Composition of SGLT2 inhibitor and menthol and application of composition in treatment of heart diseases

Abstract

The invention discloses a pharmaceutical composition of an SGLT2 inhibitor and menthol and application of the pharmaceutical composition in preparation of drugs for preventing, improving and / or treating heart diseases. The pharmaceutical composition has a myocardial cell protection effect, can synergistically reduce intracellular calcium ion concentration and synergistically promote fatty acid metabolism, has advantages compared with single medication, and can be used for preparing drugs for treating, improving and / or preventing heart failure, coronary heart disease, myocardial infarction, hypertensive heart disease and the like.

Description

A combination of an SGLT2 inhibitor and menthol and use thereof in treating heart disease Technical Field

[0001] The present invention relates to the field of pharmacotherapy, and in particular to a pharmaceutical composition of an SGLT2 inhibitor and menthol, in particular a combination of empagliflozin and L-menthol, for use in preventing and / or treating heart disease. Background Art

[0002] Heart disease is a common circulatory disease classified into various types based on different pathological mechanisms, including heart failure, coronary artery disease, myocardial infarction, myocarditis, cardiomyopathy, hypertensive heart disease, arrhythmias, and dilated cardiomyopathy. Heart failure is the terminal stage of many heart diseases. Heart failure, or HF, is a clinical condition characterized by impaired cardiac systolic and / or diastolic function, decreased ejection and filling capacity, and insufficient tissue perfusion. Common clinical symptoms include dyspnea, general fatigue, and weakness, accompanied by signs such as systemic / pulmonary congestion and peripheral edema. As a late-stage clinical symptom of various cardiovascular diseases, HF leads to extremely high morbidity, hospitalization, and mortality rates, making it a leading cause of hospitalization for people over 65 years old. According to statistics, the number of people with HF worldwide has reached 23 million, with the prevalence of HF in developed countries being approximately 1-2%. The situation in our country is not optimistic either. According to the 2015 Heart Failure Epidemiological Survey, the weighted heart failure prevalence among residents aged 35 years and above in our country is 1.3%, which means that approximately 13.7 million people suffer from heart failure, and the trend continues to rise.

[0003] Heart failure is a complex disease process caused by multiple pathogenic factors. However, neurohumoral stimulation plays the most important role in the development and progression of heart failure. Among them, chronic overactivation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system (SNS) can lead to excessive vasoconstriction, increased peripheral resistance, increased cardiac afterload, and dysfunction leading to pathological myocardial hypertrophy, fibrosis, adverse ventricular remodeling, and ultimately heart failure. Furthermore, with the development and progression of heart failure, the increased synthesis and release of factors such as natriuretic peptides, arginine vasopressin, TNF-α, and interleukins can also cause myocardial cell apoptosis and ventricular remodeling by affecting blood volume and inflammatory responses, exacerbating heart failure. Impaired myocardial energy metabolism can lead to insufficient cardiac energy production to maintain normal work requirements, resulting in dysfunction of the heart's pumping and ion transport functions, further exacerbating heart failure. With a deeper understanding of the pathogenesis of heart failure and the advancement of evidence-based medicine, treatment options for heart failure have gradually shifted from the traditional approach of "cardiotonic, diuretic, and vasodilation" to novel approaches such as neuroendocrine blockade and reversal of ventricular remodeling, including angiotensin-converting enzyme inhibitors / angiotensin II receptor inhibitors (ACEI / ARB), aldosterone inhibitors, and beta-blockers. While these newer treatments have somewhat alleviated the clinical symptoms and mortality of heart failure patients, they still have clinical limitations, such as numerous adverse reactions and the need for long-term medication, and they have not significantly improved patients' quality of life. Data show that the 5-year mortality rate for heart failure patients remains as high as over 50%.

[0004] Combination drugs are an effective method for increasing drug efficacy and reducing drug side effects by combining drugs to simultaneously act on multiple signaling pathways of the disease, and are widely used in clinical treatment. Especially for diseases with complex pathogenesis such as heart failure, combination drugs are expected to become a practical treatment strategy to improve the quality of life of heart failure patients. However, there are currently only four combination drugs for the treatment of heart failure: sacubitril-valsartan, lisinopril-torsemide, bisoprolol fumarate / perindopril arginine, and hydralazine hydrochloride / isosorbide dinitrate, which are far from meeting clinical drug needs. Therefore, the development of new combination drugs for treating heart failure is of great practical significance.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a combination of an SGLT2 inhibitor and menthol, which has therapeutic advantages over single drugs and is expected to be developed into a compound drug for preventing, improving and / or treating heart disease.

[0007] The first aspect of the present invention provides a pharmaceutical composition comprising:

[0008] (a) an SGLT2 inhibitor, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, or a prodrug ester thereof;

[0009] (b) menthol, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, or a prodrug ester thereof.

[0010] In another preferred embodiment, the SGLT2 inhibitor is selected from the following group: empagliflozin, dapagliflozin, canagliflozin, isagliflozin, rupagliflozin, togliflozin, sogliflozin, erpagliflozin, repagliflozin, empagliflozin, ganagliflozin, bepagliflozin, enagliflozin, ganagliflozin, and tigliflozin.

[0011] In another preferred embodiment, the menthol is selected from the group consisting of L-menthol, D-menthol, and (DL)-menthol.

[0012] In another preferred embodiment, "pharmaceutically acceptable salts" refer to salts formed with acids selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid; or sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed with inorganic bases; or methylamine salts, ethylamine salts or ethanolamine salts formed with organic bases.

[0013] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0014] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the components of the pharmaceutical composition of the present invention and with each other without significantly reducing the efficacy of the pharmaceutical composition. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0015] In another preferred embodiment, the molar ratio of the SGLT2 inhibitor to the menthol is 1-10000:1-10000. In another preferred embodiment, the molar ratio of the SGLT2 inhibitor to the menthol is 1-200:1-300, preferably the molar ratio of the SGLT2 inhibitor to the menthol is 5-100:5-250; more preferably the molar ratio of the SGLT2 inhibitor to the menthol is 1-8:1-22, 4-6:15-25, or 4.5-5.5:18-22.

[0016] In another preferred embodiment, the mass ratio of the SGLT2 inhibitor to the menthol is 1-10000:1-10000. In another preferred embodiment, the mass ratio of the SGLT2 inhibitor to the menthol is 1-100:1-200, preferably the mass ratio of the SGLT2 inhibitor to the menthol is 1-20:1-100; more preferably the mass ratio of the SGLT2 inhibitor to the menthol is 1-10:1-50, 2-3:5-10, 1-2:3

[0017] In another preferred embodiment, the pharmaceutical composition comprises empagliflozin and L-menthol.

[0018] In another preferred embodiment, the molar ratio of empagliflozin to L-menthol is 1-400:1-400.

[0019] In another preferred embodiment, the mass ratio of empagliflozin to L-menthol is 1-400:1-400.

[0020] In another preferred embodiment, the molar ratio of empagliflozin to L-menthol is 1-200:1-300, preferably the molar ratio of empagliflozin to L-menthol is 5-100:5-250; more preferably the molar ratio of empagliflozin to L-menthol is 1-8:1-22, 4-6:15-25 or 4.5-5.5:18-22.

[0021] In another preferred embodiment, the molar ratio of empagliflozin to L-menthol is 1:1-10, preferably 1:2-6, more preferably 1:4.5-5.5 or 1:4.

[0022] In another preferred embodiment, the mass ratio of empagliflozin to L-menthol is 1-100:1-200, preferably the mass ratio of empagliflozin to L-menthol is 1-20:1-100; more preferably, the mass ratio of empagliflozin to L-menthol is 1-10:1-50, 2-3:5-10 or 1-1:3.

[0023] In another preferred embodiment, the mass ratio of empagliflozin to L-menthol is 1:0.1-5, preferably 1:0.5-3, and more preferably 1:1-2 or 1:1.5.

[0024] In another preferred embodiment, the dosage form of the pharmaceutical composition is oral solution, tablet, pill, powder, capsule, injection and granule.

[0025] In another preferred embodiment, the two drugs in the pharmaceutical composition are administered simultaneously or separately.

[0026] The pharmaceutical composition of the present invention has a protective effect on rat myocardial cell line H9c2 cells and primary myocardial cells under injury models.

[0027] The pharmaceutical composition of the present invention can synergistically inhibit the influx of intracellular calcium ions and synergistically promote fatty acid metabolism.

[0028] The second aspect of the present invention provides the use of the pharmaceutical composition described in the first aspect for preparing a drug for treating heart disease.

[0029] In another preferred embodiment, the heart disease is selected from the group consisting of heart failure, coronary heart disease, myocardial infarction, myocarditis, myocardial disease, hypertensive heart disease, arrhythmia, and dilated cardiomyopathy.

[0030] In another preferred embodiment, the heart failure includes heart failure with decreased ejection fraction and heart failure with preserved ejection fraction.

[0031] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal, or similar purpose. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the synergistic effect of empagliflozin and menthol on cardiomyocyte protection. (A) Cell viability in the checkerboard assay; (B) Synergy index in the checkerboard assay; (C) Representative images of crystal violet staining; Scale bar, 500 μm; (D) Quantification of crystal violet staining; (E) LDH release. Data are mean ± SD: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0033] Figure 2 shows the efficacy of the empagliflozin and menthol combination in an ISO-induced heart failure model. (A) Schematic diagram of the experimental procedure; (B) Representative M-mode echocardiograms; (C) Ejection fraction; (D) Fractional shortening; (E) Left ventricular end-systolic dimension; (F) Left ventricular end-diastolic dimension; (G) Representative myocardial fibrosis images; (H) Quantification of myocardial fibrosis. Data are mean ± SD: *p < 0.05, **p < 0.01, ***p < 0.001.

[0034] Figure 3 shows the efficacy and exercise tolerance of the empagliflozin and menthol combination in a TAC-induced heart failure model. (A) Schematic diagram of the experimental procedure; (B) Survival curve; (C) Grip test; (D) Rotarod test; (E) Hanging test. Data are mean ± SD: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0035] Figure 4 shows the effects of the empagliflozin and menthol combination on cardiac function in TAC mice. (A) Heart-to-body ratio; (B) Representative heart; (C) Representative M-mode echocardiogram; (D) Ejection fraction; (E) Fractional shortening; (F) Left ventricular end-systolic dimension; (G) Left ventricular end-diastolic dimension. Data are mean ± SD: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0036] Figure 5 shows the synergistic effects of the empagliflozin and menthol combination on intracellular calcium concentration and fatty acid metabolism. (A) Intracellular calcium concentration; (B) Representative Oil Red O staining images; (C) Oil Red O staining quantification; (D) Representative Western blotting images; (E) Target band quantification. Data are mean ± SD: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0037] Figure 6 shows the safety of the empagliflozin and menthol combination. (A) Weight change curve; (B) Organ coefficients; (C) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (UREA) levels; (D) Representative images of organ appearance; (E) Representative images of heart, liver, spleen, and kidneys stained with hematoxylin and eosin.

[0038] Figure 7 shows the pharmacokinetic properties of empagliflozin alone and in the presence of menthol. (A) Changes in plasma drug concentration over time; (B) Main metabolic parameters. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0041] Example 1 Protective effect of the pharmaceutical composition on myocardial cells

[0042] Myocardial cell death plays an important role in the pathogenesis and development of heart disease, so improving the survival of myocardial cells is beneficial to the treatment of heart disease. In this example, a glucose deprivation (GD)-induced myocardial cell injury model was used to evaluate the protective effect of the drug composition on myocardial cells. Rat myocardial cells (H9c2) were purchased from the stem cell bank of the Chinese Academy of Sciences. Myocardial cells were cultured in high-glucose DMEM (containing 10% fetal bovine serum, 1% penicillin / streptomycin) culture medium and placed in an incubator at 37°C and 5% CO2. After the myocardial cells basically covered the cell culture dish (10 cm), the cells were digested with trypsin and seeded into a 96-well plate at a cell density of 8,000 cells / well, 100 μL per well. When the cells covered the bottom of the dish, the DMEM culture medium was removed and washed once with PBS. The compound was diluted to the target concentration with sugar-free and serum-free DMEM, added to the plate, and treated with GD. After 36–48 hours of GD, cell survival was observed (a survival rate of 50% was considered optimal in the model group to highlight the cytoprotective effect of the compound). Cell viability was measured using CCK8. Cell viability was measured at concentrations of 0–100 μM empagliflozin (EMPA), 0–200 μM L-menthol (MEN), and their combination. Synergy coefficients were calculated by inputting the cell viability of the individual and combined drugs into CompuSyn (http: / / www.combosyn.com). Activity data are shown in Figure 1, A and B. Menthol exhibited strong synergistic effects with empagliflozin at concentrations of 100 μM and 200 μM. In particular, the combination of 50 μM empagliflozin and 200 μM menthol exhibited superior efficacy compared to either drug alone, with a synergy index of 0.01, indicating strong synergy. Furthermore, the onset concentration of empagliflozin alone was 50 μM, but this concentration decreased to 5 μM when combined with menthol. Note: The menthol involved in the following examples is all L-menthol.

[0043] Example 2 Effect of the Preferred Equivalent Ratio of the Pharmaceutical Composition on LDH Release in Cardiac Myocytes and Crystal Violet Staining

[0044] To further validate the anti-heart failure efficacy of the drug combination, H9c2 cells were seeded into 24-well plates. After the cells had completely filled the bottom of the plate, the high-glucose DMEM medium was removed and the cells were washed once with PBS. Empagliflozin alone and various concentrations of the drug combination were prepared in sugar- and serum-free DMEM and added to the cells to induce glucose deprivation. For the GD group, sugar- and serum-free DMEM without the drug was added. For the NG group, high-glucose DMEM with serum was continued. After 48 hours of glucose deprivation, 50 μL of the culture medium was transferred to a 96-well plate, and LDH release in the culture medium was measured using an LDH assay kit. The culture medium was removed, and the cells were placed on ice and washed twice with pre-chilled PBS for 3-5 minutes each. They were then fixed with pre-chilled methanol at -20°C for 10 minutes. The methanol was removed, the cells were allowed to cool to room temperature, and a sufficient volume of 0.5% crystal violet stain was added and incubated at room temperature for 10 minutes. The crystal violet was removed, and the cells were rinsed with water until they did not fade. The cells were placed in a 37°C oven for drying, photographed under a microscope, and stained and quantified using Image J. The larger the purple area, the more surviving cells, and the greater the amount of LDH released, the more cell damage.

[0045] The experiment was divided into 6 groups, namely NG group (no glucose deprivation treatment, always cultured with high glucose DMEM containing serum), GD group (glucose deprivation treatment with glucose-free serum-free DMEM without drugs), E100 group (also known as EMPA-100μM group, glucose deprivation treatment with glucose-free serum-free DMEM containing 100μM empagliflozin), E200 group (also known as EMPA-200μM group, glucose deprivation treatment with glucose-free serum-free DMEM containing 200μM empagliflozin), The experimental results are shown in Figure 1 CE. Compared with the NG group, the cell survival rate in the GD group was significantly reduced, as shown by a decrease in the purple area and an increase in LDH release. Empagliflozin significantly increased cell survival rate in a dose-dependent manner at concentrations of 100 μM (EMPA-100 μM group) and 200 μM (EMPA-200 μM group), as shown by an increase in the purple quantitative area and a decrease in LDH release. The combination of empagliflozin 50μM and menthol 200μM (EMPA-50μM + MEM-200μM group) showed comparable efficacy to empagliflozin at 200μM alone, and after combined administration, empagliflozin exhibited significant efficacy at a concentration of 10μM. This further demonstrates that the combination of empagliflozin and menthol can enhance the efficacy of empagliflozin, reducing its effective concentration by at least 5-fold.

[0046] Example 3 Evaluation of the efficacy of the pharmaceutical composition in an isoproterenol-induced heart failure model

[0047] 3.1 Experimental Principle

[0048] Isoproterenol hydrochloride is a commonly used inducer for establishing heart failure models. Subcutaneous injection of isoproterenol hydrochloride causes mice to experience an increased heart rate, sustained and strong myocardial contractions, increased myocardial oxygen consumption, increased cardiac workload, and ultimately heart failure.

[0049] 3.2 Experimental methods

[0050] The preferred concentrations of 50 μM empagliflozin and 200 μM menthol are approximately 1:1.5 in terms of mass ratio based on the molar molecular weight. Empagliflozin doses are set at 10 mg / kg and 30 mg / kg, and the pharmaceutical composition is set as the 10 mg / kg empagliflozin + 15 mg / kg menthol group. Furthermore, to determine how much the effective dose of empagliflozin can be reduced after combining with menthol, the 5 mg / kg empagliflozin dose was combined with menthol while maintaining the 15 mg / kg dose, creating the 5 mg / kg empagliflozin + 15 mg / kg menthol group.

[0051] Specifically, C57 / 6J male mice with an average body weight of approximately 21-22 g were divided into 7 groups (sham group, ISO group, empagliflozin 30 mg / kg group (E30), empagliflozin 10 mg / kg group (E10), menthol 15 mg / kg group (M15), empagliflozin 10 mg / kg + menthol 15 mg / kg group (E10+M15), empagliflozin 5 mg / kg + menthol 15 mg / kg group (E5+M15)), with 8 mice in each group.

[0052] The drug was administered by gavage every morning, with the solvent being DMSO (content less than 5%, menthol and empagliflozin solution were mixed in equal volumes and then administered to mice by gavage). The sham group and the ISO group were gavaged with equal amounts of drinking water every morning.

[0053] Isoproterenol hydrochloride was injected subcutaneously twice daily, in the morning and evening, with a dose of 40 mg / kg on days 1-2, 20 mg / kg on days 3-7, and 10 mg / kg on days 8-14. The sham group received an equal volume of normal saline injected in the morning and evening every day ( Figure 2A ).

[0054] On day 15, cardiac structure and function were assessed in each group using a Visual-Sonics Vevo 3100 small animal high-resolution micro-ultrasound imaging system. Systolic interventricular septal thickness (IVSs), diastolic interventricular septal thickness (IVSd), left ventricular internal diameter at systole (LVIDs), left ventricular internal diameter at diastole (LVIDd), left ventricular posterior wall thickness at systole (LVPWs), and left ventricular posterior wall thickness at diastole (LVPWd) were measured during three cardiac cycles. Ejection fraction (EF, %) and fractional shortening (FS, %) were calculated. Following the experiment, mice were euthanized, and hearts were fixed, paraffin-embedded, sectioned, and Masson-stained. Fibrosis area was quantified using ImageJ, and the degree of cardiac fibrosis was calculated. Data were processed using GraphPad software, and significant differences were calculated (One-Way ANOVA was used for comparisons between multiple groups, and Student's t test was used for comparisons between two groups).

[0055] 3.3 Experimental Results

[0056] As shown in Figure 2BF, the cardiac function of mice in the ISO group was significantly impaired. The left ventricular septum thickness and posterior wall thickness during systole and diastole were significantly thinner, the inner diameter increased, and the ejection fraction and short-axis shortening rate fell below the normal range (the reference value for normal mice with ejection fraction is 55-85%, and the reference value for normal mice with short-axis shortening is 30-50%). The group receiving empagliflozin at a dose of 30 mg / kg was able to effectively improve the cardiac function impairment caused by isoproterenol hydrochloride, but the dose of 10 mg / kg did not significantly improve cardiac function. Menthol slightly improved the cardiac function of mice with heart failure at a dose of 15 mg / kg, but the difference was not significant. The combination of empagliflozin and menthol can improve the cardiac function of mice in a dose-dependent manner. The cardiac function of mice in the empagliflozin 10 mg / kg + menthol 15 mg / kg group was significantly better than the efficacy of empagliflozin 10 mg / kg alone, and was comparable to the efficacy of empagliflozin 30 mg / kg alone. Empagliflozin 5 mg / kg + menthol 15 mg / kg still showed significant efficacy. In terms of myocardial fibrosis (Figure 2G, H), the area of ​​cardiac fibrosis in the ISO group mice was significantly increased compared with the sham group mice. Empagliflozin 30 mg / kg and empagliflozin 10 mg / kg + menthol 15 mg / kg significantly reduced myocardial fibrosis caused by isoproterenol hydrochloride. Empagliflozin 5 mg / kg + menthol 15 mg / kg also significantly reduced myocardial fibrosis. The above experimental results show that empagliflozin and menthol have a significant synergistic effect, which can reduce the effective dose of empagliflozin in vivo by 6 times, and the efficacy of combined administration is better than that of empagliflozin at the same dose. The combination of empagliflozin and menthol is more effective than either drug alone in the treatment of heart failure.

[0057] Example 4 Evaluation of the efficacy of the pharmaceutical composition in aortic coarctation-induced heart failure model

[0058] 4.1 Experimental Principle

[0059] The transverse aortic constriction model (TAC) is the most commonly used disease model of chronic ventricular hypertrophy, which is used to simulate hypertrophic cardiomyopathy, heart failure, and hypertensive heart disease caused by hypertension or increased intraventricular pressure.

[0060] 4.2 Experimental methods

[0061] Nine-week-old C57 / 6J male mice were divided into five groups: sham group, TAC group, empagliflozin 30 mg / kg group (E30), empagliflozin 10 mg / kg + menthol 15 mg / kg group (E10+M15), and empagliflozin 30 mg / kg + menthol 45 mg / kg group (E30+M45). The mice in the sham group did not undergo surgical modeling, the mice in the TAC group underwent aortic arch constriction but did not receive drug treatment, the mice in the E30 group underwent aortic arch constriction and were treated with 30 mg / kg of empagliflozin, the mice in the E10+M15 group underwent aortic arch constriction and were treated with a combination of 10 mg / kg empagliflozin and 15 mg / kg menthol, and the mice in the E30+M45 group underwent aortic arch constriction and were treated with a combination of 30 mg / kg empagliflozin and 45 mg / kg menthol.

[0062] The experimental procedure for aortic arch constriction surgery: Hair was removed from the surgical area from the neck to the anterior chest of the experimental mouse, and the surgical area was disinfected with iodine. A midline thoracotomy was performed on the neck and chest, and the muscles were bluntly dissected. The incision was opened with a spreader, and the thymus was dissected to expose the aortic arch. Along the aortic arch, the innominate artery, left common carotid artery, and left subosseous artery were identified. A thread was threaded between the right innominate artery and the left common carotid artery. A constriction needle was placed parallel to the aortic arch and tied with a knot. After the thread was tightened, the constriction needle was slowly withdrawn, resulting in a quantitative constriction of the aortic arch. The chest cavity was closed, and the skin of the neck and chest was sutured and disinfected with iodine. Two weeks after surgery, cardiac function testing confirmed the presence of cardiac hypertrophy, and drug administration was initiated for four consecutive weeks. The drug-treated group received oral administration once daily in the morning, while the sham and TAC groups received the same amount of drinking water daily. The experimental procedure is shown in Figure 3A.

[0063] The survival of the mice was recorded during the experiment. After four weeks of continuous drug administration, the mice's exercise tolerance was assessed using the rotarod test, the hanging test, and the grip strength test. The rotarod test assessed the mice's maximum endurance time at 35 rpm, the hanging test assessed the mice's maximum endurance time hanging on their forearms, and the grip strength test assessed the mice's maximum forepaw grip strength. After the exercise tolerance test, the cardiac structure and function of each group of mice were evaluated using the Visual-Sonics Vevo 3100 small animal high-resolution microultrasound imaging system, using the same experimental methods as in 3.2. After the experiment, the mice were euthanized, and the hearts were weighed and the heart-to-body ratio was calculated. The hearts were then fixed, paraffin-embedded, sectioned, and Masson-stained. ImageJ was used to quantify the fibrosis area and calculate the degree of cardiac fibrosis. Graph Pad software was used for data analysis, and significant differences were calculated (One-Way ANOVA was used for comparisons between multiple groups, and Student's t test was used for comparisons between two groups).

[0064] 4.3 Experimental Results

[0065] During the experiment, one mouse died in each of the TAC group and the EMPA-30 mg / kg group, while no mouse died in the other groups (Figure 3B).

[0066] In terms of exercise tolerance (CE in Figure 3), the maximum gripping force of mice in the TAC group was significantly lower than that of mice in the sham group. The maximum rotarod tolerance time and maximum forearm suspension tolerance time were also significantly reduced, indicating that the TAC group had reduced exercise tolerance and weakened motor function, which was largely related to weakened cardiac function. Empagliflozin alone at 30 mg / kg did not improve the exercise tolerance of TAC mice, but the combination with menthol significantly improved the exercise tolerance of mice in a dose-dependent manner. After combined administration, the mice had significantly increased maximum gripping force, maximum rotarod tolerance time, and maximum forearm suspension tolerance time.

[0067] In terms of cardiac hypertrophy (Figure 4A,B), the hearts of mice in the TAC group were significantly enlarged and the heart-to-body ratio increased significantly, indicating that TAC modeling resulted in significant cardiac hypertrophy. Treatment with EMPA-30 mg / kg significantly improved cardiac hypertrophy and reduced the heart-to-body ratio. The effect was more pronounced in combination with menthol in a dose-dependent manner, with the high-dose combination (E30 + M45) showing a significant advantage over EMPA-30 mg / kg alone (P = 0.1123).

[0068] In terms of cardiac function (Figure 4, CG), the left ventricular septum and posterior wall thickness in the TAC group were significantly thinner during systole and diastole, the end-systolic and end-diastolic internal diameters were larger, and the ejection fraction and fractional shortening decreased below the normal range. The 30mg / kg dose of empagliflozin significantly improved TAC-induced cardiac dysfunction. The combination of empagliflozin and menthol significantly improved cardiac function in mice in a dose-dependent manner, and the efficacy of the E30+M45 dose group was significantly superior to that of E30 alone at the same dose.

[0069] Based on the above experimental results, the empagliflozin + menthol combination of the present invention has significant advantages over empagliflozin alone in the treatment of heart failure. In addition to having better efficacy in improving cardiac function and myocardial hypertrophy, it also has a function that empagliflozin alone does not have, namely, improving the exercise tolerance of heart failure mice, which is of great significance for improving the prognosis of heart failure patients.

[0070] Example 5 Pharmaceutical Composition Synergistically Reduces Intracellular Calcium Ion Concentration

[0071] 5.1 Experimental Principle

[0072] Intracellular calcium overload is an important factor that causes myocardial cells to go from reversible damage to irreversible damage. Intracellular calcium overload can be seen in a variety of cardiac pathological conditions, such as cardiac remodeling, myocardial ischemia-reperfusion injury, viral myocarditis, dilated cardiomyopathy, arrhythmias, and congestive heart failure. The inventors found that empagliflozin and menthol have a synergistic effect in regulating intracellular calcium. Empagliflozin can act on the sodium-hydrogen exchanger 1 (NHE1) on the myocardial cell membrane. When the myocardium is ischemic, the energy metabolism of myocardial cells is impaired, and myocardial cells switch from fatty acid metabolism to sugar-free glycolysis, producing a large amount of lactic acid, which lowers the intracellular pH. As a regulatory protein for cellular pH homeostasis, NHE1 can exchange extracellular sodium ions for H + Transport outside the cell, resulting in an increase in the concentration of sodium ions in the cytoplasm. Since the myocardial cells are already lacking ATP at this time, the excess sodium ions in the cell cannot be expelled from the cell through the sodium-potassium exchange pump that depends on ATP, and can only be transported through the sodium-calcium exchanger that does not depend on ATP, further increasing the intracellular calcium ion concentration, causing calcium overload in the cytoplasm and affecting mitochondrial function. When mitochondrial function is damaged, the release of ROS increases, and ROS can further stimulate L-type calcium channels, promoting Ca 2+ Influx of calcium into cells creates a vicious cycle. Empagliflozin inhibits NHE1 activity, indirectly reducing intracellular calcium concentration. Menthol, an L-type calcium channel blocker, can synergistically reduce intracellular calcium concentration with empagliflozin, exerting a synergistic effect. To verify this synergistic mechanism, the intracellular calcium concentration was measured using the calcium probe Fluo4-AM.

[0073] The experiment was divided into 5 groups: control group (no drug group), cariporide group (positive control group, 100 μM), EMPA-50 (empagliflozin 50 μM), M-200 (menthol 200 μM), and E50+M200 (combination of empagliflozin 50 μM and menthol 200 μM).

[0074] 5.2 Experimental steps

[0075] 1. Detection of intracellular calcium concentration under normal intracellular environment:

[0076] (1) Take an appropriate amount of Fluo4-AM stock solution (2 mM, BeyoTime, S1060) and dilute it to a 10 μM working solution with HBSS;

[0077] (2) For the cells to be tested (covering the bottom of the dish), remove the culture medium and wash with HBSS three times;

[0078] (3) Add 25 μL of Fluo4-AM working solution to each well, and then add 25 μL of compound solution to each well;

[0079] (4) Incubate at 37°C for 30 min for fluorescent probe loading;

[0080] (5) The cells were then washed three times with HBSS, and 100 μL of HBSS containing 10 mM CaCl2 was added to each well. The fluorescence of Fluo4-AM was quantitatively detected using a multifunctional fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 520 nm) to determine the changes in intracellular calcium ions.

[0081] 2. Detection of intracellular calcium concentration when the intracellular environment is acidic

[0082] Because empagliflozin indirectly affects intracellular calcium concentration by inhibiting NHE1, the intracellular environment was acidified using ammonium chloride solution to simulate the environment of myocardial ischemia, and the effect of the administered composition on intracellular calcium ions was tested. The NHE1 inhibitor cariporide was used as a positive control.

[0083] The above step (3) was changed to add 25 μL of Fluo4-AM working solution to each well, mix the test compound with 80 mM NH4Cl solution in a 1:1 ratio, and then add 25 μL of the compound and NH4Cl mixed solution to each well (the final NH4Cl concentration was 20 mM). The remaining steps were the same as above.

[0084] 5.3 Experimental Results

[0085] As shown in Figure 5A, in the absence of NH4Cl, cariporide and empagliflozin had almost no effect on intracellular calcium concentration. Menthol had a weak inhibitory effect on intracellular calcium concentration, manifested as a decrease in fluorescence value, and there was no obvious synergistic effect between empagliflozin and menthol. In the presence of NH4Cl, cariporide and empagliflozin could significantly reduce intracellular calcium concentration, indicating that empagliflozin indeed indirectly affects calcium concentration through NHE1. The effect of menthol on calcium was basically the same as in the absence of NH4Cl, with a weak effect of reducing intracellular calcium concentration. The combination of menthol and empagliflozin significantly reduced intracellular calcium concentration, indicating that the two have a synergistic effect in reducing intracellular calcium concentration.

[0086] Example 6 Pharmaceutical Composition Synergistically Promotes Fatty Acid Oxidation

[0087] In this example, the effects of empagliflozin and menthol on fatty acid degradation were evaluated by detecting the fatty acid content in cardiomyocytes and the content of upstream proteins of fatty acid metabolism.

[0088] 6.1 Experimental methods

[0089] 1. Oil Red O staining. H9c2 cells were seeded into 12-well plates (1×10 5 Cells were cultured to 80–90% confluence. Lipid droplet accumulation was induced by incubating the cells in a lipid-rich medium containing 200 μM palmitic acid (Sigma-Aldrich, P0500) for 48 hours. The lipid-rich medium was removed and the cells were cultured for an additional 24 hours with medium containing 50 μM EMPA (E50, EMPA-50), 100 μM EMPA (E100), 200 μM MEN (M200), or 50 μM EMPA plus 200 μM MEN (E50+M200) but without palmitic acid. After incubation, cells were stained with Oil Red. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The cells were then washed three times with PBS and incubated with Oil Red O working solution for 30 minutes at room temperature. After three rinses with PBS, the cells were imaged under a microscope. The red area (positive area for lipid accumulation) was measured using Image J software. The experiment was divided into 6 groups: black group (blank group, not treated with lipid culture medium and drugs), control group (control group, treated with lipid culture medium but not drugs), E50 group (treated with lipid culture medium and then treated with 50 μM EMPA), E100 (treated with lipid culture medium and then treated with 100 μM EMPA), M200 (treated with lipid culture medium and then treated with 200 μM menthol), and E50+M200 (treated with lipid culture medium and then treated with 50 μM empagliflozin + 200 μM menthol).

[0090] 2. Western blotting. H9c2 cells were seeded in 12-well plates. After 48 hours of drug treatment, the cells were harvested and total protein was extracted using RIPA lysis buffer. After BCA quantification, 4× loading buffer was added and the protein was denatured by boiling in a 95°C metal bath for 10 minutes. 5-10 μg of denatured protein sample was added to SDS-PAGE. After electrophoresis, separation, transfer to the membrane, and blocking, primary antibodies against PGC-1α, PPARα, CPT1b, and GAPDH were added and incubated overnight at 4°C. The next day, the primary antibodies were recovered and the corresponding secondary antibodies were added and incubated at room temperature for another 1 hour. After washing three times, ECL chemiluminescent solution was added to the bands, and immunoblotting analysis was performed using a Tanon gel imager. Grayscale analysis of the immunoblot bands was performed using ImageJ software, and GAPDH was used as the internal reference for correction. The relative expression levels of the proteins were calculated. The experiment was divided into DMSO group (DMSO treatment group with content not exceeding 0.5%), E50 (50 μM EMPA treatment group), M200 (200 μM menthol treatment group), and E50+M200 (50 μM empagliflozin+200 μM menthol treatment group).

[0091] 6.2 Experimental Results

[0092] As shown in Figure 5B and C, when empagliflozin and menthol are used alone, they can significantly reduce lipid accumulation in cardiomyocytes and promote fatty acid degradation, as shown by a reduction in the area of ​​Oil Red O staining. When the two drugs are combined (E50+M200), the effect of promoting fatty acid degradation is more significant and significantly better than the effect of the two single drugs at the same concentration. Menthol alone and the combination of menthol and empagliflozin can significantly increase the expression of PCG-1α / PPARα, an upstream protein of fatty acid metabolism, and upregulate the expression of CPT1b, a fatty acid transporter. Empagliflozin may promote fatty acid metabolism by increasing the activity of fatty acid oxidase. The above experiments show that empagliflozin and menthol can achieve a synergistic anti-heart failure effect by synergistically promoting fat metabolism.

[0093] Example 7 Safety Evaluation of Pharmaceutical Composition

[0094] To evaluate the safety of the pharmaceutical composition, a 14-day repeated administration toxicity test in mice was conducted.

[0095] 7.1 Experimental Methods

[0096] ICR mice (7-8 weeks) were purchased from Shanghai JSJ Laboratory Animal Co., Ltd. Mice were divided into two groups (n = 10, half male and half female): a control group (saline-treated) and a group treated with EMPA-100 mg / kg + MEN-150 mg / kg (E100 + M150) (100 mg / kg, gavage). Administration was performed once daily by gavage for 14 days. Observations focused on mortality and changes in behavior, skin, eyes, fur, and body locomotor activity, and body weight was monitored daily. After 14 days, mice were euthanized, and the heart, liver, spleen, lung, and kidney were removed for external evaluation, and organ weight (organ weight / body weight) was calculated. Liver and kidney tissues were fixed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin for histopathological analysis. Blood was collected from the orbital venous plexus, and serum was centrifuged for biochemical analysis, including creatinine, urea, alanine aminotransferase (ALT), and aspartate aminotransferase (AST).

[0097] 7.2 Experimental Results

[0098] Mice treated with EMPA-100mg / kg + MEN-150mg / kg showed no significant changes in daily food intake or body weight compared to the control group (Figure 6A). No mice died during the dosing period, no adverse reactions occurred, and no macroscopic lesions were observed in any organ, with no significant changes in body structure compared to the control group (Figure 6B). Serum biochemical markers (AST, ALT, Cr, UREA) showed no significant abnormalities (Figure 6C), and no significant pathological damage to the heart, liver, spleen, or kidneys was observed in HE staining images of the mice (Figures 6D and E). These experiments demonstrate the good safety profile of EMPA-100mg / kg + MEN-150mg / kg. Combined with the effective dose of EMPA-10mg / kg + MEN-15mg / kg in mice, the drug-delivered combination has a safe therapeutic window of at least 10 times, demonstrating its potential for successful drug development.

[0099] Example 8 Evaluation of Pharmacokinetic Properties of Pharmaceutical Compositions

[0100] In order to explore the pharmacokinetic properties of the combination of empagliflozin and menthol, this example tested the plasma kinetics of mice orally administered empagliflozin alone (100 mg / kg) and the combination of empagliflozin and menthol (EMPA-100 mg / kg + MEN-150 mg / kg), and the drug detected in the plasma was empagliflozin.

[0101] The experimental results (A and B in Figure 7) show that after mice were given a combination of empagliflozin and menthol, the degree of empagliflozin absorption and the drug metabolism rate in the body were basically the same as those of mice given empagliflozin alone, indicating that menthol has little effect on the absorption and metabolism of empagliflozin and there is no adverse drug-drug interaction.

Claims

1. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises: (a) an SGLT2 inhibitor, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, or a prodrug ester thereof; (b) menthol, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate thereof, a solvate thereof, or a prodrug ester thereof.

2. The pharmaceutical composition according to claim 1, It is characterized in that The SGLT2 inhibitor is selected from the following group: empagliflozin, dapagliflozin, canagliflozin, isagliflozin, rupagliflozin, togliflozin, sogliflozin, erpagliflozin, repagliflozin, empagliflozin, ganagliflozin, bepagliflozin, enagliflozin, ganagliflozin, tigliflozin.

3. The pharmaceutical composition according to claim 1, It is characterized in that The menthol is selected from the group consisting of L-menthol, D-menthol, and (DL)-menthol.

4. The pharmaceutical composition according to claim 1, It is characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 1, It is characterized in that The molar ratio of the SGLT2 inhibitor to the menthol is 1-10000:1-10000; or the mass ratio of the SGLT2 inhibitor to the menthol is 1-10000:1-10000.

6. The pharmaceutical composition according to claim 1, It is characterized in that The dosage form of the pharmaceutical composition is oral liquid, tablet, pill, powder, capsule, injection or granule.

7. The pharmaceutical composition according to claim 1, It is characterized in that The pharmaceutical composition comprises empagliflozin and L-menthol.

8. The pharmaceutical composition according to claim 1, It is characterized in that The molar ratio of empagliflozin to L-menthol is 1-400:1-400; or the mass ratio of empagliflozin to L-menthol is 1-400:1-400.

9. The use of the pharmaceutical composition according to claim 1, It is characterized in that Used to prepare medicines for treating heart diseases.

10. The use according to claim 9, It is characterized in that The heart disease is selected from the group consisting of heart failure, coronary heart disease, myocardial infarction, myocarditis and myocardial disease, hypertensive heart disease, arrhythmia, and dilated cardiomyopathy.

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