1.Toxicity And Compatibility With Total Ginsenoside Study Of Protoveratrine A 2.Comparative Long-term Toxicity Study Of Nesiritide With Recombinant Human B-type Natriuretic Peptide On Cynomolgus Monkey

Part One: Toxicity and Compatibility with Total Ginsenoside Study of Protoveratrine AObjective: To conduct the toxicity and compatibility study on protoveratrine A, to determine the toxic target organ of protoveratrine A and to explore its compatible relationship with ginsenoside.Methods: 1. Acute toxicity study: Thirty-two Wister rats were involved in this test, and 20.0 mg/kg(all found killed),10.0 mg/kg,5.0 mg/kg,2.5 mg/kg (no single killed) four dosage groups were set to observe the toxic reaction symptoms and the mortalities after the administration, and calculate the LD50 by Bliss method according to the mortalities. Eight Beagles were separated into four groups, each group was given the dosage of Protoveratrine A by 0.5 mg/kg (ig), 0.25 mg/kg (ig), 0.2 mg/kg (iv), 0.002 mg/kg (iv) respectively to observe the toxic reaction symptoms.2. Repeat dose toxicity study: 36 Wister rats were divided into three groups which were blank control group, 1 mg/kg group,0.5 mg/kg group. Each rat was bred in the metabolism cage. Each one is treated with drug by intragastric injection for consecutive fourteen days. Each rat was weighed every week and the urinary volume was also measured every other day. The rats were executed fourteen days later, the blood sample was collected to analyze biochemical and blood indexes. The rats were then dissected to obtain the heart, liver, spleen, lung, kidney, brain, stomach and other organs, which are then fixed for pathological observation.3. The toxicity observation of cardiovascular and respiratory system: Forty wister rats were divided into four groups, which are blank control group (vehicle), 4 mg/kg Protoveratrine A group, 2 mg/kg Protoveratrine A group, and 1mg/kg Protoveratrine A group, the rats were anesthetized later and the carotid artery catheterization was performed, and the ECG, blood pressure, respiratory change were recorded in the duration of three hours after the administration.4. The study on the antihypertensive mechanism: Lateral ventricle administration of 2.5μg/kg and tail vein administration of 0.05 mg/kg drug were applied on healthy Wistar rats, to observe its hypotensive effect. The phentolamine, atropine were given initially to block the typeαreceptors and the type M receptors, then the Protoveratrine A was given to observe the antihypertension effect. 5. The compatibility toxicity between Protoveratrine A and total ginsenoside: Fifty Wister rats were divided into five groups (ten for each group) which were blank control group (vehicle) , ginsenoside group (TG10mg/kg), Protoveratrine A group (PA 2 mg/kg), the ratio of compatibility group was TG:PA=5:1 (TG 10 mg/kg: PA 2 mg/kg) and TG:PA=5:2 (TG 5 mg/kg: PA 2 mg/kg), respectively. The rats were anesthetized later and the carotid artery catheterization was conducted and the ECG, blood pressure, respiratory change were recorded after the drug administration for three hours.Results: The LD50 of Protoveratrine A for rats by intragastric injection was 5.1 mg/kg. Repeat dose administration of Protoveratrine A could lead to body weight lose,hydrouria,the desent of HCT and %LYMPH, neutrophilic leukocytosis ,etc.And then increase the AST,ALP,CPK,LDH. The single dose administration of Protoveratrine A could significantly reduce blood pressure, slower the heart rate and breathing, and even lead to arrhythmia. The minor dose administration of Protoveratrine A by lateral ventricle injection could cause the decrease of rat blood pressure; the nadir appears in approximately three minutes which was delayed compared with intravenous administration (the nadir usually appears in one to two minutes after intravenous administration). The influences on the blood pressure, heart rate, and breathe frequency of the total ginsenoside and Protoveratrine A group which has the compatibility ratio of 5:2 is less than the one of single Protoveratrine A administration group (P<0.05), while the influences on the blood pressure, heart rate, and breathe frequency of the total ginsenoside and Protoveratrine A group which has the compatibility ratio of 5:1 is greater than the one of single Protoveratrine A administration group (P<0.05).Conclusion: The small dose of Protoveratrine A can cause relatively severe toxic reaction symptoms on animals. On rats,some toxic reactions could be observed about 10 min after the administration such as decrease of spontaneous movements, horripilation, loose stools,etc. As Beagle, Persistent vomiting, loose stools, weakness and limp could be observed. Repeat dose on rats,protoveratrine A could do something on diuresis, do harm on heart, liver, kidney by some extent. The single dose administration of Protoveratrine A could significantly reduce blood pressure, slower the heart rate and breathing, and even lead to arrhythmia. There is dose-effect relationship on the influence of blood pressure. The central nerve system and peripheral nerve system are all involved in the antihypertensive mechanism. The compatibility toxicity of Protoveratrine A and ginsenoside is related to their compatibility ratio.Part Two: Comparative long-term toxicity study of Nesiritide with Recombinant human B-type natriuretic peptide on Cynomolgus monkeyObjective: To observe the long-term toxicity on Cynomolgus monkey administrated with chemical synthetic Nesiritide by intravenous dripping for fourteen days, to compare its toxicity with the same earlier marked biotechnology drug Recombinant human B-type natriuretic peptide(rhBNP).Methods: Thirty healthy Cynomolgus monkey were divided into five groups by weight which were blank control group, positive control group, low-dosage group, medial-dosage group and high-dosage group, respectively. Each group had six Cynomolgus monkeys, half are male and the other half are female. The dosage given to low-dosage, medial-dosage group and high-dosage group was 432, 1440, 4320μg/kg, respectively. The dosage volume was 18 ml/kg and the dosage concentration was 24, 80, 240μg /ml respectively. The launched Natriuretic Peptides was employed here as positive control whose dose was equivalent to the one of medial-dosage group, the dripping velocity was 18 ml/kg/hr. The Cynomolgus monkey was treated with drug for consecutively fourteen days and half of the monkeys were then executed right after the last drug administration to perform the pathological anatomy. The drug administration of the rest of the monkeys was stopped and they were observed for another fourteen days. Then the pathological anatomy was conducted. The observation symptoms and examining indexes include body weight, appetite and other routine symptoms and indexes such as blood pressure, electrocardiogram, hematology indexes e.g. red blood cells, lactate dehydrogenase (LDH) and other blood biochemical parameters, such as complement C3, urine indexes such as urine volume, bone marrow check, pathological examination, histological examination.Results:The LDH value of both d7 and d 14 were higher than the one of d0 in high-dosage, medial-dosage and positive control group, respectively, in the meantime they were also higher than the ones in low-dosage group and control group. The differences were not found in their recovery phase. The value of creatine phosphokinase (CPK) in the high-dosage group of d 7 was higher than the one in the blank control group. The d7 value was higher than d0 value in positive control group, also higher than the d0 values in blank control group, low-dosage group, medial-dosage group and high-dosage group, still, the differences were not found in their recovery phase. The renin and aldosterone indexes of individual animal in high-dose group decreased to a certain degree, but it had no statistical significance, these indexes rose to the same levels previous to drug administration. At d1, d7, d14 after drug administration, compared with the arterial pressure measured in blank control group or pre-administration, the average arterial pressure of dosage group after drug administration decline at different levels, the average arterial pressure would generally go back to the normal level in 120 to150 minutes. And no abnormally changes were observed in the rest of the general symptoms, electrocardiogram, hematology indexes, immunology indexes, urine indexes, bone marrow examination, pathological examination and histological examination.Conclusion: after administration of Nesiritide to Cynomolgus monkey by intravenous dripping for fourteen days, decrease in blood pressure, rise in lactate dehydrogenase and creatine phosphokinase etc reactions in animals after administration were observed in high-dose group, moderate-dose group and positive control group, and all these reaction were reversible. For Cynomolgus monkey, the safe dose of v.d. Nesiritide is 432μg/kg, the toxic dose is 1440μg/kg and the main toxicity target organ lie in cardiovascular system. The positive control drug (rhBNP) has relatively similar pharmacological and toxicological effects with those of the test drug, suggesting there is no significant toxicological difference between synthetic products and recombination products.