In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195

BACKGROUND Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.背景 利什曼病（Leishmaniasis）是被忽视程度最高的疾病之一，在包括巴西在内的多个国家均为严重的公共卫生问题。当前可用的治疗手段需长期给药，且伴随严重不良反应，因此亟需开发新型治疗干预方案。由于亚马逊利什曼原虫（Leishmania amazonensis）感染细胞内的转位蛋白（translocator protein, TSPO）表达水平降低，且该蛋白参与细胞凋亡与免疫调节过程，因此TSPO可作为利什曼病化疗的潜在靶点。本研究针对该蛋白的配体PK11195的抗利什曼活性展开评估。 目的 评估PK11195在体外对感染亚马逊利什曼原虫的CBA小鼠巨噬细胞的杀利什曼活性。 方法 采用浓度范围为0.2~400 µM的PK11195处理48小时后，评估无菌培养的亚马逊利什曼原虫、硕大利什曼原虫（Leishmania major）以及巴西利什曼原虫（Leishmania braziliensis）前鞭毛体的存活率。此外，通过检测细胞内寄生虫存活率，确定PK11195（50~100 µM）处理感染巨噬细胞后的半数抑制浓度（IC50）以及活细胞内寄生虫数量。随后采用25~100 µM的PK11195处理感染巨噬细胞，以明确感染亚马逊利什曼原虫的细胞占比以及每个感染细胞内的寄生虫数量。通过电子显微镜观察PK11195诱导的形态学变化。同时检测经PK11195处理、且经干扰素γ（IFN-γ）预刺激或未预刺激的感染巨噬细胞中，氧自由基、一氧化氮以及促炎细胞因子的产生水平。 结果 PK11195的半数抑制浓度中位值分别为：亚马逊利什曼原虫14.2 µM、硕大利什曼原虫8.2 µM、巴西利什曼原虫3.5 µM。亚马逊利什曼原虫的选择性指数为13.7，提示PK11195在哺乳动物中进行后续试验具有安全性。研究观察到感染巨噬细胞的占比、每个感染巨噬细胞内的寄生虫数量以及活细胞内寄生虫数量均呈时间与剂量依赖性降低。电子显微镜结果显示存在提示自噬（autophagy）的形态学改变。值得注意的是，经PK11195处理的亚马逊利什曼原虫感染巨噬细胞中，单核细胞趋化蛋白1（MCP-1）与超氧化物水平有所降低。 主要结论 PK11195可在体外杀灭无鞭毛体，其作用机制不依赖于炎症介质，且在对巨噬细胞无毒性的剂量下即可诱导利什曼原虫出现提示自噬的形态学改变。因此，该分子具备开发为抗利什曼病药物的潜力。