| China is the birthplace of Chinese medicine, and the culture of Tradition Chinese Medicine has a long history. Compared with other traditional Chinese medicine formulation, Traditional Chinese Medicine Injection (TCMI), a new dosage forms of pharmaceutical preparations, not only retains the characteristics of Traditional Chinese Medicine, but also has fast efficacy as western medicine, easy to use, high bio-utilization, more accurate dose, and the exact characteristics of assured efficacy, thus developed rapidly. Recently years, the incidence of ADR (adverse drug reaction) is generally 5 to 20% for drugs, and there is about 10~41.5% of hospital patients occurred ADR, according to the ADR's statistics by WHO (World Health Organization). As a special material with disease prevention and regulating physiological function, drugs had been done prior to listing non-clinical safety evaluation, clinical trials. Because of the limitations of the non-pre-clinical studies and the limitations of pre-market clinical studies, some adverse reactions with frequency less than 1% and taking longer to occur and delayed could not be found. Coupled with adverse drug reactions is found hysteresis and so on. It is more important and necessity to reevaluation of post-marketing drugs.Puerarin, a kind of flavonoid glycoside extracted from Pueraria legume root of Pueraria lobata, is the main component of Puerarin Injection. As a vasodilator, there is expansion of coronary and cerebral blood vessels, reducing myocardial oxygen consumption, improve microcirculation and anti-platelet aggregation effect. Compared with other treatment drugs for cardiovascular and cerebrovascular diseases, Puerarin Injection has good efficacy, low price advantage, and has a wide range of clinical applications. Sample of hospitals across the country cardio-cerebral vascular diseases drug sales to sort the top 10 drugs, Puerarin ranked third. Total sales in 2003 reached more than 2 billion RMB. However, with the extensive application of Puerarin Injection, adverse reaction reports produced by Puerarin Injection increased gradually. Acute intravascular hemolysis is the most serious adverse reactions. Therefore, intravascular hemolysis caused by Puerarin Injection become a huge obstacle to their use. Although, National Adverse Drug Reaction Monitoring Center Bulletined acute intravascular hemolysis and the SFDA issued several informs of management and usage of Puerarin Injection from January 2003 to 2006. But there is not very clear for the mechanism of intravascular hemolysis.There is gravity of acute intravascular hemolysis. According to the traditional Chinese medicine injection safety re-evaluation of the basic technical requirements and clinical features of adverse reactions, we select acute toxicity tests and hemolysis tests in vitro for safety Re-evaluation of Puerarin Injection. And then on this basis, we had deeply studied the mechanism of hemylisis by vitro test of RBC Toxicology. There is not only the larger value and academic value, but also for further structural transformation of puerarin, reduce toxicity and adverse reactions provide the basis for the clinical use of puerarin help reduce the risk, to ensure that patients medication safety for carring out toxicity studies puerarin LD50 and other related parameters were calculated by Bliss method in acute toxicity test. There are 5 groups between 300-600 mg·kg-1, Single intravenous injection, and 15 mg?mL-1 injection volume. Results: dead animals had prone, low-voltage, ataxia, tremors, tonic seizures, startle reflex, convulsions and other symptoms and ultimately death, time of death were immediately after the injection to within 3 min. The symptoms of surviving animals gradually decreased, and get to normal after 20 min. There were found no specific target organ in all gross anatomy mice. The LD50 of male and female mice were: 415.2 (374.4 ~ 460.4), 439.2 (371.6 ~ 519.1) mg?kg-1., Experimental mice all survived in solvent propylene glycol group. Reduced to 10 mg?mL-1, poisoning symptoms and death were not significantly changed. male, female LD50 was: 466.8 (417.6 ~ 521.9) mg?kg-1, 450.4 (409.7-495.1) mg?kg-1. Puerarin Injection were diluted by using 0.9% NaCl injection and 5% glucose injection. The pH values were less than 4.0. this suggest that: there is no effect between death in acute toxicity and allergic reactions and hemolytic reactions, animal sex, drug delivery independent of volume and clinical use of solvents. Solvent of propylene glycol on Puerarin had no effect on acute toxicity. Delivery speed and solution pH could impact the acute toxicity of Puerarin.In vitro hemolysis test, whole blood of rabbits, beagle dogs and human volunteers were study system, including PI on rabbits, beagle dogs and the human body outside the hemolysis test and PI solvent propylene glycol comparison grid dogs, rabbits and human impact of foreign hemolysis tests. Puerarin Injection was diluted a series of concentration as 0.5,1,2,4, 8, and 16 mg?mL-1 by 0.9% NaCl injection. And then 2% of fresh red blood cell were mixed. The mixture was incubated at 37℃for 6 h. The absorbance value was measured and the condensation rates of red blood cell were observed. Results are as follow. The IC50 of the puerarin interacted with canine and human, and rabbit erythrocytes were 2.79, 3.96, and 5.47 mg?mL-1. The concentrations of puerarin were: 65.36, 81.89, and 135.06 mg?mL-1 when hemolysis rate is at top of three kinds of erythrocytes. The slopes of the response were: 1.45, 1.96 and 0.49. High dose puerarin can produce false red blood cell aggregation of three, while propylene glycol had no such condition.Above all, we conclusions: a certain dose of puerarin makes three kinds of red blood cells significant hemolysis. And hemolytic rate was positively correlated with dose puerarin. It is worth noting: the slope of dose - response curve of human erythrocytes is biggest. Suggesting that: once reached can cause hemolytic concentration, will soon reach the maximum rate of hemolysis. So it would was easy to severe acute intravascular hemolysis in some disease states and the infusion speed quickly. Solvent propylene glycol is not the factor of hemolysis. The hemolysis by Puerarin Injection belong to drug-induced oxidative hemolytic anemia.From the physiological and biochemical point of view, we observed erythrocyte hemolysis fragility, red blood cell oxidation, ATP energy metabolism and other effects by PI. The difference of concentration of NaCl between high-dose and others group is above 0.08% when initiation full hemolysis. Puerarin and propylene glycol increased erythrocyte membrane fluorescence polarization significantly and reduced the degree and micro-viscosity, liquidity increased significantly, depend upon PI dose. Between puerarin group and propylene glycol group, red cell membrane fluorescence polarization and micro-viscosity reduction of the value is relatively large. It was significantly increased SOD, GSH-Px activity in erythrocyte membrane by low dose puerarin. As puerarin dose was raised, SOD, GSH-Px activity decreased. The propylene glycol at low doses, the activity of erythrocyte SOD had no significant effect. Low doses, puerarin injection and propylene glycol groups are to make MDA increased, and MDA content was decreased with increasing doses of the two. SA content of propylene glycol and puerarin injection group were lower than control group. Propylene glycol group was lower than the same dose of PI group. Puerarin can increase Na+-K+-ATPase activity, and not related to dose. The Ca2+-Mg2+-ATPase activity is inhibited at high doses, while low doses increased. Solvent propylene glycol didn't direct impact on Na+-K+-ATPase, Ca2+-Mg2+-ATPase activity. High dose puerarin make significant changes morphology of red blood cell. With puerarin injection dose increased, shape of red blood cells become irregular, spine-like of membrane surface, spherical protrusions increased rupture and aggregation, adhesion gradually increased. The propylene glycol (10%) control group found no obvious abnormalities in red blood cell morphology and erythrocyte debris. Puerarin can reduce the expression of scaffolding proteins and some protein bands abnormal increase in the expression.Based on the above findings, we draw some conclusions as follow: Solvent propylene glycol, delivery volume and animal sex puerarin had no effect on acute toxicity. The solution pH value and the delivery rate may affect the acute toxicity of puerarin injection. Puerarin Injection drug-induced in vitro belong to drug-induced oxidative hemolytic anemia. Solvent propylene glycol had no effect on hemolysis puerarin. When body is in pathological conditions or rapid administration, it may likely to cause acute intravascular hemolysis and other adverse reactions. Puerarin could interact with red cell membrane proteins or membrane, chang the membrane structure and status, and increase the osmotic fragility and fluidity of red cell membrane in hypotonic solution by inhibiting the SOD, GSH-Px, Ca2+-Mg2+-ATPase activity and interfering SA, MDA metabolism. In the end, it could cause membrane proteins absence or degradation, in particular matrix proteins. And so on, there are a lot of ways by which hemolysis.
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