| The cardiovascular system is one of the most important systems in human beings, the vital physiological role of which is mataining the homeostasis and controlled the movement of blood through the thonsands of miles capillaries that permeate every tissue and reach every cell in the body. With the rapid process of industrialization, cardiovascular diseases, principally coronary heart disease, but also hypertension, stroke, congestive heart disease, and peripheral vascular diseases are the leading cause of morbidity and mortality in the western world, coronary heart disease is recognized as the leading cause of death. In china, the mortality of cardiovascular diseases is in the second position which followed the cancer, ischemic heart diseases, which can lead to myocardial infarction and heart failure, are a leading cause of morbidity and mortality in cardiovascular diseases. Therefore, the development of anti-myocardial ischemia drugs have became one of the hottest research areas in the development of cardiovascular drugs. To establish the appropriate animal models, on one hand, this can provide important information for us to understand the pathogenesis and pathophysiology changes of myocardial ischemia diseases. The most important is that which could provide effective tooles for screening therapeutic compounds in drug discovery and development and promoting new drugs for the treatment of myocardial ischemia diseases. Recently, there are two methods for the establishment of myocardial ischemia animal models, one approach is the using of the drugs which can increase the myocardial oxygen consumption or reduce the blood supply of the heart resulting in myocardial ischemia. Another way is to ligate the coronary artery of the experimental animal through the thoracic surgery. In the first part of this study, we use the pituitary induced acute myocardial ischemia model in rat to evaluate the anti-myocardial ischemia effects of CVB-D and CVB-D1, and use the rat myocardial ischemia reperfusion injury model evaluate the effects of CVB-D on myocardial ischemia reperfusion injury.As important as the effectiveness of a compound, the safety is an indispensable factor that makes a compound become a drug in the process of drug development. Whereas the ultimate species of interest is most often human beings,animal models provide critical information about the safety and efficacy of a new chemical entity prior to exposure of the first human subjects. Pharmacology studies may be conducted in either conscious or anesthetized animal models,traditionally,our knowledge of cardiovascular toxicology and pharmacology has been derived from experimentation on anesthetized animals. The use of general anesthesia provides the opportunity to conduct invasive procedures thereby gaining direct access to the cardiovascular system, and avoiding pain and distress to the animal. In addition, relatively stable baseline conditions can be eatablished during the period of of anesthesia. Conscious animal models have become more popular recently due to the promulgation of a series of safety pharmacology guidelines and the significant improvements in techniques and instrumentation devices. Compared with the anesthetized or restrained animal models, among the advantages of conducting cardiovascular experiments in conscious animals is the ability to monitor the cardiovascular parameters in long periods, the avoidance distress of handle and avoidance of drug interactions with anesthetic agents, and the more efficient use of experimental animals, and the accordance with the 3R principles. Also in 2000 and 2005, the guideline of S7A and S7B that promulgation by ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) recommended to use conscious animals in pharmaceutical studies. In the second part of the study, we establish an animal model that can monitor the cardiovascular parameters and body temperature in conscious and freely moving telemetry rat, and use the model evaluate the cardiovascular safety of AF114 injection.1. The use of myocardial ischemia rat models to evaluate the anti-myocardial ischemia effects of CVB-DIn thist part, we use the pituitary induced acute rat myocardial ischemia model and myocardial ischemia reperfusion injury model to evaluate the anti-myocardial ischemia effects of CVB-D and CVB-D1, and the effects of CVB-D on myocardial ischemia reperfusion injury of rats. 24 SD rats were randomly divided into 4 groups: contol group(saline+saline), model group(0.5u/kg pituitary+saline), CVB-D(0.5u/kg pituitary+1.6mg/kg CVB-D) group and CVB-D1 (0.5u/kg pituitary+1.6mg/kg CVB-D1) group,the mean blood pressure,heart rate and the hight of T wave on ECG were recorded during the experimental. The results showed that the CVB-D and CVB-D1 has no effect on the mean blood pressure which was increased by the pituitary; Both the CVB-D and CVB-D1 can increaseed the heart rate which was decreased by the pituitary; Compared with control group,CVB-D can significantly reduced the T wave flat in 5min, 7min, 9min, and 11min after ip pituitary (p<0.05); CVB-D1 also can reduced the T wave flat in 7min, 9min, and 11min after ip pituitary (p<0.05). 24 anesthetized rats were randomly divided into 4 groups: model group, CVB-D 1mg/kg group, CVB-D 2mg/kg group, and CVB-D 4mg/kg groups. All the rats were once treated with Cyclovirobuxine-D (CVB-D) or saline 30min before ischemia, and then subjected to ischemia for 30min followed by reperfusion for 2h. The MDA, infarct size, reperfusion arrhythmia, and hemodynamic data were measured during the experimental. The results showed that compared with the control group, CVB-D 2mg/kg and CVB-D 4mg/kg can significantly increase the mean blood pressure in the 2h of the reperfusion period (p<0.05); CVB-D 2mg/kg can significantly decrease the heart rate in the 2h of the reperfusion period (p<0.05); CVB-D 4mg/kg can significantly decrease the heart rate in the 1h and 2h of the reperfusion period (p<0.05); CVB-D 4mg/kg can significantly increased the + dp / dtmax in the 2h of the reperfusion period (p<0.05),which suggested that the CVB-D can enhancing the contractility of myocardium; Both CVB-D 2mg/kg and 4mg/kg can significantly decrease the - dp / dtmax in the 2h of the reperfusion period (p<0.05),which suggested that the CVB-D can improvement the cardiac diastolic function; CVB-D4mg/kg can significantly increased the LVDP in the 2h of the reperfusion period (p<0.05); The pressure-rate index of CVB-D 4mg/kg group in 30min, 1h, 2h of reperfusion periods were significantly lower than the control group (p<0.05), which suggest that in this dose, CVB-D can reduce oxygen demand of the myocardium; CVB-D 2mg/kg and CVB-D 4mg/kg can significantly reduce the arrhythmia score compared with control group(p<0.05); CVB -D 4mg/kg can significantly decrease the MDA levels in serum (p<0.05); Both CVB-D 2mg/kg and CVB-D 4mg/kg were Significantly decreased the myocardial infarct size compared with control group (p<0.05).Based on the results of this study, the following conclusions could be made under the present experimental condition:â‘ We successfully established the mice endure oxygen-deficiency nodel, the pituitary induced acute rat myocardial ischemia model and the rat myocardial ischemia reperfusion injury model;â‘¡Both CVB-D and CVB-D1 can prolonged the survival time of the anoxic mice;â‘¢CVB-D can prevent the myocardium from ischemia reperfusion injury through decreases the serum MDA level,decreases the myocardial infarct size,reduces the incidence of ischemia and reperfusion arrhythmia frequency and improves cardiac function .2. The establishment of telemetry conscious rat model and use the model to evaluates the cardiovascular safety of AF114 injectionIn thist part, we established an animal model that can long-term monitoring the cardiovascular parameters and body temperature in conscious and freely moving telemetry rat;And utilize this model to evaluate the effects of AF114 injection on the cardiovascular parameters and body temperature. Make a 4-6cm midline abdominal incision, exposure the abdominal artery, insert the pressure catheter of the transmitter into the artery, thoroughly dry the puncture site and surrounding tissue, apply one drop of tissue adhesive to the puncture site to prevent of bleeding. Tunnel the ECG leads subcutaneously from the abdominal incision, for proper polarity of the QRS complex in a leadâ…¡configuration, place the negative lead in the area of the right shoulder and the positive lead to the left of the xyphoid space and caudal to the rib cage. A catheter was inserted in the jugular vein for administration of saline, AF114 injection or collect blood samples. Monitor the blood pressure, heart rate, body temperature, and food intake 16d assess the recovery of the rats after the surgery. The result suggests that after 4-5d of the surgery, the telemetry rats recoveried to the normal condition. we use pituitary as a positive drug to test the reactivity of the telemetry rat model, so after a week recovery of the telemetry rat,9 animals were divided into pituitary group(n=6) and saline group(n=3), the animals were intraperitoneally 1U/kg pituitrin or equal volume of saline, administered volume is 1ml/kg weight. Test the reactivity of the cardiovascular system of the telemetry rat. Then we utilize this model evaluation the effect of AF114 injection on the cardiovascular parameters and body temperature. 20 telemetry rats divided into 4 groups, AF114 injection 9 ml / kg group, 4.5 ml / kg group, 2.25ml/kg group and saline 9ml/kg group,the AF114 injection and the saline were administrated through the jugular vein catheter. The blood pressure, heart rate, and body temperature were monitored through the experimental. At the end of the experiment, 0.2ml blood sample was collected through the jugular vein catheter; assess the platelet count and the red blood cell count. The results showed that 29 of the 32 operated rats recovery well,BP, ECG and body temperature were monitored in the recovered rats, the success rate of the surgery was 91%. After ip pituitary 1U/kg BW, the mean blood pressure is increased; ejection time of the heart is prolonged; The TTPK is also prolonged and the body temperature is decreased and heart rate is decreased, it is showed that this telemetry rat model is sensitive to the positive drug. After IV AF114 injection, the mean blood pressure, heart rate and body temperature were increased. There was no effect on the platelet count and red blood cell count.Based on the results of this study, the following conclusions could be made under the present experimental condition:â‘ We successfully built an animal model that can monitor the cardiovascular parameters and body temperature in conscious and freely moving telemetry rat, the recovery period is 7d and the telemetry rat model .â‘¡The AF114 injection can increase of the blood pressure, heart rate and body temperature of the conscious rat is sensitive to pituitary. |
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