14种抗结核药物在巨噬细胞内的抗结核活性评价

Evaluation of antituberculosis activities of 14 antituberculosis drugs in macrophage

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、硝基咪唑类药物PA-824、氯苯吩嗪的衍生物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3h,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1.00lg(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0.31lg(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0.50lg(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、4.52和1.28倍。结论各药物进入巨噬细胞的能力并不相同,巨噬细胞内抗菌活性的差异提示细胞内药物浓度及与MIC90的关系是细胞内活性的决定因素。

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 (MIC90) 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 1.00 lg (CFU/ml)(CFU, colony-forming unit) at a concentration of 0.50 μg/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 0.05 μg/ml respectively), but at the linezolid and OTB-658 concentration of 5 μg/ml, they only reduced 0.77 and 0.50 lg (CFU/ml) in macrophages. In addition, at the concentration of 20 μg/ml in cell culture medium, the concentrations 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 0.32 μg/ml. The intracellular drug concentrations were 2840.00, 70.33, 41.08, 33.00, 12.50, 5.40, 4.52, and 1.28 times that of its MIC90 (0.0005, 0.03, 0.12, 0.05, 0.04, 0.05, 0.25, 0.25 μg/ml, respectively).Conclusion The ability of each drug to enter macrophages is not the same. The difference of antibacterial activity in macrophages suggests that the concentration of drugs in cells and the relationship with MIC90 are the determining factors of intracellular activity.