WO2018126897A1

WO2018126897A1 - APPLICATION OF SODIUM 5-BROMO-2-(α-HYDROXYPENTYL) BENZOATE IN DRUGS TREATING CARDIOVASCULAR DISEASE

Info

Publication number: WO2018126897A1
Application number: PCT/CN2017/117605
Authority: WO
Inventors: 常俊标; 赵文; 宋传军
Original assignee: 郑州大学; 河南美泰宝生物制药有限公司
Priority date: 2017-01-06
Filing date: 2017-12-21
Publication date: 2018-07-12
Also published as: CN108283633A

Abstract

Disclosed is an application of sodium 5-bromo-2-(α-hydroxypentyl) benzoate (BZP) in drugs treating cardiovascular disease, belonging to the field of pharmaceutical chemistry. Experiments show that BZP has a protective effect with regard to acute and chronic myocardial ischaemia-reperfusion injury in mice, BZP oral administration and intravenous injection reduce animal overall acute myocardial infarction areas, and BZP reduces isolated perfused heart acute ischaemia-reperfusion injury myocardial infarction areas in mice, thereby improving myocardial contractile function and reducing haematological myocardial injury markers. In addition, BZP chronic oral administration delays the progress of cardiac hypertrophy changing to heart failure after acute myocardial infarction in mice, reduces ventricular remodelling caused by ischaemic heart disease in mice, and significantly improves heart function treatment effects. Furthermore, BZP temporarily reduces the blood pressure of hypertensive rats (SHR rats). These effects show that BZP has effective clinical research and development prospects for drugs protecting against acute and chronic myocardial infarction myocardial injury.

Description

Application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate in the treatment of cardiovascular diseases

Technical field

The invention relates to the application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate (BZP for short) in the preparation of a medicament for treating cardiovascular diseases, in particular to the prevention and treatment of ischemia-reperfusion injury after acute and chronic myocardial infarction by BZP. The application of diseases is in the field of medicinal chemistry.

Background technique

Cardiovascular diseases, especially coronary atherosclerosis and the resulting ischemic heart disease, are still common diseases that seriously plague human health. According to incomplete statistics, the annual number of deaths from cardiovascular diseases worldwide accounts for 30% of the total death toll. %, of which ischemic heart disease accounts for more than 40% (about 7 million people). About 1.5 million people die of ischemic heart disease every year in China, and with the improvement of people's living standards, their morbidity and mortality rate are It is on the rise, although currently clinically through thrombolysis, coronary artery bypass grafting, percutaneous coronary intervention and other means can significantly alleviate myocardial cell damage caused by ischemia-reperfusion, but myocardial cell death caused by acute myocardial infarction, Ventricular remodeling and heart failure still seriously affect the prognosis of the disease, leading to increased mortality. Moreover, with the failure of clinical trials of multiple acute myocardial infarction drugs, there has been no clinically effective treatment for acute Myocardial infarction. Therefore, the search for high-efficiency and low-toxic drugs for the treatment of acute myocardial ischemia-reperfusion injury has become a major clinical scientific problem in the field of cardiovascular disease treatment. It has received extensive attention from basic and clinical researchers at home and abroad, and is also a research hotspot in this field. .

Chinese invention patent 200810230890.X discloses for the first time the preparation method of halogen-substituted 2-(α-hydroxypentyl)benzoate compound and its application in medicine. The specification discloses the activity of a halogen-substituted potassium salt of 2-(α-hydroxypentyl)benzoate in preventing and treating cardio-cerebral ischemic diseases and improving cardiovascular and cerebrovascular disorders, anti-thrombosis and the like. However, it does not involve the detailed application of BZP in acute and chronic myocardial ischemia-reperfusion injury.

The inventors have found in a later study through detailed experiments that BZP has obvious protective effects on acute and chronic myocardial ischemia-reperfusion injury, and no relevant literature reports have been reported at present.

Summary of the invention

The object of the present invention is to provide a new application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate in the preparation of a medicament for treating cardiovascular diseases, especially in the preparation of a medicament for preventing and treating acute myocardial ischemia-reperfusion injury. application.

The chemical formula of sodium 5-bromo-2-(α-hydroxypentyl)benzoate (BZP) of the present invention is as follows:

In order to achieve the object of the present invention, the present invention has carried out experiments to confirm the protective effect of BZP on acute myocardial infarction and ischemia-reperfusion injury in ICR mice and C57BL/6 mice, and the protective effect on myocardial infarct size.

The present inventors conducted experiments to confirm that BZP oral administration of 20-40 mg/kg body weight for prophylactic and therapeutic administration can significantly reduce myocardial infarct size in acute myocardial ischemia-reperfusion injury of ICR mice.

The present inventors conducted experiments and confirmed that BZP intravenous injection of 12-24 mg/kg body weight for prophylactic and therapeutic administration can significantly reduce the myocardial infarct size of acute myocardial ischemia-reperfusion injury in ICR mice.

The present invention has carried out experiments and confirmed that BZP is orally administered at a dose of 10-200 mg/kg/day, and continuous administration for 90-120 days has a treatment for delaying myocardial hypertrophy and heart failure after chronic myocardial ischemia-reperfusion injury in ICR mice. effect.

The present inventors conducted experiments and confirmed that BZP oral administration of 20-40 mg/kg body weight for prophylactic and therapeutic administration can significantly reduce the myocardial infarct size of acute myocardial ischemia-reperfusion injury in C57BL/6 mice.

The present inventors conducted experiments and confirmed that BZP intravenous injection of 12-24 mg/kg body weight for prophylactic and therapeutic administration can significantly reduce myocardial infarct size in C57BL/6 mice with acute myocardial ischemia-reperfusion injury.

The present invention has carried out experiments and confirmed that BZP is orally administered at a dose of 10-200 mg/kg/day, and continuous administration for 90-120 days has delayed myocardial hypertrophy and heart failure in C57BL/6 mice after chronic myocardial ischemia-reperfusion injury. Therapeutic effect.

The experiment of the present invention confirmed that the systolic blood pressure, diastolic blood pressure and mean arterial pressure of SHR rats were significantly decreased after oral administration of BZP at a dose of 45 mg/kg for 30 min to 40 min.

The present inventors conducted experiments and confirmed that BZP has a significant relaxation effect on the isolated vascular rings of isolated SD rats in the concentration range of 1×10-6 mol/L to 1×10-3 mol/L.

Advantages of the invention: Through experiments, it is found that a new application of sodium 5-bromo-2-(α-hydroxypentyl)benzoate in the treatment of cardiovascular diseases proves that BZP has obvious effects on acute and chronic myocardial ischemia-reperfusion injury. Protective effect can significantly reduce the area of acute myocardial infarction, reduce myocardial damage, improve cardiac function, and significantly delay the occurrence of cardiac hypertrophy and heart failure after acute myocardial infarction. It is expected that BZP can be used as an effective acute and chronic myocardial ischemia-reperfusion injury myocardial injury. Protect the drug for development.

DRAWINGS

Figure 1 is a graphical representation of the effect of BZP on cardiac function recovery in isolated heart ischemia-reperfusion injury in ICR mice, and 1-1 is the recovery of left ventricular maximum systolic blood pressure (+dp/dtmax) in 0.24 mmol of female mice. 1-2 is the recovery of left ventricular maximum diastolic blood pressure (-dp/dt max) administered to 0.24 mmol of female mice;

Figure 2 is a photograph of the effect of BZP on the myocardial infarction area of ICR mice isolated from cardiac ischemia-reperfusion injury (* compared with the control group, P < 0.05), 2-1 for 0.24 mmol administration to male mice, 2 -2 is administered to female mice at 0.24 mmol, in the figure, a-control, b-present compound BZP;

Figure 3 is a graphical representation of the effect of BZP on LDH in isolated heart ischemia-reperfusion injury in ICR mice, (*: P<0.05 compared with the ischemia-reperfusion model control group), in the figure, a-control, b-ben Hair compound BZP;

Figure 4 is a photo of the protective effect of BZP oral (gavage) on acute ischemia-reperfusion injury in ICR mice. In the figure, a-control, b-present compound BZP (20mg/kg), c-present compound BZP (40 mg/kg), d-metoprolol BZP (10 mg/kg);

Figure 5 is a photo of the protective effect of BZP intravenous injection on acute myocardial ischemia-reperfusion injury in ICR mice. In the figure, a-control, b-present compound BZP (12mg/kg), c-present compound BZP ( 24 mg/kg), d-metoprolol BZP (10 mg/kg);

Figure 6 is a protective effect of BZP on acute myocardial ischemia-reperfusion injury in ICR mice (P<0.05 compared with model control group; #: P<0.05 compared with BZP low-dose group), , a-oral (gavage), b-intravenous injection;

Figure 7 is a photo of the protective effect of BZP oral (gavage) on myocardial injury induced by acute myocardial infarction in ICR mice. In the figure, a-control, b-present compound BZP (20mg/kg), c-present Compound BZP (40 mg/kg);

Figure 8 is a photo of the protective effect of BZP intravenous injection on myocardial injury induced by acute myocardial infarction in ICR mice. In the figure, a-control, b-present compound BZP (12mg/kg), c-present compound BZP ( 24mg/kg);

Figure 9 is a graphical representation of the protective effect of BZP on myocardial injury induced by acute myocardial infarction in ICR mice (*: P<0.05 compared with model control group; #: P<0.05 compared with BZP low-dose group), Medium, a-oral, b-intravenous; 1-control, 2-present compound BZP (20 mg/kg), 3-present compound BZP (40 mg/kg), 4-control, 5-present compound BZP ( 12mg/kg), 6-present compound BZP (24mg/kg);

Figures 10-1 and 10-2 are graphical representations of animal ultrasound evaluation of cardiac function in ICR mice;

Figure 11-1 is a representative animal ultrasound systole diagram, in the figure, a-sham control group, b-model control group, c-present compound BZP (20mg/kg), d-present compound BZP (40mg /kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 11-2 is a graphical representation of the protective effect of BZP on the important myocardial contractile function index EF% after MI in mice. In the figure, a-sham control group, b-model control group, c-present compound BZP (20 mg/ Kg), d-present compound BZP (40mg/kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 11-3 is a graphical representation of the protective effect of BZP on the important myocardial contractile function index FS% after MI in mice. In the figure, a-sham control group, b-model control group, c-present compound BZP (20 mg/ Kg), d-present compound BZP (40mg/kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 12 is a graphical representation of the protective effect of BZP oral administration on myocardial infarction area after global myocardial infarction in ICR mice, a-sham control group, b-model control group, c-present compound BZP (20 mg/kg) , d-present compound BZP (40mg/kg), e metoprolol BZP (10mg/kg); 1,2,3 represent 4,8,12 weeks after surgery;

Figure 13-1 is a graphical representation of the protective effect of BZP oral administration on myocardial remodeling after global myocardial infarction in ICR mice, a-sham control group, b-model control group, c-present compound BZP (20 mg/ Kg), d-present compound BZP (40 mg/kg), e-metoprolol BZP (10 mg/kg); 1, 2, 3, 4 represent 2, 4, 8, and 12 weeks postoperatively;

Figure 13-2 is a graphical representation of the protective effect of BZP oral administration on myocardial remodeling after global myocardial infarction in ICR mice, a-sham control group, b-model control group, c-present compound BZP (20 mg) /kg), d-present compound BZP (40mg/kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 14 is a photograph showing the protective effect of BZP oral administration on acute ischemia-reperfusion injury in C57BL/6 mice. In the figure, a-control, b-present compound BZP (20 mg/kg), c-present compound BZP ( 40 mg/kg), d-metoprolol BZP (10 mg/kg);

Figure 15 is a photo of the protective effect of BZP intravenous injection on acute myocardial ischemia-reperfusion injury in C57BL/6 mice. In the figure, a-control, b-present compound BZP (12mg/kg), c-present compound BZP (24mg/kg), d-metoprolol BZP (10mg/kg);

Figure 16 is a graphical representation of the protective effect of BZP on acute myocardial ischemia-reperfusion injury in C57BL/6 mice (*: P<0.05 compared with model control group; #: P<0.05 compared with BZP low-dose group) , in the figure, a-oral, b-intravenous; 1-control, 2-present compound BZP (20 mg/kg), 3-present compound BZP (40 mg/kg), 4-metolol BZP (10 mg) /kg), 5-control, 6-present compound BZP (12 mg/kg), 7-present compound BZP (24 mg/kg), 8-metoprolol BZP (10 mg/kg);

Figure 17 is a photograph showing the protective effect of BZP oral administration on myocardial injury induced by acute myocardial infarction in C57BL/6 mice;

In the figure, a-control, b-present compound BZP (20 mg/kg), c-present compound BZP (40 mg/kg), d-metoprolol BZP (10 mg/kg);

Figure 18 is a photo of the protective effect of BZP intravenous injection on myocardial injury induced by acute myocardial infarction in C57BL/6 mice. In the figure, a-control, b-present compound BZP (12mg/kg), c-present compound BZP (24mg/kg), d-metoprolol BZP (10mg/kg);

Figure 19 is a graphical representation of the protective effect of BZP on myocardial injury induced by acute myocardial infarction in C57BL/6 mice. In the figure, a-oral, b-intravenous; 1-control, 2-present compound BZP (20 mg/ Kg), 3-present compound BZP (40 mg/kg), 4-Metoprolol BZP (10 mg/kg), 5-control, 6-present compound BZP (12 mg/kg), 7-present compound BZP (24mg/kg), 8-Metoprolol BZP (10mg/kg); Figures 20-1, 20-2 are graphical representations of animal ultrasound evaluation of cardiac function in C57BL/6 mice;

Figure 21-1 is a representative animal ultrasound cardiac contraction legend, in the figure, a-sham control group, b-model control group, c-present compound BZP (20mg/kg), d-present compound BZP (40mg /kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 21-2 is a graphical representation of the protective effect of BZP on the important myocardial contractile function index EF% after MI in C57 mice. In the figure, a-sham control group, b-model control group, c-present compound BZP (20 mg) /kg), d-present compound BZP (40mg/kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 21-3 is a graphical representation of the protective effect of BZP on the important myocardial contractile function index FS% after MI in C57 mice. In the figure, a-sham control group, b-model control group, c-present compound BZP (20 mg) /kg), d-present compound BZP (40mg/kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent 2,4,8,12 weeks after surgery;

Figure 22 is a graphical representation of the protective effect of BZP oral administration on myocardial infarction area after chronic myocardial infarction in C57BL/6 mice. In the figure, a-sham control group, b-model control group, c-book Compound BZP (20mg/kg), d-present compound BZP (40mg/kg), e-metoprolol BZP (10mg/kg); 1,2,3,4 represent postoperative 2,4, respectively 8,12 weeks;

Figure 23-1 is a graphical representation of the protective effect of BZP oral administration on myocardial remodeling after chronic myocardial infarction in C57BL/6 mice. In the figure, a-sham control group, b-model control group, c- The present compound BZP (20 mg/kg), d-present compound BZP (40 mg/kg), e-metoprolol BZP (10 mg/kg); 1, 2, 3, 4 represent postoperative 2, 4, respectively , 8, 12 weeks;

Figure 23-2 is a graphical representation of the protective effect of BZP oral administration on myocardial remodeling after chronic myocardial infarction in C57BL/6 mice. In the figure, a-sham control group, b-model control group, c - the present compound BZP (20 mg / kg), d - the present compound BZP (40 mg / kg), e-metoprol BZP (10 mg / kg); 1, 2, 3, 4 represent postoperative 2, respectively 4,8,12 weeks;

Figure 24 is the effect of BZP on systolic blood pressure in SHR rats (* indicates P<0.05 compared with model group, ** indicates P<0.01 compared with model group);

Figure 25 is the effect of BZP on diastolic blood pressure in SHR rats (* indicates P<005 compared with model group, ** indicates P<0.01 compared with model group);

Figure 26 is the effect of BZP on mean arterial pressure in SHR rats (* indicates P<005 compared with model group, ** indicates P<0.01 compared with model group);

Figure 27 is a graphical representation of the effect of different concentrations of BZP on the tension of isolated vascular rings.

detailed description

The following examples are provided to assist those of ordinary skill in the art in a more comprehensive understanding of the invention, but are not intended to limit the invention in any way.

Example 1: Protective effect of BZP on ischemia-reperfusion injury in isolated heart of ICR mice

Experimental materials and methods

(1) Experimental materials

ICR mice were used, weighing 18-22 g, male and female, BZP (synthesized by our laboratory) was prepared with double distilled water; TTC was purchased from Sigma.

(2) Langendorff isolated heart perfusion: using the AD instrument company Powerlab Langendorff cardiac perfusion device for perfusion of the heart, the animals were anesthetized with 10% by mass of chloral hydrate (300mg/Kg), and 1000U/kg heparin was used. Injection anticoagulation. Open the chest, quickly remove the heart along the aortic root, and immediately put the KH perfusate (component mM: NaCl: 118, KCl 4.7, MgSO ₄ 1.2, KH ₂ PO ₄ 1.2, EDTA. 2Na 0.5, NaHCO ₃ 25, CaCl ₂ 2.5 , Glucose 11, pH 7.4), flush the heart, use aortic retrograde intubation, quickly place the heart in the Langendorff perfusion system, apply perfusion with KH fluid, set perfusion pressure 80cmH ₂ O, use 37 ° C circulating water bath, perfusate continued Filled with a mixture of 95% O ₂ and 5% CO ₂ in volume, the coronary outflow was maintained at 5-8 mL/min, and the entire procedure was completed in 5 minutes. After the heart is continuously perfused for 5 minutes, the left atrial appendage is cut open, the balloon is inserted into the left ventricle, and then the balloon is slowly filled with ddH ₂ O. The other end of the balloon is connected to the Powerlab multi-channel physiological recorder, and the intracapsular pressure is maintained at 4- 8mmHg.

(3) Construction of isolated heart ischemia-reperfusion model: After 15-20 minutes of stable perfusion of the heart, the perfusion system was clamped, the heart was stopped for 40 minutes, and then the perfusion was resumed for 60 minutes, resulting in isolated whole heart ischemia and reperfusion. The injury model; the Powerlab transducer was used to record the full-length recording of myocardial contractile function.

(4) Determination of myocardial infarction area in vitro myocardial ischemia-reperfusion injury: The heart was quickly removed at the end of the experiment, and frozen on dry ice for about 15 minutes, then evenly cut into 2 mm slices along the longitudinal axis of the heart. 5 tablets, dehydrated with filter paper, the heart slice was placed in a mass percentage of 1% red tetrazolium (TTC) phosphate buffer, incubated at 37 ° C for 10-15 minutes in the dark, the uninfarcted area contains intact lactate dehydrogenation The enzyme was stained red, the infarct area was white, and then photographed with a digital camera macro lens. The Med6.0 medical image analysis system measured the percentage of myocardial infarct area to the total area of the left ventricle.

(5) Determination of lactate dehydrogenase (LDH) content in coronary effluent: The LDH assay kit produced by Sigma was used to determine the LDH content of coronary effluent within 15 minutes after reperfusion to indirectly reflect the degree of myocardial damage.

(6) Experimental grouping: The experiment was divided into a model group and a 0.24 mM BZP treatment group (this dose was a pre-experimental moderate effective dose), 8 rats in each group, half of each female, and administered at the time of reperfusion.

(7) Statistical analysis: Data were expressed as mean ± standard deviation, and analyzed by pairwise t-test. Experimental result

(1) Effect of BZP on cardiac function recovery in isolated heart ischemia-reperfusion injury in ICR mice

The systolic function of the model group after ischemia-reperfusion injury was significantly lower than that before ischemia, suggesting successful modeling. Despite this, the BZP0.24 mM/L treatment group significantly improved the systolic function of the heart after ischemia-reperfusion injury in mice, including: left ventricular maximum diastolic blood pressure (-dp/dtmax) and left, compared with the model group. Recovery of maximum systolic blood pressure (+dp/dtmax) in the chamber (see Figure 1).

(2) Effect of BZP on myocardial infarction area of ICR mice isolated from ischemia-reperfusion injury

Compared with the saline group, the BZP0.24mM/L treatment group significantly reduced the area of myocardial infarction in isolated cardiac ischemia-reperfusion injury, suggesting the protective effect of BZP on acute myocardial injury (see Figure 2).

(3) Effect of BZP on LDH in isolated heart ischemia-reperfusion injury in ICR mice

Compared with the saline group, the BZP0.24mM/L treatment group significantly reduced the release of LDH (lactate dehydrogenase) from isolated heart ischemia-reperfusion injury, suggesting that BZP has significant protection against acute myocardial ischemia-reperfusion injury. Role (see Figure 3).

Example 2: Protective effect of BZP on ischemia-reperfusion injury in whole animal heart of ICR mice

Experimental materials and methods

(1) Experimental materials

(2) Preparation of myocardial ischemia and reperfusion model in whole animal mice: animal mass percentage 2-3% isoflurane was inhaled by American Viking Medical inhalation anesthesia system, and cut by the left 4-5 intercostal space about 1.2 Cm incision, blunt dissection of the pectoralis major and pectoralis minor muscle, then use the mosquito pliers to open the pleura and pericardium, expose the heart, and then gently squeeze the heart to find the left anterior descending coronary artery below the left atrial appendage At the beginning of the site about 3mm with a 6-0 silk hit a knot, the success of the surgery is that the left ventricular anterior wall below the ligation site can be seen white, ECG shows ST segment elevation, the heart will be quickly after ligation Put back into the chest, reserve the knotted ligature outside the chest, and manually close the chest and squeeze the chest wall to expel the gas, suture the chest wall with a 5-0 suture, after 40 minutes of myocardial infarction, manually loosen the knot of the knot The line (based on the feeling of loose knot) was reperfused for 24 hours. Anesthesia is removed after surgery to allow the animal to quickly return to spontaneous breathing.

(3) Determination of myocardial infarction area of myocardial ischemia-reperfusion injury in the whole animal: open the chest after the end of the experiment, and then tighten the ligature line reserved for the myocardial infarction, and pre-fill the mass percentage along the aorta. %Evan Blue, the non-infarcted area (ANAR) is blue, and the rest is called the infarct area (AAR). The heart is quickly removed, the filter paper is dehydrated, and then frozen on dry ice for about 15 minutes, then along the longitudinal axis of the heart. Evenly cut into 2mm slices, cut 5 pieces per heart, place the heart slices into 1% by mass of red tetrazolium (TTC) phosphate buffer, incubate at 37 °C for 10-15 minutes in the dark, no infarction The area was stained red by the complete lactate dehydrogenase, and the infarct area was white (MI). Then the digital camera was used to take a macro lens. The Med6.0 medical image analysis system measured the percentage of myocardial infarction area in the AAR area. The entire heart cross-sectional area is approximately 35-50% within the successful myocardial infarction model group.

(4) Determination of serum lactate dehydrogenase (LDH) content: blood was taken from the common carotid artery, serum was taken by centrifugation, and LDH content in serum was determined using LDH assay kit produced by Sigma to indirectly reflect myocardial ischemia and reperfusion. The extent of the damage.

(5) Experimental group: BZP oral 0, 20, 40 mg/kg group, 12 mice per female, half male and half female; and

BZP

0, 12, 24 mg/kg intravenous group, 12 mice per dose Only, male and female; select metoprolol alginate 10mg/kg as a positive control, this group selected 12 mice, male and female; the selected experimental doses are pre-experimental effective dose, in animal model surgery Pre-administered.

(6) Statistical analysis: Data were expressed as mean ± standard deviation, and statistical analysis was performed by pairwise t-test and One-Way ANOVA. P < 0.05 was considered statistically significant.

Experimental result

(1) Protective effect of BZP oral administration on acute ischemia-reperfusion injury in whole animal of ICR mice

BZP oral administration of 20mg/kg, 40mg/kg administration group compared with the saline control group, myocardial infarct size was significantly reduced, and the treatment effect was better than the positive drug (see Figure 4).

(2) Protective effect of BZP intravenous injection on acute myocardial ischemia-reperfusion injury in whole animal of ICR mice

BZP intravenous injection of 12mg/kg, 24mg/kg administration group compared with the saline control group, myocardial infarct size was significantly reduced, and better than the positive drug (see Figure 5).

(3) Protective effect of BZP on acute myocardial ischemia-reperfusion injury in whole animal of ICR mice

Specific quantitative indicators showed that both BZP intravenous and oral administration significantly reduced myocardial infarct size in acute whole animal ischemia-reperfusion (see Figure 6).

(4) Protective effect of BZP oral administration on myocardial injury induced by acute myocardial infarction in ICR mice

BZP oral administration of 20 mg / kg, 40 mg / kg administration group compared with the saline control group, myocardial infarct size was significantly reduced (see Figure 7).

(5) Protective effect of BZP intravenous injection on myocardial injury induced by acute myocardial infarction in whole animal of ICR mice

BZP was intravenously injected at 12 mg/kg, and the myocardial infarct size was significantly lower in the 24 mg/kg administration group than in the saline control group (see Figure 8).

(6) Protective effect of BZP on myocardial injury induced by acute myocardial infarction in ICR mice

Specific quantitative indicators show that both BZP intravenous and oral administration can significantly reduce the infarct size of acute global myocardial infarction (see Figure 9).

Conclusion: BZP oral administration or intravenous injection can significantly reduce myocardial injury induced by acute myocardial ischemia and reperfusion in ICR mice and acute myocardial infarction, and can be used as a protective drug for acute myocardial ischemic injury.

Example 4: Protective effect of BZP on delayed cardiac hypertrophy and heart failure in ICR mice after global ischemia and reperfusion injury

experimental method:

(1) Experimental materials

ICR mice, half male and half, BZP powder was configured with double distilled water.

(2) Experimental grouping

A total of 4 groups, 12 in each group, male and female, a total of 48. They were sham operation group; myocardial infarction model group (MI); BZP high (40 mg/kg) and low dose group (20 mg/kg). The positive drug was selected from metoprolol alginate 10 mg/kg.

(3) Long-term administration method

Treatment group: After the establishment of the MI model, BZP was administered by intragastric administration at doses of 20 mg/kg and 40 mg/kg, respectively. The administration volume was 0.01 ml/g.

Positive drug group: After the establishment of the MI model, metoprolol succinate was administered by gavage at a dose of 10 mg/kg. The administration volume is the same as above.

Control group: After the establishment of the MI model, saline was administered by the stomach, and the administration volume was the same as above.

Preparation of MI model: preoperative weighing, inhalation anesthesia was performed with 1% by mass of isoflurane, and the supine position was fixed on the rat plate. Percentage of mass 75% alcohol disinfects the skin of the precordial area. Cut the skin in the front area, bluntly separate the muscles, and cut the chest wall in the third and fourth intercostal space. In the myocardial infarction model group, the left anterior border of the left atrial appendage was 3-4 mm, and the left coronary artery (coronary artery) anterior descending branch was ligated with a 6-0 suture. The myocardial dysfunction in the area below the ligation site was successful in ligation; as a sham operation group, The needle passes through the left coronary artery and the descending branch is not ligated. The chest cavity was closed along the third and fourth ribs with a 4-0 line, the chest muscles were sutured layer by layer, and the chest skin was continuously sutured.

(4) Non-invasive ultrasound evaluation of cardiac function: 2 weeks, 4 weeks, 8 weeks and 12 weeks after MI operation, the heart function of the mice was measured using an ultrasonic diagnostic apparatus and a 30 MHz high-frequency probe, and the mass percentage was 1% different. The halothane was inhaled and anesthetized in mice, and the hair in the anterior region was shaved. The mouse was placed in a supine position on a hot plate to keep the body temperature constant. Apply appropriate amount of ultrasonic couplant in the anterior region, use the 30MHz probe to start the paraparusal long axis view on the chest wall of the mouse, and take the left ventricular short axis of the sternum to obtain the M-shaped slice image to measure left ventricular diastolic and systolic Front and rear wall thickness, left ventricular ejection fraction (EF%), etc. A B-section image was obtained to measure left ventricular diastolic and systolic volume. The ultrasound diagnostics set the same parameters for different individuals. For each measurement index of M super, five consecutive cardiac cycles were selected and the average values of EF%, FS%, etc. were obtained. B-ultrasound selects the left ventricular maximum volume and the minimum volume, ie, the left ventricular section of the diastolic and systolic phases. The circumference was drawn along the left inner wall to determine FAC% for evaluation of overall cardiac function (see Figure 10).

(5) Statistical analysis: Data were expressed as mean ± standard deviation, and statistical analysis was performed by pairwise t-test and One-Way ANOVA. P < 0.05 was considered statistically significant.

Experimental result

1. The protective effect of oral administration of BZP on cardiac function in the whole animal after chronic myocardial infarction in ICR mice.

The myocardial infarction model (MI) was constructed by ligation of the anterior descending coronary artery. The systolic function of the mice was recorded by small animal ultrasound at 2, 4, 8 and 12 weeks after surgery. The results showed that BZP was orally administered at 20 mg/kg/day. And 40mg/kg/day, continuous administration for 12 weeks, compared with the model group (MI group), can significantly improve the systolic function after myocardial infarction, including: ejection fraction (EF%), percent shrinkage (FS%) To delay the onset of heart failure (see Figure 11). *: P<0.05 compared with the time of the sham control group (Sham); #: P<0.05 compared with the corresponding time of the model control group (MI) FC. And the therapeutic effect of the BZP administration group was slightly better than that of the positive drug metoprolol.

2. The protective effect of oral administration of BZP on myocardial infarction area after chronic myocardial infarction in ICR mice.

The myocardial infarction model (MI) was constructed by ligation of the anterior descending coronary artery. The myocardial infarction area (FAC%) was recorded by small animal ultrasound at 2, 4, 8 and 12 weeks after surgery. The results showed that BZP was orally administered 20 mg. /kg/day and 40mg/kg/day, continuous administration for 12 weeks, compared with the model group (MI group), can significantly improve the myocardial infarction area after myocardial infarction, reduce the degree of myocardial damage (Figure 12: *: and false The corresponding time of the surgical control group (Sham) was compared with P<0.05; #: compared with the corresponding time of the model control group (MI), P<0.05).

3. The protective effect of oral administration of BZP on myocardial remodeling after chronic myocardial infarction in ICR mice.

The myocardial infarction model (MI) was constructed by ligation of the anterior descending coronary artery. The myocardial remodeling of the mice was recorded by small animal ultrasound at 2, 4, 8 and 12 weeks after surgery (LV Mass and LV Mass corrected). The results showed that BZP oral administration of 20mg/kg/day and 40mg/kg/day, continuous administration for 12 weeks, compared with the model group (MI group), can significantly improve LV Mass and LV Mass corrected after myocardial infarction, reduce myocardial remodeling The occurrence of delayed heart failure (Figure 13: *: compared with the sham control group (Sham) corresponding time P <0.05; #: compared with the model control group (MI) corresponding time P <0.05).

Conclusion: BZP oral administration can significantly improve cardiac function damage caused by chronic myocardial infarction in mice, reduce the occurrence of myocardial remodeling, and delay the progression of heart failure.

Example 4: Protective effect of BZP on myocardial ischemia-reperfusion injury in C57BL/6 mice

Experimental materials and methods

(1) Experimental materials

C57BL/6 mice, weighing 18-22g, male and female, BZP (synthesized by our laboratory), were prepared with double distilled water; TTC was purchased from Sigma, and metoprolol was used as a positive control.

(2) Preparation of myocardial ischemia and reperfusion model in whole animal mice: animal mass percentage 2-3% isoflurane, inhalation anesthesia using American Viking Medical inhalation anesthesia system, through the left 4-5 intercostal scissors 1.2cm incision, blunt dissection of the pectoralis major and pectoralis minor muscle, then use the mosquito pliers to open the pleura and pericardium, expose the heart, and then gently squeeze the heart to find the left coronary artery below the left atrial appendage Branch, at the beginning of about 3mm with a 6-0 silk hit a knot, the success of the surgery is that the left ventricular anterior wall of the ligation site can be seen white, ECG shows ST segment elevation, the heart after ligation Quickly put back into the chest, reserve the knotted ligature outside the chest, and manually close the chest and squeeze the chest wall to expel the gas, suture the chest wall with a 5-0 suture, and gently soften the knot after 40 minutes of myocardial infarction. The ligature line (based on the feeling of loosening of the slip) was reperfused for 24 hours. Anesthesia is removed after surgery to allow the animal to quickly return to spontaneous breathing.

(3) Determination of myocardial infarction area of myocardial ischemia-reperfusion injury in the whole animal: open the chest after the end of the experiment, and then tighten the ligature line reserved for the myocardial infarction, and pre-fill the mass percentage along the aorta. %Evan Blue, the non-infarct area (ANAR) field is blue, and the rest is called the infarct area (AAR). The heart is quickly removed, dehydrated with filter paper, and frozen on dry ice for about 15 minutes, then along the longitudinal axis of the heart. Evenly cut into 2mm slices, cut 5 pieces per heart, place the heart slices into 1% by mass of red tetrazolium (TTC) phosphate buffer, incubate at 37 °C for 10-15 minutes in the dark, no infarction The area was stained red by the complete lactate dehydrogenase, and the infarct area was white (MI). Then the digital camera was used to take a macro lens. The Med6.0 medical image analysis system measured the percentage of myocardial infarction area in the AAR area. The entire heart cross-sectional area is approximately 35-50% within the successful myocardial infarction model group.

(5) Experimental group: BZP oral 0mg/kg, 20mg/kg, 40mg/kg group, 12 mice per female, male and female; and BZP 0mg/kg, 12mg/kg, 24mg/kg intravenous group 12 mice were selected for each dose, half male and half female; the positive drug group was treated with metoprolol 10 mg/kg orally, and 12 mice in this group were selected, male and female; the selected experimental doses were all valid for pre-experiment. The dose is administered prior to the animal model surgery.

Experimental result

(1) Protective effect of BZP oral administration on acute ischemia-reperfusion injury in C57BL/6 mice

BZP oral administration of 20 mg/kg, 40 mg/kg administration group compared with the saline control group, myocardial infarct size was significantly reduced, and slightly better than the positive drug group (see Figure 14).

(2) Protective effect of BZP intravenous injection on acute myocardial ischemia-reperfusion injury in C57BL/6 mice

BZP intravenous injection of 12mg/kg, 24mg/kg administration group compared with the saline control group, myocardial infarct size was significantly reduced, and slightly better than the positive drug group (see Figure 15).

(3) Protective effect of BZP on acute myocardial ischemia-reperfusion injury in C57BL/6 mice

Specific quantitative indicators showed that both BZP intravenous and oral administration significantly reduced myocardial infarct size in acute whole animal ischemia-reperfusion (see Figure 16).

(4) Protective effect of BZP oral administration on myocardial injury induced by acute myocardial infarction in C57BL/6 mice

BZP was orally administered at 20 g/kg, and the 40 mg/kg administration group showed a significant decrease in myocardial infarct size compared with the saline control group (see Figure 17).

(5) Protective effect of BZP intravenous injection on myocardial injury induced by acute myocardial infarction in C57BL/6 mice

BZP was intravenously administered at 12 mg/kg, and the 24 mg/kg administration group showed a significant decrease in myocardial infarct size compared with the saline control group (see Figure 18).

(6) Protective effect of BZP on myocardial injury induced by acute myocardial infarction in C57BL/6 mice

Specific quantitative indicators showed that both BZP intravenous and oral administration significantly reduced the infarct size of acute global myocardial infarction (Figure 19, *: P < 0.05 compared with the model control group).

Conclusion: BZP oral administration or intravenous injection can significantly reduce acute myocardial ischemia-reperfusion and myocardial infarction caused by acute myocardial infarction in C57BL/6 mice, and can be used as a protective drug for acute myocardial ischemic injury.

Example 6: Protective effect of BZP on delayed cardiac hypertrophy and heart failure in C57BL/6 mice after global ischemia and reperfusion injury

experimental method:

(1) Experimental materials

C57BL/6 mice, half male and half, BZP powder was configured with double distilled water.

(2) Experimental grouping

A total of 4 groups, 12 in each group, male and female, a total of 48. They were sham operation group; myocardial infarction model group (MI); BZP high (40 mg/kg) and low dose group (20 mg/kg).

(3) Long-term administration method

Preparation of MI model: preoperative weighing, inhalation anesthesia was performed with 1% by mass of isoflurane, and the supine position was fixed on the rat plate. Percentage of mass 75% alcohol disinfects the skin of the precordial area. Cut the skin in the front area, bluntly separate the muscles, and cut the chest wall in the third and fourth intercostal space. In the myocardial infarction model group, the left anterior border of the left atrial appendage was 3-4 mm, and the left coronary artery (coronary artery) anterior descending branch was ligated with a 6-0 suture. The myocardial dysfunction in the area below the ligation site was successful in ligation; as a sham operation group, The needle passes through the left coronary artery and the descending branch is not ligated. The chest cavity was closed along the third and fourth ribs with a 5-0 line, the chest muscles were sutured layer by layer, and the chest skin was continuously sutured.

Non-invasive ultrasound evaluation of cardiac function: 2 weeks, 4 weeks, 8 weeks, and 12 weeks after MI surgery, the heart function of the mice was measured using an ultrasonic diagnostic apparatus and a 30 MHz high-frequency probe. The mass percentage of isoflurane was 1%. The mice were anesthetized by inhalation and the hair in the anterior region was shaved. The mice were placed on the hot plate in the supine position to keep the body temperature constant. Apply appropriate amount of ultrasonic couplant in the anterior region, use the 30MHz probe to start the paraparusal long axis view on the chest wall of the mouse, and take the left ventricular short axis of the sternum to obtain the M-shaped slice image to measure left ventricular diastolic and systolic Front and rear wall thickness, left ventricular ejection fraction (EF%), etc. A B-section image was obtained to measure left ventricular diastolic and systolic volume. The ultrasound diagnostics set the same parameters for different individuals. For each measurement index of M super, five consecutive cardiac cycles were selected and the average values of EF%, FS%, etc. were obtained. B-ultrasound selects the left ventricular maximum volume and the minimum volume, ie, the left ventricular section of the diastolic and systolic phases. The circumference was drawn along the left inner wall to find FAC% for evaluation of overall cardiac function (see Figure 20).

Experimental result

1. The protective effect of oral administration of BZP on cardiac function in chronic animals after C57BL/6 mice.

The myocardial infarction model (MI) was constructed by ligation of the anterior descending coronary artery. The systolic function of the mice was recorded by small animal ultrasound at 2, 4, 8 and 12 weeks after surgery. The results showed that BZP was orally administered at 20 mg/kg/day. And 40mg/kg/day, continuous administration for 12 weeks, compared with the model group (MI group), can significantly improve the systolic function after myocardial infarction, including: ejection fraction (EF%), percent shrinkage (FS%) , delayed heart failure (Figure 21: *: compared with the sham control group (Sham) corresponding time P <0.05; #: compared with the model control group (MI) FC corresponding time P <0.05).

2. The protective effect of oral administration of BZP on the myocardial infarction area of chronic cerebral infarction in C57BL/6 mice.

The myocardial infarction model (MI) was constructed by ligation of the anterior descending coronary artery. The myocardial infarction area (FAC%) was recorded by small animal ultrasound at 2, 4, 8 and 12 weeks after surgery. The results showed that BZP was orally administered 20 mg. /kg/day and 40mg/kg/day, continuous administration for 12 weeks, compared with the model group (MI group), can significantly improve the myocardial infarction area after myocardial infarction, reduce the degree of myocardial damage, and slightly better than the positive drug. (Fig. 22: *: P<0.05 compared with the time of the sham control group (Sham); #: P<0.05 compared with the corresponding time of the model control group (MI).

3. The protective effect of oral administration of BZP on myocardial remodeling after chronic myocardial infarction in C57BL/6 mice.

The myocardial infarction model (MI) was constructed by ligation of the anterior descending coronary artery. The myocardial remodeling of the mice was recorded by small animal ultrasound at 2, 4, 8 and 12 weeks after surgery (LV Mass and LV Mass corrected). The results showed that BZP oral administration of 20mg/kg/day and 40mg/kg/day, continuous administration for 12 weeks, compared with the model group (MI group), can significantly improve LV Mass and LV Mass corrected after myocardial infarction, reduce myocardial remodeling The occurrence of delayed heart failure, and the effect is better than the positive drug control group. (Fig. 23: *: P<0.05 compared with the time of the sham control group (Sham); #: P<0.05 compared with the corresponding time of the model control group (MI).

Experimental conclusion: Oral administration of BZP can significantly improve cardiac function damage caused by chronic myocardial infarction in C57BL/6 mice, reduce the occurrence of myocardial remodeling, and delay the progression of heart failure.

Example 7: Antihypertensive effect of BZP on SHR rats

(1) Experimental materials

35 SHR rats, weighing 200g-250g, male and female, BZP (synthesized by our laboratory) were prepared with double distilled water; SHR was purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and isosorbide mononitrate was also selected. As a positive control. The non-invasive blood pressure monitor is Kent Scientific Corporation of the United States.

Laboratory Animal Production License No.: SCXK (Beijing) 2012-0001

Laboratory animal quality certificate number: 11400700114418

(2) Experimental method: The rats were repeatedly trained before the test to adapt to the environment of the non-invasive blood pressure tester. The blood pressure values of the rats were measured before administration, and the rats with blood pressure were randomly divided into three groups according to blood pressure and body weight (model control group, positive control group, administration group), and an average of 10 animals in each group. The experiment was administered by intragastric administration. The positive control group was given isosorbide mononitrate 25 mg/kg, BZP was administered 45 mg/kg, and the model control group was given the same volume of physiological saline. The blood pressure was measured three times before administration, and the SHR rats were fully adapted to the environment of the measuring instrument. The tail artery pressure of the animals was measured by a non-invasive blood pressure meter at 20 min, 40 min, and 60 min after the administration. Blood pressure was measured at the same time every day for one week, and the average was taken.

(3) Statistical analysis: Data were expressed as mean ± standard deviation. One-Way ANOVA statistical analysis was performed with SPSS 17.0. P < 0.05 was considered statistically significant.

(4) Experimental results: Isosorbide mononitrate (5mg/kg) can significantly reduce the systolic blood pressure of SHR rats when administered for 40min. BZP 45mg/kg can significantly reduce the systolic blood pressure of SHR rats 40min after intragastric administration. Diastolic blood pressure and mean arterial pressure (see Figures 24, 25, 26).

(5) Experimental conclusion: BZP (45mg/kg) can significantly reduce systolic blood pressure, diastolic blood pressure and mean arterial pressure in SHR rats 30-40min after intragastric administration. The antihypertensive effect is slightly better than isosorbide mononitrate.

Example 8: Effect of BZP on the in vitro vascular ring tension of SD rats

(1) Experimental materials

Thirty SHR rats were used, weighing 200g-250g, male and female, and the positive drug was propranolol. The vasoconstriction was used to confirm the norepinephrine of tartaric acid. The isolated vascular ring device was Chengdu Instrument Factory.

(2) Experimental method:

Preparation of PSS solution: Weigh 280mg of CaCl _{2 and} dissolve it in 100mL of distilled water. Weigh 4.47mg of EDTA, 6962mg of NaCl, 2100mg of NaHCO ₃ , 350mg of KCl, 163.2mg of KH ₂ PO _{4 ,} 14.4mg of MgSO ₄ , add 800mL of distilled water to dissolve, slowly add The above dissolved CaCl ₂ solution was adjusted to a volume of 1000 mL and placed in a refrigerator for use. Add 2178 mg of glucose before use.

Preparation of BZP solution: Weigh 30.4mg of BZP powder, add 10mL of ultrapure water, prepare a mother liquor with a concentration of 1×10 ^-2 mol/L, take a certain amount of mother liquor, and then dilute it to a concentration of 1×10 ^{- 3} mol/L, 1×10 ^-4 mol/L, 1×10 ^-5 mol/L, 1×10 ^-6 mol/L BZP solution.

The normal SD rats were sacrificed by cervical dislocation, the thoracic cavity was opened, and the thoracic aorta was carefully removed. Two self-made vascular ring hooks were used to pass through the vascular lumen, one fixed in the water bath with the PSS solution and the other with the thin wire. Connected to the tension transducer to maintain a water temperature of 37 °C. After the vascular ring is equilibrated, norepinephrine is added to the water bath to make the final concentration in the bath reach 6×10 ^-6 mol/L. The vasoconstriction is observed and the tension is increased. After the vasoconstriction value is stable, the different concentrations are added. The concentration of BZP was observed to observe the change in tension.

(3) Experimental results

BZP has a relaxing effect on the isolated vascular rings of SD rats in the concentration range of 1×10 ^-6 mol/L～1×10 ^-3 mol/L, and the best effect is obtained at the concentration of 1×10 ^-3 mol/L. See Figure 27.

(4) Experimental conclusion

BZP has obvious diastolic effect on the vascular ring, which can significantly reduce the contraction amplitude caused by heavy tartaric acid adrenaline on the vascular ring. The antihypertensive effect of BZP may be related to the role of diastolic blood vessels.

Claims

The use of sodium 5-bromo-2-(α-hydroxypentyl)benzoate in the treatment of cardiovascular diseases, characterized in that it is used as an active ingredient for the preparation of a medicament for treating or ameliorating acute and chronic myocardial ischemia-reperfusion injury in.
The use of sodium 5-bromo-2-(α-hydroxypentyl)benzoate according to claim 1 for the treatment of a medicament for cardiovascular diseases, which is formulated into an oral preparation or an intravenous preparation.
The use of sodium 5-bromo-2-(α-hydroxypentyl)benzoate according to claim 2 for the treatment of a medicament for cardiovascular diseases, characterized in that 5-bromo-2-(α-hydroxypentyl)benzene Sodium formate is administered orally to an acute myocardial ischemia-reperfusion injury at a dose of 12-24 mg/kg.
The use of sodium 5-bromo-2-(α-hydroxypentyl)benzoate according to claim 2 for the treatment of a medicament for cardiovascular diseases, characterized in that 5-bromo-2-(α-hydroxypentyl)benzene Sodium formate is administered intravenously to acute myocardial ischemia-reperfusion injury at a dose of 20-40 mg/kg.
The use of sodium 5-bromo-2-(α-hydroxypentyl)benzoate according to claim 2 for the treatment of a medicament for cardiovascular diseases, characterized in that 5-bromo-2-(α-hydroxypentyl) Sodium benzoate is administered orally in a dose of 10-200 mg/kg/day for delayed myocardial hypertrophy and heart failure after chronic myocardial ischemia-reperfusion injury.