Compound for treating myocardial infarction

Abstract

The invention belongs to the technical field of biology, and particularly relates to a compound for treating myocardial infarction. The chondroitin sulfate oligosaccharide is extracted from natural chondroitin sulfate, and after the chondroitin sulfate oligosaccharide is treated into an oligosaccharide chain through a chemical enzyme method, experiments prove that the chondroitin sulfate oligosaccharide is beneficial to the survival of myocardial cells. Compared with other products with similar functions, the glycopeptide polymer hydrogel formed after introduction of the polymer skeleton overcomes the problem of batch-to-batch difference of natural products and is clear in structure, and the hydrogel has excellent biological activity and does not need to entrap other active ingredients such as drugs. The effects of supporting the ventricular wall of the infarction region and promoting the survival of myocardial cells are obvious, the myocardial infarction area can be effectively limited, the thickness of the ventricular wall of the infarction region is increased, the damage of myocardial infarction to the heart is reduced, and the application prospect is very good.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a compound for treating myocardial infarction. Background Art

[0002] Myocardial infarction caused by coronary artery occlusion is one of the most common cardiovascular diseases with the highest mortality rate. In the initial stage of myocardial infarction, the local microenvironment of ischemia and hypoxia will lead to irreversible necrosis of most cardiomyocytes and produce an inflammatory response. Subsequently, these problems usually extend further to other non-infarcted areas, causing more serious cardiomyocyte loss, impaired contractile function, destruction of ventricular wall integrity, and even heart rupture.

[0003] Clinically, the main measures used for myocardial infarction are drug therapy, stent implantation and coronary artery bypass surgery. But except for heart transplantation, there is no method that can regenerate or repair damaged heart tissue. Therefore, in recent years, biomaterials such as injectable hydrogels, heart patches and nanocarriers have shown great application prospects 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 enters the human body in liquid form and is converted into a gel state under specific conditions. Compared with other materials, hydrogels have two obvious advantages in the treatment of myocardial infarction: 1. They can provide mechanical support for the ventricular wall, and 2. They can simulate the extracellular matrix and myocardial microenvironment to further exert their therapeutic and recovery functions (Xu, Q., et al., Hydrogel-based cardiac repair and regeneration function in the treatment of myocardial infarction. Materials Today Bio 2024, 25, 100978.). Hydrogels can be divided into three categories according to their sources, namely natural biomaterial-based hydrogels, synthetic hydrogels, and hybrid hydrogels. Among them, natural biomaterial-based hydrogels and hybrid hydrogels have better biocompatibility and degradability, while synthetic hydrogels often have higher mechanical strength.

[0004] At present, 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 Algisylin 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 have excellent safety and improved cardiac function in patients with heart failure. In addition, extracellular matrix has also been shown to be used as an injectable scaffold in cardiovascular tissue engineering. For example, VentriGel hydrogel is an extracellular matrix extracted from porcine cardiomyocytes. This product has been proven to be safe, feasible and initially effective in patients with early and late myocardial infarction through phase I clinical trials (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 in post-myocardial infarction tissue repair 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 have to face problems such as large batch differences, low mechanical strength and possible immune response.Synthetic hydrogels generally only provide support, and actual use often requires loading of 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 natural bioactive macromolecule belonging to the glycosaminoglycan (GAG) class. It is not only the main component of the extracellular matrix (ECM), but also usually connects with membrane proteins to form chondroitin sulfate proteoglycan (CSPG) distributed on the surface of almost all cells (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 that interacts 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), which has a profound impact on many physiological processes such as inflammation, tumor progression and metastasis, vascular remodeling, and anti-oxidation. As a biopolysaccharide, CS has multivalency, controllable molecular weight, and strong designability (Lin, K. and Kasko, AM Carbohydrate-Based Polymers for Immune Modulation. ACS Macro Letters, 2014, 3, 652-657.), and is favored by polymer science researchers. CS has good adhesion, biocompatibility, biodegradability and cell targeting (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.), making it widely used in the preparation of targeted delivery systems, aiming to embed drugs, cells or genes into composite biomaterials for targeted delivery and release under specific conditions, thereby reducing toxic side effects, prolonging the drug action time, improving pharmacodynamic function, or overcoming the immunogenicity of inhibitory tissues and reducing the risk of rejection (Yang, JY, Jiang, S., et al. Pancreatic Islet Surface Engineering with astarPEG-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 nerve 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] The present invention uses chemical synthesis to construct glycopeptide polymers existing in nature, thereby improving the problem of large batch differences in natural materials and achieving the desired effect of reducing cardiac tissue damage without encapsulating other components.

[0007] In a first aspect, the present invention provides a use of chondroitin sulfate oligosaccharide in preparing a glycopeptide hydrogel material, wherein the glycopeptide hydrogel material is used for treating myocardial infarction.

[0008] Furthermore, the chondroitin sulfate oligosaccharide is extracted from cartilage and collagen tissues of various animals such as shark and bovine cartilage, fermented by microorganisms or chemically synthesized.

[0009] Furthermore, the chondroitin sulfate oligosaccharide includes 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] In a second aspect, the present invention provides a glycopeptide hydrogel, wherein the hydrogel is composed of a polymer skeleton and chondroitin sulfate oligosaccharides through covalent bonds, and the structural formula of the glycopeptide hydrogel is shown in formula (I):

[0012]

[0013] Wherein, n=1-10000; R is chondroitin sulfate oligosaccharide, and R' is a compound containing cis-diol.

[0014] Furthermore, the chondroitin sulfate oligosaccharide includes one or more of chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide and / or chondroitin sulfate octasaccharide.

[0015] Preferably, the chondroitin sulfate oligosaccharide is chondroitin sulfate tetrasaccharide (CS-T) and / or chondroitin sulfate hexasaccharide (CS-H).

[0016] Furthermore, the compound containing cis-diol includes but is not limited to one or more of catechol, malic acid, glucose and / or mannose.

[0017] Furthermore, the connection modes of the chondroitin sulfate oligosaccharide and the polymer backbone include but are not limited to N connection, O connection, S connection or triazole connection.

[0018] Furthermore, the connection modes of the compound containing cis-diol and the polymer backbone include but are not limited to N connection, O connection, S connection or triazole connection.

[0019] 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:

[0020]

[0021] In one embodiment of the present invention, when the chondroitin sulfate oligosaccharide is CS-H, the structural formula of the glycopeptide hydrogel material is as follows:

[0022]

[0023] In a third aspect, 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.

[0024] Furthermore, the pharmaceutical composition may also contain stereoisomers of the chondroitin sulfate oligosaccharide described in the first aspect or the glycopeptide hydrogel described in the second aspect, or pharmaceutically acceptable salts thereof, or tautomers thereof, or isotope-labeled compounds thereof.

[0025] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0026] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0027] Furthermore, the pharmaceutical composition can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

[0028] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration is nasal administration.

[0029] In a fourth aspect, the present invention provides use of the chondroitin sulfate oligosaccharide or glycopeptide hydrogel described in the first aspect in preparing a product for repairing cardiac tissue.

[0030] Furthermore, the product is a biopharmaceutical or a bioscaffold.

[0031] In a fifth aspect, the present invention provides 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 drug for treating myocardial infarction.

[0032] 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.

[0033] Beneficial Effects

[0034] 1. The chondroitin sulfate oligosaccharide in the present invention is extracted from natural chondroitin sulfate, which is generally used in the treatment of arthritis. However, after being processed into oligosaccharide chains by chemical enzyme method, experiments have shown that it helps the survival of myocardial cells. After the introduction of polymer skeleton, this effect has been better developed.

[0035] 2. The design of the glycopeptide polymer hydrogel in the present invention is mainly based on the reversible interaction between natural sugar molecules and phenylboronic acid groups. Compared with other products with similar functions, it overcomes the problem of batch differences in natural products, has a clear structure, and the hydrogel itself has excellent biological activity, without the need to encapsulate other active ingredients such as drugs, and has the potential for further development.

[0036] 3. The glycopeptide hydrogel in the present invention has obvious effects on supporting the ventricular wall in the infarcted area and promoting the survival of myocardial cells. It can effectively limit the area of ​​myocardial infarction, enhance the thickness of the ventricular wall in the infarcted area, and reduce the damage of myocardial infarction to the heart, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 High-resolution mass spectrometry characterization of CS-T.

[0038] Figure 2 High-resolution mass spectrometric characterization of CS-H.

[0039] Figure 3 NMR characterization of polymer 4.

[0040] Figure 4 NMR characterization of polymer 7.

[0041] Figure 5 NMR characterization of polymer 8.

[0042] Figure 6 The effects of CS-T and CS-H treatment were evaluated on the cardiac ejection fraction of mice.

[0043] Figure 7 Characterization of the physical properties of polymer 4+7.

[0044] Figure 8 Characterization of the physical properties of polymer 4+8.

[0045] Fig. 9 The effect of hydrogel treatment was evaluated for the ejection fraction of the mouse heart. Note: Left: 1 week; Right: 4 weeks.

[0046] Fig.10 Evaluation of hydrogel therapeutic effects on mouse heart slices. DETAILED DESCRIPTION

[0047] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, 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.

[0048] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0049] Explanation of terms

[0050] Type 1 myocardial infarction: spontaneous myocardial infarction. Due to rupture, ulceration, cracking, erosion or dissection of atherosclerotic plaques, one or more coronary artery thrombosis occurs, resulting in myocardial reduction or distal platelet embolism with myocardial necrosis. Most patients have severe coronary artery lesions, while a few patients have only mild coronary artery stenosis or even normal coronary arteries.

[0051] Type 2 myocardial infarction: Myocardial infarction secondary to an imbalance between myocardial oxygen supply and demand. Conditions other than coronary artery disease cause an imbalance between myocardial oxygen demand and supply, leading to myocardial damage and necrosis, such as coronary endothelial dysfunction, coronary artery spasm or embolism, tachycardia / bradyarrhythmia, anemia, respiratory failure, hypotension, hypertension with or without left ventricular hypertrophy.

[0052] Type 3 myocardial infarction: sudden cardiac death. Cardiac death with symptoms of myocardial ischemia and new ischemic ECG changes or left bundle branch block, but no results of myocardial injury markers.

[0053] Type 4a myocardial infarction: Myocardial infarction related to percutaneous coronary intervention (PCI). Patients with normal baseline cardiac troponin (cTn) have an increase of more than 5 times the upper limit of normal after PCI, or patients with elevated baseline cTn have an increase of >= 20% after PCI and then a steady decrease. Concurrently occurring: (1) symptoms of myocardial ischemia; (2) ischemic changes on electrocardiogram or new-onset left bundle branch block; (3) angiography showing obstruction of the main or branch coronary arteries or persistent slow blood flow or no reflow or embolism; (4) imaging findings of new loss of viable myocardium or segmental wall motion abnormalities.

[0054] Type 4b myocardial infarction: Myocardial infarction caused by stent thrombosis. Coronary angiography or autopsy revealed thrombotic obstruction at the site of stent implantation, and the patient had symptoms of myocardial ischemia and / or at least one myocardial injury marker was higher than the upper limit of normal.

[0055] Type 5 myocardial infarction: myocardial infarction related to coronary artery bypass grafting (CABG). Patients with normal baseline cTn and cTn elevation exceeding 10 times the upper limit of normal after CABG, with the occurrence of: (1) new pathological Q waves or left bundle branch block; (2) angiographic evidence of new graft vessels or native coronary artery occlusion; (3) imaging evidence of new loss of viable myocardium or segmental wall motion abnormalities.

[0056] Example 1 Synthesis of Chondroitin Sulfate Oligosaccharides

[0057]

[0058] Synthetic routes of CS-T and CS-H

[0059] 1.1 Synthesis of CS-DS

[0060] In a 1L flask, add 1L of anhydrous methanol, disperse acetyl chloride (5mL) dropwise in methanol, add natural chondroitin sulfate (10g), stir and react for 7 days, and change the solution every other day. After the reaction is completed, remove the methanol by suction, dry and weigh.

[0061] 1.2 Synthesis of CS-T and CT-H

[0062] Take an appropriate amount of CS-DS in a flask, add 0.1M sodium hydroxide solution and stir for 24 hours, then use cation exchange resin to neutralize, filter and concentrate, and precipitate and dry the concentrate in ethanol. The light yellow powder is added to sodium acetate buffer (pH=5.0), placed in a 37°C thermostat, and after the temperature stabilizes, Hyaluronidas is added and the enzyme digestion reaction is carried out for 7 days. The enzyme digestion product is precipitated in ethanol, passed through an anion exchange resin, and CS-T and CS-H are separated. The corresponding characterization is as follows Figure 1-2 shown.

[0063] Example 2 Synthesis of Glycopeptide Hydrogel

[0064] 2.1 Synthesis of polymer backbone

[0065]

[0066] Synthesis of 4: Add phenylboronic acid (Bob, 0.14 g), NaBH4 (0.11 g), and 3 (210 mg) to a flask. Add 2.8 mL of methanol in an ice bath, remove the ice bath, and stir at room temperature for 6 hours. Dialyze in a sodium hydroxide solution at pH = 9, and freeze-dry to obtain a light yellow solid product 4, which is the polymer backbone of the present invention. The NMR characterization of polymer 4 is as follows Figure 3 shown.

[0067] 2.2 Sugar modification of polymers

[0068]

[0069] 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 completely dissolved in DMF or water in turn, and stirred at 50 ° C for 2 days after deoxygenation. After the reaction, the product was dialyzed in EDTA aqueous solution for 2 days and then dialyzed in pure water for 2 days. The corresponding product 7 was obtained by freeze drying. The corresponding NMR image is shown in Figure 4 shown.

[0070] Synthesis of 8: Refer to the synthesis of 7, the corresponding NMR images are as follows Figure 5 shown.

[0071] Example 3 Evaluation of the biological activity of chondroitin sulfate oligosaccharides in the treatment of myocardial infarction in mice

[0072] The experiment selected 8-10 week old male mice, and surgically ligated the left anterior descending coronary artery to obtain a mouse model of left ventricular wall infarction. Subsequently, 20 μL of CS-T and CS-H were injected into the myocardium using a microsyringe. Ultrasound was performed to observe the heart recovery after one and four weeks.

[0073] like Figure 6 As shown, ultrasound tests showed that the ejection fraction of mice after myocardial infarction was generally around 20%, and natural chondroitin sulfate (CS) did not play any role in the recovery of the heart, while the oligosaccharide groups CS-T and CS-H both showed a certain effect on the survival of cardiomyocytes. This shows that this type of chondroitin sulfate oligosaccharide compound has excellent biosafety and helps cardiomyocyte survival.

[0074] Example 4 Physical properties and biosafety evaluation of glycopeptide hydrogels

[0075] In order 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.

[0076] like Figure 7 and 8 As shown, after polymer 4 and polymer 7 are mixed, rheological tests are immediately performed, and the storage modulus of the hydrogel is about 800Pa and the loss modulus is about 250Pa, which meets the mechanical performance requirements for supporting the growth of myocardial cells in the case of myocardial infarction. This shows that the synthesized glycopeptide hydrogel has excellent physical properties and biosafety, and is suitable for physical support of the infarct site after myocardial infarction.

[0077] Example 5 Evaluation of the biological activity of glycopeptide hydrogel in the treatment of myocardial infarction in mice

[0078] The experiment selected 8-10 week old male mice, and surgically ligated the left anterior descending coronary artery to obtain a mouse model of left ventricular wall infarction. Subsequently, 10 μL of polymers 4 and 7 were injected into the myocardium using a microsyringe. Ultrasound observation was performed after one and four weeks, and sections were taken after four weeks.

[0079] like Fig. 9 As shown, ultrasound tests show that the ejection fraction of mice after myocardial infarction is generally around 20%. The hydrogel obtained by mixing polymers 4 and 7 has a certain supporting effect on the ventricular wall of the infarcted area of ​​mice (AB), and the hydrogel obtained by mixing polymers 4 and 7 (AB-CS-T) shows better therapeutic effects. HE staining and Masson staining of mouse myocardial sections after four weeks show ( Fig.10 ), the infarct tissue area and heart cavity of infarcted mice (MI) were larger, and the fibrosis was severe. The infarct tissue area and fibrosis degree of mice injected with polymers 4 and 7 were lower. The infarct area and fibrosis degree of mice injected with polymers 4 and 8 were the mildest. This shows that the synthesized glycopeptide hydrogel showed good biological activity in the treatment of myocardial infarction mice, which can effectively support the ventricular wall in the infarct area and is conducive to the survival of myocardial cells.

Claims

1. An application of chondroitin sulfate oligosaccharide in the preparation of a glycopeptide hydrogel material, wherein the glycopeptide hydrogel material is used to treat myocardial infarction.

2. A glycopeptide hydrogel, wherein the hydrogel is composed of a polymer skeleton and chondroitin sulfate oligosaccharides through covalent bonds, and the structural formula of the glycopeptide hydrogel is shown in formula (I): in, n=1-10000; R is chondroitin sulfate oligosaccharide, and R' is a compound containing cis-diol.

3. The glycopeptide hydrogel according to claim 2, wherein the chondroitin sulfate oligosaccharide comprises one or more of chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide and / or chondroitin sulfate octasaccharide, preferably chondroitin sulfate tetrasaccharide (CS-T) and / or chondroitin sulfate hexasaccharide (CS-H); the compound containing cis-diol includes but is not limited to one or more of catechol, malic acid, glucose and / or mannose.

4. The glycopeptide hydrogel according to any one of claims 2 or 3, wherein the connection mode of the chondroitin sulfate oligosaccharide to the polymer backbone includes but is not limited to N connection, O connection, S connection or triazole connection, etc.; the connection mode of the compound containing cis-diol to the polymer backbone includes but is not limited to N connection, O connection, S connection or triazole connection, etc.

5. The glycopeptide hydrogel according to any one of claims 2 to 4, when the chondroitin sulfate oligosaccharide is CS-T, the glycopeptide hydrogel material has the following structural formula: When the chondroitin sulfate oligosaccharide is CS-H, the structural formula of the glycopeptide hydrogel material is as follows:

6. A pharmaceutical composition comprising the chondroitin sulfate oligosaccharide according to claim 1 or the glycopeptide hydrogel according to claim 2.

7. Use of the chondroitin sulfate oligosaccharide according to claim 1 or the glycopeptide hydrogel according to claim 2 in preparing a product for repairing cardiac tissue.

8. The use according to claim 7, wherein the product is a biopharmaceutical or a bioscaffold.

9. Use of the chondroitin sulfate oligosaccharide according to claim 1 or the glycopeptide hydrogel according to claim 2 in the preparation of a medicament for treating myocardial infarction.

10. The use according to claim 9, 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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