The discovery of novel anti-leishmanial compounds remains essential as current treatments have known limitations and there are insufficient novel compounds in development. We have investigated three complex and physiologically relevant in vitro assays, including: (i) a media perfusion based cell culture model, (ii) two 3D cell culture models, and (iii) iPSC derived macrophages in place of primary macrophages or cell lines, to determine whether they offer improved approaches to anti-leishmanial drug discovery and development. Using a Leishmania major amastigote-macrophage assay the activities of standard drugs were investigated to show the effect of changing parameters in these assays. We determined that drug activity was reduced by media perfusion (EC₅₀ values for amphotericin B shifted from 54 (51–57) nM in the static system to 70 (61–75) nM under media perfusion; EC₅₀ values for miltefosine shifted from 12 (11–15) µM in the static system to 30 (26–34) µM under media perfusion) (mean and 95% confidence intervals), with corresponding reduced drug accumulation by macrophages. In the 3D cell culture model there was a significant difference in the EC₅₀ values of amphotericin B but not miltefosine (EC₅₀ values for amphotericin B were 34.9 (31.4-38.6) nM in the 2D and 52.3 (46.6–58.7) nM in 3D; EC₅₀ values for miltefosine were 5.0 (4.9–5.2) µM in 2D and 5.9 (5.5–6.2) µM in 3D (mean and 95% confidence intervals). Finally, in experiments using iPSC derived macrophages infected with Leishmania, reported here for the first time, we observed a higher level of intracellular infection in iPSC derived macrophages compared to the other macrophage types for four different species of Leishmania studied. For L. major with an initial infection ratio of 0.5 parasites per host cell the percentage infection level of the macrophages after 72 h was 11.3% ± 1.5%, 46.0% ± 1.4%, 66.4% ± 3.5% and 75.1% ± 2.4% (average ± SD) for the four cells types, THP1 a human monocytic cell line, mouse bone marrow macrophages (MBMMs), human bone marrow macrophages (HBMMs) and iPSC derived macrophages respectively. Despite the higher infection levels, drug activity in iPSC derived macrophages was similar to that in other macrophage types, for example, amphotericin B EC₅₀ values were 35.9 (33.4–38.5), 33.5 (31.5–36.5), 33.6 (30.5—not calculated (NC)) and 46.4 (45.8–47.2) nM in iPSC, MBMMs, HBMMs and THP1 cells respectively (mean and 95% confidence intervals). We conclude that increasing the complexity of cellular assays does impact upon anti-leishmanial drug activities but not sufficiently to replace the current model used in HTS/HCS assays in drug discovery programmes. The impact of media perfusion on drug activities and the use of iPSC macrophages do, however, deserve further investigation.

中文翻译：

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确定抗利什曼原虫药物活性的新型 2D 和 3D 分析。

新型抗利什曼原虫化合物的发现仍然必不可少，因为目前的治疗方法具有已知的局限性，并且开发中的新型化合物不足。我们研究了三种复杂且生理相关的体外测定，包括：(i) 基于培养基灌注的细胞培养模型，(ii) 两种 3D 细胞培养模型，以及 (iii) iPSC 衍生的巨噬细胞代替原代巨噬细胞或细胞系，以确定它们是否为抗利什曼病药物的发现和开发提供了改进的方法。使用主要利什曼原虫无鞭毛体-巨噬细胞试验研究了标准药物的活性，以显示在这些试验中改变参数的影响。我们确定介质灌注降低了药物活性 (EC ₅₀两性霉素 B 的值从静态系统中的 54 (51–57) nM 变为培养基灌注下的 70 (61–75) nM；米替福新的EC ₅₀值从静态系统中的 12 (11–15) µM 变为培养基灌注下的 30 (26–34) µM（平均和 95% 置信区间），相应地减少了巨噬细胞的药物积累。在 3D 细胞培养模型中，两性霉素 B的 EC ₅₀值存在显着差异，但米替福新不存在（两性霉素 B 的EC ₅₀值在 2D 中为 34.9 (31.4-38.6) nM，在 3D 中为 52.3 (46.6-58.7) nM ; 欧共体₅₀米替福新的值在 2D 中为 5.0 (4.9–5.2) µM，在 3D 中为 5.9 (5.5–6.2) µM（平均和 95% 置信区间）。最后，在使用感染利什曼原虫的 iPSC 衍生巨噬细胞的实验中，我们观察到 iPSC 衍生巨噬细胞的细胞内感染水平高于所研究的四种不同利什曼原虫物种的其他巨噬细胞类型。对于初始感染率为每个宿主细胞 0.5 个寄生虫的 L. Major，72 小时后巨噬细胞的感染百分比为 11.3% ± 1.5%、46.0% ± 1.4%、66.4% ± 3.5% 和 75.1% ± 2.4% (四种细胞类型的平均值 ± SD)，THP1 是人单核细胞系，小鼠骨髓巨噬细胞 (MBMM)，人骨髓巨噬细胞 (HBMM​​) 和 iPSC 衍生的巨噬细胞。尽管感染水平较高，_{50 个}值分别为 35.9 (33.4–38.5)、33.5 (31.5–36.5)、33.6 (30.5 — 未计算 (NC)) 和 46.4 (45.8–47.2) nM，分别在 iPSC、MBMM、HBMM 和 THP95% 细胞中置信区间）。我们得出的结论是，增加细胞分析的复杂性确实会影响抗利什曼原虫药物的活性，但不足以取代药物发现计划中 HTS/HCS 分析中使用的当前模型。然而，培养基灌注对药物活性和 iPSC 巨噬细胞使用的影响确实值得进一步研究。