Use of a blm helicase inhibitor in the treatment of cardiac hypertrophy

Abstract

The application discloses application of a BLM helicase inhibitor in treating myocardial hypertrophy. The application firstly finds that the BLM helicase inhibitor ML216 can significantly inhibit cardiac hypertrophy and myocardial cell fibrosis, thereby providing a new drug for treating myocardial cell hypertrophy diseases in the field, and providing practical experimental evidence and scientific basis for treating myocardial cell hypertrophy diseases, and having a good application prospect in treating myocardial hypertrophy and / or cardiac hypertrophy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of BLM helicase inhibitors in the treatment of myocardial hypertrophy. Background Technology

[0002] There are many causes of cardiac hypertrophy, including hypertensive left ventricular hypertrophy, congestive heart failure, anterior wall myocardial infarction, mitral regurgitation, aortic stenosis, hypertrophic cardiomyopathy, pulmonary hypertension, cor pulmonale, dilated cardiomyopathy, endocarditis, left ventricular aneurysm, and mitral stenosis. Cardiac hypertrophy can be divided into two main categories: enlargement and hypertrophy. ① Enlargement refers to a condition where the heart muscle does not thicken; sometimes, the myocardium even thins. This condition is usually caused by the heart muscle losing its original elasticity, leading to increased volume and pressure, resulting in heart enlargement. Common causes include myocarditis, valvular insufficiency, hyperthyroidism, and vitamin B1 deficiency. ② Hypertrophy refers to a condition where the heart muscle thickens, but the heart chambers do not enlarge; sometimes, they even shrink, but the overall appearance of the heart is larger than normal. Common causes of cardiac hypertrophy include narrowing of the heart outlet, such as aortic stenosis; increased peripheral vascular resistance, such as hypertension; and cardiomyopathy, such as hereditary hypertrophic cardiomyopathy. The main danger of cardiac hypertrophy is that the heart's contractility decreases after it enlarges, thus leading to heart failure.

[0003] Currently, anti-cardiac hypertrophy methods are mainly divided into three categories: ① Reversing cardiac hypertrophy: Commonly used drugs include angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and aldosterone receptor antagonists, which reverse cardiac chamber enlargement to some extent through neuro-humoral regulation; ② Reducing cardiac load: Correcting heart failure by reducing cardiac load. Treatment methods include diuresis and vasodilators, with loop diuretics being the most commonly used; ③ Using vasodilators: ACEIs (angiotensin-converting enzyme inhibitors), ARBs (angiotensin II receptor blockers), and nitrates. The role of ML216, as a BLM helicase inhibitor, in cardiac hypertrophy has not yet been studied. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a new use for the BLM helicase inhibitor ML216, specifically, to provide the application of the BLM helicase inhibitor ML216 in the treatment of myocardial hypertrophy.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides the use of BLM helicase inhibitors in the preparation of products for the prevention and / or treatment of cardiomyocyte hypertrophy.

[0007] Furthermore, the BLM helicase inhibitor is ML216.

[0008] Furthermore, the aforementioned cardiomyocyte hypertrophy disease includes myocardial hypertrophy and cardiac hypertrophy.

[0009] Furthermore, the cardiac hypertrophy includes ventricular hypertrophy, cardiac enlargement, and atrial enlargement.

[0010] Furthermore, the cardiac hypertrophy includes heart diseases induced by increased peripheral vascular resistance, narrowing of the cardiac outlet, or cardiomyopathy.

[0011] Furthermore, the product includes pharmaceuticals.

[0012] Furthermore, the drug can be used directly or in the form of a pharmaceutical composition.

[0013] In some embodiments, the BLM helicase inhibitor has the following activities: inhibiting the binding of BLM helicase to DNA, inhibiting the unwinding activity and ATPase activity of BLM helicase, and disrupting the secondary structure of BLM helicase. In a specific embodiment of the present invention, the BLM helicase inhibitor is ML216. ML216 is an effective, selective, and cell-penetrating BLM helicase inhibitor. Its IC50 values ​​for BLM full-length and BLM636-1298 are 2.98 μM and 0.97 μM, respectively, and it can inhibit the ssDNA-dependent ATPase activity of BLM. It is mainly used in the field of cell cycle and DNA damage research, especially targeting DNA / RNA synthesis. In a specific embodiment of the present invention, the inventors have experimentally demonstrated that ML216 can significantly inhibit cardiac hypertrophy, cardiomyocyte hypertrophy, and cardiomyocyte fibrosis, and has a therapeutic effect on cardiomyocyte hypertrophy.

[0014] In some implementations, hypertrophic cardiomyopathy has multiple precipitating types, including hypertrophic cardiomyopathy induced by hypertension, aortic stenosis, or increased intraventricular pressure.

[0015] In this invention, cardiomyocyte hypertrophy disease also includes complications caused by cardiomyocyte hypertrophy, such as endocarditis, heart failure, arterial embolism, and arrhythmia induced by cardiomyocyte hypertrophy. By slowing down cardiomyocyte hypertrophy, the complications can also be treated accordingly.

[0016] A second aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of cardiomyocyte hypertrophy.

[0017] Furthermore, the pharmaceutical composition comprises a BLM helicase inhibitor.

[0018] Furthermore, the BLM helicase inhibitor is ML216.

[0019] Furthermore, the aforementioned cardiomyocyte hypertrophy disease includes myocardial hypertrophy and cardiac hypertrophy.

[0020] Furthermore, the cardiac hypertrophy includes ventricular hypertrophy, cardiac enlargement, and atrial enlargement.

[0021] Furthermore, the cardiac hypertrophy includes heart diseases induced by increased peripheral vascular resistance, narrowing of the cardiac outlet, or cardiomyopathy.

[0022] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

[0023] In some embodiments, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants.

[0024] In some implementations, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, water, etc.

[0025] In some embodiments, the adhesive includes, but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginate, xanthan gum, hydroxypropylcellulose and hydroxypropylmethylcellulose, etc.

[0026] In some embodiments, the surfactant includes, but is not limited to: polyethylene oxide sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, hexadecyl alcohol, etc.

[0027] In some embodiments, the humectant includes, but is not limited to, glycerin, starch, etc.

[0028] In some embodiments, the adsorbent carrier includes, but is not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay.

[0029] In some embodiments, the lubricant includes, but is not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0030] In some embodiments, the filler includes, but is not limited to: mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, and sodium bicarbonate, etc.

[0031] In some embodiments, the disintegrant includes, but is not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0032] In some embodiments, the pharmaceutically acceptable carrier and / or excipients may additionally contain liquids such as water, saline, glycerin, and ethanol. The dosage forms of the pharmaceutical compositions include, but are not limited to: tablets, pills, powders, granules, capsules, lozenges, syrups, solutions, emulsions, suspensions, controlled-release formulations, aerosols, films, injections, intravenous infusions, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, nasal preparations, pulmonary preparations, eye drops, etc., for patient ingestion.

[0033] In some embodiments, suitable routes of administration of the pharmaceutical composition include any of a variety of methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical composition of the present invention into a subject, including but not limited to: oral administration, non-gastrointestinal administration, administration via inhalation spray, topical administration, rectal administration, nasal administration, buccal administration, vaginal administration, or administration via an implanted storage device. When the pharmaceutical composition is intended for injection, it may be in the form of a suspension, solution, or emulsion in an oily or aqueous medium and may contain formulation agents such as suspending agents, preservatives, stabilizers, and / or dispersants.

[0034] In some embodiments, the pharmaceutical composition or active ingredient of the present invention (the BLM helicase inhibitor of the present invention) may also be used in combination with other drugs for treating hypertrophic cardiomyocyte disease, including simultaneous or sequential use, such that a therapeutically effective amount of the pharmaceutical composition or active ingredient of the present invention and one or more other drugs for treating hypertrophic cardiomyocyte disease are present in the patient.

[0035] In some embodiments, the pharmaceutical composition or active ingredient of the present invention (the BLM helicase inhibitor of the present invention) may be used in combination, including administering a unit dose of the pharmaceutical composition or active ingredient of the present invention before, after, or simultaneously with the administration of a unit dose of one or more other drugs for treating hypertrophic cardiomyopathy. For example, the pharmaceutical composition or active ingredient of the present invention may be administered within seconds, minutes, or hours after the administration of one or more other drugs for treating hypertrophic cardiomyopathy; for example, a unit dose of the pharmaceutical composition or active ingredient of the present invention may be administered within seconds, minutes, or hours after the administration of a unit dose of the pharmaceutical composition or active ingredient of the present invention; for example, a unit dose of one or more other drugs for treating hypertrophic cardiomyopathy and the pharmaceutical composition or active ingredient of the present invention may be administered simultaneously.

[0036] Furthermore, other drugs for treating cardiomyocyte hypertrophy that are used in combination with the pharmaceutical composition or active ingredient (the BLM helicase inhibitor described in this invention) provided by this invention include, but are not limited to: diuretics such as furosemide, spironolactone, etc.; β-blockers such as metoprolol, bisoprolol, etc.; angiotensin-converting enzyme inhibitors such as captopril, ramipril; and calcium channel blockers such as verapamil, diltiazem.

[0037] A third aspect of this invention provides the use of BLM helicase inhibitors in the preparation of reagents for inhibiting cardiomyocyte enlargement and / or cardiomyocyte fibrosis in vitro.

[0038] Furthermore, the BLM helicase inhibitor is ML216.

[0039] The fourth aspect of the present invention provides a method for inhibiting cardiomyocyte enlargement and / or inhibiting cardiomyocyte fibrosis in vitro for non-therapeutic purposes.

[0040] Furthermore, the method includes the following steps: treating cardiomyocytes with an effective amount of BLM helicase inhibitor or a pharmaceutical composition containing an effective amount of BLM helicase inhibitor.

[0041] Furthermore, the BLM helicase inhibitor is ML216.

[0042] In this invention, the method is purely biological and does not involve any therapeutic purpose. For example, this method can be used to study the pathogenesis of cardiac hypertrophy.

[0043] The fifth aspect of the present invention provides a system for inhibiting cardiomyocyte enlargement and / or inhibiting cardiomyocyte fibrosis.

[0044] Furthermore, the system includes a treatment unit for treating cardiomyocytes with a therapeutically effective amount of a BLM helicase inhibitor.

[0045] Furthermore, the BLM helicase inhibitor is ML216.

[0046] In this invention, the term "effective amount" refers to the therapeutic amount required to alleviate at least one or more symptoms of a disease or condition, and relates to a sufficient amount of medicine to provide the desired effect. Therefore, the term "therapeutic effective amount" refers to a therapeutic amount sufficient to cause a specific effect when administered to a typical subject. In various contexts, the effective amount, as used herein, also includes an amount sufficient to delay the development of disease symptoms, alter the course of the disease (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. It should be understood that many methods are known in the art for determining the effective amount for a given application. For example, pharmacological methods for dosage determination can be used in a therapeutic context. In the context of therapeutic or preventative application, the amount of composition administered to the subject will depend on the type and severity of the disease and individual characteristics such as general health status, age, sex, weight, and tolerance to the drug. It also depends on the degree, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. For example, the therapeutically effective amount of Daidzin can be determined by referring to its current safe dosage for treating arterial thrombotic disease in patients and through clinical trials. Appropriate and effective dosage also needs to take into account treatment factors such as drug formulation, individual constitution, weight, age, disease progression, and administration site.

[0047] In some specific embodiments, the system provided by the present invention includes a computer-readable storage medium for storing a computer-readable program or instructions, which, when executed by a processor, enable the therapeutic unit in the system of the present invention to perform its functions.

[0048] The sixth aspect of the present invention provides a method for evaluating whether a drug to be screened has the effect of preventing and / or treating cardiomyocyte hypertrophy.

[0049] Furthermore, the method includes using a BLM helicase inhibitor as a positive control to evaluate the efficacy of the drug to be screened.

[0050] Furthermore, the BLM helicase inhibitor is ML216.

[0051] In some implementations, the drugs to be screened include, but are not limited to, nucleic acid inhibitors designed to target BLM helicase, small molecule compounds, etc.

[0052] Preferably, the nucleic acid inhibitor is selected from: dsRNA, antisense nucleic acid, small interfering RNA, microRNA; or constructs that can express or form said dsRNA, antisense nucleic acid, small interfering RNA, microRNA.

[0053] Preferably, the source of the small molecule compound is selected from: newly synthesized or existing databases; wherein existing databases include, but are not limited to, general natural product databases (COCONUT, Super Natural II, NPASS), plant natural product databases (KNApSaCK, CMAUP, TriForC, Alkamid, NPACT DB, BioPhytMol), traditional Chinese medicine natural product databases (TCM@Taiwan, CEMTDD, CHDD, ETCM, TM-MC, TCMID, YaTCM), microbial natural product databases (StreptomeDB, NP Altas, ProCarDB, PAMDB, Lichen Database), marine natural product databases (MNPD, SWMD), natural product databases from different countries and regions (IMPPAT, NeMedPlant, MedPServer, TlPdb, AfroDB, ANPDB, BIOFACQUIM, NUBBEDB), and food natural product databases (FooDB, BitterDB, Phenol-Explorer, PhytoHub, SuperSweet). Databases include: toxic natural product databases (Exposome-Explorer, T3DB, Snake Neurotoxin Database, TPPT), natural product industry directories (Greenpharma, AnalytiConDiscovery, InterBioScreen, Indofine Chemical Company, Pi Chemicals Systems\Specs, TargetMol), databases deduplicated using MS data (MoNA, MassBank, METLIN, HMDB, YMDB, ReSpect, GNPS), and databases deduplicated using NMR data (NMRShiftDB, NAPROC-13), etc.

[0054] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0055] This invention is the first to discover that the BLM helicase inhibitor ML216 has an inhibitory effect on cardiac hypertrophy, and can prevent and / or treat the occurrence and development of cardiac hypertrophy; the BLM helicase inhibitor ML216 can also be used as a product for the prevention and / or treatment of cardiomyocyte hypertrophy. Attached Figure Description

[0056] Figure 1 The figure shows the effect of ML216 on cardiac hypertrophy; among which... Figure 1A is a diagram showing the mouse model of myocardial hypertrophy using isoproterenol and the operation mode of intervention using ML216. Figure 1 B shows the cardiac images of mice in each group; Figure 1 C shows the results of the ratio of heart weight to body weight and the ratio of heart weight to tibia length for each group of mice.

[0057] Figure 2 The results of the effect of ML216 on cardiac ejection capacity are shown in the figure (EF: ejection fraction; FS: fractional shortening; LVVol-d: diastolic left ventricular volume; LVVol-s: systolic left ventricular volume).

[0058] Figure 3 The figure shows the effect of ML216 on cardiac cells; Figure 3 A shows the size results of heart cells in each group of mice; Figure 3 B shows the results of fibrosis in the cardiac cells of mice in each group. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Simple modifications made to the present invention based on its essence are all within the scope of protection claimed by the present invention.

[0060] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0062] The experimental materials used in the following examples were sourced as follows: ML216 was purchased from MCE (HY-K0010), isoproterenol was purchased from Thoso (T1056), SBE-β-CD was purchased from Sigma (H-107), and DMSO was purchased from Sigma (D2650).

[0063] Example 1: Effects of ML216 on cardiac hypertrophy

[0064] 1. Experimental Methods

[0065] (1) Mouse feeding and drug treatment: Healthy adult mice were selected, usually 8-week-old C57BL / 6 experimental mice (purchased from Spefair), and were housed in a standard environment to ensure good health before the experiment. Mice were randomly divided into a control group (injected with PBS, labeled as PBS group), a modeling group (injected with isoproterenol, labeled as ISO group), and a treatment group (injected with isoproterenol and ML216, labeled as ISO+ML216 group). Isoproterenol was diluted with PBS and injected at a dose of 10 mg / kg once a day for 3 weeks; ML216 (solvent: 10% DMSO + 90% (20% SBE-β-CD in Saline)) was injected subcutaneously at a dose of 0.5 mg / kg twice a week for 3 weeks. Mouse weight was measured twice a week. After 3 weeks of drug treatment, 6-8 mice from each group were sacrificed and their hearts were weighed, while 10-12 mice were retained for echocardiography.

[0066] 2. Experimental Results

[0067] The effect of ML216 on cardiac hypertrophy in mice, such as Figure 1 As shown in Figure B, compared with the control group (Ctrl), the heart volume of mice injected with isoproterenol (ISO) was significantly larger, while the heart volume of mice injected with ML216 (ISO+ML) was significantly smaller. The ratio of heart weight to body weight and the ratio of heart weight to tibia length were calculated for each group of mice, and the results are shown below. Figure 1 As shown in Figure C, compared with the ISO group mice, the ratio of heart weight to body weight and the ratio of heart weight to tibia length were significantly reduced in the ISO+ML group mice. These results indicate that ML216 has the effect of reducing cardiac hypertrophy.

[0068] Example 2: Effect of ML216 on cardiac ejection capacity

[0069] 1. Experimental Methods

[0070] After drug administration, mice underwent ultrasound examination using a high-resolution small animal echocardiography device provided by the Laboratory Animal Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. To ensure the mice remained stationary during the scan, they were lightly anesthetized with isoflurane, and a heating pad was used to maintain their body temperature (approximately 37°C). The anesthetized mice were placed back-down on the experimental table, with their limbs gently secured so that the ultrasound probe could contact their chests. The chest hair was shaved to improve the clarity of the ultrasound images. A high-frequency probe was used to locate the mouse heart. Common observation angles included the long-axis section, short-axis section, and apical four-chamber section. Multiple cardiac cycles were recorded to capture the heart's systolic and diastolic functions. Dedicated software was used to analyze the recorded video data and calculate various cardiac function parameters (such as EF, FS, and ventricular wall thickness).

[0071] 2. Experimental Results

[0072] The effects of ML216 on cardiac ejection fraction, fractional shortening, diastolic left ventricular volume, and systolic left ventricular volume are as follows: Figure 2 As shown, compared with the ISO group mice, the ISO+ML group mice had significantly increased cardiac ejection fraction and short axis shortening rate, indicating that ML216 has the effect of improving the ejection capacity of hypertrophic hearts.

[0073] Example 3: Effects of ML216 on cardiac cells

[0074] 1. Experimental Methods

[0075] After drug administration, mice underwent ultrasound examination. Following ultrasound scanning, anesthesia was discontinued, and the mice were allowed to awaken on a heating pad. After recovery, they were returned to their cages. The mice were euthanized the following day by cervical dislocation. After euthanasia, the heart was weighed, then longitudinally divided in half. The left half was collected and rapidly frozen in liquid nitrogen for subsequent molecular experiments; the right half was fixed in 4% paraformaldehyde for subsequent immunohistochemical experiments. The specific steps for immunohistochemical experiments were as follows: frozen tissue blocks were placed in a cryostat for sectioning, with sections of 5 μm in size. The sections were then stored at -80°C and allowed to slowly recover on ice at 4°C before use. H&E staining was performed at the facilities of the State Key Laboratory of Medical Molecular Biology. Cardiac fibrosis was detected using a Sirius Red staining kit (Solepro #G1472). The paraformaldehyde-fixed heart was routinely dehydrated and embedded, sectioned at 5 μm, routinely dewaxed, and then stained and mounted according to the kit instructions. Immunohistochemical images were captured using an Evos FL Auto 2.

[0076] 2. Experimental Results

[0077] The effect of ML216 on the size of heart cells, such as Figure 3As shown in Figure A, compared with the ISO group mice, the ISO+ML group mice had significantly smaller cardiac cells; the effect on the degree of cardiac cell fibrosis is as follows. Figure 3 As shown in Figure B, compared with the ISO group mice, the ISO+ML group mice showed a significant reduction in the degree of cardiac cell fibrosis; these results indicate that ML216 has the effect of inhibiting cardiomyocyte hypertrophy and fibrosis.

[0078] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of BLM helicase inhibitors in the preparation of products for the prevention and / or treatment of cardiac hypertrophy, characterized in that, The BLM helicase inhibitor is ML216.

2. The application according to claim 1, characterized in that, The term "cardiac hypertrophy" includes ventricular hypertrophy, cardiac enlargement, and atrial enlargement.

3. The application according to claim 1, characterized in that, The term "cardiac hypertrophy" includes heart diseases induced by increased peripheral vascular resistance, narrowing of the heart outlet, or cardiomyopathy.

4. The application according to claim 1, characterized in that, The products include pharmaceuticals.

5. The application of BLM helicase inhibitors in the preparation of reagents for inhibiting cardiac cell enlargement and / or cardiac cell fibrosis in vitro, characterized in that, The BLM helicase inhibitor is ML216.

6. A method for inhibiting cardiac cell enlargement and / or cardiac cell fibrosis in vitro for non-therapeutic purposes, characterized in that, The method includes the following steps: treating cardiac cells with an effective amount of a BLM helicase inhibitor or a pharmaceutical composition containing an effective amount of a BLM helicase inhibitor; wherein the BLM helicase inhibitor is ML216.

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