新药心脏毒性体外检测方法的建立

Establishment of an in vitro detection method for new cardiotoxicity drugs

目的:建立适合于药物心脏毒性评价的人心肌细胞模型,并采用此模型评价新药中化合物潜在的心脏毒性。方法:选用浓度为0.33、1、3μmol/mL的他莫昔芬(Tamoxifen)和浓度为0.11、0.33、1μmol/mL的氟哌利多(Droperidol)分别与人心肌细胞孵育48 h。给药后1、2、3、6、9、12、18、24、30、36、42和48 h采用实时细胞分析法和高内涵细胞成像技术监测心肌细胞搏动的频率、振幅、规律性及线粒体膜电位等指标在给药前后的变化,建立人心肌细胞体外心脏毒性评价模型。应用此模型评价化合物他莫昔芬和氟哌利多的心脏毒性。结果:他莫昔芬和氟哌利多与人心肌细胞共同孵育后,3μmol/mL的他莫昔芬在处理3 h时使人源性心肌细胞场电位时程(FPD)和人源性心肌细胞收缩频率降为0,18 h后恢复,其他浓度与对照组基本一致;心肌细胞FPD和心肌细胞收缩频率对氟哌利多均具有浓度依赖性,浓度越高FPD(Max)数值越大而收缩频率数值越小,24 h后进行全换液,换液后各浓度处理的心肌细胞均可恢复。结论:利用人心肌细胞可以建立一种可行的体外心脏毒性评价模型,结合实时细胞分析法以及高内涵细胞成像技术可评价药物的心脏毒性。

OBJECTIVE:To develop a human cardiomyocyte model for cardiotoxicity drug evaluation,and to evaluate its potential for screening new cardiotoxicity drugs.METHODS:Human cardiomyocytes in cultures were treated with tamoxifen(0.33,1,3μmol/mL)and haloperidol(0.11,0.33,1μmol/mL)for 48 h.At 1,2,3,6,9,12,18,24,30,36,42 and 48 h after the initiation of treatments real-time cell analyses and high-content cell imaging technology were used to monitor changes of cardiac myocyte pulse frequency,amplitude,regularity and mitochondrial membrane potential before and after administration.The data were used to evaluate the usefulness of this model as an in vitro cardiotoxicity assay.RESULTS:After tamoxifen and haloperidol were co-incubated with human cardiomyocytes,the FPD and systolic frequencies reduced to 0 after 3 h treatment with 3μmol/mL tamoxifen but recovered after 18 h treatment.Results from the other treatment concentrations were similar to those in the control group.Both FPD and systolic frequency of cardiomyocytes were concentration-dependent on fluperidol.The higher the concentrations,the higher were the values of FPD(Max),and the smaller were the values of systolic frequency.After 24 h,complete fluid exchange was conducted and all cardiomyocytes treated with different concentrations recovered.CONCLUSION:Our data suggest that human cardiomyocytes can be used to establish a feasible model for evaluating cardiotoxicity in vitro.In addition,real-time cell analysis and high-content cell imaging can be used to evaluate cardiotoxicity of drugs.