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

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.

Macrophages Derived From Human Induced Pluripotent Stem Cells: The Diversity of Protocols, Future Prospects, and Outstanding Questions

“…Macrophages play a pivotal role in pathogen clearance. The fact that iMphs are infectable with various intracellular bacteria (e.g., S. typhimurium , P. aeruginosa , M. tuberculosis ) and viruses (e.g., HIV, ZIKA, and dengue) allows using them as a standardized model to study Mφ-pathogen interactions and to search for key targets to reinforce a Mφ-mediated immune defense ( van Wilgenburg et al, 2013 ; Hale et al, 2015 ; Ackermann et al, 2018 ; Lang et al, 2018 ; Bernard et al, 2020 ; Haake et al, 2020 ; Nenasheva et al, 2020 ; O’Keeffe et al, 2020 ).…”

Pharmacokinetics and pharmacodynamics in the treatment of cutaneous leishmaniasis – challenges and opportunities

“…Modifications to investigate the impact of rate of flow of culture media and different scaffold support for macrophages on drug efficacy showed shifts in EC 50/90 values for amphotericin B and miltefosine in cultures under RSC Medicinal Chemistry Review medium flow. 52,92 Further changes such as the addition of a second medium reservoir introducing fresh medium into the assay design, similar to the hollow fibre infection model, would enable the opportunity to test continuous versus intermittent drug exposure. 93 Recent investigations demonstrate how the hollow fibre infection model can be used to reproduce pharmacokinetic profiles of clinically relevant anti-mycobacterial drug combinations, simulating drug levels at the target lung tissue.…”

“…However, this is an in vitro parameter, and like in all antimicrobial studies 51 it has several critical limitations: (i) the set time period and concentration of drug exposure, which is critically different from the transient nature of the in vivo PK exposure in animal models and humans, (ii) the different properties of host cells subject to differences in nutrient supply, O 2 tensions and flow (see ref. 17 for further discussion on impact on Leishmania and 52 for effect of flow on drug activity) and (iii) does not take into account the in vivo survival and division of pathogens which may have fast dividing as well as quiescent populations. 53,54 Hence the determination of the in vivo PD parameter, must determine skin amastigote burden over a dose range using qPCR or other methods.…”

The Stem Cell Revolution Revealing Protozoan Parasites’ Secrets and Paving the Way towards Vaccine Development

“…Over twenty morphologically indistinguishable species of Leishmania cause this disease that is transmitted by the bite of infected female phlebotomine sand flies [ 53 ]. The promastigotes are injected into the host by the fly during blood meals and are engulfed by phagocytic cells, mainly macrophages.…”

“…Though the objective of this study was to highlight the necessity for screening for this parasite before stem cell transplantation in endemic countries, it showed that stem cells can be a versatile model to study this parasite [ 60 ]. In a recent study, hiPS-derived macrophages were shown to be much better at supporting infection by L. major and L. amazonensis and very good for L. mexicana , as compared with THP1 and primary macrophages [ 53 ]. Several compounds were tested in this system with a similar outcome, though IPS-derived macrophages displayed the highest effect in some cases.…”