Application of OpiCa1 or OpiCa1 nano-liposome in preparation of medicine for treating and / or preventing myocardial fibrosis related diseases

Abstract

The invention relates to the technical field of medicines, in particular to application of OpiCa1 or a nano-liposome thereof in preparation of medicines for treating and / or preventing myocardial fibrosis related diseases. The research on modeling and administration of myocardial infarction mice finds that the OpiCa1 and the nano-liposome thereof can inhibit myocardial fibrosis and improve myocardial remodeling in the myocardial remodeling period after myocardial infarction, and have a better antagonistic effect on myocardial infarction and subsequent heart failure. Further transcriptomics analysis shows that in the process of treating the myocardial infarction of mice, the OpiCa1 and the nano-liposome thereof may participate in extracellular matrix degradation in myocardial remodeling and fibrosis pathways (such as TGF-beta signal pathways) to inhibit fibrosis and improve myocardial remodeling, and the purpose of relieving deterioration of the myocardial infarction is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of OpiCal or its nanoliposomes in the preparation of drugs for treating and / or preventing myocardial fibrosis-related diseases. Background Art

[0002] For a long time, the mortality rate of myocardial infarction (hereinafter referred to as MI) among cardiovascular diseases has remained high. Early diagnosis is difficult, the incubation period is long, the rescue time is short, and there are few patients who can be cured. The current treatment is still a comprehensive treatment with surgery as the main method and drugs as the auxiliary method. Myocardial fibrosis is an important feature of pathological remodeling after cardiac injury, and it is also an important clinical problem in cardiac repair, regeneration and functional recovery. Although fibrosis can play a protective role in preventing heart rupture during wound healing in the acute phase of myocardial infarction, if the fibrosis pathway is activated for a long time after myocardial infarction, it will lead to excessive scar formation and loss of tissue compliance. At present, there is no effective treatment in clinical practice that can significantly improve the occurrence and development of long-term fibrosis after myocardial infarction.

[0003] Calcin is a scorpion toxin transmembrane polypeptide that can specifically bind to and activate the RyR2 receptor. Its family currently has more than 20 members, among which Opicalcin1 (hereinafter referred to as OpiCal) has the strongest binding activity. In response to the defects of OpiCal alone, the applicant's prior patent application "Preparation of an Opicalcin1-lipo-Opicalcin1 Nanoliposome and Its Application in Antagonizing Sudden Cardiac Death" (Publication No. CN117582523A) combined the advantages of liposomes to synthesize the OpiCal-lipo-OpiCal liposome nano-delivery system. Under the premise of maintaining the main biological functions of OpiCal unchanged, it improves the water solubility of the drug, reduces toxic side effects and immunogenicity, enhances in vitro and in vivo stability, prolongs its half-life in the body, and thus increases the binding time of OpiCal to the RyR2 receptor to reduce cardiac Ca. 2+ Overload antagonizes the potential of sudden cardiac death.

[0004] Although myocardial infarction and sudden cardiac death are both heart diseases, there are significant differences in pathogenesis, clinical manifestations, treatment, prognosis and prevention. In terms of pathogenesis, sudden cardiac death is mostly caused by abnormal cardiac electrophysiology, while myocardial infarction is caused by obstruction of the coronary arteries, leading to myocardial ischemia, hypoxia, and local necrosis. Chronic myocardial infarction that cannot be recovered for a long time will turn into heart failure. In terms of clinical manifestations, the symptoms of sudden cardiac death are sudden loss of consciousness, respiratory arrest, and disappearance of heartbeat, and the patient's condition deteriorates rapidly. The symptoms of myocardial infarction are chest pain, chest tightness, sweating, nausea, etc., and the disease progresses relatively slowly. In terms of treatment, the key to the treatment of sudden cardiac death is to restore the normal beating of the heart and abnormal cardiac electrophysiology. The purpose of treating myocardial infarction is to restore coronary blood flow and save the myocardium on the verge of necrosis. Although OpiCa1 and its nanoliposomes have been developed to antagonize sarcoplasmic reticulum Ca 2+ OpiCa1 has potential medicinal value in treating sudden cardiac death caused by overload. However, given the significant differences between myocardial infarction and sudden cardiac death, from the pathogenesis to treatment strategies, it is uncertain whether OpiCa1 and its nanoliposomes will be effective in treating myocardial infarction and subsequent myocardial fibrosis, or even heart failure. The risk of sudden death from myocardial infarction is high, and long-term efficacy remains poor. Therefore, the search for a safe, highly effective, and minimally toxic treatment (e.g., medication and form) remains urgent. Summary of the Invention

[0005] In view of the current technical problem of lack of efficient, low-toxic and safe treatment methods for post-myocardial infarction heart failure, the purpose of the present invention is to study the therapeutic effect of OpiCa1 and its nanoliposomes on myocardial infarction models and provide a new treatment plan for post-myocardial infarction heart failure.

[0006] The present invention studies the effects and molecular mechanisms of OpiCa1 and its nanoliposomes in antagonizing myocardial infarction. A mouse model was established by ligating the left anterior descending artery of the heart, and then intervention with OpiCa1 and its nanoliposomes was performed (PBS was used as a blank control). Transcriptome sequencing and genome enrichment analysis were performed to identify significantly enriched pathways and their genes, providing a new therapeutic approach for the treatment and prevention of heart failure after myocardial infarction.

[0007] The first object of the present invention is to provide a use of OpiCal or its nanoliposomes in the preparation of a drug for treating and / or preventing myocardial fibrosis-related diseases.

[0008] The results of drug administration studies on myocardial infarction mice model of the present invention found that OpiCa1 and its nanoliposomes can inhibit myocardial fibrosis and improve myocardial remodeling during the myocardial remodeling period after myocardial infarction, and have a good antagonistic effect on myocardial infarction and subsequent heart failure.

[0009] In some embodiments, the drug is a drug that inhibits myocardial fibrosis-related pathways.

[0010] In some embodiments, the myocardial fibrosis-related pathway includes the TGF-β signaling pathway and / or the HIF-1 signaling pathway.

[0011] In some embodiments, the drug is a drug that inhibits myocardial fibrosis and / or improves myocardial remodeling.

[0012] In some embodiments, the drug is a drug for treating and / or preventing heart failure after myocardial infarction.

[0013] The amino acid sequence of OpiCal is as follows: GDCLPHLKRCKENNDCCSKKCKRR GTNPEKRCR.

[0014] In some embodiments, the nanoliposomes are OpiCal-lipo-OpiCal liposomes, and the synthesis process thereof is as follows: encapsulating the OpiCal polypeptide into PGE-modified PLGA liposomes, then mixing the OpiCal polypeptide and 2-iminothiolane for thiolation, and after the two react together (about 12 hours), the OpiCal polypeptide is stably connected to the liposome surface through a disulfide bond. Phospholipids are used as an emulsifier, and the synthesis is performed by a heated emulsifier evaporation method.

[0015] The second object of the present invention is to provide a drug for treating and / or preventing myocardial fibrosis-related diseases, comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes OpiCal or its nanoliposomes.

[0016] In some embodiments, the drug is a drug that inhibits myocardial fibrosis and / or improves myocardial remodeling.

[0017] In some embodiments, the drug is a drug for treating and / or preventing heart failure after myocardial infarction.

[0018] In some embodiments, the auxiliary material is one or more of a diluent, an excipient, a binder, a filler, a disintegrant, a flavoring agent, and a sweetener.

[0019] The dosage form of the medicine is oral preparation or injection.

[0020] The beneficial effects of the present invention are:

[0021] The results of drug administration studies in mice with myocardial infarction (MI) revealed that OpiCa1 and its nanoliposomes can inhibit myocardial fibrosis and improve myocardial remodeling during the post-MI myocardial remodeling phase, demonstrating a potent antagonistic effect against MI and subsequent heart failure. Further transcriptomic analysis revealed that OpiCa1 and its nanoliposomes, when used to treat MI in mice, may participate in extracellular matrix degradation in myocardial remodeling and fibrosis pathways (such as the TGF-β signaling pathway), thereby inhibiting fibrosis and improving myocardial remodeling, ultimately alleviating the worsening of MI. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The antagonistic effects of OpiCa1 and its nanoliposomes on myocardial infarction mice are shown in the figure: (A) Survival curve analysis of OpiCa-lipo-OpiCa1 antagonistic MI mice; (B) HE staining grading score; (C) Statistical chart of myocardial fibrosis in mice; (D) Pathological sections of mice.

[0023] Figure 2 This is a statistical diagram of myocardial infarction area in mice with myocardial infarction.

[0024] Figure 3 These are pathological sections of the heart, liver, kidney and other tissues and organs of mice with myocardial infarction.

[0025] Figure 4 The results of transcriptomic analysis are shown in the figure: (A) Volcano plot of differentially expressed genes in the hearts of OpiCa-lipo-OpiCa1-treated MI mice; (B) Bubble plot of enrichment of differentially expressed genes in the hearts of OpiCa-lipo-OpiCa1-treated MI mice; (C) Venn diagram of upregulated pathways in the hearts of OpiCa-lipo-OpiCa1-treated MI mice; (D) Network diagram of cytokine changes in the hearts of OpiCa-lipo-OpiCa1-treated MI mice; (E) Heat map of upregulated and downregulated cytokines in the hearts of OpiCa-lipo-OpiCa1-treated MI mice.

[0026] Figure 5 These are the key molecules and verification results of OpiCa1-lipo-OpiCa1 nanoliposomes in antagonizing myocardial infarction. DETAILED DESCRIPTION

[0027] The following embodiments of the present invention are described in detail with reference to the accompanying drawings. The following examples are implemented based on the technical solution of the present invention and provide detailed implementation methods, but the scope of protection of the present invention is not limited to the following examples. The following reagents are all commercially available unless otherwise specified.

[0028] 1. Materials and Experimental Subjects

[0029] 1.1 Main Reagents

[0030] Table 1 Main reagents and sources

[0031]

[0032] 1.2 Main consumables

[0033] Table 2 Main instruments and manufacturers

[0034]

[0035]

[0036] 1.3 Experimental subjects

[0037] Table 3 Experimental subjects and manufacturers

[0038]

[0039] 2. Experimental Methods

[0040] 2.1 Myocardial infarction mouse model

[0041] First, depilatory cream was used to remove hair from the mouse's chest. The mouse was then anesthetized with isoflurane and placed in a supine position. The isoflurane and oxygen flow rates were adjusted to maintain anesthesia at a final isoflurane concentration of 1% to 2%.

[0042] Surgical Method: The mouse was placed in the right lateral decubitus position. After applying a sterile drape, an oblique incision approximately 1 cm long was made 0.5 cm from the parasternal bone. The pectoralis major and minor muscles were dissected with a scalpel. Once the ribs were visible, they were grasped with forceps. The intercostal muscles were gently punctured with another forceps at the third intercostal space. The surgical field was then expanded with hemostats until the pinkish-white left anterior descending coronary artery running beneath the myocardium was visible. The artery was then ligated 1.5 mm from the inferior margin of the left atrial appendage using an 8-0 manikin. After ligation, the blood supply area of the left anterior descending artery rapidly turned white under the microscope, forming a clear demarcation line from the normal myocardium. The wound was then quickly sutured.

[0043] 2.2 Grouped drug administration experiment

[0044] C57 mice were randomly divided into four groups: control group, MI group (myocardial infarction model group), OpiCa1+MI group and OpiCa1-lipo-OpiCa1+MI group, with 30 mice in each group. After successful modeling, OpiCa1 and OpiCa1-lipo-OpiCa1 were injected into the tail vein of the OpiCa1+MI group and the OpiCa1-lipo-OpiCa1+MI group every other day for one month, and the survival of the mice was observed; after one month, the dosing interval was changed to once a week, and the survival rate of the mice was observed after three consecutive months.

[0045] 2.3 Echocardiographic evaluation

[0046] After one week, one month, and four months of treatment, the mice were depilated on their chests with depilatory cream to expose their hearts. The depilatory cream was then wiped off with an alcohol cotton ball, and the mice were put back on the machine for echocardiography. Subsequently, the mouse hearts were removed for HE staining, Masson staining, and WGA staining.

[0047] 2.4 Transcriptomic analysis of OpiCa1-lipo-OpiCa1 liposomes against myocardial infarction in mice

[0048] The surviving mice in each group were anesthetized with isoflurane, dissected, and blood and hearts were collected. The samples were then sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for transcriptomic sequencing. The remaining samples were stored in EP tubes, quickly frozen in liquid nitrogen, and stored at -80°C.

[0049] Western blot analysis of 2.5 OpiCa1-lipo-OpiCa1 liposomes against myocardial infarction in mice

[0050] To confirm that the key molecules identified in the transcriptome analysis had a significant antagonistic effect on MI in mice, western blot analysis was performed to investigate changes in classic markers of angiogenesis and oxidative stress. Mice were dissected and perfused with saline to flush out any remaining blood. After the liver turned white, the heart was excised and a rice-grain-sized portion was placed in an EP tube. The remaining portion was snap-frozen in liquid nitrogen and stored at -80°C. 100 μL of SDS lysis mixture containing protease inhibitors, phosphatase inhibitors, and PMSF was added to each tube. The tissue lysate was disrupted using a 65W cell sonicator for three consecutive 3-second cycles. After removal, the cells were placed on ice for 5 minutes, centrifuged at 14,000 g for 5 minutes at 4°C, and the supernatant was collected to determine total cellular protein. Protein was quantified by BCA analysis, and loading buffer was added. The cells were heated at 100°C for 10 minutes. A 10% SDS-polyacrylamide gel was prepared, and the calculated volume of sample was loaded for electrophoresis. Equal separation was achieved by applying a constant voltage of 80V for 30 minutes, followed by 120V until bromophenol blue reached the bottom 1 cm of the gel. The gel was then transferred to the NC membrane using a "sandwich" process: positive electrode, sponge, NC membrane, gel, sponge, and negative electrode. The membrane was then transferred at 100V for 90 minutes, keeping the membrane in an ice bath throughout the entire process. Following transfer, the membrane was incubated with protein-free rapid blocking buffer for 1 hour at room temperature. Primary antibodies against target proteins (including GAPDH, Tgfb1, Mmp9, Hmox1, Egln3, Casp7, and Smad3) were added and incubated overnight at 4°C. The primary antibody was then recovered, and after three washes with 1xTBST, a secondary antibody was added and incubated at room temperature for 2 hours. Protein expression levels were detected using an ECL luminescence kit, and band analysis was performed using imag J software.

[0051] 3. Results Analysis

[0052] 3.1 Antagonistic effect of OpiCa1-lipo-OpiCa1 nanoliposomes on myocardial infarction in mice

[0053] Figure 1 The results show the antagonistic effect of OpiCa1 and its nanoliposomes on myocardial infarction in mice. Myocardial infarction model was established by surgical intervention. The left anterior descending branch of the mouse heart was ligated to block the blood supply to the mouse heart. The effectiveness of OpiCa1 and its nanoliposomes was preliminarily evaluated by injection into the tail vein. Figure 1 Figure A, center, shows that injecting OpiCa1 and nanoliposomes into the tail vein of mice one week after ligation temporarily accelerated their mortality. Preliminary evidence suggests that OpiCa1 increases cardiac workload and accelerates death during the recovery phase of myocardial infarction. However, after 2-3 weeks, the mortality rate significantly decreased, indicating that OpiCa1 plays a key role in myocardial remodeling in MI mice. H&E staining reveals that three months after myocardial infarction, myocardial necrosis due to ischemia results in a disordered fiber state, with centrally located nuclei and blurred striations. Masson staining reveals the development of myocardial fibrosis due to cell death and excessive proliferation of fibrotic cells. Whether OpiCa1 and its nanoliposomes can significantly reduce the extent of fibrosis is a key indicator of their ability to combat MI.

[0054] Figure 1 Figure D in the middle shows pathological sections of the heart, liver, and kidney of mice with myocardial infarction. H&E staining of the sections reveals significant thinning of the ventricular wall and enlargement of the ventricles in these mice. One month after injection of OpiCa1 and its nanoliposomes, ventricular wall thickness has significantly recovered. Masson staining reveals an increasing trend of myocardial fibrosis in the MI group. Comparison with WGA staining reveals that while myocardial cell volume in this group was not significantly enlarged, the myocardial fibers were irregularly arranged. Injection of OpiCa1 and its nanoliposomes significantly improved the arrangement of myocardial fibers in the mouse hearts.

[0055] 3.2 Statistics of myocardial infarction area and echocardiography results in mice

[0056] Evans blue and TCC double staining effectively assesses infarct size in mice with myocardial infarction and provides intuitive statistical analysis, providing an experimental basis for evaluating drug efficacy. Its advantage lies in its simplicity. Later, combined with echocardiography to record key parameters such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), it allows for objective evaluation of the effectiveness of OpiCa1 and OpiCa1-lipo-OpiCa1 nanoliposomes in treating myocardial infarction in mice.

[0057] Figure 2Figure 2 shows the myocardial infarction area of mice undergoing myocardial infarction. After dissecting the mouse hearts and sectioning and staining, the vascularized areas stained dark red with Evan's blue and TTC, while the completely infarcted areas remained unstained and appeared white. The remaining viable myocardium within the infarcted area was bright red. Software mapping revealed an IA / AAR ratio of 74±9.64%, compared to 35.7±4.73% in mice injected with OpiCa1-lipo-OpiCa1 nanoliposomes, demonstrating that OpiCa1-lipo-OpiCa1 nanoliposomes have a potent antagonistic effect on myocardial infarction in mice.

[0058] In echocardiography, LVEF reflects the shortening capacity of left ventricular myocardial fibers, and LVFS reflects the relationship between stress and shortening. After myocardial infarction, if both LVEF and LVFS are significantly reduced (P>0.05), it indicates that the heart function of mice after myocardial infarction has deteriorated. If it is not recovered for a long time, the mice will have chronic myocardial infarction and turn to heart failure. Figure 3 As shown in the figure, echocardiography results showed that two months after ligation of the left anterior descending artery of the mouse heart, EF and FS decreased significantly (P < 0.05), indicating that the left ventricular cavity was enlarged and the left ventricular anterior wall was thinned, indicating that the mouse heart had undergone morphological and structural changes after myocardial infarction. Compared with the group injected with OpiCa1-lipo-OpiCa1 nanoliposomes, EF and FS showed an upward trend (P < 0.05), indicating that the mouse ventricular myocardium was in a state of slow recovery during this period.

[0059] 3.3 Transcriptomic Mechanism Analysis

[0060] The therapeutic mechanism of OpiCa1 and its nanoliposomes in myocardial infarction is still unclear. Therefore, after 4 months of continuous administration of OpiCa1 to MI mice, the hearts were dissected for transcriptomic analysis. Figure 4 The results showed that 34,486 genes were detected in the heart, of which 161 were common genes. Moreover, functional enrichment analysis of differentially expressed genes revealed that, after annotation in the KEGG database, 51 signaling pathways were enriched in the hearts of MI mice injected with OpiCa1 and its nanoliposomes via the tail vein, including 5 common signaling pathways, including TGF-β signaling pathway, FoxO signaling pathway, Calcium signaling pathway, PI3K-Akt signaling pathway, and other signaling pathways related to heart disease. Analyzing the specific differentially expressed genes, OpiCa1 and its nanoliposomes may achieve the purpose of treating myocardial infarction in mice by downregulating a series of signaling pathways in the mouse heart, such as matrix metalloproteinases (MMP-9), transforming growth factor (TGF-β), and heme oxygenase (Hmox1).

[0061] 3.4 Screening of key molecules for OpiCa1-lipo-OpiCa1 nanoliposomes to antagonize myocardial infarction

[0062] Transcriptomic screening revealed that OpiCa1 and its nanoliposomes, in the treatment of myocardial infarction in mice, activated myocardial remodeling and fibrosis pathways (such as the TGF-β signaling pathway), particularly TGFβ1 and MMP-9, which may participate in extracellular matrix degradation, inhibiting fibrosis and improving myocardial remodeling, thereby alleviating the worsening of myocardial infarction. Furthermore, Hmox1, Egln3 (a regulator of hypoxia-inducible factor), Casp7, and TGFβ1 and its downstream targets (such as Smad3) in oxidative stress and metabolic pathways (such as the HIF-1 signaling pathway) may play an important role in regulating core molecules of fibrosis, thus inhibiting the TGF-β pathway can alleviate myocardial fibrosis.

[0063] 3.5 Validation of key molecules in OpiCa1-lipo-OpiCa1 nanoliposomes for antagonizing myocardial infarction

[0064] Figure 5 The key molecules and validation results of OpiCa1-lipo-OpiCa1 nanoliposomes in antagonizing myocardial infarction are shown. Experimental results show that after tail vein injection of OpiCa1-lipo-OpiCa1, the expression of TGFβ1 and MMP-9 in the TGF-β signaling pathway in the hearts of mice with myocardial infarction decreased. Furthermore, Hmox1 and Smad3 in the HIF-1 signaling pathway were also downregulated to varying degrees. Although CXCL2 gene expression was not statistically significant compared to the MI group, it still showed an overall downward trend.

[0065] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of OpiCa1 or its nanoliposomes in the preparation of drugs for treating and / or preventing myocardial fibrosis-related diseases.

2. The use according to claim 1, characterized in that The drug is a drug that inhibits myocardial fibrosis-related pathways.

3. The use according to claim 1, characterized in that The myocardial fibrosis-related pathways include the TGF-β signaling pathway and / or the HIF-1 signaling pathway.

4. The use according to claim 1, characterized in that The drug is a drug for inhibiting myocardial fibrosis and / or improving myocardial remodeling.

5. The use according to claim 1, characterized in that The medicine is a medicine for treating and / or preventing heart failure after myocardial infarction.

6. The use according to any one of claims 1 to 5, characterized in that The amino acid sequence of OpiCa1 is as follows: GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR.

7. The use according to any one of claims 1 to 5, characterized in that The nanoliposomes are OpiCal-lipo-OpiCal liposomes, and their synthesis process is as follows: OpiCal polypeptide is encapsulated into PGE-modified PLGA liposomes, and then the OpiCal polypeptide and 2-iminothiolane are mixed and thiolated. After the two react together, the OpiCal polypeptide is stably connected to the liposome surface through a disulfide bond. Phosphatidylcholine is used as an emulsifier and the synthesis is performed by a heated emulsifier evaporation method.

8. A drug for treating and / or preventing myocardial fibrosis-related diseases, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises OpiCal or its nanoliposomes.

9. The drug according to claim 8, characterized in that The drug is a drug for inhibiting myocardial fibrosis and / or improving myocardial remodeling.

10. The drug according to claim 8, characterized in that The medicine is a medicine for treating and / or preventing heart failure after myocardial infarction.

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