Compound for preventing and treating diabetic cardiomyopathy and application thereof

Abstract

The invention relates to a compound, which belongs to a fungal metabolite, can inhibit a ceramide synthesis pathway, is used for preventing and treating diabetic cardiomyopathy, can target a diabetic cardiomyopathy ceramide metabolic pathway and specifically alleviate the occurrence and development of diabetic cardiomyopathy conditions, does not interfere with non-related pathways, and is wider in applicability compared with traditional medicines. And meanwhile, the blank of targeted treatment of diabetic cardiomyopathy at present is filled.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of a compound, in particular to a compound having the effect of preventing and treating diabetic cardiomyopathy, and its application in pharmaceuticals and drug-containing medical devices. BACKGROUND

[0002] Diabetic cardiomyopathy (DbCM) refers to the abnormality of cardiac function in diabetic patients without coronary atherosclerotic heart disease, valvular disease and other underlying heart diseases. The early pathological changes mainly manifest as slight increase in left ventricular stiffness, decrease in compliance and impaired diastolic function. With disease progression, patients can gradually develop arrhythmia, angina pectoris and eventually heart failure, which seriously affects the quality of life. However, the current treatment for diabetic cardiomyopathy is still mainly focused on controlling blood glucose and improving insulin utilization, and there is a lack of effective drugs directly targeting cardiac toxicity. In addition, the treatment mainly for reducing blood sugar is often accompanied by adverse reactions such as hypoglycemia. Therefore, developing a treatment strategy directly targeting secondary cardiac toxicity will likely bring significant benefits to patients with diabetic cardiomyopathy (Cell Death Discov. 2023; 9(1): 256.).

[0003] In the state of type 2 diabetes, due to the decreased sensitivity of adipocytes to insulin, the inhibitory effect of insulin on lipolysis is weakened, and a large amount of fatty acids are released from adipose tissue into the blood. In cardiomyocytes, with the increase of exogenous fatty acid uptake, the fatty acids that fail to be metabolized will accumulate in the form of lipid droplets. The excessive accumulation of lipid droplets will disrupt the balance of cellular fatty acid metabolism, generate lipid metabolism intermediates such as ceramide and diacylglycerol, and induce lipid toxicity through pathways such as oxidative stress and apoptosis. The occurrence of lipid toxicity will lead to decreased myocardial cell function, impaired excitation-contraction coupling and aggravated cardiac fibrosis, and eventually manifest as diabetic cardiomyopathy characterized by diastolic dysfunction (Cardiovasc Res. 2011; 92(1): 10-8.).

[0004] Ceramides are important mediators of lipotoxicity. First, serine and palmitoyl-CoA react under the catalysis of serine-palmitoyltransferase (SPT) to produce ketosphingosine, which is then rapidly converted to sphingosine by 3-ketosphingosine reductase. This sphingosine scaffold subsequently acquires fatty acids, producing dihydroceramide. A dihydroceramide desaturase inserts a double bond into the sphingosine base of dihydroceramide, thus producing ceramide. During lipotoxicity, ceramides can directly induce various pathological processes in diabetic cardiomyocytes, including abnormal autophagy and apoptosis. Specifically, ceramides are believed to induce widespread autophagy, leading to autophagy-mediated cell death. Furthermore, ceramides can induce a damaged phenotype characterized by mitochondrial dysfunction, oxidative stress, and interference with normal cellular physiological functions by inducing the widespread expression of inflammatory cytokines. These pathological processes escalate, ultimately leading to cardiomyocyte death. Following extensive cardiomyocyte death, cardiac function declines significantly (FASEB J. 2018;32:1403–1416). Therefore, targeting ceramides could help alleviate this cardiac dysfunction. However, current therapeutic drugs targeting ceramides remain insufficient. Research on drugs that can lower ceramide concentrations to alleviate diabetic cardiomyopathy dysfunction is lacking. Summary of the Invention

[0005] One object of the present invention is to provide a compound as an inhibitor for use in binding the functional group of serine-palmitoyltransferase and thereby inhibiting the ceramide synthesis pathway mediated by it.

[0006] Another object of the present invention is to provide a compound that acts as an inhibitor to suppress ceramide synthesis.

[0007] Another object of the present invention is to provide a compound for improving cardiac dysfunction in diabetic cardiomyopathy, and its use in the preparation of a medicament for treating diabetic cardiomyopathy.

[0008] Another object of the present invention is to provide an application of a compound in the preparation of drug-containing medical devices.

[0009] This invention will use the inhibition of ceramide synthesis as a basis to design a highly selective small molecule inhibitor targeting the bioactive region of serine-palmitoyltransferase, in order to achieve the biological objective of inhibiting the development of diabetic cardiomyopathy.

[0010] A compound, belonging to fungal metabolites, was used as a chemical reagent to bind to the functional group of serine-palmitoyltransferase in order to study the biological effects of inhibiting serine-palmitoyltransferase.

[0011] The compound of this invention is a polysaccharide, which has been verified to improve cardiac dysfunction in diabetic cardiomyopathy. It is highly specific, does not interfere with unrelated pathways, and can be used for the prevention and treatment of diabetic cardiomyopathy.

[0012] In a mouse model of diabetic cardiomyopathy, ceramides were significantly upregulated, and this pathway could be further reversed by the compound of this invention (denoted as Myriocin) by inhibiting the biological activity of endogenous and exogenous serine-palmitoyltransferase. Similarly, diastolic dysfunction in diabetic cardiomyopathy was effectively alleviated by Myriocin, and cardiac dysfunction was significantly relieved. This indicates that Myriocin can directly inhibit the core pathway mediated by serine-palmitoyltransferase by binding to its functional group, thereby improving diabetic cardiomyopathy.

[0013] Therefore, the compounds of the present invention can specifically alleviate the occurrence and development of diabetic cardiomyopathy and improve cardiac dysfunction in diabetic cardiomyopathy. They have a stronger and more specific regulatory effect on serine-palmitoyltransferase and do not interfere with unrelated pathways. They are more widely applicable than traditional drugs and fill the current gap in etiological treatment of ceramide accumulation in diabetic cardiomyopathy.

[0014] The application of a polysaccharin as an experimental inhibitor in the inhibition of serine-palmitoyltransferase activity.

[0015] A polysaccharin, used as a chemical reagent in the preparation of drugs for treating diabetic cardiomyopathy.

[0016] Polysaccharin is mixed with other excipients to form a formulation, which is then used to manufacture drugs for the prevention and treatment of diabetic cardiomyopathy, such as, but not limited to, tablets, capsules, granules, emulsions, implants, or injections.

[0017] These pharmaceutical excipients can be those commonly used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for use to be compatible with the substances in the formulation, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the body and enhancing the drug delivery effect of the composition.

[0018] In aqueous injections, excipients generally include isotonic agents and buffer solutions, as well as necessary emulsifiers (such as Tweeen-80, Pluronic, and Poloxamer), solubilizers, and antibacterial agents. In addition, they may include other pharmaceutically acceptable excipients, such as antioxidants, pH adjusters, and analgesics.

[0019] Excipients used in the preparation of oral liquid formulations generally include solvents, as well as necessary flavoring agents, antibacterial agents, emulsifiers, and coloring agents.

[0020] Excipients used in tablet manufacturing generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium), and lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate).

[0021] The excipients used in the preparation of emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents.

[0022] The excipients used to produce granules are similar to those used for tablets, but the granulation process is different. Depending on the requirements, the produced granules are mixed with a gliding agent and then encapsulated to obtain capsules.

[0023] Various excipients and compounds are used to formulate dosage forms that facilitate drug delivery, such as, but not limited to, aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release formulations, and controlled-release formulations. Furthermore, excipients may be used to achieve specific drug delivery purposes or methods, such as sustained-release, controlled-release, and pulsatile administration, including, but not limited to, gelatin, albumin, chitosan, polyethers, and polyester polymers, such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers. The main manifestations of "facilitating drug delivery" include, but are not limited to, improved therapeutic efficacy, increased bioavailability, reduced toxicity and side effects, and improved patient compliance.

[0024] The compounds shown in this invention can be combined with other excipients, such as through chemical coupling, to further improve the efficacy of the compounds, reduce toxicity, and prolong the dosing cycle. These excipients are typically polymers, such as polyesters, polyethers, and polyamides.

[0025] Drug-eluting medical devices, which combine drugs and medical devices, are already quite common. Examples include compound-containing stents, where the compound is loaded or coated onto the stent material as an active ingredient, used to create medical devices for the prevention and treatment of atherosclerosis. Common stent materials include PLA, PLGA, and metals. Attached Figure Description

[0026] Figure 1 To assess the effect of Myriocin on ceramide levels in vitro using immunofluorescence assays; Figure 2 Random blood glucose statistics in diabetic cardiomyopathy model mice (ns in the figure indicates no statistical difference); Figure 3 A statistical graph of body weight in diabetic cardiomyopathy model mice (ns indicates no statistically significant difference). Figure 4 A schematic diagram illustrating the assessment of cardiac ceramide levels in a diabetic cardiomyopathy model mouse using an immunofluorescence assay. Figure 5 A statistical graph showing the cardiac ceramide levels in a diabetic cardiomyopathy model mouse as assessed by immunofluorescence assay (** indicates...). P <0.01); Figure 6 A statistical graph showing the effect of Myriocin on cardiomyocyte diastolic dysfunction as assessed by echocardiography (** indicates...). P <0.01). Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0028] Unless otherwise specified, all other reagents used in the embodiments of the present invention were purchased from MCE (MedChemExpress).

[0029] Example 1: In vitro validation of candidate compounds in living organisms The candidate compounds were validated using primary mouse cardiomyocytes.

[0030] The compound Myriocin (purchased from MCE) and its working solution were prepared as follows: Myriocin was dissolved in dimethyl sulfoxide (DMSO) to obtain a 1 mM stock solution.

[0031] Mouse cardiomyocytes were cultured in DMEM + 10% serum + 1% penicillin antibody (serum present) until the cell density reached approximately 80%. Myriocin (working concentration 0.1 μM) was added, and the cells were induced for 24 hours until the cell density reached approximately 90%-100%. Primary mouse cardiomyocytes were then fixed in cell fixation medium (Beyotime, catalog number: P0098) for 15 minutes, followed by cell permeation medium (Beyotime, catalog number: P0096) to penetrate the cells for 15 minutes. The cells were then blocked with goat serum (Beyotime, catalog number: C0265) for one hour. Subsequently, anti-ceramide antibody (Sigma-Aldrich, catalog number: C8104) was added and incubated for 24 hours, followed by incubation with the corresponding fluorescent secondary antibody for one hour, and then mounted for observation. Figure 1 As shown, the addition of Myriocin significantly decreased the concentration of ceramide. This indicates that the compound Myriocin can act on the ceramide synthesis pathway.

[0032] Example 2: In vivo validation of the compound Myriocin The experimental validation of Myriocin in diabetic cardiomyopathy in mice is as follows: C57BL / 6J mice were fed a high-fat diet (60% fat) for 3 months (7 weeks of fasting). Starting from the third month of the high-fat diet, streptozotocin (STZ) (50 mg / kg dissolved in sodium citrate buffer) was administered intraperitoneally for 5 consecutive days to induce a diabetic model. After induction, part of the mouse tail was removed to measure random blood glucose levels. Mouse weight was also recorded. Figure 2 and Figure 3 As shown, the mice exhibited random blood glucose levels exceeding the diagnostic threshold for diabetes and significant weight gain, indicating a successful establishment of a diabetes model in mice.

[0033] The successfully modeled diabetic mice were then divided into two groups: a diabetic control group and a diabetic intervention group. The diabetic intervention group was injected with Myriocin (Myriocin stock solution was mixed with corn oil and administered at a dose of 1 mg / kg / day for two consecutive weeks, five days a week), while the diabetic control group was injected with saline at the same time and frequency. Two weeks after the injections were completed, the regulatory effect of Myriocin on the occurrence and development of diabetic cardiomyopathy in mice was comprehensively evaluated using techniques such as histopathological staining and small animal echocardiography.

[0034] The method for assessing the regulatory effect of myriocin on diabetic cardiomyopathy using ceramide staining is as follows: Two groups of mouse heart tissues were collected, covered with OCT embedding medium for 20 minutes, and frozen at -80°C for fixation. The mouse heart tissues were then sliced ​​into 10 μm thick sections using a cryostat and transferred to glass slides for ceramide staining. The ceramide staining reagent was from Sigma-Aldrich (catalog number C8104). According to the reagent manual, tissue fixative (brand: Beyotime, catalog number: P0098) was added for fixation for 15 minutes, followed by permeabilization buffer (brand: Beyotime, catalog number: P0096) to penetrate the tissue for 15 minutes, and then blocked with goat serum (brand: Beyotime, catalog number: C0265) for one hour. Subsequently, anti-ceramide antibody (brand: Sigma-Aldrich, catalog number: C8104) was added for incubation for 24 hours, followed by incubation with the corresponding fluorescent secondary antibody for 1 hour. After staining, the slides were covered and observed under a wide-field microscope. Figure 4 and Figure 5 As shown, the number of ceramide-positive areas in the diabetic control group was significantly greater than that in the diabetic intervention group, indicating that small molecule compounds can act on ceramides in cardiomyocytes of diabetic cardiomyopathy.

[0035] The method for assessing the regulatory effect of Myriocin on diabetic cardiomyopathy using echocardiography is as follows: One week after intervention with Myriocin and saline, mice were placed in a Vevo 3100 small animal cardiac ultrasound tracking system for cardiac function assessment. First, mice were anesthetized with 2% isoflurane and their heart rate was maintained in the 350-400 bpm range. The mice were then fixed to a detection plate, and an ultrasound probe was placed on the mouse's heart. The detection plate was rotated to display a four-chamber view on the monitor. Pulse Doppler and tissue Doppler images of transvalvular blood flow were acquired and exported to the Vevo ultrasound analysis system to analyze the E / e' ratio. Results are as follows: Figure 6 As shown, the diastolic function of the heart in the diabetic control group mice was significantly worse than that in the diabetic intervention group, indicating that the small molecule compound can inhibit the deterioration of cardiac function in diabetic cardiomyopathy and exert a therapeutic effect.

Claims

1. The use of a compound as a chemical reagent in binding with serine-palmitoyltransferase to study the biological effects of inhibiting the ceramide synthesis pathway, said compound being polysaccharin.

2. The application of an experimental inhibitor in a reagent for inhibiting serine-palmitoyltransferase activity, wherein the compound is polysaccharin.

3. The use of a compound in the preparation of a drug that inhibits serine-palmitoyltransferase, said compound being polysaccharin.

4. The use of a compound in the preparation of a medicament for treating diabetic cardiomyopathy, said compound being polysaccharin.

5. The use of a compound in the preparation of a medicament for improving cardiac dysfunction in diabetic cardiomyopathy, said compound being polysaccharin.

6. The application according to any one of claims 3 to 5, characterized in that... The medicines mentioned also include pharmaceutical excipients.

7. The application according to any one of claims 3 to 5, characterized in that... The drug is further formulated with pharmaceutical excipients, and the formulation is a tablet, capsule, granule, emulsion, implant, or injection.

8. The use of a compound in the preparation of a drug-containing medical device for diabetic cardiomyopathy, said compound being polysaccharin.

9. The use of a compound in the preparation of a drug-containing medical device for improving cardiac dysfunction in diabetic cardiomyopathy, said compound being polysaccharin.

10. The application according to claim 8 or 9, wherein the medical device includes a stent.

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