Application of EP3 receptor antagonist L-798106 in the preparation of drugs for preventing myocardial ischemia-reperfusion injury

Abstract

The present invention relates to the field of biomedicine and provides an application of an EP3 receptor antagonist L-798106 in the preparation of a drug for preventing myocardial ischemia-reperfusion injury. After long-term research, the present invention unexpectedly found that L-798106 can significantly reduce the myocardial infarction area and reduce the serum myocardial enzyme spectrum levels in mice after myocardial ischemia-reperfusion surgery, improve cardiac contractile function, and alleviate myocardial ischemia-reperfusion injury. The results of the present invention provide a theoretical basis for L-798106 to improve myocardial ischemia-reperfusion injury, and in particular provide a foundation for the research and development of related drugs. In summary, the present invention has found that the EP3 receptor antagonist L-798106 can be used to prevent and treat diseases related to myocardial ischemia-reperfusion injury, and has clinical research and development value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an EP3 receptor antagonist, L-798106 ((2E)-N-[(5-bromo-2-methoxyphenyl)sulfonyl]-3-[2-(2-naphthalenylmet-hyl)phenyl]-2-propenamide; CAS No.: 244101-02-8), in improving diseases related to myocardial ischemia-reperfusion injury. Background Art

[0002] Cardiovascular disease (CVD) is the leading cause of death. Among these, ischemic heart disease (IHD), particularly coronary heart disease (CHD), has the highest morbidity and mortality rates. Acute myocardial infarction (AMI) is the leading cause of death among CHD patients. Therefore, effective treatment of CHD is crucial to reducing CVD-related mortality.

[0003] In recent years, treatments such as intravenous thrombolysis, percutaneous coronary intervention, and coronary artery bypass grafting have been widely used clinically, reducing mortality in hyperacute and acute myocardial infarction (AMI) to a certain extent. However, rapid restoration of blood flow often leads to acute and chronic myocardial injury, causing adverse events such as no-reflow, myocardial stunning, and arrhythmias, thereby overshadowing the benefits of reperfusion. This is known as myocardial ischemia / reperfusion injury (MIRI). The cellular mechanisms of MIRI are complex, involving multiple signaling pathways and molecular mechanisms, including oxidative stress, intracellular calcium overload, expanded inflammation, rapid pH recovery during reperfusion, and mitochondrial damage. The occurrence of MIRI severely impacts patients' long-term prognosis, and currently, there is a lack of effective intervention strategies and therapeutic agents to mitigate MIRI.

[0004] EP3 is a prostaglandin receptor widely and stably expressed in various tissues and organs throughout the body, and L-798106 is a highly selective EP3 receptor antagonist. Studies have shown that L-798106 has effects on brain tissue oxidative stress, neuronal apoptosis, and prostate cancer cell proliferation, providing new insights into the prevention and treatment of certain diseases. However, there are currently no studies or patents reporting the use of L-798106 in the prevention and treatment of MIRI. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of the EP3 receptor antagonist L-798106 in the preparation of drugs for preventing and treating MIRI, providing ideas for new drug research and development and innovative therapies.

[0006] In order to solve the above technical problems, the present invention provides an application of an EP3 receptor antagonist in the preparation of a drug for preventing and treating MIRI, wherein the antagonist is L-798106.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] Use of an EP3 receptor antagonist in the preparation of a drug for preventing myocardial ischemia-reperfusion injury.

[0009] Preferably, the EP3 receptor antagonist is used in the preparation of a drug for reducing the area of myocardial infarction after myocardial ischemia-reperfusion surgery.

[0010] Preferably, the EP3 receptor antagonist is used in the preparation of a drug for reducing serum myocardial enzyme levels after myocardial ischemia-reperfusion surgery.

[0011] Preferably, the enzyme comprises one or more of lactate dehydrogenase, creatine kinase, creatine kinase and isoenzymes thereof.

[0012] Preferably, the EP3 receptor antagonist is used in the preparation of a drug for improving cardiac contractile function after myocardial ischemia-reperfusion surgery.

[0013] Preferably, the EP3 receptor antagonist comprises L-798106.

[0014] Preferably, the medicine includes one or more of an oral preparation and an injection.

[0015] Preferably, the drug comprises an oral agent, and the concentration of L-798106 is 14 mg / kg / day.

[0016] Preferably, the L-798106 is dissolved in DMSO at 10 mg / ml to obtain an L-798106 solution, and the L-798106 solution is then dissolved in drinking water.

[0017] Preferably, the L-798106 solution is dissolved in the drinking water at a volume ratio of less than 0.75%.

[0018] L-798106 was dissolved in DMSO, and the working solution was dissolved in drinking water.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] During the invention process, after long-term research, it was unexpectedly discovered that L-798106 can significantly reduce the size of myocardial infarction and lower serum myocardial enzyme levels in mice after MIR surgery, improving cardiac contractile function and alleviating MIRI. The results of this invention provide a theoretical basis for L-798106 to improve MIRI, and in particular, provide a foundation for the development of related drugs.

[0021] In summary, the present invention discovered that the EP3 receptor antagonist L-798106 can be used to prevent and treat MIRI-related diseases and has clinical research and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 L-798106 reduced the infarct size in mice after MIR.

[0023] Figure 2 L-798106 reduced the levels of serum lactate dehydrogenase (LDH), creatine kinase (CK), and creatine kinase and its isoenzymes (CK-MB) in mice after MIR.

[0024] Figure 3 L-798106 improves cardiac function in mice after MIR. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and can implement it, but the embodiments are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0026] Example 1

[0027] 1. Materials and Methods

[0028] 1.1 Experimental Animals

[0029] C57BL / 6N wild-type male mice (8–12 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and maintained in an SPF-grade environment. The room temperature was controlled at 24 ± 1°C and the humidity was 50–60%. They had free access to food and water and were maintained under a 12-h light / 12-h dark regime.

[0030] 1.2 Drugs and treatment

[0031] Compound L-798106 was purchased from a Cayman company and dissolved in DMSO at 10 mg / ml, then added to the mice's drinking water at a concentration of less than 0.75%. The mice were fed a dose of 14 mg / kg / day starting 24 hours before modeling and continuing until postoperative sampling.

[0032] 1.3 Establishment of a Myocardial Ischemia-Reperfusion Model in Mice Mice were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital (70 mg / kg) and fixed in a supine position on a temperature-controlled small animal laboratory table. The mouse body temperature was maintained at approximately 37°C, and depilatory cream was applied to the mouse chest to remove hair. A tongue depressor was used to elevate the mouse's mandible and perform endotracheal intubation. The ventilator parameters were adjusted to a tidal volume of 150 μL and a frequency of 150 breaths / min. After connecting the ventilator, the rise and fall of the mouse's chest was observed to be consistent with the ventilator rhythm. During the experiment, the mouse's respiratory changes should be observed at all times, and adjustments should be made at any time based on individual conditions.

[0033] The mouse was placed in the right lateral decubitus position. After disinfection with complex iodine, a transverse incision was made along the left sternal margin at the third or fourth intercostal space to expose the pectoralis major muscle. The chest muscles were then bluntly dissected layer by layer. The intercostal muscles were bluntly dissected at the third or fourth intercostal space. The ribs were then pulled apart with a homemade retractor to enter the thoracic cavity and fully expose the heart. The pericardium was then dissected, and the left atrial appendage and pulmonary conus were identified. A small round needle (8-0) with suture was inserted 1 mm below the left atrial appendage (the left anterior descending coronary artery leading to the apex can be found through the accompanying great cardiac vein). The needle passed through approximately one-third of the myocardium and exited lateral to the pulmonary conus. After the mouse acclimated for 1-2 minutes, a 2 mm long plastic tubing was placed over the ligated artery to assist in ligation and tying the slipknot. The ligature was tightened to the point where the tubing collapsed. Cyanosis of the anterior wall of the left ventricle was observed after ligation, indicating successful ligation. Finally, gauze moistened with saline was placed over the incision to close and keep it moist.

[0034] After 30 minutes of ischemia, the slipknot ligated above the left anterior descending coronary artery was loosened, and the small plastic tube was removed to allow reperfusion. Successful reperfusion was indicated by a change from pale to bright red in the corresponding area of the left ventricular anterior wall. A 5-0 suture needle was used to suture the intercostal spaces, muscle layers, and skin layers layer by layer. Mice were weaned from the ventilator and placed in a PVC cage on a normal diet. After 24 hours of reperfusion, blood collection, Evan's blue-TTC double staining, and small animal ultrasound experiments were performed.

[0035] Note: Except for the lack of ligation, the other operations of the sham operation group were the same as those of the ischemia-reperfusion group. An equal amount of DMSO was used in the drinking water of the sham operation group instead of L-798106, and the same experimental treatment was performed.

[0036] 1.4 Measurement of myocardial infarct size

[0037] After 24 hours of reperfusion, mice were anesthetized and immobilized with an intraperitoneal injection of 1% sodium pentobarbital (70 mg / kg). After intubation and connection to a ventilator, the chest cavity was reopened and the heart exposed. A suture was threaded through the original ligature without ligating. The abdominal cavity was opened, and 0.3 mL of 1% TTC dye was injected through the inferior vena cava. After 10 seconds of blood circulation, the left anterior descending coronary artery was ligated. Again, 0.4 mL of 2% Evan's blue was slowly injected through the inferior vena cava. Perfusion was stopped when half of the heart turned dark blue. The mice were quickly sacrificed, and the hearts were excised and washed in pre-chilled PBS. The heart surface was thoroughly aspirated and placed in an EP tube. The heart, along with the heart sectioning mold, was frozen at -20°C for 30 minutes. After cryofixation was complete, the heart was placed in the mold and sliced continuously along its long axis from the ligature into five slices approximately 2 mm thick. Finally, heart sections were fixed in 4% paraformaldehyde for 6 hours. Each slice of heart tissue was weighed and photographed. Sections were analyzed using Photoshop. After staining, the infarcted area was colored white, the risk area red, and the non-risk area blue. The risk area was expressed as the percentage of risk area / left ventricular area, and the infarct size was expressed as the percentage of infarct area / risk area.

[0038] Note: The ischemia-reperfusion group and the ischemia-reperfusion+L-798106 group were stained, while the sham operation group was not stained or measured because it had no ischemia or infarction.

[0039] 1.5 Detection of changes in serum myocardial enzyme spectrum

[0040] Blood was collected from the inferior vena cava of mice and allowed to stand at room temperature for 2 hours. Serum was then centrifuged at 3000 rpm for 10 minutes at 4°C to collect serum. Serum levels of LDH, CK, and CK-MB were measured using a Beckman Coulter biochemical assay system.

[0041] 1.6 Echocardiography to assess cardiac function in mice

[0042] Mouse echocardiograms were acquired using the Vevo® LAZR Small Animal Multimodal Imaging System. Mice were first placed in an anesthesia box filled with 3% isoflurane gas to induce rapid anesthesia. Mice were then immobilized on a 37°C thermostat and maintained anesthesia with 0.5%-1% isoflurane. Ultrasound coupling gel was applied to the chest of the mice, and an MS-400 ultrasound probe was used to acquire dynamic cardiac videos in both long- and short-axis views in M-Mode and B-Mode for five consecutive cardiac cycles. Data were processed using Vevo LAB 3.0.0 software. Cardiac systolic function was assessed using left ventricular ejection fraction (LVEF%) and left ventricular fractional shortening (LVFS%): LVEF% = (left ventricular end-diastolic volume - left ventricular end-systolic volume) / left ventricular end-diastolic volume; LVFS% = (left ventricular end-diastolic diameter - left ventricular end-systolic diameter) / left ventricular end-diastolic diameter).

[0043] 1.7 Statistical methods

[0044] Experimental data are expressed as mean ± standard deviation (SD). Single-factor analyses between two groups were performed using independent t-tests, and single-factor analyses between three groups were performed using one-way ANOVA. Statistical analysis was performed using GraphPad Prism 6 (San Diego, CA, USA). P < 0.05 indicated a statistically significant difference; *P < 0.05; **P < 0.01; NS indicates no significant difference.

[0045] Example 2

[0046] Experimental results

[0047] 1. The infarct area of myocardial tissue after myocardial ischemia-reperfusion, such as Figure 1 shown.

[0048] from Figure 1 As can be seen, the myocardial area at risk was approximately 45% in both the control and experimental groups, indicating that while the ligation location and technique were essentially the same, the myocardial infarction area in the experimental group was significantly smaller, suggesting that L-798106 can effectively mitigate myocardial infarction caused by MIRI.

[0049] 2. Changes in serum myocardial enzyme spectrum after myocardial ischemia reperfusion, such as Figure 2 As shown:

[0050] from Figure 2As can be seen, in the sham-operated group, serum LDH, CK, and CK-MB levels remained at physiological levels due to the lack of ligation. However, in the control group, all three indicators increased significantly after ischemia-reperfusion. Furthermore, in the experimental group, the myocardial enzyme profile, which was upregulated by ischemia-reperfusion, was effectively suppressed. This suggests that L-798106 can effectively reduce myocardial tissue damage caused by reperfusion.

[0051] 3. Changes in cardiac function after myocardial ischemia-reperfusion, such as Figure 3 As shown:

[0052] from Figure 3 As can be seen, the LVEF% and LVFS% in the sham-operated group remained stable at physiological levels. Twenty-four hours after ischemia-reperfusion, both cardiac function indicators decreased significantly, while those in the experimental group recovered significantly. This suggests that L-798106 can improve cardiac contractile function after ischemia-reperfusion.

[0053] Based on the above experiments, the application of L-798106 to inhibit EP3 receptor is a new therapy for preventing and treating myocardial ischemia-reperfusion injury.

[0054] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Use of an EP3 receptor antagonist in the preparation of a drug for preventing myocardial ischemia-reperfusion injury; the EP3 receptor antagonist is L-798106.

2. The use according to claim 1, characterized in that The EP3 receptor antagonist is used in the preparation of a medicine for reducing the myocardial infarction area after myocardial ischemia-reperfusion surgery.

3. The use according to claim 1, characterized in that The EP3 receptor antagonist is used in the preparation of a drug for reducing serum myocardial enzyme spectrum levels after myocardial ischemia reperfusion surgery.

4. The use according to claim 3, characterized in that The enzymes include one or more of lactate dehydrogenase, creatine kinase, creatine kinase and isoenzymes thereof.

5. The use according to claim 1, characterized in that The EP3 receptor antagonist is used in the preparation of a drug for improving cardiac contractile function after myocardial ischemia-reperfusion surgery.

6. The use according to claim 1, characterized in that The medicine includes one or more of oral preparations and injections.

7. The use according to claim 1, characterized in that The drug comprises an oral agent, and the dosage of L-798106 is 14 mg / kg / day.

8. The use according to claim 1, characterized in that The L-798106 was dissolved in DMSO at 10 mg / ml to obtain an L-798106 solution, which was then dissolved in drinking water.

9. The use according to claim 8, characterized in that The L-798106 solution is dissolved in the drinking water at a volume ratio of less than 0.75%.

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