Studies On Cardiotoxicity Of Bufalin And Its Mechanisms

Venenum Bufonis（VB） is a traditional Chinese medicine,obtained from the parotid venom glands and skin of the toad,which has been used in the clinics for many years in China.It has been reported that Venenum Bufonis and its preparations induce adverse drug reaction/adverse drug event（ADR/ADE）in the cardiovascular system（e.g.arrhythmia）.therefore the cardiotoxicity of VB has clearly limits its clinical applications.Bufalin is one of the main pharmacological and toxicological components of VB and many traditional Chinese medicine preparations,such as cinobufotalin injection.Hence,an in vitro investigation of its toxicological mechanism would bring insights into the development of new Chinese medicine with VB and the treatment of its related clinical adverse reactions.Cinobufotalin injection is a traditional Chinese medicine preparation on market in China.It is an intravenous injection by extracting dried skin of the toad with sterile hot water.There have been clinical reports of cardiac adverse drug reaction to cinobufotalin injection.The aim of this study is to use modern toxicology techniques to systematically study the cardiotoxic mechanism and toxic manifestations of cinobufacin injection,and to explore the preventive effect of phenytoin on cardiotoxicity caused by cinobufacin injection.This study is divided into three parts:The first part:we investigated the effect of bufalin on cardiomyocyte pulses(0.003-0.1μmol·L^-1),action potential(0.03-0.3μmol·L^-1),and seven human ion channels and human sodium-calcium exchanger current(0.01-100μmol·L^-1)using human induced pluripotent stem cells-derived cardiomyocytes（hi PSC-CMs）,HEK293 cells and CHO cells combined with an impedance-based bioanalytical method and patch clamp recordings.The second part:with 4×4 Latin square design,we investigated the effect of different dosage cinobufotalin injection on indicators of myocardial injury and electrocardiogram（ECG）parameters in conscious Beagle dogs with telemetry system.Furthermore,we explored the cardiotoxicity-antagonistic effect of phenytoin sodium in conscious Beagle dogs following single intravenous injection of 3 g·kg^-1 cinobufotalin injection+cardiotoxicity antidote（phenytoin sodium）10 mg kg^-1 in treatment phase V.The third part:building upon the results from the second part,we assessed the cardiotoxicity of cinobufotalin injection in Sprague Dawley（SD）rats.Three groups of SD rats,each consisting of 5 males and 5 females,were administered with cinobufotalin injection at 0 g·kg^-1（control group）,1,and 5 g·kg^-1 via intravenous injection for two weeks.The dose volume was 10 m L·kg^-1 for all dose levels.Since no remarkable cardiotoxicity was detected,an additional experiment,when administered to Sprague Dawley（SD）rats via intravenous infusion for one weeks,was conducted.Two groups of SD rats,each consisting of 5 males and 5 females,were administered with cinobufotalin injection at 0,and 10 g·kg^-1.The dose volume was 20 m L·kg^-1 for all dose levels.The following parameters and endpoints were assessed:morbidity and mortality,clinical observations,body weight,indicators of myocardial injury,organ weights,gross and microscopic pathologyThe major results from our study are:In the in vitro studies,we discovered a biphasic effect of bufalin on the contractility in hi PSC-CMs.In the early stage,the administration of bufalin strengthened myocardial contractility,accelerated conduction,and increased beating rate,whereas in the late stage,bufalin weakened myocardial contractility,abolished conduction,and ceased beating rate.Bufalin decreased the action potential duration（Action potential duration at 30%,50%and 90%repolarization）,and maximal depolarization rate of hi PSC-CMs.Spontaneous beating rates of hi PSC-CMs were markedly increased at 0.03μmol·L^-1,while were weakened at 0.3μmol·L^-1 after application.Bufalin blocks I_Nav1.5 in a concentration-dependent manner with half maximal inhibitory concentration of 74.5μmol·L^-1.Bufalin increased the late sodium current and Na⁺-Ca²⁺exchange current with a concentration for 50%of maximal effect of 2.48μmol·L^-1 and 66.06μmol·L^-1.Whereas,bufalin showed no significant effects on other cardiac ion channels.In the Beagle dogs study,the cinobufotalin injection-related changes were observed in clinical observations（rapid breathing pattern）,indicators of myocardial injury（increased cardiac troponin I,creatine kinase isoenzymes,and aspartate aminotransferase）,and electrocardiogram graphics（ventricular arrhythmia）at 3 g·kg^-1concentration in treatment phases I-IV.The cardiotoxicity of cinobufotalin injection was attenuated by sodium phenytoin in treatment phase V.In the SD rats studies,we did not observe any cinobufotalin-related deaths.In the 1g·kg^-1 group,no test article-related changes in clinical observation,organ weight,macroscopic and microscopic pathological findings were noted.The cinobufotalin injection-related changes were only observed in 5 g·kg^-1 group（male）and 10 g·kg^-1group（male and female）,with signs of myocardial injury.Cardiac troponin I increased at 1.5 and/or 3 hours after administration,and nearly recovered at 24 hours)The primary conclusions can be drawn as below:In the in vitro studies,the inhibition of I_Nav1.5,the enhancement of Na-late and Na⁺-Ca²⁺exchanger current is one of the main mechanisms for cardiotoxicity of bufalin.In the in vivo studies,the results confirms the cardiotoxicity of cinobufotalin injection,.This study might bring information about the monitoring time points and cardiotoxicity parameters in clinical practices,and shed light on the treatment of cardiovascular adverse reactions.