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Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs

O’Keeffe, A; Hale, C; Cotton, JA; Yardley, V; Gupta, K; Ananthanarayanan, A; Murdan, S; (2020) Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs. Microorganisms , 8 (6) , Article 831. 10.3390/microorganisms8060831. Green open access

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Abstract

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.

Type: Article
Title: Novel 2D and 3D Assays to Determine the Activity of Anti-Leishmanial Drugs
Open access status: An open access version is available from UCL Discovery
DOI: 10.3390/microorganisms8060831
Publisher version: https://doi.org/10.3390/microorganisms8060831
Language: English
Additional information: This is an open access article distributed under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Leishmania; macrophages; drug assays; media perfusion; 3D cellular models; iPSC macrophages
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > UCL School of Pharmacy > Pharmaceutics
URI: https://discovery.ucl.ac.uk/id/eprint/10099661
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