As Inabogliflozin has a Tmax of 1 to 2 hours, the drug absorption rate in the stomach is considered important. The above dissolution rate was measured under conditions 1.2 of the dissolution solution by the Korean Pharmacopoeia Dissolution Test Method 2 (Paddle Method). Specific conditions can be referenced in the experimental examples below.

[0082] The present invention also

[0083] The present invention provides a pharmaceutical composition comprising a pre-mixed granule containing inabogliflozin or a pharmaceutically acceptable salt thereof and a post-mixed granule containing a mixed portion.

[0084] The inventors confirmed that, during the formulation research of inabogliflozin, preparing granules and formulating them into tablets or the like is advantageous in terms of drug content uniformity and formulation uniformity.

[0085] In the above pharmaceutical composition, the granules are prepared by mixing the pre-mixed granules and the post-mixed part.

[0086] The above-mentioned premixed granules may include inabogliflozin or its pharmaceutically acceptable salt, excipients, binders, and lubricants.

[0087] In addition, the above-mentioned post-mixing part may include excipients, disintegrants, and lubricants.

[0088] The descriptions of excipients, binders, disintegrants, lubricants, etc. are identical to those described above, so they are omitted to avoid duplication.

[0089] In a specific embodiment of the present invention, the pre-mixed granules and the post-mixed part may each include an excipient. More specifically, the pre-mixed granules and the post-mixed part may each include microcrystalline cellulose as an excipient.

[0090] According to the following examples, it was found that the microcrystalline cellulose included in the pre-mixed granules and post-mixed parts affects the uniformity of the drug content depending on its particle size and bulk density.

[0091] In a specific embodiment of the present invention, the particle size of the microcrystalline cellulose in the premixed granules may be 130 µm or less, preferably 60 to 130 µm. The bulk density of the microcrystalline cellulose in the premixed granules may be 0.26 to 0.33. When the particle size and bulk density of the microcrystalline cellulose in the premixed granules are under the above conditions, a formulation with a low deviation (SD) in content uniformity can be secured. When the particle size of the microcrystalline cellulose in the premixed granules is 130 µm or less, the content uniformity of the premixed granules, the content uniformity of the final granules, and the formulation all showed good levels, and the Carr's index value, which indicates the physical properties of the final granules, was also good, confirming that the flowability of the formulation was also excellent. On the other hand, when the particle size of microcrystalline cellulose in the premixed granules exceeded 130 µm, both the content uniformity of the premixed granules and the content uniformity of the final granules showed large deviations and were unsuitable, and the formulation uniformity was also found to be poor.

[0092] Meanwhile, the particle size of the microcrystalline cellulose in the post-mixing section may be 130 µm or more, preferably 130 to 250 µm. The bulk density of the excipient in the post-mixing section may be 0.28 to 0.37. When the particle size of the microcrystalline cellulose in the post-mixing section is less than 130 µm, it was confirmed that the Carr's index value, which indicates the physical properties of the final granules, was not appropriate and the granule flowability became weak.

[0093] When comparing the microcrystalline cellulose in the pre-mixed granules and the microcrystalline cellulose in the post-mixed section relatively, it was found that, unlike the microcrystalline cellulose contained in the pre-mixed granules which is desirable to have a small particle size, the microcrystalline cellulose in the post-mixed section which is desirable to have a relatively larger particle size compared to the microcrystalline cellulose contained in the pre-mixed granules.

[0094] According to the following examples, it was confirmed that not only the particle size of microcrystalline cellulose in the pre-mixed granules and microcrystalline cellulose in the post-mixed portion, but also the weight ratio of excipients in the pre-mixed granules and excipients in the post-mixed portion affects the uniformity of the drug content.

[0095] In a specific embodiment of the present invention, the weight ratio of the excipient in the pre-mixed granules and the excipient in the post-mixed portion may be 4:1 to 1:1. It was found that as the proportion of microcrystalline cellulose in the post-mixed portion increases, the flowability of the granules improves, while the variation in content increases, so it is desirable to match the weight ratio within the appropriate range.

[0096] Meanwhile, in the pharmaceutical composition according to the present invention, the binder may be one or more selected from the group consisting of hydroxypropylcellulose, povidone, copovidone, and hypromellose.

[0097] In one embodiment of the present invention, the binder is hydroxypropylcellulose and may have a weight-average molecular weight of less than 200,000. If hydroxypropylcellulose with a weight-average molecular weight of 200,000 or more is used, both the 5-minute dissolution rate and the 30-minute dissolution rate are low, which is undesirable in terms of bioavailability.

[0098] Meanwhile, regarding Carr's index used as a measure of flowability in formulation, it is preferable that the Carr's index of the granules be 21 to 25.

[0099] Although not limited thereto, the granules in the pharmaceutical composition of the present invention may be dry granules. In other embodiments, the granules may be wet granules.

[0100] In the present invention, the pharmaceutical composition may have a formulation for oral administration, such as a tablet or capsule. In one embodiment of the present invention, the pharmaceutical composition may have a tablet formulation.

[0101] In a preferred embodiment, the pharmaceutical composition may contain inabogliflozin in a dose of 0.3 mg to 0.5 mg.

[0102] The pharmaceutical composition according to the present invention may be administered orally once a day, but is not limited thereto.

[0103] According to the present invention, inabogliflozin significantly reduces valve calcification caused by calcium deposition through a mechanism that inhibits the expression of RUNX2 and BMP2, which are biomarkers of calcification. Accordingly, a pharmaceutical composition containing inabogliflozin of the present invention as an active ingredient can provide a new clinical means of non-invasive drug treatment for calcific valve disease, which previously required reliance on surgical procedures or invasive treatments due to the lack of effective drug therapies. Furthermore, it offers high clinical utility by providing the effect of inhibiting the progression of valve calcification in addition to managing existing diseases in patients with type 2 diabetes, who are a major high-risk group for calcific valve disease.

[0104] Figure 1 is a diagram showing the results of measuring the inhibitory efficacy of inabogliflozin on calcification of human valve interstitial cells (VICs) according to an embodiment of the present invention using Alizarin Red S staining and CPC quantitative analysis. Figure 1a is a micrograph of Alizarin Red S staining showing the degree of calcium deposition in each experimental group (top: before staining, bottom: after staining), and (b) is a graph quantitatively comparing the degree of calcification through CPC analysis. (CM: normal control medium; PCM-CM: calcification-inducing medium)

[0105] FIG. 2 is a diagram illustrating the effect of inabogliflozin according to an embodiment of the present invention on the expression of RUNX2 and BMP2, which are calcification biomarkers of human valve interstitial cells. FIG. 2a shows the results of confirming the protein-level expression of these markers via Western blot, and FIG. 2b is a graph quantifying the mRNA-level expression of these markers via Real-time PCR. (CM: Normal control medium; PCM-CM: Calcification-inducing medium)

[0106] One or more specific examples are described in more detail below through embodiments. However, these embodiments are intended to illustrate one or more specific examples and the scope of the present invention is not limited to these embodiments.

[0107]

[0108] [Example]

[0109] <Example 1> Confirmation of the inhibitory effect of inabogliflozin on valve interstitial cell calcification

[0110] In this example, we confirmed whether inabogliflozin directly inhibits calcium deposition in a calcification model of human valve interstitial cells.

[0111] 1-1. Experimental Method

[0112] Human valve interstitial cells (1x10 4( / mL) was dispensed into 96-well culture vessels. The experimental group was divided into three groups as follows and cultured for a total of 10 days.

[0113] (1) Normal control medium (CM): DMEM (high-glucose; Gibco) culture medium containing 0.1% amphotericin B, 1% penicillin-streptomycin and 5% FBS.

[0114] (2) Pro-calcifying medium (PCM-CM): A culture medium to which 2 mM NaH2PO₄ was added to the normal control (CM) culture medium. 10% DMSO was administered to the pro-calcifying medium (PCM-CM) instead of inabogliflozin, and the final DMSO concentration was maintained at 0.1%, the same as in the inabogliflozin treatment group.

[0115] (3) Inabogliflozin treatment group (PCM-ENA): A group treated with 1 μM inabogliflozin in the culture medium of the calcification-induced group (PCM-CM).

[0116] The medium was replaced every 2 days, and the cells were cultured in a 37℃ cell culture incubator maintained at a 5% CO2 concentration.

[0117] After culturing for 10 days, the medium was removed and the cells were washed with PBS buffer. The cells were then fixed with 4% paraformaldehyde for 20 minutes and washed with dH2O. To qualitatively confirm the deposition of calcification in valve interstitial cells, Alizarin Red S staining was performed at room temperature (in a dark state) for 20 minutes, and the cells were observed under a microscope after washing 2 to 4 times with triple-distilled water.

[0118] In addition, to quantify Alizarin Red stain, the sample was washed twice with PBS and fixed with cold 70% ethanol at 4°C for 1 hour. After washing with dH2O, the stained calcium precipitate was eluted by treating with 10% CPC (cetylpyridinium chloride, Sigma-Aldrich) for 1 hour (shake and blackout), transferred to a new measuring vessel, and the absorbance was measured at 550 nm.

[0119]

[0120] 1-2. Experimental Results

[0121] Figure 1 shows the results of measuring the inhibitory effect of inabogliflozin on human valve interstitial cells (VICs) calcification according to one embodiment of the present invention using Alizarin Red S staining and CPC quantitative analysis.

[0122] Alizarin Red staining results (Fig. 1a, scale bar=200 μm) showed that almost no calcification deposition was observed in the normal control group (CM), whereas red calcium deposition was observed in the calcification-induced group (PCM-CM). In contrast, it was confirmed that calcium deposition was significantly inhibited in the inabogliflozin 1 μM treatment group (PCM-ENA 1 μM) compared to the calcification-induced group. Quantitative analysis of CPC (Fig. 1b) was also consistent with this, showing that the inabogliflozin 1 μM treatment group statistically significantly reduced the degree of calcification (absorbance) compared to the calcification-induced group (PCM-CM).

[0123]

[0124] <Example 2> Inhibitory Effect of Inabogliflozin on Calcification Biomarker Expression (Western Blot and Real-time PCR)

[0125] In this example, to confirm the mechanism of inabogliflozin’s inhibition of calcification, changes in the expression of key biomarkers of calcification indicators were confirmed at the protein and mRNA levels, respectively.

[0126]

[0127] 2-1. Experimental Method

[0128] Human valve interstitial cells (5x10⁶) in a 6-well plate 4 / mL) was dispensed and cultured for 10 days under the same conditions as in <Example 1> [(1) normal control group (CM), (2) calcification-induced group (PCM-CM), (3) inabogliflozin-treated group (PCM-ENA)].

[0129] (1) Western blot analysis

[0130] After 10 days of culture, 1X RIPA buffer was added to each well and lysed for 10 minutes to lyse the cells. The cells were scraped with a scraper and centrifuged at 14,000 rpm at 4°C for 15 minutes. Protein quantification of the obtained cell lysates was performed using Bio-Rad DC protein reagent. Analysis was performed using an equal loading of 20 μg of protein. RUNX2 (1:1,000), BMP2 (1:1,000), or beta-actin (1:3,000) were reacted as the primary antibody at 4°C for 24 hours, followed by 3 to 5 washes with 1X TBS-T buffer for 10 minutes each. The secondary antibody was reacted at a 1:10,000 ratio at room temperature for 1 hour, followed by 3 to 5 washes with 1X TBS-T buffer for 10 minutes each. Protein bands were visualized using an Amersham Imager 680 after treatment with ECL solution.

[0131] (2) Real-time PCR

[0132] After culturing for 10 days, the cells were washed with PBS, 800 μL of RNAiso Plus (Takara, Tokyo, Japan) was added to lyse the cells, and the solution was transferred to a 1.5 ml test tube (EP-tube). 160 μL of chloroform was added and the mixture was shaken about 15 times, then incubated at room temperature for 3 minutes. The supernatant was centrifuged at 12,000 rpm at 4°C for 15 minutes, mixed with 300 μL of isopropanol, left at room temperature for 20 minutes, and centrifuged again at 12,000 rpm at 4°C for 15 minutes to precipitate total RNA. The precipitated total RNA pellet was washed with 70% ethanol, and the total RNA was quantified using a nanodrop.

[0133] cDNA was synthesized by performing a reverse transcription reaction with 1 µg of RNA added to the extracted RNA using AccuPower RT PreMix (Bioneer, Republic of Korea). Subsequently, TB Green Premix Ex Taq III (Takara, Tokyo, Japan) was added, and a reverse transcription polymerase chain reaction was performed using the CFX connect Real-Time system (Bio-Rad). The expression of the RUNX2 and BMP2 genes was confirmed using beta-actin as the overgene, and gene expression was evaluated using the cycle threshold (Ct) method (2 -△△Ct Quantified using ).

[0134] Real-time PCR primer sequencesGeneForward (5'→3')Reverse (5'→3')RUNX2GGT TAA TCT CCG CAG GTC ACT (SEQ ID NO.: 1)CAC TGT GCT GAA GAG GCT GTT (SEQ ID NO.: 2)BMP2AGA ATG CAA GCA GGT GGG AA (SEQ ID NO.: 3)TGT TTC TCC TCC AAG TGG GC (SEQ ID NO.: 4)beta-actinCTC TTC CAG CCT TCC TTC CT (SEQ ID NO.: 5)AGC ACT GTG TTG GCG TAC AG (SEQ ID NO.: 6)

[0135] 2-2. Experimental Results

[0136] Figure 2 shows the effect of inabogliflozin according to one embodiment of the present invention on the expression of RUNX2 and BMP2, which are calcification biomarkers of human valve interstitial cells.

[0137] Western blot (Fig. 2a) and Real-time PCR analysis results (Fig. 2b) showed that the calcification-induced group (PCM-CM) had significantly increased protein and mRNA expression of calcification biomarkers RUNX2 (runt-related transcription factor 2) or BMP2 (bone morphogenic protein 2) compared to the normal control group (CM). On the other hand, the inabogliflozin-treated group showed significantly reduced expression of RUNX2 and BMP2 compared to the calcification-induced group (PCM-CM).

[0138] The results of Examples 1 and 2 above show that inabogliflozin significantly reduces valve calcification caused by calcium deposition through a mechanism that inhibits the expression of RUNX2 and BMP2, which are biomarkers of calcification. This strongly suggests that inabogliflozin can be usefully employed in the prevention or treatment of calcific valve disease.

Claims

1. A pharmaceutical composition for the prevention or treatment of calcific valvular disease comprising inabogliflozin as an active ingredient.

2. A pharmaceutical composition for the prevention or treatment of calcific valve disease, wherein the calcific valve disease is calcific aortic valve disease.

3. A pharmaceutical composition for the prevention or treatment of calcific valve disease according to paragraph 2, wherein the calcific aortic valve disease is aortic valve sclerosis or aortic valve stenosis.

4. A pharmaceutical composition for the prevention or treatment of calcific valve disease according to claim 1, wherein the calcific valve disease is mitral anterior calcification.

5. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered to a patient with Type 2 Diabetes Mellitus, for the prevention or treatment of calcific valvular disease.

6. A pharmaceutical composition for the prevention or treatment of calcific valve disease, wherein, in any one of claims 1 to 5, the pharmaceutical composition is intended for single administration.

7. A pharmaceutical composition for the prevention or treatment of calcific valve disease, wherein, in any one of claims 1 to 5, the pharmaceutical composition is intended for co-administration with metformin.

8. A pharmaceutical composition for the prevention or treatment of calcific valvular disease, wherein, in any one of claims 1 to 5, the pharmaceutical composition is intended for co-administration with a DPP4 inhibitor.

9. A pharmaceutical composition for the prevention or treatment of calcific valvular disease, wherein the DPP4 inhibitor in claim 8 is gemigliptin.

10. A pharmaceutical composition for the prevention or treatment of calcific valvular disease, wherein, in any one of claims 1 to 5, the pharmaceutical composition is intended for co-administration with metformin and a DPP4 inhibitor.

11. A pharmaceutical composition for the prevention or treatment of calcified valve disease, wherein the pharmaceutical composition according to any one of claims 1 to 5 is administered in combination with a sulfonylurea-based insulin secretion promoter.

12. In any one of claims 1 to 5, the pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of calcific valve disease administered in combination with insulin.

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