摘要
目的测定14种抗结核药物在巨噬细胞内的抗结核活性,并评价体外巨噬细胞内抗结核活性测定对新药筛选的作用。方法采用结核分枝杆菌标准株H37Rv感染J774.1巨噬细胞,建立体外巨噬细胞感染模型。分别检测利福平、异烟肼、左氧氟沙星、吡嗪酰胺、贝达喹啉、氯苯吩嗪、利奈唑胺、硝基眯唑类药物PA-824、氯苯吩嗪的術生物TBI-166、利奈唑胺的同类药物OTB-658,以及苯并噻嗪类药物PBTZ-169和BTZ-043、TZY-5-84、NTB-3119H作用后的巨噬细胞中的细菌载量,评价抗结核药物的胞内活性;并采用微孔培养显色法(MABA法)测定_上述药物的体外最低抑菌浓度。从中选取利福平、异烟肼、左氧氟沙星、贝达喹啉、氯苯吩嗪、利奈唑胺、TBI-166、PBTZ-169分别作用J774.1巨噬细胞3 h,利用高效液相色谱-质谱/质谱联用技术测定细胞内药物浓度。结果细胞培养基中药物浓度为0.50μg/ml时,异烟肼、PBTZ-169、贝达喹啉、BTZ-043、NTB-3119H、TZY-5-84和PA-824降低巨噬细胞内结核分枝杆菌1.60、1.33、1.31、1.25、1.13、1.01和1.00 lg(CFU/ml)(CFU:菌落形成单位),胞内活性最强的是异烟肼和PBTZ-169,其次是贝达喹啉;但吡嗪酰胺、利奈唑胺、TBI-166和OTB-658仅降低巨噬细胞内结核分枝杆菌0.65、0.55、0.33和0.31 lg(CFU/ml)。经体外90%最低抑菌浓度(MIC90)和胞内活性比较,发现利奈唑胺和OTB-658虽然具有良好的体外抗菌活性(MIC90分别为0.25和0.05μg/ml),但是二者在细胞培养基中浓度为5μg/ml时仅降低巨噬细胞内结核分枝杆菌0.77和0.50 lg(CFU/ml)。在培养基中药物浓度为20μg/ml时,PBTZ-169、贝达喹啉、氯苯吩嗪、利福平、TBI-166、异烟肼、左氧氟沙星和利奈唑胺在巨噬细胞内的浓度为1.42、2.11、4.93.1.65.0.50、0.27.1.13和0.32μg/ml,胞内药物浓度是其MIC90(分别为0.0005、0.03、0.12、0.05、0.04、0.05、0.25、0.25μg/ml)的2840.00、70.33、41.08、33.00、12.50、5.40、4
Objective To determine the activities of 14 drugs against Mycobacterium tuberculosis in macrophages,and to evaluate the effect of detection the anti-tuberculosis activity in macrophages on screening of new drugs.Methods J774.1 macrophages were infected with Mycobacterium tuberculosis standard strain H37Rv to establish an in vitro macrophage infection model.Rifampicin,isoniazid,levofloxacin,pyrazinamide,bedaquiline,clofazimine,linezolid,PA-824,TBI-166,OTB-658,PBTZ-169,BTZ-043,TZY-5-84 and NTB-3119H were detected respectively to evaluate the intracellular activity.The minimal inhibitory concentration(MICg)of the above drugs was determined by MABA method.In addition,rifampicin,.isoniazid,levofloxacin,bedaquiline,clofazimine,linezolid,TBI-166 and PBTZ-169 were selected to treat on J774.1 macrophages for 3 h respectively,and the concentrations of these drugs in the macrophages were determined by high performance liquid chromatography-mass spectrometry/mass spectrometry.Results Isoniazid,PBTZ-169,bedaquiline,BTZ-043,NTB-3119H,TZY-5-84 and PA-824 reduced Mycobacterium tuberculosis in macrophages by 1.60,1.33,1.31,1.25,1.13,1.01 and 1.00lg(CFU/ml)(CFU,colony-forming unit)at a concentration of 0.50μg/ml,isoniazid and PBTZ-169 were the most active in the cell,followed by bedaquiline.However,pyrazinamide,linezolid,TBI-166 and OTB-658 only reduced Mycobacterium tuberculosis in macrophages by 0.65,0.55,0.33 and 0.31 lg(CFU/ml).Comparing the minimal inhibitory concentration(MIC90)with intracellular activity,linezolid and OTB-658 had good antibacterial activity in vitro(MIC90 is 0.25 and 0.05μg/ml respectively),but at the linezolid and OTB-658 concentration of 5μg/ml,they only reduced 0.77 and 0.50 lg(CFU/ml)in macrophages.In addition,at the concentration of 20 ug/ml in cell culture medium,the concentations of PBTZ-169,bedaquiline,clofazimine,rifampicin,TBI-166,isoniazid,levofloxacin and linezolid in macrophages were 1.42,2.11,4.93,1.65,0.50,0.27,1.13 and 0.32μg/ml.The intracellular drug concentrations were 2840.00,70.33,41
作者
陈曦
刘忠泉
王彬
朱慧
付雷
李媛媛
陆宇
Chen Xi;Liu Zhongquan;Wang Bin;Zhu Hui;Fu Lei;Li Yuanyuan;Lu Yu(Beijing Key Laboratory of Drug-resistant Tuberculosis Research,Department of Pharmacology,Beijing Chest Hospital,Capital Medical University,Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China)
出处
《结核病与胸部肿瘤》
2020年第1期1-6,共6页
Tuberculosis and Thoracic Tumor
关键词
抗结核药
巨噬细胞
微生物敏感性试验
色谱法
高压液相
串联质谱法
评价研究
Antubercular agents
Macrophages
Microbial sensitivity tests
Chromatography,high pressure liquid
Tandem mass spectrometry
Evaluation studies
