Application of glutathione peroxidase mimetics in treatment of ejection fraction retention type heart failure

Abstract

So far, there is no specific treatment method for reducing the morbidity and mortality of patients with ejection fraction retention type heart failure (HFpEF). The disclosure proves that the glutathione peroxidase simulant is expected to be used as a clinical treatment drug to improve the life quality of HFpEF patients, and the glutathione peroxidase simulant has good application prospects and clinical values.

Description

Technical Field

[0001] The present application relates to the field of medicine, and specifically to the application of glutathione peroxidase mimics in the treatment of heart failure with preserved ejection fraction. Background Art

[0002] Heart failure with preserved ejection fraction (HFpEF) refers to the presence of symptoms and signs of heart failure, but a normal or near-normal left ventricular ejection fraction (LVEF) (LVEF ≥ 50%). It is characterized by diastolic dysfunction and is a syndrome involving multiple organs and systems (Sharma K, Kass DA. Heart failure with preserved ejection fraction: mechanisms, clinical features, and therapies. Circ Res. 2014 Jun 20;115(1):79-96). Among all types of heart failure (HF), HFpEF accounts for at least 50%. The 5-year all-cause mortality rate of HFpEF > 50%, and the readmission rate within 1 year > 50%. Nearly 50% of HFpEF patients have 5 or more comorbidities, such as obesity, diabetes, pulmonary hypertension, etc., so it is heterogeneous and complex. So far, the pathophysiological mechanism of HFpEF is not fully understood, so it has always been a difficult point in the treatment of heart failure.

[0003] Clinical drugs effective in treating heart failure with reduced ejection fraction (HFrEF), such as angiotensin-converting enzyme inhibitors (ACEI) (Cleland JG, Tendera M, Adamus J, et al. The perindopril in elderly people with chronic heart failure (PEP-CHF) study. Eur Heart J. 2006;27(19):2338-2345.), angiotensin receptor inhibitors (Massie BM, Carson PE, McMurray JJ, et al. Irbesartan in patients with heart failure and preserved ejection fraction. N Engl J Med. 2008;359(23):2456-246.), angiotensin receptor / neprilysin inhibitors (ARNI) (Solomon SD, McMurray JJV, Anand IS, et al. Angiotensin-Neprilysin Inhibition in Heart Failure with Preserved Ejection Fraction. N Engl J Med. 2019;381(17):1609-1620.), β-blockers (Cleland JGF, Bunting KV, Flather MD, et al. Beta-blockers for heart failure with reduced, mid-range, and preserved ejection fraction: an individual patient-level analysis of double-blind randomized trials. Eur Heart J. 2018;39(1):26-35), etc., have not effectively reduced clinical endpoints in large randomized controlled trials of HFpEF. Therefore, drugs that can be used to treat HFrEF may not necessarily be used to treat HFpEF. Trials of other types of drugs (such as drugs targeting the nitric oxide-cyclic guanosine monophosphate pathway) have failed to improve the clinical condition or have been neutral in terms of the primary endpoint. The 2022 ESC Heart Failure Guidelines and the 2022 AHA / ACC / HFSA Guidelines recommend sodium-glucose cotransporter 2 (SGLT2) inhibitors as a class IIa recommendation for patients with HFpEF.In the three clinical trials of EMPEROR-Preserved, DELIVER, and SOLOIST-WHF DELIVER, the SGLT2 inhibitor effectively reduced the composite endpoint of cardiovascular death or hospitalization for heart failure, becoming the only drug currently proven to improve the prognosis of patients with HFpEF. However, in these clinical trials, only some of the heart failure patients included had an ejection fraction ≥ 50%. As the LVEF increased, the effect of the drug in reducing mortality weakened (Anker SD, Butler J, Filippatos G, et al. Empagliflozin in Heart Failure with a Preserved Ejection Fraction. N Engl J Med. 2021;385(16):1451-1461.). It can be seen that the SGLT2 inhibitor only benefits some patients with HFpEF and is not an effective treatment strategy.

[0004] From the results of the above clinical trials, it can also be found that although HFpEF is one of the classifications of heart failure, due to its relatively special and complex pathogenesis, the traditional drugs available for treating heart failure have very limited therapeutic effects on HFpEF. Therefore, so far, there is no specific treatment method for reducing the morbidity and mortality of patients with HFpEF, and it is very necessary to explore a drug that can effectively treat HFpEF.

[0005] It should be noted that the methods described in this section are not necessarily methods that have been previously conceived or adopted. Unless otherwise specified, no method described in this section should be considered prior art solely because it is included in this section. Similarly, unless otherwise specified, the problems mentioned in this section should not be considered to have been recognized in any prior art. Summary of the Invention

[0006] To solve the above technical problems, the present application provides the use of glutathione peroxidase mimetics in the preparation of a drug or pharmaceutical composition for preventing, alleviating or treating heart failure with preserved ejection fraction.

[0007] According to an embodiment of the present application, there is also provided the use of glutathione peroxidase mimetics in the preparation of a drug or pharmaceutical composition for improving cardiac diastolic function, improving pathological ventricular hypertrophy, improving left ventricular volume reduction, improving hypertension symptoms, improving glucose tolerance disorder, improving liver lipid accumulation, reducing the weight of obese heart failure patients, and reducing the fat content of obese heart failure patients.

[0008] According to an embodiment of the present application, there is also provided a method for preventing, alleviating or treating heart failure with preserved ejection fraction, the method comprising administering to a subject in need thereof a drug or pharmaceutical composition comprising a glutathione peroxidase mimetic.

[0009] According to an embodiment of the present application, there is also provided a method for improving cardiac diastolic function, improving pathological ventricular hypertrophy, improving left ventricular volume reduction, improving hypertension symptoms, improving glucose tolerance impairment, improving hepatic lipid accumulation, reducing the body weight of patients with obese heart failure, and reducing the fat content of patients with obese heart failure, the method comprising administering to a subject in need thereof a drug or pharmaceutical composition comprising a glutathione peroxidase mimetic.

[0010] By constructing an HFpEF animal model, the applicant found that the glutathione peroxidase mimetic can treat HFpEF and improve various clinical symptoms caused by HFpEF. The disclosure of the present application confirms that the glutathione peroxidase mimetic is expected to be used as a clinical therapeutic drug in the future to improve the quality of life of HFpEF patients, and it has good application prospects and clinical value.

[0011] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the present application, nor is it used to limit the scope of the present application. Other features of the present application will become readily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings exemplarily show the embodiments and form a part of the specification, and are used together with the written description of the specification to explain the exemplary embodiments of the embodiments. The shown embodiments are for illustrative purposes only and do not limit the scope of the claims. In all the drawings, the same reference numerals refer to similar but not necessarily identical elements.

[0013] Figure 1 It is a therapeutic effect diagram of the glutathione peroxidase mimetic on mice with heart failure with preserved ejection fraction in Example 1. Among them, Figure 1 A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L are respectively the result diagrams of body weight, food intake, heart weight, lean body mass, fat content, echocardiogram, ejection fraction, E / E’, interventricular septum thickness, left ventricular diastolic diameter, systolic blood pressure, and glucose tolerance of the model mice before and after treatment.

[0014] Figure 2 It is the cardiac tissue pathological WGA staining ( Figure 2 A, Figure 2 B) and hepatic tissue pathological HE staining (Figure 2 C) and pathological HE staining of white adipose tissue ( Figure 2 C, Figure 2 D) result diagrams. Detailed implementation manners

[0015] Unless otherwise specified, all numbers representing contents, concentrations, ratios, masses, volumes, times, temperatures, thicknesses, technical effects, etc. used in this specification and claims shall be understood to be modified by the term "about" or "substantially" in any case. Therefore, unless there is a contrary indication, the numerical parameters listed in the following specification and appended claims are approximate values. For those skilled in the art, they can vary according to the desired properties and effects sought through this disclosure and should be interpreted according to the number of significant digits and the conventional rounding method or the way understood by those skilled in the art for each numerical parameter.

[0016] Although the numerical ranges and parameters of the broad scope of this disclosure are approximate values, the values set forth in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain certain errors, which are necessarily caused by the standard deviations found in their corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that broader numerical range, as if these narrower numerical ranges were all explicitly written herein.

[0017] Unless otherwise specified or inconsistent with the context, the terms or expressions used herein shall be read in conjunction with the overall content of this article and as understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0018] When used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0019] When used herein, the terms "alleviate", "treat" and their synonyms refer to the improvement of diseases, disorders and / or conditions. "Alleviate" and "treat" can be the improvement of at least one measurable physical parameter, which may not necessarily be recognizable by the patient. "Alleviate" and "treat" can also physically (e.g., stabilize recognizable symptoms), physiologically (e.g., stabilize physical parameters) or both inhibit the development of diseases, disorders and / or conditions. "Alleviate" and "treat" can also slow down or reverse the development of diseases, disorders and / or conditions.

[0020] As used herein, the term "prevention" and its synonyms refer to delaying the onset of a particular disease, disorder, and / or condition or symptoms associated with these diseases, disorders, and / or conditions or reducing the risk of acquiring these diseases, disorders, and / or conditions.

[0021] As used herein, the term "pharmaceutical composition" refers to the glutathione peroxidase mimetic described in this application and other components such as pharmaceutically or physiologically acceptable carriers and / or pharmaceutically acceptable excipients.

[0022] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier, diluent, or adjuvant used for the formulation or administration of a glutathione peroxidase mimetic, which is not itself an essential active ingredient and has no excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art and include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20.

[0023] As used herein, the term "physiologically acceptable carrier" refers to a carrier, diluent, or adjuvant that does not cause significant irritation to the organism and does not eliminate the pharmaceutical activity and properties of the administered glutathione peroxidase mimetic. Suitable physiologically acceptable carriers are also well known to those of ordinary skill in the art.

[0024] To make the above objects, features, and advantages of the present application more obvious and understandable, the following detailed description of the specific embodiments of the present application is provided.

[0025] According to an embodiment of the present application, there is provided the use of a glutathione peroxidase mimetic in the preparation of a drug or pharmaceutical composition for preventing, alleviating, or treating heart failure with preserved ejection fraction.

[0026] According to an embodiment of the present application, there is also provided the use of a glutathione peroxidase mimetic in the preparation of a drug or pharmaceutical composition for improving cardiac diastolic function, improving pathological ventricular hypertrophy, improving left ventricular volume reduction, improving hypertension symptoms, improving glucose tolerance impairment, improving liver lipid accumulation, reducing the body weight of patients with obese heart failure, and reducing the fat content of patients with obese heart failure.

[0027] According to an embodiment of the present application, there is also provided a method for preventing, alleviating, or treating heart failure with preserved ejection fraction, the method comprising administering to a subject in need thereof a drug or pharmaceutical composition comprising a glutathione peroxidase mimetic.

[0028] According to an embodiment of the present application, there is also provided a method for improving cardiac diastolic function, improving pathological ventricular hypertrophy, improving left ventricular volume reduction, improving hypertension symptoms, improving glucose tolerance impairment, improving hepatic lipid accumulation, reducing the body weight of patients with obese heart failure, and reducing the fat content of patients with obese heart failure. The method includes administering to a subject in need thereof a drug or pharmaceutical composition comprising a glutathione peroxidase mimetic.

[0029] In some embodiments, the glutathione peroxidase includes glutathione peroxidase 1 (GPx1). Glutathione peroxidase 1 is an important cellular antioxidant enzyme present in the cytoplasm and mitochondria of mammalian cells and can regulate the balance between the necessary and harmful levels of reactive oxygen species.

[0030] In some specific embodiments of the present application, ebselen is used as a representative of GPx mimetics. Other suitable GPx mimetics include, but are not limited to: MitoEbselen-2chloride, cyclic selenenylamides having Se-N bond, diaryl diselenides, aromatic or aliphatic monoselenides, tellurium compounds. Since these GPx mimetics have the same target or similar mechanism of action, those skilled in the art can learn from the disclosure of the present application that other suitable GPx mimetics can also achieve the technical effects of the present application.

[0031] In some preferred embodiments, the glutathione peroxidase mimetic includes at least one of ebselen or its stereoisomers, racemates, solvates, isotope derivatives, or pharmaceutically acceptable salts. In some embodiments, the structure of ebselen includes the compound structure shown in formula (I):

[0032]

[0033] Among them, the definitions of terms such as stereoisomers, racemates, solvates, isotope derivatives, or pharmaceutically acceptable salts are well known to those skilled in the art. From the disclosure of the present application, those skilled in the art can learn that any configuration, crystal form, or salt form of ebselen can achieve the technical effects of the present application. Therefore, any variants such as any configuration, crystal form, or salt form having the activity of ebselen are within the protection scope of the present application and are not limited herein.

[0034] In some embodiments, the left ventricular ejection fraction (LVEF) of the patients with heart failure with preserved ejection fraction is greater than or equal to 50%.

[0035] In some embodiments, the prevention, alleviation or treatment of heart failure with preserved ejection fraction includes at least one of improving cardiac diastolic function, improving left ventricular volume reduction, improving pathological ventricular hypertrophy, improving hypertension symptoms, improving glucose intolerance, improving hepatic lipid accumulation, reducing the body weight of patients with obesity-related heart failure, and reducing the fat content of patients with obesity-related heart failure.

[0036] In some embodiments, the prevention, alleviation or treatment of heart failure with preserved ejection fraction includes at least one of the following groups: (1) reducing the E / E’ ratio to improve cardiac diastolic function; (2) increasing the left ventricular end-diastolic diameter (LVID,d) to improve left ventricular volume reduction; (3) reducing the end-diastolic interventricular septum thickness (IVS,d) to improve pathological ventricular hypertrophy; (4) reducing the cardiomyocyte area to improve pathological ventricular hypertrophy; (5) reducing the ratio of heart weight to tibia length to improve pathological ventricular hypertrophy; (6) reducing systolic blood pressure to improve hypertension symptoms; (7) improving glucose intolerance; (8) improving hepatic lipid accumulation; (9) reducing the body weight of patients with obesity-related heart failure; and / or (10) reducing the fat content of patients with obesity-related heart failure. Wherein, E is the peak blood flow velocity through the mitral valve in early diastole, and E’ is the peak velocity of mitral annulus early diastolic motion.

[0037] In the application described in the present application, the GPx mimetic can be administered in the form of a single drug or in the form of a pharmaceutical composition.

[0038] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable carrier. The carrier can be any compatible physiologically acceptable non-toxic substance suitable for delivering the GPx mimetic provided in the present application to the gastrointestinal tract of mammals (such as humans).

[0039] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. In some embodiments, the excipients include at least one of solubilizers, disintegrants, wetting agents, stabilizers, thickeners, diluents, buffers, and flavoring agents.

[0040] In some non-limiting embodiments, the carrier and / or excipients for the pharmaceutical composition used in the present application may comprise, for example, liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending agents, dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0041] For further illustration, aqueous vehicles may include, such as, sodium chloride injection, Ringer's injection, isosmotic dextrose injection, sterile water injection or dextrose and lactated Ringer's injection; non-aqueous vehicles may include, such as, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil; antimicrobial agents include phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl paraben, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents may include, such as, sodium chloride or dextrose; buffering agents may include, such as, phosphate or citrate buffers; antioxidants may include, such as, sodium bisulfate; suspending and dispersing agents may include, such as, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone; chelating agents may include, such as, ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Suitable excipients may include, for example, water, normal saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0042] In the applications described in the present application, the GPx mimetic can be formulated into any suitable dosage form (e.g., liquids, capsules, sachets, hard capsules, soft capsules, tablets, enteric-coated tablets, suspension powders, granules or matrix sustained-release formulations) for oral administration, and administered in any suitable manner (e.g., orally, by injection, immediate release, pulsed release, delayed release, sustained release). The dosage form of the pharmaceutical composition will depend on the intended mode of administration, and any suitable dosage form is well known to those skilled in the art. In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solutions, powders, powders, tablets, dragees, capsules, granules, suspensions, syrups, drops, sublingual tablets.

[0043] In some specific embodiments of the present application, the GPx mimetic is formulated for oral administration. In some non-limiting embodiments, the GPx mimetic can be directly incorporated into the diet of the subject; it can also be enclosed in a hard or soft shell capsule or compressed into a tablet and then incorporated into the diet of the subject; it can also be orally administered, for example, together with an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the compound can be mixed with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0044] In the applications described in the present application, the dosage of the GPx mimetic can depend on several factors, including the severity and responsiveness of the disease, the route of administration, the duration of treatment (from a few days to several months to several years), and the time to disease improvement. Those skilled in the art can adjust the dosage regimen according to the specific situation of the patient to provide a therapeutic response. For example, a single dose can be administered, several separate doses can be administered over a predetermined period of time, or the dose can be decreased or increased as indicated by the treatment situation. The specification of the dose is determined by the unique characteristics of the active compound and the specific therapeutic effect to be achieved. The dose value can vary with the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the treating clinician.

[0045] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments thus formed are regarded as part of the disclosure of the present application.

[0046] Exemplary embodiments of the present application will be described below in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered to be merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined only by the claims. Therefore, those of ordinary skill in the art should recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0047] Example

[0048] For those not specified with specific techniques or conditions in this embodiment, follow the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0049] I. Experimental Instruments and Reagents

[0050] Table 1 Biochemical Reagents and Kits

[0051]

[0052]

[0053] Table 2 Experimental Instruments

[0054]

[0055] II. Experimental Animals

[0056] Male wild-type mice (C57BL / 6J) were all purchased from Beijing Huafukang Bioscience Co., Ltd. All animals were raised and bred in the SPF-level environmental animal house of Beijing Institute of Heart, Lung and Blood Vessel Diseases. The wild mice should be male mice at 10-12 weeks of age and weighing about 25-30 g. All experimental operations were carried out according to the "Guide for the Care and Use of Laboratory Animals" formulated by NIH in 1996 and the experimental procedures stipulated by the Laboratory Animal Management Committee of Capital Medical University. All experimental animals were grouped by random method.

[0057] III. Experimental Methods

[0058] 1. Measurement of animal body composition

[0059] A QMR06-090H-PRO small animal body composition analyzer (Suzhou Newmai Analytical Instruments Co., Ltd., China) was used for measurement and data collection. First, the body weight of the mouse was weighed, the mouse number and weight were entered on the instrument screen, and the mouse was fixed in the analyzer. Using the principle of low-field nuclear magnetic resonance, the fat mass, lean mass and their corresponding content ratios of the mouse were measured.

[0060] 2. Non-invasive blood pressure measurement

[0061] A smart non-invasive blood pressure monitor BP-2010A (Beijing Ruanlong Biotechnology Co., Ltd., China) was used for blood pressure measurement. The process was as follows: In a standardized animal house, the pulse-type tail cuff pressure measurement system was turned on. After calibration, the mouse was placed in a special cage for pressure measurement and placed on a thermostatic blanket (37 °C) for 5-10 min. After the mouse was in a stable state, the tail pulse of the mouse was sensed through the tail pulse sensor. After the pulse was stable, blood pressure measurement began. During the measurement process, the temperature of the thermostatic blanket and the number of pressure measurements could be appropriately adjusted according to the mouse state. The BP value (≥6 times) was read through the instrument, the highest and lowest values were removed, and the average value was taken as the final BP value of the mouse.

[0062] 3. Intraperitoneal injection glucose tolerance test

[0063] After the mice fasted for 12 h, the fasting blood glucose level (mmol / L) was first measured by a blood glucose meter using the tail vein blood sampling method, and then the blood glucose was measured again at 15, 30, 60, 90, and 120 minutes after intraperitoneal injection of glucose (2 g / kg dissolved in sterile water).

[0064] 4. Small animal ultrasound

[0065] At 0, 10 weeks after the construction of the HFpEF model and 5 weeks after drug administration, cardiac ultrasound data were collected using a small animal ultrasound Vevo2100 high-resolution imaging instrument (Vevo2100; Visual Sonics). The process was as follows: Mice were anesthetized with isoflurane (2%) in 100% oxygen, and the heating plate was maintained at 37°C to keep the heart rate within the range of 400 - 500 beats / min. The chest hair of the mice was removed using depilatory cream, and coupling agent was applied. An M-mode ultrasound of the left ventricular outflow tract of the mouse heart was collected using an ultrasound probe, and LVEF and other systolic function indexes were obtained by scanning in the short-axis M-mode at the ventricular level; pulsed wave and tissue Doppler imaging were used to obtain the apical four-chamber view of the anesthetized mice at the mitral valve level to measure diastolic function. Subsequently, the left ventricular end-diastolic diameter (LVIDd), end-diastolic interventricular septum thickness (IVSd), left ventricular ejection fraction (LVEF), peak blood flow velocity through the mitral valve in early diastole (E wave), peak blood flow velocity caused by atrial contraction in late diastole (A wave), and peak velocity of early diastolic movement of the mitral annulus (E' wave) were calculated through analysis software. All parameters were measured at least 3 times and the mean values were given.

[0066] 5. Cardiac wheat germ agglutinin (WGA) staining

[0067] Place the baked slides in a microwave oven, then add the prepared sodium citrate solution for antigen retrieval. Heat in the microwave at high power until boiling for 10 minutes (avoid drying out), then place at room temperature and wait for the temperature to slowly drop to room temperature (do not use running water to cool, as it is likely to reduce the antigen retrieval effect). After drying the tissue edges, block with goat serum for blocking or 5% BSA for 1 hour, then wash with PBS for 5 minutes, 3 times. Dilute the 100X WGA stock solution to 1X, and add about 100 μL of the diluted WGA to each slide. Place in a wet box and incubate in the dark for 1 hour, then wash with PBS for 5 minutes, 3 times. Then mount with DAPI and immediately take pictures under the microscope in the dark.

[0068] 6. Hematoxylin-eosin (HE) staining of liver and adipose tissue

[0069] (1) Take out the paraffin sections and wash 3 times with 1×PBS, 5 minutes each time;

[0070] (2) Immerse in absolute ethanol (I) for 5 minutes; absolute ethanol (I) for 5 minutes; 80% ethanol for 5 minutes; rehydrate in a gradient of distilled water for 5 minutes;

[0071] (3) Stain with hematoxylin solution for 5 minutes; rinse off the hematoxylin solution with running water for 1 - 3 seconds;

[0072] (4) Immerse in 1% hydrochloric acid ethanol for 1 - 3 seconds; rinse slightly with water for 10 - 30 seconds; wash with distilled water for 1 - 2 seconds;

[0073] (5) Stain with 0.5% eosin solution for 1 - 3 min; Wash with distilled water for 1 - 2 s;

[0074] (6) Pass the slide through 80% ethanol for 1 - 2 s; 95% ethanol (I) for 2 - 3 s; 95% ethanol (II) for 3 - 5 s; absolute ethanol for 5 - 10 min for dehydration;

[0075] (7) Clear with xylene (I) for 2 min; xylene (II) for 2 min; xylene (III) for 2 min, and finally mount with neutral balsam.

[0076] Example 1: Construction of a heart failure with preserved ejection fraction model (HFpEF "two-hit" mouse model)

[0077] 1.1 Model construction method

[0078] Randomly divide male C57BL / 6J mice aged 8 - 12 weeks into two groups, place them in the animal room for feeding, maintain a 12 - hour light / dark cycle, and allow free access to food and water.

[0079] One group is the control group (n = 12): Given normal diet and water;

[0080] The other group is the HFpEF group (n = 20): Given a high - fat diet (D12492), and dissolve L - NAME in drinking water at a concentration of 0.5 g / L.

[0081] 1.2 Model evaluation method and results

[0082] The phenotypic evaluation plan for constructing the HFpEF model is as follows:

[0083] (1) At the 0th and 10th weeks of feeding, measure the body weights of the two groups of mice.

[0084] (2) At the 0th and 10th weeks of feeding, perform small - animal cardiac ultrasound on the two groups of mice to measure cardiac function conditions such as the cardiac ejection fraction and the degree of left ventricular diastolic dysfunction.

[0085] (3) At the 0th and 10th weeks of feeding, measure the blood pressure of the two groups of mice using the tail - cuff plethysmography method to measure the blood pressure changes.

[0086] (4) At the 0th and 10th weeks of feeding, perform an intraperitoneal glucose tolerance test on the two groups of mice to measure the glucose tolerance.

[0087] At the 10th week of feeding, the following phenomena can be observed:

[0088] (1) The body weight of the HFpEF group is significantly increased compared with the control group, showing an obese state;

[0089] (2) In the HFpEF group, the left ventricular ejection fraction was ≥ 50%, indicating preserved ejection fraction; in the HFpEF group, E / E’ ≥ 40, indicating left ventricular diastolic dysfunction.

[0090] (3) In the HFpEF group, the systolic blood pressure was ≥ 130 mmHg, indicating hypertension.

[0091] (4) Glucose intolerance occurred more frequently in the HFpEF group than in the control group.

[0092] The above experimental phenomena indicate successful model construction.

[0093] Example 2: Therapeutic effect of administering a GPx mimetic (ebselen) on heart failure with preserved ejection fraction

[0094] 2.1 Animal grouping and administration

[0095] The glutathione peroxidase mimetic ebselen was ordered from MCE (USA), and the catalog number is HY-13750. Using the successfully constructed HFpEF mouse model as in Example 1, the HFpEF group of mice that had been continuously fed for 10 weeks was randomly divided into two groups:

[0096] (1) HFpEF group (n = 10): Continued to be fed a high-fat diet (D12492), and L-NAME was dissolved in drinking water at a concentration of 0.5 g / L.

[0097] (2) GPx mimetic treatment group (n = 10): Continued to be fed a high-fat diet (D12492), and L-NAME was dissolved in drinking water at a concentration of 0.5 g / L. At the same time, ebselen was mixed in the high-fat diet feed and orally administered at a dose of 10 mg / kg / d.

[0098] At the same time, wild-type mice without the constructed HFpEF model were used as the control group (n = 10), and were given ordinary drinking water and feed.

[0099] 2.2 Detection of treatment effect

[0100] After 5 weeks of continuous experiment, the treatment effect was detected, and the observation indicators included:

[0101] (1) After 5 weeks of administration, small animal body composition analysis was performed on the mice to measure the changes in total body weight, lean body mass (fat-free body mass), and fat content. Among the detection indicators, the total body weight was divided into two components: fat and non-fat. The former is called fat body weight (or fat mass), and the latter is called lean body mass (or fat-free body mass). In this experiment, the lean body mass index was detected.

[0102] (2) After 5 weeks of administration, small animal cardiac ultrasound was performed on the mice to measure cardiac function such as the cardiac ejection fraction and the degree of left ventricular diastolic dysfunction.

[0103] (3) After 5 weeks of drug administration, the blood pressure of the mice was measured using the pulse tail-cuff method to measure the changes in blood pressure.

[0104] (4) After 5 weeks of drug administration, an intraperitoneal glucose tolerance test was performed on the mice to measure glucose tolerance.

[0105] (5) After 5 weeks of drug administration, the hearts of all the mice were collected, the heart weight was measured, and the tibia length of the mice was measured. The heart tissue was stained with WGA to observe the changes in the size of cardiomyocytes, and the liver tissue was collected for pathological staining (HE staining) to observe the changes in liver lipid accumulation. The white adipose tissue was collected for pathological staining (HE staining) to observe the changes in the size of adipose tissue area.

[0106] 2.3 Analysis of experimental results

[0107] The results showed that the phenotype of HFpEF mice was significantly improved after treatment with the GPx mimetic (ebselen).

[0108] Specifically:

[0109] (1) The body weight of HFpEF mice treated with ebselen for 5 weeks was significantly improved ( Figure 1 A), and the food intake returned to the original level similar to that of the HFpEF group after a significant decrease during the first week of adaptation ( Figure 1 B);

[0110] (2) The ratio of heart weight to tibia length (HW / TL) was significantly decreased ( Figure 1 C);

[0111] (3) The ratio of lean body mass (fat-free body mass) to fat content was significantly improved ( Figure 1 D, E);

[0112] (4) The results of echocardiography showed ( Figure 1 F, the upper, middle, and lower parts are representative M-mode echocardiogram of the left ventricle (upper), pulsed wave Doppler map (middle), and tissue Doppler map (lower)), the ejection fraction (LVEF) of the three groups of mice in the control group, HFpEF group, and ebselen treatment group was > 50%, at the normal level, showing preserved ejection fraction ( Figure 1 G); At the same time, compared with the HFpEF group, the diastolic function of the mice in the ebselen treatment group was restored to near normal (significantly decreased E / E') ( Figure 1 H), and the end-diastolic interventricular septum thickness (IVS,d) was significantly decreased after ebselen treatment ( Figure 1 I), and the left ventricular end-diastolic diameter (LVID,d) was significantly increased ( Figure 1 J).

[0113] (5) The systolic blood pressure (SBP) of mice in the ebselen treatment group was significantly decreased ( Figure 1 K);

[0114] (6) Glucose tolerance impairment was significantly improved ( Figure 1 L).

[0115] (7) WGA staining of the heart (at 400x magnification) showed that the area of cardiomyocytes was significantly reduced and myocardial hypertrophy was alleviated after ebselen treatment ( Figure 2 A, B).

[0116] (8) HE staining of the liver (at 100x magnification) showed that lipid accumulation in the liver was significantly reduced after ebselen treatment and was significantly restored to normal ( Figure 2 C).

[0117] (9) HE staining of adipose tissue (at 100x magnification) showed that the area of adipocytes was reduced after ebselen treatment ( Figure 2 C, D).

[0118] In summary, the GPx mimetic (ebselen) can significantly improve various clinical symptoms of the HFpEF model, including: improving cardiac diastolic function, improving left ventricular volume reduction, improving pathological ventricular hypertrophy, improving hypertension symptoms, improving glucose tolerance impairment, improving liver lipid accumulation, reducing the body weight of individuals with obese heart failure, reducing the fat content of individuals with obese heart failure, etc.

[0119] It should be stated that the above are only the preferred embodiments of this application and are not used to limit this application. For those skilled in the art, this application can have various changes and modifications. Although the specific implementation manners have been described, for the applicant or other skilled persons in this field, there may be or currently unforeseen alternatives, modifications, changes, improvements, and substantial equivalents to the above implementation manners. Therefore, the appended claims submitted and the claims that may be modified are intended to cover all such alternatives, modifications, changes, improvements, and substantial equivalents. Importantly, with the evolution of technology, many of the elements described herein can be replaced by equivalent elements that emerge after this application.

Claims

1. Use of a glutathione peroxidase mimetic in the preparation of a medicament or a pharmaceutical composition for preventing, alleviating or treating heart failure with preserved ejection fraction (HFpEF).

2. The use according to claim 1, wherein the glutathione peroxidase comprises glutathione peroxidase 1 (GPx1).

3. The use according to claim 1, wherein the glutathione peroxidase mimetic comprises ebselen or at least one of its stereoisomers, racemates, solvates, isotopic derivatives, or pharmaceutically acceptable salts.

4. The use according to claim 3, wherein the structure of the ebselen comprises the compound structure shown in formula (I):

5. The use according to claim 1, wherein the left ventricular ejection fraction (LVEF) of the patient with heart failure with preserved ejection fraction is greater than or equal to 50%.

6. The use according to claim 1, wherein the prevention, alleviation or treatment of heart failure with preserved ejection fraction comprises at least one of improving cardiac diastolic function, improving left ventricular volume reduction, improving pathological ventricular hypertrophy, improving hypertension symptoms, improving glucose tolerance disorders, improving liver lipid accumulation, reducing the body weight of patients with obese heart failure, and reducing the fat content of patients with obese heart failure.

7. The use according to claim 1, wherein the prevention, alleviation or treatment of heart failure with preserved ejection fraction comprises at least one of the following groups: (1) Reduce the E / E' ratio and improve cardiac diastolic function; (2) Increase left ventricular end-diastolic diameter (LVID,d) and improve left ventricular volume reduction; (3) Reduce the end-diastolic interventricular septum thickness (IVS,d) and improve pathological ventricular hypertrophy; (4) Reduce the area of ​​myocardial cells and improve pathological ventricular hypertrophy; (5) Reduce the ratio of heart weight to tibia length and improve pathological ventricular hypertrophy; (6) Reduce systolic blood pressure and improve hypertension symptoms; (7) Improve glucose tolerance; (8) Improve liver lipid accumulation; (9) reducing body weight in obese patients with heart failure; and / or (10) Reduce fat content in obese patients with heart failure.

8. The use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically or physiologically acceptable carrier.

9. The use according to claim 8, wherein the dosage form of the pharmaceutical composition comprises at least one of a solution, a powder, a granule, a tablet, a sugar-coated tablet, a capsule, a granule, a suspension, a syrup, drops, and a sublingual tablet.

10. The use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, wherein the excipient comprises at least one of a solubilizer, a disintegrant, a wetting agent, a stabilizer, a thickener, a diluent, a buffer, and a flavoring agent.

11. Use of glutathione peroxidase mimetics in the preparation of drugs or pharmaceutical compositions for improving cardiac diastolic function, improving left ventricular volume reduction, improving pathological ventricular hypertrophy, improving hypertension symptoms, improving impaired glucose tolerance, improving liver lipid accumulation, reducing the body weight of patients with obese heart failure, and reducing the fat content of patients with obese heart failure.

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