Use of usp32 as a therapeutic target in the preparation of a medicament for treating diabetic cardiomyopathy

Abstract

The application discloses application of USP32 as a therapeutic target in preparation of an anti-diabetic cardiomyopathy drug, relates to the diabetic cardiomyopathy, and particularly relates to a therapeutic drug for the diabetic cardiomyopathy. In a mouse diabetic cardiomyopathy model, knockout of USP32 obviously improves the heart function of the mouse, reduces myocardial hypertrophy and reduces the degree of myocardial fibrosis, indicating that down-regulation of USP32 can obviously improve the heart function of the mouse with the diabetic cardiomyopathy and reverse myocardial remodeling.

Description

Technical Field

[0001] This invention relates to diabetic cardiomyopathy, and more particularly to a treatment for diabetic cardiomyopathy. Background Technology

[0002] Diabetic cardiomyopathy (DCM) is a specific type of myocardial damage caused by diabetes, independent of hypertension and coronary atherosclerosis. Long-term high glucose toxicity promotes interstitial fibrosis of the heart tissue, cardiac stiffness, diastolic dysfunction, and subsequently systolic dysfunction, leading to a clinical syndrome of heart failure.

[0003] To date, the basic mechanism of DCM has not been fully elucidated. Currently, clinical intervention relies solely on general anti-heart failure drugs such as hypoglycemic agents, SGLT2 inhibitors, and beta-blockers for symptomatic treatment. There are no specific targeted drugs for the core pathological pathways of DCM, which can only relieve symptoms and cannot block or reverse the continuous myocardial remodeling.

[0004] Therefore, exploring new therapeutic targets for treating DCM disease is of great clinical significance. Summary of the Invention

[0005] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide the application of USP32 as a therapeutic target in the preparation of drugs for diabetic cardiomyopathy. In a mouse DCM model, knocking out USP32 significantly improved cardiac function, reduced myocardial hypertrophy, and decreased the degree of myocardial fibrosis, indicating that downregulating USP32 can significantly improve cardiac function and reverse myocardial remodeling in DCM mice.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the use of USP32 inhibitors in the preparation of drugs for the prevention and / or treatment of diabetic cardiomyopathy.

[0007] In one embodiment, the USP32 inhibitor includes an antibody, shRNA, siRNA, miRNA, antisense oligonucleotide, antagonist, and / or blocker targeting USP32 or a nucleic acid molecule encoding USP32.

[0008] In another aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of diabetic cardiomyopathy, comprising: (1) A therapeutically effective dose of a USP32 inhibitor; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0009] In one embodiment, the USP32 inhibitor includes an antibody, shRNA, siRNA, miRNA, antisense oligonucleotide, antagonist, and / or blocker targeting USP32 or a nucleic acid molecule encoding USP32.

[0010] In another aspect, the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for the prevention and / or treatment of diabetic cardiomyopathy.

[0011] In another aspect, the present invention provides a pharmaceutical preparation for the prevention and / or treatment of diabetic cardiomyopathy, comprising the above-described pharmaceutical composition.

[0012] In another aspect, the present invention also provides the use of the above-mentioned pharmaceutical preparation as a drug for the prevention and / or treatment of diabetic cardiomyopathy.

[0013] In another aspect, the present invention provides a pharmaceutical product for the prevention and / or treatment of diabetic cardiomyopathy, comprising the above-described pharmaceutical preparation.

[0014] In another aspect, the present invention also provides the use of the above-mentioned pharmaceutical product as a preparation of a drug for the prevention and / or treatment of diabetic cardiomyopathy.

[0015] In one embodiment, the pharmaceutical product includes a vial or box.

[0016] In another aspect, the present invention also provides the use of USP32 inhibitors in combination with diabetic cardiomyopathy treatment drugs as a preparation of drugs for the prevention and / or treatment of diabetic cardiomyopathy.

[0017] In one embodiment, the diabetic cardiomyopathy treatment includes an SGLT2 inhibitor (sodium-glucose cotransporter 2 inhibitor) and / or a GLP-1 receptor agonist (GLP-1RA).

[0018] In one embodiment, the SGLT2 inhibitor includes empagliflozin, dapagliflozin, soragliflozin, and / or canagliflozin.

[0019] In one embodiment, the GLP-1 receptor agonist includes liraglutide, smegglutide, dulaglutide, and / or telpoglutide.

[0020] In one embodiment, the subjects of the application are mammals.

[0021] In one embodiment, the examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs.

[0022] In one embodiment, the mammal includes a mouse.

[0023] In one embodiment, the subjects of the application are healthy.

[0024] In one embodiment, the subjects of the application are unhealthy.

[0025] In one embodiment, the subject of the application suffered from diabetic cardiomyopathy.

[0026] In one embodiment, the subject of the application does not have diabetic cardiomyopathy.

[0027] (III) Beneficial Effects This invention provides the application of USP32 as a therapeutic target in the preparation of drugs for treating diabetic cardiomyopathy. Compared with the prior art, it has the following beneficial effects: 1. In the mouse DCM model, knocking out USP32 significantly improved cardiac function, reduced myocardial hypertrophy and myocardial fibrosis, indicating that downregulating USP32 can significantly improve cardiac function and reverse myocardial remodeling in DCM mice. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a graph showing the results of USP32 expression level detection; Figure 2 This is a graph showing the effect of USP32 knockdown on diabetic cardiomyopathy mice; Figure 3 This is a graph showing the effect of USP32 overexpression on diabetic cardiomyopathy mice. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Terms and Definitions As used herein, the term “USP32” refers to ubiquitin-specific protease (USP) 32, a member of deubiquitinases (DUB).

[0032] As used herein, the terms “USP32 inhibitor” or “inhibitor of USP32 or its encoding nucleic acid molecule” are used interchangeably and refer to substances that can reduce the level or activity of USP32 or its encoding nucleic acid molecule. Inhibitors that can be used in this disclosure include, but are not limited to, antibodies against USP32 or nucleic acid molecules encoding the protein, shRNA, siRNA, miRNA, antisense oligonucleotides, antagonists, and blocking agents.

[0033] The inhibitor disclosed in this invention can inhibit USP32, and can thus be further used to prevent or treat diseases and / or related symptoms associated with diabetic cardiomyopathy.

[0034] As used herein, the term "pharmaceutical composition" refers to a composition comprising a USP32 inhibitor formulated with one or more pharmaceutically acceptable carriers.

[0035] The formulation of the pharmaceutical composition can be adjusted according to the application.

[0036] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0037] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0038] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0039] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0040] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0041] As used herein, the term “therapeutic effective dose” refers to a dose that is adequately suited for medical treatment with a reasonable benefit / risk ratio for treating the disease, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant medications, and other factors known in the medical field.

[0042] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to accomplishing one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0043] As used in this article, the term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0044] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0046] Example 1: Expression of USP32 in diabetic cardiomyopathy: 1. Construction of a diabetic cardiomyopathy model: C57BL / 6 background USP32 gene knockout (USP32fl / fl) and overexpression (LSL-USP32fl / fl) mice, Cx3cr1-Cre tool mice, and C57BL / 6 wild-type (WT) mice were all purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. They were housed in the Standardized Experimental Animal Center (SPF grade) of the Big Health Research Institute of the Hefei Comprehensive National Science Center. Housing conditions included: temperature between 22~24℃, humidity between 40~70%, alternating light and dark lighting for 12 hours, and free access to water and food.

[0047] Five-week-old male mice were acclimatized for one week, and at six weeks of age, they were given a high-fat diet (HFD) for eight weeks. Following this, they were intraperitoneally injected with streptozotocin (STZ, 30 mg / kg / day) for seven consecutive days. Successful model establishment was defined as a random blood glucose level ≥16.7 mmol / L sustained for two weeks. The control group was given a normal diet and injected with an equal volume of citrate buffer. All mice continued to be fed until week 14 after successful model establishment for endpoint testing.

[0048] 2. Detection of USP32 expression level in myocardial tissue: 2.1 RT-qPCR: 2.1.1 Design the layout of the 96-well plate according to the number of samples and genes in the experimental and control groups.

[0049] 2.1.2. Prepare the reaction system: Remove the primers, qPCR mix, and cDNA from the freezer and thaw them on ice. Prepare the premixed reagents according to the SYBR Green I (Vazyme, Q712-02) instructions, and dilute the cDNA 5-10 times as needed. Vortex the primers multiple times to prevent primer dimer formation. Place eight-tube strips on an eight-tube rack and add the qPCR mix, primers (sequences shown in Table 1), water, and cDNA in sequence. After ensuring that all reagents are free of residue, cap the tubes, label the beginning and end of the caps, and centrifuge to mix until no air bubbles remain.

[0050] 2.1.3 Perform PCR cycling: Place the prepared reaction solution into a real-time qPCR instrument, set the appropriate temperature cycling and acquisition mode, and perform amplification and detection.

[0051] 2.1.4 Data Analysis: After the reaction, export the qPCR data. Analyze the melting curve for a single peak and the amplification curve for a normal "S"-shaped curve. The ideal Ct value is usually between 15 and 30. Perform relative or absolute quantification analysis.

[0052] Table 1 RT-qPCR primer sequences 2.2 Preparation of paraffin sections for tissue: 2.2.1. Sampling and fixation: Immerse 10mm×10mm×2mm tissue samples in a fixative solution containing 4% paraformaldehyde for 48 hours, then rinse thoroughly with water.

[0053] 2.2.2 Dehydration: The tissues were placed in 70%, 80%, 90%, and 95% ethanol for 1 hour each, followed by anhydrous ethanol for 30 minutes for dehydration.

[0054] 2.2.3. Transparency: After dehydration, place the product in a 1:1 mixture of ethanol and xylene, xylene I, and xylene II for 20 minutes each.

[0055] 2.2.4. Wax Impregnation: Take out the tissue from the previous step and soak it in liquid paraffin at 60°C for 1 hour. Repeat this step twice.

[0056] 2.2.5 Embedding: After pouring the molten paraffin into the embedding frame, quickly place the tissue into the embedding frame with heated forceps. After cooling and solidifying, it will become a wax block, which should be trimmed neatly.

[0057] 2.2.6 Sectioning: Place the wax block on a microtome and cut it into thin sections with a thickness of about 10μm.

[0058] 2.2.7 Gluing the slide: Take the cut, wrinkle-free wax slide and glue it onto the glass slide.

[0059] 2.2.8. Drying the slides: Place the glass slides on a table at 40°C and dry for 1.5 hours.

[0060] 2.3 Immunofluorescence: 2.3.1 Dewaxing and hydration: The paraffin sections were soaked in xylene I, xylene II and xylene III for 10 minutes each, then removed and soaked in 100%, 90%, 80% and 70% ethanol for 5 minutes each, and then rinsed with running water for 5 minutes.

[0061] 2.3.2 Antigen retrieval: The slides were placed in a citrate retrieval solution prepared with distilled water and kept at a constant temperature (100℃) for 20 minutes for antigen retrieval. After that, they were allowed to cool naturally to room temperature and then washed with PBS for 3 minutes each time.

[0062] 2.3.3. Covering: Add 5% BSA to cover the tissue, incubate at room temperature for 30 minutes, then spin dry without washing.

[0063] 2.3.4 Incubation of primary antibody: Add diluted mixed primary antibody: antibody dilution concentration USP32 1:400 (immunoway, YT4835), F4 / 80 1:200 (Cell Signaling Technology, 69361S) to fully cover the tissue, incubate overnight (>16h) in a 4°C refrigerator, and then wash with PBS for 3min × 3 times.

[0064] 2.3.5 Add fluorescent secondary antibody: Add diluted mixed secondary antibody (the dilution ratio of the two secondary antibodies is 1:100) to fully cover the tissue and incubate at room temperature in the dark for 1 hour, then wash with PBS for 3 minutes × 3 times.

[0065] 2.3.6. Nucleus staining: Add DAPI dropwise, react at room temperature in the dark for 5 min, then wash with PBS for 3 min × 3 times.

[0066] 2.3.7 Sealing: Add fluorescence quencher to seal the slide in the dark and let it air dry.

[0067] 2.3.8. Observe and photograph under a fluorescence microscope, and analyze the images.

[0068] The expression level and localization results of USP32 in myocardial tissue are as follows: Figure 1 As shown, USP32 is highly expressed in the myocardial tissue of DCM mice, and is specifically expressed mainly on macrophages in the myocardial tissue.

[0069] Figure 1 In the diagram, A: qRT-PCR was used to detect the relative mRNA expression level of USP32 in the myocardial tissue of mice in the sham group and the DCM group; B: Immunofluorescence co-staining showed the colocalization of USP32 (green), macrophage marker F4 / 80 (red), and nuclear DAPI (blue) in the myocardial tissue of mice in the sham group and the DCM group; sham: control group; DCM: diabetic cardiomyopathy group.

[0070] Example 2: Effect of inhibiting USP32 on diabetic cardiomyopathy mice: 1. Construction and grouping of macrophage-specific USP32 knockout model: Using Cx3cr1-Cre tool mouse and USP32 flox / flox (USP32) fl / fl Through hybridization with mice, macrophage-specific USP32 knockout mice (Cx3cr1-CreUSP32) were obtained. flox / flox USP32 (abbreviated as USP32) macKO ); USP32 from the same nest flox / flox As a comparison.

[0071] Six-week-old male mice (20±2g) were randomly divided into 4 groups (n=10): ①Sham+USP32 flox / flox ②Sham+USP32 macKO ③DCM+USP32 flox / flox ④DCM+USP32 macKO .

[0072] 2. Cardiac function and structural analysis: Mice were shaved of hair in the anterior chest region, anesthetized with 4-5% isoflurane inhalation, and fixed in a supine position. M-mode, B-mode, and pulsed Doppler images were acquired using a small animal ultrasound system, and key cardiac function parameters were recorded, including: LVEF, LVFS, LVIDs, LVmass, E / A, and E / E' ratio.

[0073] To ensure data accuracy, each mouse was observed for at least three consecutive cardiac cycles, and the average of these cycles was used for calculation. Throughout the experiment, the mice were kept in a stable state of anesthesia to reduce errors caused by motion artifacts. Simultaneously, the ultrasound probe was kept in a stable position to obtain clear and consistent images.

[0074] 3. Cardiac remodeling assessment: Body weight (BW), heart weight (HW), and tibia length (TL) were recorded before sacrifice, and HW / BW and HW / TL were calculated. Hearts were fixed by perfusion with 4% paraformaldehyde, embedded in paraffin, and serially sectioned (5 μm). HE and WGA staining were performed; ImageJ was used to measure the cross-sectional area of ​​cardiomyocytes; Masson staining was used to calculate the fibrosis area; RT-qPCR was used to detect the mRNA expression levels of ANP, BNP, MYH7, Collagen I, Collagen III, and Acta2 in ventricular tissue.

[0075] Results of cardiac function and myocardial damage in mice as follows Figure 2 As shown, in the mouse DCM model, knocking out USP32 significantly improved cardiac function, reduced myocardial hypertrophy and myocardial fibrosis, indicating that downregulating USP32 can significantly improve cardiac function and reverse myocardial remodeling in DCM mice.

[0076] Figure 2 A: Schematic diagram of the strategy for constructing macrophage-specific USP32 knockout and DCM mice; B: Representative B-mode and M-mode echocardiographic images of the left ventricle of macrophage-specific USP32 knockout mice and their littermates with DCM mice, using echocardiography to assess left ventricular ejection fraction, fractional shortening, and left ventricular mass; representative pulsed Doppler images and the ratio of mitral valve E wave to A wave (E / A), representative tissue Doppler trajectories, and the ratio of mitral valve E wave to E' wave (E / E'); C: Heart weight / body weight (HW / BW) and heart weight / tibia length (HW / TL); D: HE staining, WGA staining, and cardiomyocyte size; E: RT-PCR detection of ANP, BNP, and MYH7 expression in mouse heart tissue; F: Masson staining and quantification of fibrosis area, and RT-PCR detection of COL1, COL3, and Acta2 expression in mouse heart tissue.

[0077] Example 3: Effects of USP32 overexpression on diabetic cardiomyopathy mice: The USP32 was overexpressed using methods well known to those skilled in the art, and the rest remained consistent with Example 2.

[0078] Results of cardiac function and myocardial damage in mice as follows Figure 3 As shown, in the mouse DCM model, overexpression of USP32 significantly aggravated the deterioration of cardiac function and promoted the progression of myocardial hypertrophy and myocardial fibrosis, indicating that upregulation of USP32 can exacerbate the deterioration of cardiac function and myocardial remodeling in DCM mice.

[0079] Figure 3A: Schematic diagram of the strategy for constructing macrophage-specific USP32 overexpression and DCM mice; B: Representative B-mode and M-mode echocardiographic images of the left ventricle of macrophage-specific USP32 knockout DCM mice from the same littermate, with left ventricular ejection fraction, shortening fraction, and left ventricular mass assessed by echocardiography; representative pulsed Doppler images and the mitral valve E-wave to A-wave ratio (E / A), representative tissue Doppler trajectories, and the mitral valve E-wave to E'-wave ratio (E / E'); C: Heart weight / body weight (HW / BW) and heart weight / tibia length (HW / TL); D: HE staining, WGA staining, and cardiomyocyte size; E: RT-PCR detection of ANP, BNP, and MYH7 expression in mouse heart tissue; F: Masson staining and quantification of fibrosis area, and RT-PCR detection of COL1, COL3, and Acta2 expression in mouse heart tissue.

[0080] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of USP32 inhibitors in the preparation of drugs for the prevention and / or treatment of diabetic cardiomyopathy.

2. The application according to claim 1, characterized in that, The USP32 inhibitors include antibodies, shRNA, siRNA, miRNA, antisense oligonucleotides, antagonists, and / or blockers that target USP32 or nucleic acid molecules encoding USP32.

3. A pharmaceutical composition for the prevention and / or treatment of diabetic cardiomyopathy, characterized in that, include: (1) A therapeutically effective dose of a USP32 inhibitor; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The USP32 inhibitors include antibodies, shRNA, siRNA, miRNA, antisense oligonucleotides, antagonists, and / or blockers that target USP32 or nucleic acid molecules encoding USP32.

5. The use of the pharmaceutical composition of claim 3 or 4 as a preparation of a medicament for the prevention and / or treatment of diabetic cardiomyopathy.

6. A pharmaceutical preparation for the prevention and / or treatment of diabetic cardiomyopathy, characterized in that, Includes the pharmaceutical composition according to claim 3 or 4.

7. The use of the pharmaceutical preparation of claim 6 as a preparation of a drug for the prevention and / or treatment of diabetic cardiomyopathy.

8. A pharmaceutical product for the prevention and / or treatment of diabetic cardiomyopathy, characterized in that, Includes the pharmaceutical preparation described in claim 6.

9. The use of the pharmaceutical product of claim 8 as a preparation of a drug for the prevention and / or treatment of diabetic cardiomyopathy.

10. The use of USP32 inhibitors in combination with drugs for the treatment of diabetic cardiomyopathy as a means of preparing drugs for the prevention and / or treatment of diabetic cardiomyopathy.

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