In Vitro Characterization of the Anti-Bacterial Activity of SQ109 against Helicobacter pylori

The most evident challenge to treatment of Helicobacter pylori, a bacterium responsible for gastritis, peptic ulcers and gastric cancer, is the increasing rate of resistance to all currently used therapeutic antibiotics. Thus, the development of novel therapies is urgently required. N-geranyl-N'-(2-adamantyl) ethane-1, 2-diamine (SQ109) is an ethylene diamine-based antitubercular drug that is currently in clinical trials for the treatment of tuberculosis (TB). Previous pharmacokinetic studies of SQ109 revealed that persistently high concentrations of SQ109 remain in the stomach 4 hours post oral administration in rats. This finding, combined with the need for new anti-Helicobacter therapies, prompted us to define the in vitro efficacy of SQ109 against H. pylori. Liquid broth micro-dilution was used for susceptibility studies to determine the antimicrobial activity of SQ109 against a total of 6 laboratory strains and 20 clinical isolates of H. pylori; the clinical isolates included a multi-drug resistant strain. All strains tested were susceptible to SQ109 with MIC and MBC ranges of 6-10 µM and 50-60 µM, respectively. SQ109 killing kinetics were concentration- and time-dependent. SQ109 killed H. pylori in 8-10 h at 140 µM (2MBCs) or 4-6 h at 200 µM (~3MBCs). Importantly, though the kinetics of killing were altered, SQ109 retained potent bactericidal activity against H. pylori at low pH. Additionally, SQ109 demonstrated robust thermal stability and was effective at killing slow growing or static bacteria. In fact, pretreatment of cultures with a bacteriostatic concentration of chloramphenicol (Cm) synergized the effects of typically bacteriostatic concentrations of SQ109 to the level of five-logs of bacterial killing. A molar-to-molar comparison of the efficacy of SQ109 as compared to metronidazole (MTZ), amoxicillin (AMX), rifampicin (RIF) and clarithromycin (CLR), revealed that SQ109 was superior to MTZ, AMX and RIF but not to CLR. Finally, the frequency of resistance to SQ109 was low and electron microscopy studies revealed that SQ109 interacted with bacterial inner membrane and cytoplasmic content(s). Collectively, our in vitro data demonstrate that SQ109 is an effective monotherapy against susceptible and multi-drug resistant strains of H. pylori and may be useful alone or in combination with other antibiotics for development as a new class of anti-Helicobacter drugs.

幽门螺杆菌（Helicobacter pylori）是引发胃炎、消化性溃疡及胃癌的致病菌，其临床治疗面临的最显著挑战是当前所有常用治疗性抗生素的耐药率持续攀升，因此亟须开发新型治疗方案。N-香叶基-N'-(2-金刚烷基)乙烷-1,2-二胺（SQ109）是一款基于乙二胺的抗结核药物，目前正处于结核病（tuberculosis, TB）的临床试验阶段。此前针对SQ109的药代动力学研究显示，大鼠经口给药4小时后，胃部仍可持续维持较高的SQ109浓度。结合抗幽门螺杆菌治疗的新药需求，本研究团队着手探究SQ109抗幽门螺杆菌的体外抗菌活性。本研究采用肉汤微量稀释法开展药敏试验，以检测SQ109对总计6株实验室菌株及20株临床分离株（含1株多重耐药菌株）的抗菌活性。结果显示，所有受试菌株对SQ109均敏感，其最低抑菌浓度（minimum inhibitory concentration, MIC）范围为6~10 μM，最低杀菌浓度（minimum bactericidal concentration, MBC）范围为50~60 μM。SQ109的杀菌动力学呈浓度与时间依赖性：在140 μM（2倍MBC）浓度下，SQ109可在8~10小时内完成幽门螺杆菌杀灭；在200 μM（约3倍MBC）浓度下，杀灭时长缩短至4~6小时。值得注意的是，尽管杀菌动力学有所改变，但SQ109在低pH环境下仍保留强效杀菌活性。此外，SQ109具备优异的热稳定性，对缓慢生长或处于静止期的细菌同样具有杀菌效果。尤为关键的是，采用抑菌浓度的氯霉素（chloramphenicol, Cm）预处理细菌培养物，可将常规抑菌浓度下SQ109的杀菌效力提升至5 log级细菌杀灭水平。通过摩尔浓度对比，本研究评估了SQ109与甲硝唑（metronidazole, MTZ）、阿莫西林（amoxicillin, AMX）、利福平（rifampicin, RIF）及克拉霉素（clarithromycin, CLR）的疗效差异，结果显示SQ109的活性优于甲硝唑、阿莫西林及利福平，但与克拉霉素相当。此外，SQ109的耐药突变频率较低；电子显微镜研究表明，SQ109可与细菌内膜及胞内物质相互作用。综上，本研究的体外实验数据证实，SQ109对敏感株及多重耐药幽门螺杆菌菌株均具有高效单药杀菌活性，有望单独或与其他抗生素联合开发为新型抗幽门螺杆菌药物。