吡法齐明与贝达喹啉协同作用及机制的初步研究

doi:10.19982/j.issn.1000-6621.20220007

摘要/Abstract

摘要： 目的评价吡法齐明(pyrifazimine,TBI-166)与贝达喹啉(bedaquiline,Bdq)、莫西沙星(moxifloxacin,Mfx)、德拉马尼(delamanid,Dlm)、SQ109及PBTZ169的两药组合对结核分枝杆菌(MTB)临床分离株的相互作用,并初步探讨TBI-166与Bdq的协同作用机制,为TBI-166联合用药提供依据。方法选取MTB标准株H37Rv(ATCC 27294)、2株药物敏感型MTB临床分离株(菌株编号:30031和30091),以及1株前广泛耐药结核分枝杆菌临床分离株(菌株编号:13385),应用棋盘法测定TBI-166与Bdq、Mfx、Dlm、SQ109、PBTZ169两药组合对临床分离菌株的相互作用;应用时间杀菌曲线法评估具有协同作用的两药组合的抗菌活性;采用细菌活性氧(ROS)检测试剂盒及三磷酸腺苷(ATP)检测试剂盒分别测定不同浓度的TBI-166和Bdq处理H37Rv标准株24h后菌体内的ROS及ATP水平的变化。结果与Bdq联用后,TBI-166对MTB临床分离株30031、30091及13385的最低抑菌浓度(minimum inhibitory concentration,MIC)分别为0.08、0.05、0.08μg/ml,均降至TBI-166单药MIC值的1/2。TBI-166与Bdq的两药组合对菌株30031、30091及13385的分级抑菌浓度指数(fractional inhibitory concentration index,FICI)值分别为1.00、0.75、1.00。时间杀菌曲线法结果显示,TBI-166与Bdq的两药组合与菌株30031、30091及13385作用14d后与TBI-166单药应用时相比活菌量分别减少1.59log₁₀CFU/ml、1.27log₁₀CFU/ml、1.70log₁₀CFU/ml。0.4μg/ml TBI-166与0.3μg/ml Bdq联用作用于H37Rv标准株24h后菌体内ATP含量(相对光单位值为115160.67±19129.79)与0.3μg/ml Bdq(相对光单位值为208599.20±24078.74)相比,明显下降,差异有统计学意义(F=75.109,P=0.013);而ROS含量(荧光值为21014.33±1189.19)与0.3μg/ml Bdq(荧光值为13715.00±907.93)相比明显升高,差异有统计学意义(F=115.403,P<0.001)。结论 TBI-166与Bdq联用具有协同杀菌活性,并且其协同杀菌机制可能与ROS进一步累积及ATP合成受阻有关。

关键词: 抗结核药, 药物相互作用, 分枝杆菌, 结核, 吡法齐明, 贝达喹啉

Abstract: Objective: To evaluate the interaction between pyrifazimine (TBI-166) and bedaquiline (Bdq), moxifloxacin (Mfx), delamanid (Dlm), SQ109 and PBTZ169 on clinical isolates of Mycobacterium tuberculosis (MTB), and primarily explore the synergistic mechanism of TBI-166 and Bdq, so as to provide basis for TBI-166 combination therapy.Methods: MTB standard strain H37Rv (ATCC 27294), two drug-sensitive MTB clinical isolates (strain numbers: 30031 and 30091), and one clinical isolate of pre-extensively drug-resistant Mycobacterium tuberculosis (strain number: 13385) were selected. Checkerboard method was used to determine the interaction between TBI-166 and Bdq, Mfx, Dlm, SQ109, and PBTZ169 on MTB clinical isolates clinical isolates. Time sterilization curve method was used to evaluate the antibacterial activity of the synergistic of the two drug combinations. Bacterial reactive oxygen species (ROS) detection kit and adenosine triphosphate (ATP) detection kit were used to determine the ROS and ATP levels in H37Rv standard strain 24 hours after being treated with different concentrations of TBI-166 and Bdq.Results: After combining with Bdq, the minimum inhibitory concentration (MIC) of TBI-166 on MTB clinical isolates No. 30031, 30091 and 13385 were 0.08, 0.05 and 0.08 μg/ml, respectively, all of which decreased to 1/2 of the MIC values of TBI-166 alone. The fractional inhibitory concentration index (FICI) values of the two-drug combination of TBI-166 and Bdq on strains No. 30031, 30091 and 13385 were 1.00, 0.75 and 1.00, respectively. The time sterilization experiment showed that compared with TBI-166 alone, after being treated with TBI-166 and Bdq for 14 days, the viable bacteria of strains No. 30031, 30091 and 13385 decreased by 1.59log₁₀ CFU/ml, 1.27log₁₀ CFU/ml and 1.70log₁₀ CFU/ml, respectively. Compared with the 0.3 μg/ml Bdq group, the ATP content of H37Rv standard strain 24 h after being treated with 0.4 μg/ml TBI-166 in combination with 0.3 μg/ml Bdq had a significant decrease (relative light unit value, 115160.67±19129.79 vs. 208599.20±24078.74; F=75.109, P=0.013), while the ROS content increased significantly (the fluorescence value, 21014.33±1189.19 vs. 13715.00±907.93; F=115.403, P<0.001).Conclusion: The combination of TBI-166 and Bdq had synergistic bactericidal activity, and the mechanism of synergistic bacteria might be related to the augmenting accumulation of ROS and the block of ATP synthesis.

Key words: Antitubercular agents, Drug interactions, Mycobacterium tuberculosis, Pyrifazimine, Bedaquiline

中图分类号:

R969.2

刘海婷, 李东硕, 张蕾, 王宁, 王彬, 丁杨明, 姚蓉, 陆宇. 吡法齐明与贝达喹啉协同作用及机制的初步研究[J]. 中国防痨杂志, 2022, 44(7): 646-653. doi: 10.19982/j.issn.1000-6621.20220007

LIU Hai-ting, LI Dong-shuo, ZHANG Lei, WANG Ning, WANG Bin, DING Yang-ming, YAO Rong, LU Yu. A preliminary study on the synergy and mechanism of pyrifazimine and bedaquiline[J]. Chinese Journal of Antituberculosis, 2022, 44(7): 646-653. doi: 10.19982/j.issn.1000-6621.20220007

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参考文献 13

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药品	H37Rv	菌株30031	菌株30091	菌株13385
异烟肼	0.040	0.060	0.052	>10.000
利福平	0.030	0.023	0.016	2.464
吡法齐明	0.08	0.16	0.10	0.16
贝达喹啉	0.06	0.12	0.06	0.06
莫西沙星	0.15	0.08	0.08	2.50
SQ109	0.50	0.45	0.54	0.50
PBTZ169	0.0002	0.0004	0.0004	0.0004
德拉马尼	0.002	0.020	0.010	0.020

药物组合	组合MIC(μg/ml)			组合FICI
药物组合	菌株30031	菌株30091	菌株13385	菌株30031	菌株30091	菌株13385
吡法齐明/贝达喹啉	0.08/0.060	0.05/0.015	0.08/0.030	1.00	0.75	1.00
吡法齐明/莫西沙星	0.005/0.08	0.003/0.08	0.040/1.25	1.03	1.06	0.75
吡法齐明/SQ109	0.005/0.45	0.025/0.27	0.040/0.25	1.03	0.75	0.75
吡法齐明/PBTZ169	0.080/0.0001	0.025/0.0002	0.080/0.0002	0.75	0.75	1.00
吡法齐明/德拉马尼	0.04/0.0100	0.10/0.0003	0.08/0.0100	0.75	1.03	1.00

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