Use of OpiCal or nano-liposome thereof in the preparation of a drug for treating and / or preventing myocardial fibrosis related diseases

Abstract

The present application relates to the technical field of medicine, and particularly relates to application of OpiCa1 or a nano-liposome thereof in preparation of a drug for treating and / or preventing myocardial fibrosis related diseases. The present application is found that 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 good antagonistic effect on myocardial infarction and subsequent heart failure. Further transcriptomic analysis shows that in the process of treating myocardial infarction in mice, OpiCa1 and the nano-liposome thereof can inhibit fibrosis and improve myocardial remodeling by possibly participating in extracellular matrix degradation in myocardial remodeling and fibrosis pathways (such as TGF-beta signaling pathway), so as to achieve the purpose of relieving myocardial infarction deterioration.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of OpiCa1 or its nanoliposomes in the preparation of drugs for the treatment and / or prevention of myocardial fibrosis-related diseases. Background Technology

[0002] Myocardial infarction (MI) has long been a leading cause of death among cardiovascular diseases. Early diagnosis is difficult, the incubation period is long, and the rescue time is short, resulting in a limited number of patients who can be cured. Current treatment primarily relies on surgery, supplemented by medication. Myocardial fibrosis is a key characteristic of pathological remodeling after cardiac injury and a significant clinical challenge for cardiac repair, regeneration, and functional recovery. While fibrosis can play a protective role in the wound healing process during the acute phase of MI, preventing cardiac rupture, prolonged activation of fibrotic pathways after MI can lead to excessive scarring and loss of tissue compliance. Currently, there are no effective treatments to significantly improve the long-term development of fibrosis after MI.

[0003] Calcin is a scorpion venom transmembrane polypeptide that can specifically bind to and activate the RyR2 receptor. Its family currently includes more than 20 members, among which Opicalcin1 (hereinafter referred to as Opical) exhibits the strongest binding activity. Addressing the limitations 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 number CN117582523A), combined the advantages of liposomes to synthesize an Opical-lipo-OpiCal liposome nanodelivery system. This system aims to improve drug water solubility, reduce toxicity and immunogenicity, enhance in vitro and in vivo stability, and prolong its in vivo half-life while maintaining the main biological functions of Opical. This, in turn, increases the binding time of Opical to the RyR2 receptor to reduce cardiac calcinemia. 2+ Overload has the potential to antagonize sudden cardiac death.

[0004] Although both myocardial infarction and sudden cardiac death are heart diseases, they differ significantly 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 originates from coronary artery obstruction, leading to myocardial ischemia, hypoxia, and local necrosis. Chronic myocardial infarction, if left untreated, can progress to heart failure. Clinically, sudden cardiac death presents with sudden loss of consciousness, respiratory arrest, and cessation of heartbeat, with rapid deterioration of the patient's condition. Myocardial infarction, on the other hand, presents with chest pain, chest tightness, sweating, nausea, etc., with a relatively slow progression. Regarding treatment, the key to treating sudden cardiac death is restoring normal heartbeat and correcting cardiac electrophysiological abnormalities, while the goal of myocardial infarction treatment is to restore coronary blood flow and salvage the myocardium on the verge of necrosis. Although OpiCa1 and its nanoliposomes are developing antagonistic effects against sarcoplasmic reticulum Ca2+... 2+ OpiCa1 and its nanoliposomes have potential medicinal value in treating sudden cardiac death caused by overload. However, given the significant differences between myocardial infarction and sudden cardiac death in terms of pathogenesis and treatment strategies, the efficacy of OpiCa1 and its nanoliposomes in treating myocardial infarction and long-term myocardial fibrosis or even heart failure is unpredictable. Myocardial infarction carries a high risk of sudden death, and its long-term efficacy remains uncertain. Therefore, finding a highly effective, low-toxicity, and safe treatment method (such as drugs and formulations) remains urgent. Summary of the Invention

[0005] In view of the current technical problem of lacking efficient, low-toxicity, and safe treatment methods for heart failure after myocardial infarction, the purpose of this invention is to study the therapeutic effect of OpiCa1 and its nanoliposomes on a myocardial infarction model and provide a new treatment plan for heart failure after myocardial infarction.

[0006] This invention investigates 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 branch of the heart, and then OpiCa1 and its nanoliposomes were used for intervention (PBS was used as a blank control). Transcriptome sequencing and genome enrichment analysis were performed to identify significantly enriched pathways and genes, providing a new treatment approach for the treatment and prevention of heart failure after myocardial infarction.

[0007] The first objective of this invention is to provide the use of OpiCa1 or its nanoliposomes in the preparation of medicaments for the treatment and / or prevention of myocardial fibrosis-related diseases.

[0008] The results of the drug administration study in a mouse model of myocardial infarction showed 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 pathways include 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 OpiCa1 is as follows: GDCLPHLKRCKENNDCCSKKCKRR GTNPEKRCR.

[0014] In some embodiments, the nanoliposomes are OpiCa1-lipo-OpiCa1 liposomes, and their synthesis process is as follows: the OpiCal peptide is encapsulated in a PGE-modified PLGA liposome, and then the OpiCal peptide and 2-iminothione are mixed and thiolated. After the two react together (about 12 h), the OpiCal peptide is stably linked to the surface of the liposome through disulfide bonds. Lecithin is used as an emulsifier, and the synthesis is carried out by heating emulsifier evaporation method.

[0015] A second objective of the present invention is to provide a medicament for treating and / or preventing myocardial fibrosis-related diseases, comprising an active ingredient and pharmaceutically acceptable excipients, said active ingredient comprising OpiCa1 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 excipients are one or more of the following: diluent, excipient, binder, filler, disintegrant, flavoring agent, and sweetener.

[0019] The drug is available in oral or injectable form.

[0020] The beneficial effects of this invention are as follows:

[0021] The results of this study on mice with myocardial infarction showed that OpiCa1 and its nanoliposomes can inhibit myocardial fibrosis and improve myocardial remodeling during the post-myocardial infarction period, exhibiting good antagonistic effects on myocardial infarction and subsequent heart failure. Further transcriptomic analysis revealed that OpiCa1 and its nanoliposomes may participate in extracellular matrix degradation in myocardial remodeling and fibrosis pathways (such as the TGF-β signaling pathway) during the treatment of myocardial infarction in mice, thereby inhibiting fibrosis and improving myocardial remodeling, thus achieving the goal of alleviating the deterioration of myocardial infarction. Attached Figure Description

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

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

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

[0025] Figure 4 The transcriptomics analysis results are shown in the figure. (A) Volcano plot of differentially expressed genes in the heart of OpiCa-lipo-OpiCa1 antagonistic MI mice; (B) Bubble plot of enriched differentially expressed genes in the heart of OpiCa-lipo-OpiCa1 antagonistic MI mice; (C) Venn plot of upregulated pathways in the heart of OpiCa-lipo-OpiCa1 antagonistic MI mice; (D) Network diagram of changed cytokines in the heart of OpiCa-lipo-OpiCa1 antagonistic MI mice; (E) Heatmap of upregulated and downregulated cytokines in the heart of OpiCa-lipo-OpiCa1 antagonistic MI mice.

[0026] Figure 5 Key molecules for OpiCa1-lipo-OpiCa1 nanoliposomes to antagonize myocardial infarction and validation results. Detailed Implementation

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

[0028] 1. Materials and Experimental Subjects

[0029] 1.1 Main Reagents

[0030] Table 1. Main Reagents and Their 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 Mouse model of myocardial infarction

[0041] First, hair removal cream was used to remove hair from the chest of the mice. Then, the mice were anesthetized with isoflurane, placed in a supine position, and the flow rate of isoflurane and oxygen was adjusted. The final concentration of isoflurane was 1% to 2% for maintenance anesthesia.

[0042] Surgical procedure: The mouse was positioned on its right lateral decubitus position and draped with a sterile sheet. A 1cm oblique incision was made approximately 0.5cm from the sternum. The pectoralis major and pectoralis minor muscles were cut open with a scalpel. Once the ribs were visible, they were grasped with small forceps. At the third intercostal space, the intercostal muscles were gently punctured with another forceps. Hemostatic forceps were then used to expand the surgical field until the pinkish-white left anterior descending coronary artery running beneath the myocardium was visible. The artery was ligated with 8-0 MANI sutures 1.5mm below the lower edge of the left atrial appendage. After ligation, the area supplied by the left anterior descending coronary artery rapidly turned white under the microscope, forming a clear boundary with the normal myocardium. The wound was then quickly sutured.

[0043] 2.2 Grouped Dosing 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, the OpiCa1+MI group and the OpiCa1-lipo-OpiCa1+MI group were injected with OpiCa1 and OpiCa1-lipo-OpiCa1 via the tail vein every other day for one month to observe the survival of the mice. 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 dehaired on their chests with hair removal cream to expose their hearts. The hair removal cream was then wiped off with an alcohol swab, and the mice were re-echoic for examination. Subsequently, the mice's hearts were harvested for HE staining, Masson staining, and WGA staining.

[0047] 2.4 Transcriptomic analysis of OpiCa1-lipo-OpiCa1 liposome antagonism in mice with myocardial infarction

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

[0049] 2.5 Western blot analysis of the protein immunoblotting of OpiCa1-lipo-OpiCa1 liposome antagonistic mice with myocardial infarction.

[0050] To confirm the significant antagonistic effect of key molecules screened from the transcriptome on mice with myocardial infarction, Western blotting analysis was used to investigate changes in classic angiogenesis and oxidative stress indicators. Mice were first dissected, perfused with physiological saline to flush out residual blood, and after the liver turned white, the heart was removed. A rice-grain-sized sample was placed in an EP tube, and the remainder was flash-frozen in liquid nitrogen and stored at -80°C. 100 μL of SDS lysis buffer containing protease inhibitors, phosphatase inhibitors, and PMSF was added to each tube. The tissue lysate was continuously disrupted three times for 3 seconds each time using a 65W cell sonicator. After removal, the cells were placed on ice for 5 minutes, then centrifuged at 14,000g for 5 minutes at 4°C. The supernatant was collected as total cellular protein. Proteins were quantified using BCA, and then loading buffer was added, followed by heating at 100°C for 10 minutes. A 10% SDS-polyacrylamide gel was prepared and loaded to the calculated volume for electrophoresis. Initially, the gel was separated at 80V for 30 minutes, then at a constant voltage of 120V until the bromophenol blue reached 1 cm from the bottom of the gel. The gel was then transferred to the NC membrane in a "sandwich" sequence: positive electrode-sponge-NC membrane-gel-sponge-negative electrode. The membrane was then transferred at 100V for 90 minutes, and kept in an ice bath throughout the transfer. After transfer, the membrane was incubated with protein-free rapid blocking buffer at room temperature for 1 hour. Primary antibodies against the target proteins (GAPDH, Tgfb1, Mmp9, Hmox1, Egln3, Casp7, and Smad3) were added and incubated overnight at 4°C. The primary antibodies were then recovered, and the membrane was washed three times with 1xTBST. Secondary antibodies were then added and incubated at room temperature for 2 hours. Finally, protein expression levels were detected using an ECL luminescence assay kit, and band analysis was performed using imagJ software.

[0051] 3. Results Analysis

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

[0053] Figure 1 This study demonstrates the antagonistic effect of OpiCa1 and its nanoliposomes on mice with myocardial infarction. Myocardial infarction was induced through surgical intervention, by ligating the left anterior descending branch of the mouse heart to block cardiac blood supply. The effectiveness was preliminarily evaluated by tail vein injection of OpiCa1 and its nanoliposomes. See also... Figure 1 Figure A shows that, through experiments, injection of OpiCa1 and nanoliposomes into the tail vein of mice one week after ligation temporarily accelerated mouse mortality. Preliminary analysis suggests that OpiCa1 increases cardiac load and accelerates death during the recovery period from myocardial infarction. However, after 2-3 weeks, the mortality rate significantly decreased, indicating that OpiCa1 plays a crucial role in the myocardial remodeling period of MI mice. H&E staining revealed that three months later, the mouse myocardium was necrotic due to ischemia, resulting in disordered fibers, with cell nuclei located centrally and striations becoming indistinct. Masson staining showed myocardial fibrosis due to cell death and excessive proliferation of fibrotic cells. Whether OpiCa1 and its nanoliposomes can significantly improve the area of ​​fibrosis is a key indicator of their ability to antagonize myocardial infarction.

[0054] Figure 1 The middle D figure shows pathological sections of the heart, liver, kidney, and other organs of mice with myocardial infarction (MI). Observation of the pathological sections of MI mice revealed that H&E staining clearly showed that the ventricular walls of MI mice were significantly thinner and the ventricles were significantly enlarged. One month after injection of OpiCa1 and its nanoliposomes, the ventricular wall thickness was significantly restored. Masson staining images showed that the myocardium in the MI group showed a trend of increased fibrosis. Comparison with WGA staining results showed that although the volume of cardiomyocytes in this group was not significantly hypertrophic, the myocardial fibers were irregularly arranged. However, after injection of OpiCa1 and its nanoliposomes, the arrangement of myocardial fibers in the mouse heart was significantly improved.

[0055] 3.2 Statistical analysis of myocardial infarction area and echocardiographic results in mice

[0056] Evans blue and TCC double staining can effectively assess the infarct area in mice with myocardial infarction and provide intuitive statistical analysis, offering an experimental basis for evaluating drug efficacy. Its advantages lie in its simple operation. Subsequent echocardiographic recording of key parameters such as left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in mice allows for an objective evaluation of the efficacy of OpiCa1 and OpiCa1-lipo-OpiCa1 nanoliposomes in treating myocardial infarction in mice.

[0057] Figure 2The statistical analysis of myocardial infarction area in mice with myocardial infarction is shown. After dissecting and staining the mouse heart sections, areas with blood supply were stained dark red by Evans blue and TTC, while completely infarcted areas remained unstained and white. Residual viable myocardium in the infarcted area appeared bright red. Software mapping revealed an IA / AAR ratio of 74±9.64%, while the IA / AAR ratio in mice injected with OpiCa1-lipo-OpiCa1 nanoliposomes was 35.7±4.73%, indicating that OpiCa1-lipo-OpiCa1 nanoliposomes have a good antagonistic effect on myocardial infarction in mice.

[0058] In echocardiography, LVEF reflects the shortening capacity of left ventricular myocardial fibers, while LVFS reflects the relationship between stress and shortening. After myocardial infarction, a significant decrease in both LVEF and LVFS (P > 0.05) indicates impaired cardiac function in mice, and prolonged lack of recovery may lead to chronic myocardial infarction progressing to heart failure. Figure 3 As shown in the figure. Echocardiographic results showed that two months after ligation of the left anterior descending branch of the mouse heart, EF and FS decreased significantly (P<0.05), indicating that the left ventricular cavity was enlarged and the anterior wall of the left ventricle was thinned, indicating that the morphology and structure of the mouse heart changed after myocardial infarction. Compared with the group injected with OpiCa1-lipo-OpiCa1 nanoliposomes, EF and FS showed an increasing trend (P<0.05), indicating that the mouse ventricular myocardium was in a slow recovery state during this period.

[0059] 3.3 Transcriptomics Mechanism Analysis

[0060] The therapeutic mechanism of OpiCa1 and its nanoliposomes for myocardial infarction remains unclear. Therefore, after continuous administration to MI mice for 4 months, the hearts were dissected for transcriptomic analysis. Figure 4 The results showed that 34,486 genes were detected in the heart, including 161 common genes. Furthermore, 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 via the tail vein with OpiCa1 and its nanoliposomes. Among these, five identical signaling pathways were enriched, including TGF-β, FoxO, Calcium, and PI3K-Akt signaling pathways, all of which are associated with heart disease. Specific differential gene analysis suggests that OpiCa1 and its nanoliposomes may achieve therapeutic effects in mouse myocardial infarction by downregulating a series of genes in the mouse heart, such as matrix metalloproteinase-9 (MMP-9), transforming growth factor-β (TGF-β), and heme oxygenase (Hmox1).

[0061] 3.4 Screening of key molecules for OpiCa1-lipo-OpiCa1 nanoliposome antagonism of myocardial infarction

[0062] Transcriptomic screening revealed that OpiCa1 and its nanoliposomes, during the treatment of myocardial infarction in mice, may participate in extracellular matrix degradation and inhibit fibrosis by influencing myocardial remodeling and fibrosis pathways (such as the TGF-β signaling pathway), particularly TGFβ1 and MMP-9, thereby improving myocardial remodeling and alleviating the deterioration of myocardial infarction. Simultaneously, Hmox1, Egln3 (hypoxia-inducible factor regulator), 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 important roles in core molecules regulating fibrosis, thus inhibiting the TGF-β pathway can alleviate myocardial fibrosis.

[0063] 3.5 Validation of key molecules in the antagonism of myocardial infarction by OpiCa1-lipo-OpiCa1 nanoliposomes

[0064] Figure 5 This study illustrates the key molecules involved in the antagonistic effect of OpiCa1-lipo-OpiCa1 nanoliposomes on myocardial infarction and the validation results. Experimental results show that in mice with myocardial infarction, intravenous injection of OpiCa1-lipo-OpiCa1 via the tail vein resulted in decreased expression levels of TGFβ1 and MMP-9 in the TGF-β signaling pathway in the heart. Simultaneously, Hmox1 and Smad3 in the HIF-1 signaling pathway were also downregulated to varying degrees. Although the expression level of the CXCL2 gene was not statistically significant compared to the MI group, it still showed an overall decreasing trend.

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

Claims

1. The use of OpiCa1 or OpiCa1-lipo-OpiCa1 liposomes in the preparation of drugs for the treatment and / or prevention of myocardial fibrosis-related diseases, characterized in that, The myocardial fibrosis-related disease is post-myocardial infarction heart failure. The amino acid sequence of OpiCa1 is as follows: GDCLPHLKRCKENNDCCSKKCKRRGTNPEKRCR.

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

3. The application according to claim 2, characterized in that, The myocardial fibrosis-related pathways include the TGF-β signaling pathway.

4. The application according to claim 1, characterized in that, The drug is a drug that inhibits myocardial fibrosis and / or improves myocardial remodeling.

5. The application according to any one of claims 1-4, characterized in that, The OpiCa1-lipo-OpiCa1 liposome is synthesized as follows: the OpiCal peptide is encapsulated in a PGE-modified PLGA liposome, and then the OpiCal peptide and 2-iminothion are mixed and thiolated. After the two react together, the OpiCal peptide is stably linked to the surface of the liposome through disulfide bonds. Lecithin is used as an emulsifier, and the synthesis is carried out by heating emulsifier evaporation method.

Images