新型先导化合物E体外抗结核分枝杆菌活性的研究

摘要

本文旨在评价抗结核分枝杆菌先导化合物E在体外抗H37Ra的活性,为新型抗结核药物筛选提供参考依据.应用96孔板微稀释法测定化合物E单独及与异烟肼(INH)、利福平(RIF)联合应用时对H37Ra的最小抑菌浓度(MIC).从肉眼未见细菌生长孔取培养物涂Middlebrook 7H10-ADC平板,以没有细菌生长所对应孔的浓度为化合物E的最小杀菌浓度(MBC).并采用棋盘滴定法计算分级抑菌指数(FICI),评价化合物E与其他抗结核药物之间是否存在协同作用.采用药物暴露法,将培养10 d、6周的H37Ra暴露于不同浓度的化合物E中3 d,涂板测定化合物E对不同生长时期H37Ra的杀菌作用.同法测定化合物E在酸性缺氧条件下对不同生长时期H37Ra的杀菌作用.测定H37Ra对化合物E、INH的耐药频率并分离耐药菌株.结果显示,化合物E对H37Ra的MIC为0.078 μg/ml,MBC为0.312 μg/ml;与一线抗结核药物INH和RIF联合应用时,MIC分别降至单独应用化合物E的1/2 MIC、1/10 MIC,FICI分别为0.6、0.27,表明化合物E与INH联用有相加作用,与RIF联用有协同作用.药物暴露实验结果表明,在正常条件下新型小分子化合物E对生长活跃期(10 d)结核分枝杆菌有明显的杀菌活性;在酸性缺氧条件下,化合物E对生长2个月的结核分枝杆菌具有较好的杀菌活性.3轮实验均未筛选出耐化合物E的H37Ra菌株,而H37Ra对INH的耐药频率为3.4×10-6.这些结果显示,化合物E具有较好的体外抗结核分枝杆菌的活性,值得进一步研究.%Tn order to evaluate the antituberculous activities of leading compound E in vitro and provide the evidence for further studies, the minimum inhibitory concentration (MTC) and minimum bactericidal concentration (MBC) of compound E, isoniazid (TNH) and rifampicin (RTF) against Mycobacterium tuberculosis H37Ra were determined by standard methods. Synergy between compound E and TNH/RTF was determined by chequerboard titration in 96-well microtitre plates. According to MTCs of the drugs alone and in combination, the fractional inhibitory concentration index (FTCT) was calculated to find out that if there was synergy between the drugs. The elimination rate of non-growing H37Ra under normal as well as hypoxic and acidic conditions were determined by drug exposure in 3 days. Drug-resistant mutants of H37Ra and drug-resistant frequencies were obtained by Middlebrook 7H10-ADC agar containing compound E or TNH.rnThe results indicated that compound E was active against H37Ra with MTC of 0.078 μg/ml, and MBC of 0.312 fig/ml. There was an additive effect between compound E and TNH (FTCT = 0.6) and a synergistic effect between E and RTF (FTCT = 0. 27). The results also indicated that compound E showed better antituberculous activity against 10-day-old H37Ra than 6-week-old H37Ra under normal growth condition, but more active against 2-month-old H37Ra than 3-week-old H37Ra under hypoxic and acidic condition. No H37Ra resistant to compound E was detected after three independent repeats while the TNH-resistant frequency was 3. 4 × 10 -6.