The in vitro assay platform presented here evaluates the effects of the drugs on different steps of the entire process of gametocytogenesis from early and late gametocyte development in in vitro culture to oocyst development in Anopheles mosquitoes. In these assays, the spiroindolone drug candidate NITD609 shows potent dose-dependent inhibition against all steps in gametocyte and sporogonic development of P. falciparum in vitro. Significant inhibitory activity was also found for lumefantrine, while such activity was present only at the highest tested concentration of 500 nM for artemether. Under our assay conditions, primaquine did not show any significant activity at concentrations as high as 5 μM.
The spiroindolones represent a novel class of antimalarial drug candidates with robust activity against Plasmodium
symptomatic asexual stages (37
). Consistent with its subnanomolar potency for asexual stages, NITD609 was found to be the most potent compound in our early and late gametocyte development assays, with very significant gametocytemia reduction at the lowest tested concentration of 5 nM, which is still 10 times the IC50
for asexual parasites. Remarkably, NITD609 also shows potent dose-dependent activity in the SMFA, which is unprecedented for (candidate) antimalarial drugs; NITD609 completely blocks transmission of the parasite at 500 nM, suggesting a further inhibiting effect on the (remaining) stage V mature gametocytes going into fertilization in the mosquito midgut and/or on the production of ookinetes or oocysts. The data underline the potency of NITD609 as a potential transmission-blocking drug with inhibiting activity in several critical steps of this part of the life cycle.
Artemisinin derivatives are known for the fastest parasite clearance time in vivo
of all known antimalarial drugs (2
). Some in vivo
studies conducted with artemisinin derivatives have also shown reducing effects on gametocyte prevalence (18
). This has been speculated to be the consequence of fast killing of asexual stages, although in vitro
data suggest that dihydro-artemisinin may target early rather than late gametocyte development (7
). Our results are consistent with this hypothesis but contrast with results reported for artesunate (9
). We saw an inconsistent but, in both experiments, insignificant effect on late gametocyte development at the lower doses of artemether, likely due to variations between culture flasks. Furthermore, there was no inhibitory effect on mature gametocytes with artemether in the SMFA, even at the highest tested concentration of 500 nM, while Chotivanich et al. reported potent activity for artesunate in a similar assay (9
). The explanation for this discrepancy may lie in methodological differences; in the study by Chotivanich et al., mosquito vectors were fed with mature gametocytes that had been cultured in a drug solution for 24 h prior to analysis using the SMFA, whereas the protocol used in our study entails adding drugs into a mature gametocyte culture immediately before the SMFA.
Although with reduced in vitro
potency compared to NITD609, lumefantrine also inhibits both early and late gametocyte development and significantly reduces transmission at a high tested concentration (5 μM). As previously reported (26
), the transmission-reducing activity of lumefantrine-artemether is speculated to be borne largely by artemether. However, our in vitro
data, as well as data recently published by Adjalley et al. (1
), suggest that lumefantrine may contribute significantly to this activity.
The only antimalarial drug reported to have a gametocytocidal effect on P. falciparum in vivo
is the 8-aminoquinolone primaquine. While accelerating gametocyte clearance, primaquine did not affect gametocyte development (23
). A dosing regimen of primaquine is currently being further tested as a potential transmission-reducing therapy (25
). Although it has been previously shown that it effectively clears gametocytes in human patients (16
), primaquine did not show significant activity in any of our assays. We observed only a small, statistically insignificant reduction of the formation of stages I and II gametocytes and only at a relatively high concentration (5,000 nM). The observation that primaquine did not affect P. falciparum
gametocytes in vitro
is in agreement with previous reports (7
) and can be explained by the fact that primaquine needs to be bioactivated or metabolized to exploit its gametocytocidal properties (29
In conclusion, our data support the notion that, if the drug is shown to be safe and efficacious at doses providing plasma levels in the range of 50 to 500 nM in humans, NITD609 could be used to exert transmission-blocking activity through direct and cidal activity against mature gametocytes. This is particularly important, because this suggests that spiroindolones could also be an important tool to treat asymptomatic carriers, which constitute a constant reservoir of mature gametocytes in areas of high transmission. Collectively, our data support the notion that if NITD609 is successful in the clinics as an treatment against acute malaria, this compound could also provide transmission-blocking activity to a novel combination therapy.