Abstract:

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.

Key words: Antitubercular agents, Macrophages, Microbial sensitivity tests, Chromatography, high pressure liquid, Tandem mass spectrometry, Evaluation studies