Compound for treating myocardial infarction

Abstract

The present invention belongs to the technical field of biology, and particularly relates to a compound for treating myocardial infarction. A chondroitin sulfate oligosaccharide in the present invention is extracted from natural chondroitin sulfate. After the chondroitin sulfate oligosaccharide is treated by a chemically enzymatic process into an oligosaccharide chain, experiments prove that the chondroitin sulfate oligosaccharide contributes to the survival of cardiomyocytes. Compared with other products with similar functions, a glycopeptide polymer hydrogel formed after the introduction of a polymer backbone overcomes the problem of batch-to-batch differences of natural products and has a definite structure. Moreover, the hydrogel has excellent inherent biological activity, and does not need to load active ingredients such as other drugs. The hydrogel exhibits an obvious effect on supporting the ventricular wall in an infarcted area and promoting the survival of cardiomyocytes, and can effectively limit the area of myocardial infarction, increase the ventricular wall thickness of the infarcted area, and alleviate cardiac damage caused by myocardial infarction, and therefore has a good application prospect.

Description

A compound used to treat myocardial infarction Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a compound for the treatment of myocardial infarction. Background Technology

[0002] Myocardial infarction caused by coronary artery occlusion is one of the most common and deadliest cardiovascular diseases. In the initial stages of myocardial infarction, the ischemic and hypoxic local microenvironment leads to irreversible necrosis of most cardiomyocytes and an inflammatory response. Subsequently, these problems often extend to other non-infarcted areas, resulting in more severe cardiomyocyte loss, impaired contractile function, disruption of ventricular wall integrity, and even cardiac rupture.

[0003] Clinically, the main treatments for myocardial infarction include drug therapy, stent implantation, and coronary artery bypass surgery. However, apart from heart transplantation, there is no other method to regenerate or repair damaged heart tissue. Therefore, in recent years, biomaterials such as injectable hydrogels, cardiac patches, and nanocarriers have shown great promise in repairing heart tissue (Tariq, U., et al., Role of Biomaterials in Cardiac Repair and Regeneration: Therapeutic Intervention for Myocardial Infarction. ACS Biomaterials Science & Engineering 2022, 8(8), 3271-3298.). Injectable hydrogels are a type of hydrophilic three-dimensional polymer network that, after entering the human body in liquid form, transforms into a gel state under specific conditions. Compared to other materials, hydrogels have two significant advantages in treating myocardial infarction: 1. They can provide mechanical support for the ventricular wall; 2. They can mimic the extracellular matrix and myocardial microenvironment to further enhance their therapeutic and regenerative functions (Xu, Q., et al., Hydrogel-based cardiac repair and regeneration function in the treatment of myocardial infarction. Materials Today Bio 2024, 25, 100978.). Based on their origin, hydrogels can be classified into three categories: natural biomaterial-based hydrogels, synthetic hydrogels, and hybrid hydrogels. Natural biomaterial-based hydrogels and hybrid hydrogels exhibit better biocompatibility and biodegradability, while synthetic hydrogels often possess higher mechanical strength.

[0004] Currently, two alginate-based hydrogels (Algisyl-LVR (Mann,DL, et al., One-year follow-up results from AUGMENT-HF: a multicentre randomized controlled clinical trial of the efficacy of left ventricular augmentation with Algisyl in the treatment of heart failure. European Journal of Heart Failure 2016, 18(3), 314-325.) and IK-5001 (Rao,SV, et al., Bioabsorbable Intracoronary Matrix for Prevention of Ventricular Remodeling After Myocardial Infarction. Journal of the American College of Cardiology 2016, 68(7), 715-723.)) have successfully entered the clinical trial stage. Clinical results show that they exhibit excellent safety and improve cardiac function in heart failure patients. In addition, extracellular matrix has also been shown to be useful as an injectable scaffold in cardiovascular tissue engineering. For example, VentriGel hydrogel is an extracellular matrix extracted from porcine cardiomyocytes. This product has demonstrated its safety, feasibility, and preliminary efficacy in patients with early and late myocardial infarction through a phase I clinical trial (Traverse, JH, et al., First-in-Man Study of a Cardiac Extracellular Matrix Hydrogel in Early and Late Myocardial Infarction Patients. JACC: Basic to Translational Science 2019, 4(6), 659-669.). Synthetic hydrogels are more promising for tissue repair after myocardial infarction due to their excellent mechanical properties, morphology, and degradability. The required mechanical properties can be adjusted by changing the molecular weight and composition. Hydrogels derived from natural biomaterials often face problems such as large batch-to-batch variability, low mechanical strength, and potential induction of immune responses.Synthetic hydrogels generally only provide support, and in practical applications they often need to be loaded with active ingredients such as cytokines and drugs (Zhang, F., et al., Injectable and Conductive Nanomicelle Hydrogel with α-Tocopherol Encapsulation for Enhanced Myocardial Infarction Repair. ACS Nano 2024, 18(14), 10216-10229; Yue, K., et al., Synthesis, properties, and biomedical applications of gelatin methacryloyl(GelMA)hydrogels. Biomaterials 2015, 73, 254-271.).

[0005] Chondroitin sulfate (CS) is a naturally derived bioactive macromolecule belonging to the glycosaminoglycan (GAG) class. It is not only a major component of the extracellular matrix (ECM), but also typically binds with membrane proteins to form chondroitin sulfate proteoglycan (CSPG), distributed across almost the entire cell surface (du Souich, P., García, AG, Vergés, J., et al. Immunomodulatory and Anti-Inflammatory Effects of Chondroitin Sulphate. Journal of Cellular and Molecular Medicine, 2009, 13, 1451-1463.). It is a multifunctional signaling molecule and regulator, interacting with cytokines, growth factors, and enzymes. A., Zhou, G., et al. Medical Application of Glycosaminoglycans: A Review. Journal of Tissue Engineering and Regenerative Medicine, 2018, 12, e23-e41. Glycosaminoglycans have a profound impact on many physiological processes, including inflammation, tumor progression and metastasis, angiogenesis, and antioxidation. As a biological polysaccharide, CS possesses multivalent properties, controllable molecular weight, and strong designability (Lin, K. and Kasko, AMCarbohydrate-Based Polymers for Immune Modulation. ACS Macro Letters, 2014, 3, 652-657), making it popular among polymer scientists. The excellent adhesion, biocompatibility, biodegradability, and cell targeting properties of CS (Lee, JY, Lee, et al. Blood Component Ridable and CD44 Receptor Targetable Nanoparticles Based on a Maleimide-Functionalized Chondroitin Sulfate Derivative. Carbohydrate Polymers, 2020, 230, Article ID: 115568.) make it widely used in the preparation of targeted delivery systems. These systems aim to embed drugs, cells, or genes into composite biomaterials for targeted delivery and release under specific conditions, thereby reducing toxic side effects, prolonging drug action time, improving pharmacodynamics, or overcoming the immunogenicity of inhibiting tissues and reducing the risk of rejection (Yang, JY, Jiang, S., et al. Pancreatic Islet Surface Engineering with a starPEG-Chondroitin Sulfate Nanocoating. Biomaterials Science, 2019, 7, 2308-2316.).In addition, CS is also widely used as a biological scaffold in the fields of bone, cartilage, cornea, skin and neural tissue engineering (Farrugia, BL, Lord, et al. Harnessing Chondroitin Sulphate in Composite Scaffolds to Direct Progenitor and Stem Cell Function for Tissue Repair. Biomaterials Science, 2018, 6, 947-957.). Summary of the Invention

[0006] This invention uses chemical synthesis to construct naturally occurring glycopeptide polymers, which improves the problem of large batch-to-batch variations in natural materials, and achieves the desired effect of reducing damage to heart tissue without the need to encapsulate other components.

[0007] In a first aspect, the present invention provides the application of chondroitin sulfate oligosaccharide in the preparation of glycopeptide hydrogel materials, wherein the glycopeptide hydrogel materials are used to treat myocardial infarction.

[0008] Furthermore, the chondroitin sulfate oligosaccharide is extracted from the cartilage and collagen tissues of various animals such as sharks and bovine cartilage, through microbial fermentation, or chemical synthesis.

[0009] Furthermore, the chondroitin sulfate oligosaccharides include one or more of chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, and / or chondroitin sulfate octasaccharide.

[0010] Preferably, the chondroitin sulfate oligosaccharide is chondroitin sulfate tetrasaccharide (CS-T) and / or chondroitin sulfate hexasaccharide (CS-H).

[0011] Secondly, the present invention provides a glycopeptide hydrogel, wherein the hydrogel is composed of a polymer backbone and chondroitin sulfate oligosaccharides via covalent bonds, and the structural formula of the glycopeptide hydrogel is shown in formula (I):

[0012] Where n = 1-10000; R is chondroitin sulfate oligosaccharide, and R' is a compound containing cis-diol.

[0013] Furthermore, the chondroitin sulfate oligosaccharides include one or more of chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, and / or chondroitin sulfate octasaccharide.

[0014] Preferably, the chondroitin sulfate oligosaccharide is chondroitin sulfate tetrasaccharide (CS-T) and / or chondroitin sulfate hexasaccharide (CS-H).

[0015] Furthermore, the compounds containing cis-diols include, but are not limited to, one or more of catechol, malic acid, glucose, and / or mannose.

[0016] Furthermore, the chondroitin sulfate oligosaccharide is linked to the polymer backbone in ways including but not limited to N-linking, O-linking, S-linking, or triazole-linking.

[0017] Furthermore, the linkage between the compound containing cis-diol and the polymer backbone includes, but is not limited to, N-linkage, O-linkage, S-linkage, or triazole linkage.

[0018] In one embodiment of the present invention, when the chondroitin sulfate oligosaccharide is CS-T, the structural formula of the glycopeptide hydrogel material is as follows:

[0019] In one embodiment of the present invention, when the chondroitin sulfate oligosaccharide is CS-H, the glycopeptide hydrogel material has the following structural formula:

[0020] Thirdly, the present invention provides a pharmaceutical composition comprising the chondroitin sulfate oligosaccharide described in the first aspect or the glycopeptide hydrogel described in the second aspect.

[0021] Furthermore, the pharmaceutical composition may also contain a stereoisomer of the chondroitin sulfate oligosaccharide of the first aspect or the glycopeptide hydrogel of the second aspect, or a pharmaceutically acceptable salt thereof, or a tautomer thereof, or an isotopically labeled compound thereof.

[0022] Furthermore, the pharmaceutical composition can be formulated into various dosage forms, including but not limited to one or more of the following: tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, and / or lyophilized powder injections.

[0023] Furthermore, colorants, preservatives, flavorings, tasters, sweeteners, or other materials may be added to the pharmaceutical preparations if necessary.

[0024] Furthermore, the pharmaceutical composition can be administered by injection, cavity administration, respiratory administration, or mucosal administration.

[0025] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; the cavity administration includes rectal or vaginal administration; and the respiratory administration is nasal administration.

[0026] Fourthly, the present invention provides the use of the chondroitin sulfate oligosaccharide or glycopeptide hydrogel described in the first aspect in the preparation of products for repairing heart tissue.

[0027] Furthermore, the product is a biopharmaceutical or a biological scaffold.

[0028] Fifthly, the present invention provides the use of the chondroitin sulfate oligosaccharide described in the first aspect or the glycopeptide hydrogel described in the second aspect in the preparation of a medicament for treating myocardial infarction.

[0029] Furthermore, the myocardial infarction includes type 1 myocardial infarction, type 2 myocardial infarction, type 3 myocardial infarction, type 4a myocardial infarction, type 4b myocardial infarction, and type 5 myocardial infarction. Beneficial effects

[0030] 1. The chondroitin sulfate oligosaccharides in this invention are extracted from natural chondroitin sulfate, which is commonly used in the treatment of arthritis. However, after being processed into oligosaccharide chains by chemical enzymatic methods, experiments have shown that they help the survival of cardiomyocytes. This effect is further enhanced by the introduction of a polymer backbone.

[0031] 2. The design of the glycopeptide polymer hydrogel in this invention is primarily based on the reversible interaction between natural sugar molecules and phenylboronic acid groups. Compared to other products with similar functions, it overcomes the problem of batch-to-batch variability in natural products, has a well-defined structure, and the hydrogel itself possesses excellent bioactivity, eliminating the need to encapsulate other active ingredients such as drugs, thus demonstrating potential for further development.

[0032] 3. The glycopeptide hydrogel in this invention has a significant effect on supporting the ventricular wall in the infarct area and promoting the survival of cardiomyocytes. It can effectively limit the area of ​​myocardial infarction, increase the thickness of the ventricular wall in the infarct area, and reduce the damage of myocardial infarction to the heart, and has a good application prospect. Attached Figure Description

[0033] Figure 1 shows the high-resolution mass spectrometry characterization of CS-T.

[0034] Figure 2 shows the high-resolution mass spectrometry characterization of CS-H.

[0035] Figure 3 shows the NMR characterization of polymer 4.

[0036] Figure 4 shows the NMR characterization of polymer 7.

[0037] Figure 5 shows the NMR characterization of polymer 8.

[0038] Figure 6 shows the therapeutic effects of CS-T and CS-H on mouse cardiac ejection fraction.

[0039] Figure 7 shows the physical properties characterization of polymer 4+7.

[0040] Figure 8 shows the physical properties characterization of polymer 4+8.

[0041] Figure 9 shows the effect of hydrogel treatment on cardiac ejection fraction in mice. Note: Left image: 1 week; Right image: 4 weeks.

[0042] Figure 10 shows a mouse heart slice to evaluate the therapeutic effect of hydrogel. Detailed Implementation

[0043] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0044] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0045] Terminology Explanation

[0046] Type 1 myocardial infarction: Spontaneous myocardial infarction. It occurs due to the rupture, ulceration, cracking, erosion, or dissection of atherosclerotic plaques, leading to thrombosis in one or more coronary arteries, resulting in reduced myocardial density or distal platelet embolism accompanied by myocardial necrosis. Most patients have severe coronary artery disease, while a few have only mild stenosis or even normal coronary arteries.

[0047] Type 2 myocardial infarction: Myocardial infarction secondary to an imbalance between myocardial oxygen supply and demand. Other conditions besides coronary artery disease can cause an imbalance between myocardial oxygen demand and supply, leading to myocardial damage and necrosis. These conditions include coronary endothelial dysfunction, coronary artery spasm or embolism, tachycardia / bradycardia, anemia, respiratory failure, hypotension, and hypertension with or without left ventricular hypertrophy.

[0048] Type 3 myocardial infarction: sudden cardiac death. Cardiac death with symptoms of myocardial ischemia and new ischemic electrocardiographic changes or left bundle branch block, but without myocardial injury markers.

[0049] Type 4a myocardial infarction: Percutaneous coronary intervention (PCI)-related myocardial infarction. In patients with normal baseline cardiac troponin (cTn), cTn increases more than 5 times the upper limit of normal after PCI, or in patients with elevated baseline cTn, cTn increases by ≥20% after PCI and then stabilizes and decreases. Concurrent occurrences include: (1) symptoms of myocardial ischemia; (2) ischemic changes on electrocardiogram or new-onset left bundle branch block; (3) angiography showing occlusion or persistent slow flow or no-reflow or embolism in the main or branch coronary arteries; (4) new loss of viable myocardium or radiographic manifestations of segmental wall motion abnormalities.

[0050] Type 4b myocardial infarction: Myocardial infarction caused by stent thrombosis. Coronary angiography or autopsy reveals thrombotic obstruction at the stent implantation site, the patient has symptoms of myocardial ischemia and / or at least one myocardial injury marker is above the upper limit of normal.

[0051] Type 5 myocardial infarction: surgical coronary artery bypass grafting (CABG)-related myocardial infarction. In patients with normal baseline cTn, cTn increases more than 10 times the upper limit of normal after CABG, and the following occurs simultaneously: (1) new pathological Q waves or left bundle branch block; (2) angiography showing new bypass graft or autologous coronary artery occlusion; (3) new loss of viable myocardium or radiographic evidence of segmental wall motion abnormalities.

[0052] Example 1: Synthesis of Chondroitin Sulfate Oligosaccharides

[0053] 1.1 Synthesis of CS-DS

[0054] Add 1L of anhydrous methanol to a 1L flask, then disperse acetyl chloride (5mL) dropwise in the methanol. Add natural chondroitin sulfate (10g), stir and react for 7 days, changing the solution every other day. After the reaction is complete, filter to remove methanol, dry and weigh.

[0055] 1.2 Synthesis of CS-T and CT-H

[0056] An appropriate amount of CS-DS was placed in a flask, and 0.1M sodium hydroxide solution was added. After stirring for 24 hours, the mixture was neutralized and concentrated using a cation exchange resin. The concentrate was then precipitated in ethanol and dried. A pale yellow powder was obtained and added to sodium acetate buffer (pH = 5.0). The mixture was placed in a 37°C incubator, and after the temperature stabilized, Hyaluronidas was added. The enzymatic digestion reaction was carried out for 7 days. The digestion product was precipitated in ethanol and passed through an anion exchange resin to separate CS-T and CS-H. The corresponding characterization is shown in Figure 1-2.

[0057] Example 2 Synthesis of Glycopeptide Hydrogel

[0058] 2.1 Synthesis of Polymer Backbone

[0059] Synthesis of 4: Phenylated boric acid (Bob, 0.14 g), NaBH4 (0.11 g), and 3 (210 mg) were added to a flask. 2.8 mL of methanol was added in an ice bath, the ice bath was removed, and the mixture was stirred at room temperature for 6 hours. Dialysis was performed in a sodium hydroxide solution at pH 9, and the product was lyophilized to obtain a pale yellow solid, product 4, which is the polymer backbone described in this invention. The NMR characterization of polymer 4 is shown in Figure 3.

[0060] 2.2 Sugar Modification of Polymers

[0061] Synthesis of 7: 6 (15 mg, 0.035 mmol), CS-T (40 mg, 0.033 mmol), copper sulfate pentahydrate (2 mg, 0.008 mmol), and sodium ascorbate (14 mg, 0.071 mmol) were dissolved completely in DMF or water sequentially. After deoxygenation treatment, the mixture was stirred at 50 °C for 2 days. After the reaction was complete, the product was dialyzed against EDTA aqueous solution for 2 days, then against pure water for 2 days. Lyophilization yielded the corresponding product 7, and the corresponding NMR spectrum is shown in Figure 4.

[0062] Synthesis of 8: Refer to the synthesis of 7, the corresponding NMR spectrum is shown in Figure 5.

[0063] Example 3: Evaluation of the bioactivity of chondroitin sulfate oligosaccharides in the treatment of myocardial infarction in mice.

[0064] Male mice aged 8-10 weeks were selected for the experiment. A mouse model of left ventricular wall infarction was established by surgically ligating the left anterior descending coronary artery. Subsequently, 20 μL of CS-T and CS-H were injected into the myocardium using a microsyringe. The recovery of the heart was observed by ultrasound one week and four weeks later.

[0065] As shown in Figure 6, ultrasound testing revealed that the ejection fraction of mice after myocardial infarction was generally around 20%. Natural chondroitin sulfate (CS) did not contribute to cardiac recovery, while the oligosaccharide groups CS-T and CS-H both showed some effect on cardiomyocyte survival. This indicates that this class of chondroitin sulfate oligosaccharide compounds has excellent biocompatibility and contributes to cardiomyocyte survival.

[0066] Example 4: Evaluation of the physical properties and biocompatibility of glycopeptide hydrogels

[0067] To test the physical properties of the synthesized glycopeptide hydrogel, we used a rheometer to test it, mainly determining the gel modulus through frequency scanning and strain scanning.

[0068] As shown in Figures 7 and 8, rheological tests were performed immediately after mixing polymer 4 and polymer 7. The results showed that the hydrogel had a storage modulus of approximately 800 Pa and a loss modulus of approximately 250 Pa, which meets the mechanical performance requirements for supporting cardiomyocyte growth during myocardial infarction. This indicates that the synthesized glycopeptide hydrogel has excellent physical properties and biocompatibility, making it suitable for providing physical support to the infarcted site after myocardial infarction.

[0069] Example 5: Evaluation of the bioactivity of glycopeptide hydrogels in the treatment of myocardial infarction in mice.

[0070] Male mice aged 8-10 weeks were used in the experiment. A mouse model of left ventricular wall infarction was established by surgically ligating the left anterior descending coronary artery. Subsequently, 10 μL each of polymers 4 and 7 were injected into the myocardium using a microsyringe. Ultrasound observation was performed at one and four weeks, and tissue sections were taken at four weeks.

[0071] As shown in Figure 9, ultrasound testing revealed that the ejection fraction of mice after myocardial infarction treatment was generally around 20%. The hydrogel obtained by mixing polymers 4 and 7 provided some support to the ventricular wall in the infarct area (AB), while the hydrogel obtained by mixing polymers 4 and 7 (AB-CS-T) showed better efficacy. Four weeks later, myocardial sections from mice stained with HE and Masson's staining (Figure 10) showed that the infarcted mice (MI) had a larger infarct area and more cardiac chambers, with severe fibrosis. Mice injected with polymers 4 and 7 had lower infarct area and fibrosis. Mice injected with polymers 4 and 8 had the least infarct area and fibrosis. This indicates that the synthesized glycopeptide hydrogel exhibited good bioactivity in the treatment of myocardial infarction in mice, effectively supporting the ventricular wall in the infarct area and promoting the survival of cardiomyocytes.

Claims

1. A glycopeptide hydrogel, wherein the hydrogel is prepared by mixing a sugar-modified polymer and a polymer 4, wherein the sugar-modified polymer is composed of a polymer backbone and chondroitin sulfate oligosaccharides via covalent bonds, and when the chondroitin sulfate oligosaccharides are chondroitin sulfate tetrasaccharides, the polymer structure modified by the glycopeptide hydrogel material is as follows: When the chondroitin sulfate oligosaccharide is chondroitin sulfate hexasaccharide, the polymer structure modified by the sugar is as follows: The structural formula of polymer 4 is as follows:

2. A pharmaceutical composition comprising the glycopeptide hydrogel of claim 1.

3. The use of the glycopeptide hydrogel of claim 1 in the preparation of products for repairing heart tissue.

4. The application as described in claim 3, wherein the product is a biological scaffold.

5. The use of the glycopeptide hydrogel of claim 1 in the preparation of a medicament for treating myocardial infarction.

6. The application as described in claim 5, wherein the myocardial infarction includes type 1 myocardial infarction, type 2 myocardial infarction, type 3 myocardial infarction, type 4a myocardial infarction, type 4b myocardial infarction, and type 5 myocardial infarction.

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