Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.
Int J Parasitol Drugs Drug Resist. 2019 Aug;10:125-132. doi: 10.1016/j.ijpddr.2019.08.004. Epub 2019 Aug 21.
Visceral leishmaniasis (VL) is associated with treatment complications due to the continued growth of resistant parasites toward currently available pathogen-directed therapeutics. To limit the emergence and combat resistant parasites there is a need to develop new anti-leishmanial drugs and alternative treatment approaches, such as host-directed therapeutics (HDTs). Discovery of new anti-leishmanial drugs including HDTs requires suitable in vitro assay systems. Herein, we modified and evaluated a series of resazurin assays against different life-stages of the VL causing parasite, Leishmania donovani to identify novel HDTs. We further analyzed the synergy of combinatorial interactions between traditionally used pathogen-directed drugs and HDTs for clearance of intracellular L. donovani. The inhibitory concentration at 50% (IC) of the five evaluated therapies [amphotericin B (AMB), miltefosine, paromomycin, DNER-4, and AR-12 (OSU-03012)] was determined against promastigotes, extracellular amastigotes, and intracellular amastigotes of L. donovani via a resazurin-based assay and compared to image-based microscopy. Using the resazurin-based assay, all evaluated therapies showed reproducible anti-leishmanial activity against the parasite's different life-stages. These results were consistent to the traditional image-based technique. The gold standard of therapy, AMB, showed the highest potency against intracellular L. donovani, and was further evaluated for combinatorial effects with the HDTs. Among the combinations analyzed, pathogen-directed AMB and host-directed AR-12 showed a synergistic reduction of intracellular L. donovani compared to individual treatments. The modified resazurin assay used in this study demonstrated a useful technique to measure new anti-leishmanial drugs against both intracellular and extracellular parasites. The synergistic interactions between pathogen-directed AMB and host-directed AR-12 showed a great promise to combat VL, with the potential to reduce the emergence of drug-resistant strains.
内脏利什曼病（VL）与治疗并发症相关，这是由于目前可用的针对病原体的治疗方法对耐药寄生虫的持续生长。为了限制耐药寄生虫的出现并对抗它们，需要开发新的抗利什曼病药物和替代治疗方法，例如宿主定向治疗（HDTs）。包括 HDTs 在内的新抗利什曼病药物的发现需要合适的体外测定系统。在此，我们针对引起 VL 的寄生虫利什曼原虫的不同生命阶段修改并评估了一系列 Resazurin 测定法，以确定新的 HDTs。我们进一步分析了传统的针对病原体的药物与 HDTs 之间组合相互作用的协同作用，以清除细胞内的 L. donovani。通过 Resazurin 测定法测定了五种评估疗法[两性霉素 B（AMB）、米替福新、巴龙霉素、DNER-4 和 AR-12（OSU-03012）]对前鞭毛体、细胞外无鞭毛体和细胞内无鞭毛体的 50%抑制浓度（IC），并与基于图像的显微镜进行了比较。使用 Resazurin 测定法，所有评估的疗法均针对寄生虫的不同生命阶段显示出可重复的抗利什曼病活性。这些结果与传统的基于图像的技术一致。治疗的金标准 AMB 对细胞内 L. donovani 显示出最高的效力，并进一步评估了与 HDTs 的组合效果。在分析的组合中，与单独治疗相比，针对病原体的 AMB 和针对宿主的 AR-12 显示出协同减少细胞内 L. donovani 的作用。本研究中使用的改良 Resazurin 测定法证明是一种有用的技术，可用于测量针对细胞内和细胞外寄生虫的新抗利什曼病药物。针对病原体的 AMB 和针对宿主的 AR-12 之间的协同相互作用为对抗 VL 带来了巨大的希望，有可能减少耐药菌株的出现。
