Oral activity of the antimalarial endoperoxide 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Leishmania donovani complex

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL.

For in vitro and in vivo infectivity assays, L. donovani D10 was used because it is constantly maintained in mice in our lab, therefore, highly infective. The usual procedure is to use freshly isolated parasites that have undergone 1-3 cycles of passages in M-199 complete medium to ensure high infectivity rates in our experiments.

In vitro inhibition activity against intracellular amastigotes
The leishmanicidal effect of N-251 on L. donovani amastigotes was evaluated in murine macrophages. First, 4 x 10 4 RAW 264.7 macrophage cells in 200 μl of DMEM (containing 1% penicillin/streptomycin, supplemented with 10% Hi-FBS) were seeded in the wells of an 8-well chamber slide incubated at 37˚C in 5% CO 2 for 2 hours to allow cell attachment. Next, stationary phase L. donovani promastigotes in fresh DMEM were added to the macrophages at a ratio of 50:1 (parasites:macrophage) and incubated for 6 h at 37˚C in 5% CO 2 . During this period, the parasites invaded the macrophages and then transformed into amastigotes. Free promastigotes were subsequently removed by successive washes with DMEM. Infected macrophages were then treated with N-251 at concentrations ranging from 0.78 to 50 μM, and incubated for 24, 48 and 72 h. Miltefosine was used as a reference drug control. Infected treated macrophages were then washed with 1x phosphate-buffered saline (PBS) and fixed with methanol for 10 min. Finally, the macrophages were stained with 5% Giemsa in PBS for 25 min and observed under a light microscope. Anti-leishmanial activity was evaluated by observing 300 macrophages within each treatment group. The percentage of infected macrophages was calculated using the following formula: [(Number of infected macrophages/300 macrophages observed) x 100]. Infection index values were then calculated according to the following formula: [Percentage of infected macrophages x average number of intracellular amastigotes per infected macrophage]. Infection index values were then converted to percentage survival values relative to the untreated parasite population. IC 50 values were eventually obtained by sigmoidal dose-response curve analysis using the scatter plot option of Microsoft Excel 2016 (Microsoft Corporation, Washington, USA) expressed as the mean of samples ± stard deviation (SD) from three independent experiments conducted in duplicates.

Cytotoxicity evaluation selectivity index determination
The cytotoxicity of N-251 against murine macrophage cell lines was assessed using the Invitrogen alamar blue assay kit (ThermoFisher Scientific, Japan) according to the manufacturer's instructions with modifications. Both RAW 264.7 and J774 cell lines were used in this assay. First, 5 × 10 3 cells were seeded into each well of a 96-well plate. Varying concentrations of N-251, ranging from 0.195 μM to 200 μM, were then added to the cells and incubated for 48 h at 37˚C in 5% CO 2 . Miltefosine was used as a positive control. Next, 10% alamar blue dye was added to all wells and the plate was incubated for another 24 h in darkness. After a total of 72 h, fluorescence intensity was measured at a wavelength of 600 nm using the SpectraMax Paradigm Multi-Mode Detection Platform (Molecular devices LLC, CA, USA). All experiments were carried out 4 times in duplicates. Fluorescence intensity, which is directly proportional to the concentration of surviving parasites, was converted to percentage survival. Cytotoxic concentrations at 50% (CC 50 ) were eventually obtained by sigmoidal dose-response curve analysis using the scatter plot option of Microsoft Excel 2016 and expressed as mean of samples ± standard deviation (SD). The selectivity index (SI) was calculated as the ratio of the CC 50 obtained for both RAW 264.7 and J774 macrophage cells and the IC 50 for Leishmania donovani D10 amastigotes.

In vitro leishmanicidal activity against promastigotes
The effect of N-251 was also evaluated in logarithmic phase promastigotes of the L. donovani complex using the Invitrogen alamar blue assay kit. The experimental procedure carried out was essentially the same as described above. However, 5 × 10 4 promastigotes were seeded per well in this case. Treated and untreated promastigotes were incubated at 25˚C. Miltefosine was used a reference compound. IC 50 values were also obtained as described above.

Anti-leishmanial efficacy of N-251 in vivo
To evaluate the anti-leishmanial efficacy of N-251, L. donovani D10-infected mice, randomly allocated into experimental groups of 5 animals each, were treated orally with 68 mg/kg body weight of N-251 in olive oil. This dose was determined as the maximum concentration at which no toxicity (ruffled fur, severe weight loss, reduced activity and death) was observed in previous antimalarial studies [21]. Miltefosine (10 mg/kg body weight) and olive oil were used as positive and negative controls, respectively. First, 6-week-old BALB/cA mice (CLEA Japan, Inc. Tokyo, Japan) were infected intraperitoneally with 1x10 8 stationary phase promastigotes. Four weeks post-infection, treatment was administered through a feeding gavage at 12-hour intervals for 14 consecutive days. Initially, treatment efficacy was expressed as Leishman-Donovan Units (LDU), which was determined by sterilely harvesting and weighing the spleen and liver of euthanized mice. The macrophages in the spleen and liver were imprinted on glass slides and then fixed with methanol for 10 minutes. Imprints were then stained with 5% Giemsa in PBS for 25 mins and examined microscopically. LDU was calculated based on the formula: [(Number of Leishmania amastigotes per 1000 macrophage cells) x organ weight (g)] [28-33]. Percentage reduction was also calculated as follows: 100-[(LDU of treatment group/LDU of untreated group) x 100] Determination of LDU was done before treatment at 1-day post-treatment.
To further evaluate the anti-leishmanial efficacy of N-251, post-treatment parasite burden levels were also assessed by limiting dilution analysis, modified for L. donovani in our laboratory. Briefly, portions of harvested mice liver and spleen were cut, weighed and homogenized in tissue grinders sterilely. The homogenate was suspended in a final volume of 2 ml of M-199 complete medium supplemented with 10% Hi-FBS and 1% penicillin/streptomycin. Five-fold serial dilutions (ranging from 1 to 6.5 x 10 −12 ) of the homogenate were made in M-199 complete medium plated (100 ul/well) in 96-well flat-bottom tissue culture plates. Plates were stored in a humidified incubator at 25˚C for 14 days after which wells were visually examined for growth with an inverted microscope. The presence or absence of motile promastigotes was recorded in each well. The final titer was the last dilution for which the well contained at least one parasite. The number of parasites per gram organ (parasite burden) was calculated as follows: parasite burden = (geometric mean of reciprocal titers from each duplicate/ weight of homogenized cross section) x 20, where 20 is the reciprocal fraction of the homogenized organ inoculated into the first well. Percentage reduction was also calculated as follows: 100-[(parasite burden in treatment group/ parasite burden of control group) x 100]. LDU and limiting dilution results were analyzed using Microsoft Excel 2016 are expressed as mean ± stard error of the mean (SEM) from 5 mice per group. Comparison of means was done using two-tailed Mann-Whitney U-test and differences were considered significant when p � 0.05.
In addition to assessing post-treatment parasite burden levels, samples of harvested mice liver and spleen were also collected for histological studies. Tissue samples were fixed with formaldehyde for 48 hours and gradually dehydrated with increasing concentrations of ethanol. The tissues were then embedded in paraffin 5 μm thick and sections were cut using a microtome. Thin tissue sections on glass slides were stained with Hematoxylin and Eosin (HE) and analyzed by visualization under light microscope. The number of granulomas was determined with quantification of twenty microscope optic fields using a 40x objective of a light microscope. The experimental plan to evaluate the anti-leishmanial efficacy of N-251 has been summarized in Fig 1

N-251 selectively exhibits leishmanicidal activity against L. donovani D10 amastigotes
To evaluate the leishmanicidal activity of N-251, we used the amastigote form of L. donovani D10 parasites as the model parasite because the intracellular amastigote, which lives within the  Table 1). Although most parasites had been cleared by 24 hours, a few macrophages were observed with about one or two intracellular parasites that were probably dead. By 48 and 72 hours, intracellular parasites were completely cleared from all macrophages (Fig 2). The leishmanicidal activity of N-251 was comparable to that of miltefosine (reference drug control). The cytotoxicity of N-251 was evaluated on both  Table 1), suggesting that N-251 is highly specific in its activity against L. donovani amastigotes, as biological efficacy cannot be attributed to cytotoxicity when the selectivity index is �10. The SI values of miltefosine were about twice that of N-251, which were quite comparable ( Table 1). The results therefore suggest that N-251 selectively and specifically exhibits leishmanicidal activity by targeting L. donovani intracellular amastigotes within host macrophage cells, without having any cytotoxic effect on host cells.

N-251 exhibits leishmanicidal activity against various L. donovani complex parasites
To Mice were then treated orally through feeding gavages at 12-hour intervals for 14 days. LDU was then determined in harvested spleen liver tissues before and after treatment. Before 14 and 3.09 ± 1.29 within the liver and spleen, respectively, relative to the untreated group at 6 weeks post-infection (wpi). These results clearly show that after 14 days of treatment, parasites were significantly cleared by N-251 (Fig 3a and 3b). Similarly, in miltefosine-treated mice, parasites were cleared at levels that were comparable to those obtained by N-251. To further confirm the therapeutic effect of N-251, we also evaluated its efficacy by limited dilution analysis to determine parasite burden levels post-treatment. Results here also showed that N-251 significantly cleared L. donovani parasites. After treatment, parasite burden in the untreated group were determined at 4.15 x 10 8 and 2.59 x 10 8 parasites/g organ in liver and spleen respectively. However, in N-251-treated mice, parasite burden decreased significantly (p < 0.05) by 85.89% and 97.41% to 5.86 x 10 7 and 6.68 x 10 6 parasites/g organ in liver and spleen respectively (Fig 3c and 3d). In vivo data obtained by both LDU and limiting dilution analyses therefore support our in vitro observation that N-251 has leishmanicidal activity against L. donovani amastigotes. Data herein therefore demonstrates the therapeutic effect of N-251 as an oral drug in L. donovani-infected mice.
One of the major signs of histopathological damage known to be associated with L donovani infection in mice humans are granuloma formation in the liver [38]. Hence, we investigated efficacy of N-251 in suppressing tissue damage by VL. To accomplish this, samples of compound-treated untreated mice liver tissues were collected and processed for histological examinations. The results clearly showed that N-251 is able to suppress damage due to granuloma formation in L. donovani-infected liver. The untreated group revealed high levels of granuloma formation (an average of about 4 per field) within mice liver. In contrast, when mice were treated with N-251, no granuloma formation was observed within the liver (Fig 4). These results demonstrate that N-251 significantly suppresses and improves the conditions of L. donovani-infected mice liver tissues as a result of significant parasite reduction.

Discussion
The demand for novel, orally administered alternatives to miltefosine for the treatment of VL has become more urgent. This is particularly true as existing drugs are plagued with several limitations, such as their invasive means of administration and long duration regimens [39]. Currently, miltefosine is the sole orally-administered drug for the treatment of VL. Recently, the widespread use of miltefosine, coupled with recent reports of increasing treatment relapses, have raised concerns of possibility of the rapid emergence of resistance due to its long half-life [40,41], thus, highlighting the urgent need for novel, oral antileishmanial drugs. In the present study, we showed that N-251, a novel, synthetic, orally-administered endoperoxide, selectively and specifically kills L. donovani amastigotes with no toxicity to host cells. Even after a progressive infection was established in mice, N-251 eliminated intracellular amastigotes, resulting in the suppression of hyper-granuloma formation in mice liver. The formation of hepatic hypergranulomas is known to be mostly elicited by the presence of intracellular amastigotes in Leishmania-infected hosts [38, 42,43]. This therefore suggest that the significant reduction of Oral activity of N-251 against L. donovani complex intracellular amastigotes by N-251 observed in this study, may have resulted in the absence of granuloma, indicating the potential of N-251 as a lead compound for the development of an oral chemotherapy against VL.
The cleavage of the endoperoxide bridge (C-O-O-C, S1 Fig) generates short-lived, cytotoxic oxyradicals in the presence of heme iron or free Fe 2+ . Fenton degradation of the oxyradical intermediates can produce hydroxyl radicals (OH) that are highly reactive against a wide variety of molecules such as amino acids, enzymes, lipids and nucleic acids [44,45]. The released radicals then oxidize these molecules, thereby inhibiting their functions, which eventually leads to parasite death. This explains why endoperoxides exhibit a wide spectrum of anti-infective activity. N-251 is also characterized by a peroxide bridge within its structure that can be cleaved and activated in the presence of a heme iron or free Fe 2+ [20, 46,47], suggesting that this is the basis for its antileishmanial activity observed in both promastigotes and amastigotes screened in this study. Leishmania are known to forage for iron from host macrophages for their growth for defense against the macrophage's oxidative assault by providing iron to the antioxidant enzyme superoxide dismutase [19,48,49]. The accumulation of iron within the parasite therefore results in the selective killing of the parasite by N-251. This also explains the 4-fold increase in activity observed against intracellular amastigotes relative to that of promastigotes.
Some of the current antileishmanial drugs, including pentavalent antimonials and paromomycin, have been reported to exhibit a limited spectrum of antileishmanial activity, which is mostly dependent on the species of Leishmania, the geographical location, as well as the clinical presentation of the disease [50][51][52][53][54]. However, in this study, N-251 exhibited leishmanicidal effects against various L donovani complex parasites from different parts of the world. This therefore shows the reproducibility of N-251's activity in different L. donovani complex parasites, suggesting that N-251 may exhibit leishmanicidal activity against different VL parasites, regardless of their geographical origin. In addition, in previous studies, N-251 was observed to be active against other non-Leishmania parasites such as Plasmodium, T. gondii and Schistosoma [20,21,23,47,55]. It is therefore quite clear that N-251 exhibits a wide spectrum of activity. In general, advantages of wide spectrum compounds include cost effectiveness in using one drug to treat different infectious diseases as well as serving as good options for Mass drug administration exercises in poor endemic regions [56,57]. This inherent characteristic of N-251 appears to be one of the several advantages it has over other existing antileishmanial drugs.
Miltefosine, the only oral drug currently available against VL, affects parasites by the disruption of parasitic Ca 2+ homeostasis via opening of the sphingosine-activated plasma membrane Ca 2+ channel, together with the impairment of the acidocalcisomes [58]. However, it is limited by its teratogenicity and several side effects. There are also concerns that its use as a monotherapy can eventually lead to the rapid emergence of resistant parasites [40,59]. In fact, treatment relapses among VL patients [60], as well as the identification of miltefosine-resistant clinical strains, have already been reported [61,62]. Our compound, N-251, is also orallyadministered and acts through a different mode of action from miltefosine, making it a good candidate for possible combination with miltefosine. According to the WHO, combination therapy is an important strategy to improve leishmaniasis therapy and also delay the emergence of resistance [63,64], as can be seen in the case of malaria, tuberculosis, and HIV [65][66][67]. In future studies, we will therefore evaluate the combinatory effect of N-251 and miltefosine in experimental VL.
In conclusion, results herein demonstrates the in vitro and in vivo antileishmanial effect of the novel orally administered synthetic endoperoxide, N-251. Also, the broad-spectrum activity of N-251 against various parasites of L. donovani complex from different geographical locations was established. More importantly, this study highlights the importance of N-251 as an oral drug for monotherapy its possible combination with miltefosine. Finally, N-251 may be a promising lead compound for the development of new oral chemotherapy against visceral leishmaniasis.