http://orcid.org/0000-0003-0133-9471
Figures
Abstract
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.
Author summary
Visceral Leishmaniasis remains a serious health problem in many developing countries with thousands of new cases recorded annually. Novel oral therapies are required as existing drugs are limited by their invasive means of administration long duration of treatment regimens. Moreover, with miltefosine as the sole oral drug, there are concerns of the eventual development of parasite resistance with its continuous use. In this study, we report on the in vitro and in vivo leishmanicidal effect of orally administered N-251 on the Leishmania donovani complex parasites in mice. Our results suggest that N-251 may be a potential lead compound for the development of a new oral chemotherapy against VL
Citation: Kwofie KD, Sato K, Sanjoba C, Hino A, Shimogawara R, Amoa-Bosompem M, et al. (2019) 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. PLoS Negl Trop Dis 13(3): e0007235. https://doi.org/10.1371/journal.pntd.0007235
Editor: Laura-Isobel McCall, University of Oklahoma, UNITED STATES
Received: November 1, 2018; Accepted: February 12, 2019; Published: March 25, 2019
Copyright: © 2019 Kwofie et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by grant from the Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED)(https://www.amed.go.jp/program/list/01/06/001.html), awarded to S. I. (15650501) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Visceral leishmaniasis (VL), also known as kala azar, is a disease with a worldwide distribution. It is endemic in more than 62 countries, with over 90% of cases occurring in Brazil, Ethiopia, India, Somalia, South Sudan Sudan [1]. An estimated 200,000 to 400,000 new cases are reported annually [1], making VL a serious public health problem. It is caused by members of the Leishmania donovani complex, which consists of four species: L. archibaldi, L. chagasi, L. donovani, and L. infantum, which are distinguished by their vectors, reservoir hosts, and their associated pathology [2,3]. Parasites are transmitted to humans by the female phlebotomine sandfly. VL is typically presented as a chronic infection, characterized by irregular bouts of fever, weight loss, splenohepatomegaly, anemia, and a high mortality rate of almost 100% if left untreated [1]. At present, there is no licensed vaccine, therefore, chemotherapy remains the main form of control.
Currently, treatment for VL relies on the use of drugs such as pentavalent antimonials, paromomycin, pentamidine, and amphotericin B. Most of these drugs are, however, either limited by their invasive methods of administration and/or their long periods of high dose treatment. Miltefosine remains the sole oral anti-leishmanial drug since it was approved for use in 2002 [4–6]. Recently, the absence of alternate oral drugs has become a major concern, particularly with recent reports of increasing treatment relapses as well as the identification of miltefosine-resistant clinical strains [7–9]. There is, therefore, an urgent need for the development of novel oral chemotherapeutics as alternatives to miltefosine for the effective treatment of VL cases.
Recently, natural and synthetic endoperoxide compounds have received increasing attention due to their fast action and ability to target drug-resistant parasite strains [10–19]. This class of compound is known to be activated within host cells, as a result of the iron-mediated cleavage of the characteristic endoperoxide bridge. Moreover, as anti-infective agents, they are easily administered via the oral route. Previously, we synthesized a novel endoperoxide compound, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) (S1 Fig), that possesses antimalarial [20,21], anti-Toxoplasma gondii [22], anti-schistosomal [23–25], anti-viral [26,27] activities in vitro when administered to mice orally. These findings suggest that N-251 possesses a broad spectrum of anti-infective activities.
In the present study, we report on the in vitro leishmanicidal effects of N-251 against amastigotes of L. donovani, as well as the promastigotes of various L. donovani complex parasites from different geographical locations. In addition, we report on the in vivo efficacy of N-251 as an oral drug against L. donovani amastigotes. Results suggest that N-251 may be a promising lead compound for the development of a new oral chemotherapy against VL.
Methods
Reagents parasites
N-251 was chemically synthesized as described previously [20,21]. It was dissolved in dimethyl sulfoxide (DMSO) as 100 mM stock solutions stored at −20 °C. Miltefosine was purchased from Sigma-Aldrich (MO, USA). Medium 199 was purchased from Nissui Pharmaceuticals Co., Ltd, Japan. Dulbecco’s Modified Eagle’s Medium (DMEM) Roswell Park Memorial Institute (RPMI) medium were purchased from Sigma-Aldrich. HEPES Buffer (1 M) was purchased from MP Biomedicals, LLC (Ohio, USA). All reagents were maintained at 4°C unless otherwise stated. Five strains of L. donovani complex from different geographical locations with high VL burden (Brazil, Nepal, India, Sudan Turkey) were used in this study. These include L. chagasi PP75 (MHOM/BR/74/PP75), L. donovani D10 (MHOM/NP/03/D10), L. donovani DD8 (MHOM/IN/80/DD8), L. donovani KH (MHOM/SU/43/KH) (ATCC 30503), and L. infantum EP173. Promastigotes were cultured in vitro in M-199 complete medium (containing 25 mM HEPES at 25°C) supplemented with 10% heat-inactivated fetal bovine serum (Hi-FBS). 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.
Ethics statement
All animal experiments were reviewed and approved by the Animal Experiment Committee at the Graduate School of Agricultural and Life Sciences, University of Tokyo (Ref. No. P16-254). The experiments were performed in accordance with the Regulations for Animal Care Use of the University of Tokyo, which are based on the Law for the Humane Treatment and Management of Animals, Stards Relating to the Care and Management of Laboratory Animals Relief of Pain (the Ministry of the Environment), Fundamental Guidelines for Proper Conduct of Animal Experiment Related Activities in Academic Research Institutions (the Ministry of Education, Culture, Sports, Science Technology) and the Guidelines for Proper Conduct of Animal Experiments (the Science Council of Japan). At the end of the experiments, the animals were euthanized by exsanguination under anesthesia with isoflurane followed by cervical dislocation.
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 104 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% CO2 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% CO2. 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. IC50 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 × 103 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% CO2. 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% (CC50) 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 CC50 obtained for both RAW 264.7 and J774 macrophage cells and the IC50 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 × 104 promastigotes were seeded per well in this case. Treated and untreated promastigotes were incubated at 25 °C. Miltefosine was used a reference compound. IC50 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 1x108 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
Results
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 host macrophage, is the form of the parasite responsible for clinical symptoms of VL observed in the human host. Therefore, targeting this parasite form in vitro provides a more direct insight into the effect of N-251 in vivo. Stationary phase promastigotes and RAW 264.7 macrophages were incubated for 6 h to allow the invasion and transformation of promastigotes within the macrophages. Intracellular amastigotes were then challenged with 0–50 μM of N-251 for 24, 48 and 72 h to investigate the dose-dependent leishmanicidal effect of N-251. In addition, the IC50 of N-251 was determined at 72 h by microscopy. Our results clearly show the leishmanicidal effect of N-251 on intracellular amastigotes. When treated with varying concentrations of N-251, L. donovani amastigotes were eliminated in a dose dependent manner (S2 and S3 Figs) at an IC50 of 6.69 ± 0.82, (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 RAW 264.7 and J774 murine macrophage cell lines with an alamar blue assay kit for 72 hours. The toxicity of the N-251 treatments was very low among both RAW 264.7 and J774 cells; within the concentration ranges tested, the IC50 values were 66.41 ± 4.15 μM and 138.25 ± 24.27 μM, respectively (Table 1). These IC50 values correspond to SI values of ≥10 for N-251 (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.
Representative Giemsa-stained images (Bar- 20 μm) showing the leishmanicidal effect of N-251 on L. donovani D10 intracellular amastigotes (A) within RAW 264.7 macrophages (M) at 24, 48 and 72 hours.
N-251 exhibits leishmanicidal activity against various L. donovani complex parasites
To examine the effect of N-251 on different L. donovani complex parasites, we screened N-251 against the promastigotes of five parasites belonging to the L. donovani complex. The parasites used in this study were selected specifically from different geographical locations known to be highly endemic for VL. They include: L. chagasi PP75 (Brazil), L. donovani D10 (Nepal) [34,35], L. donovani DD8 (India), L. donovani KH (ATCC 30503) (Sudan) and L. infantum EP173 (Turkey). Our results clearly show the leishmanicidal effect of N-251 on various parasites of the L. donovani complex. When treated with N-251, leishmanicidal activity was observed against all three L. donovani (D10, Dd8 KH) parasites, as well as L. chagasi and L. infantum, in a dose dependent manner (S4 Fig). The IC50 values obtained ranged from 6.12 ± 1.64 μM to 26.90 ± 2.51 μM (Table 2). The results shows that the activity of N-251 is reproducible in promastigotes of different L. donovani complex parasites including L. donovani D10, which had its amastigote forms eliminated by N-251 in our in vitro infection assays. This suggests that N-251 will probably exhibit leishmanicidal activity against their respective amastigote forms, regardless of their geographic origin.
Therapeutic effect of N-251 in L. donovani D10-infected BALB/cA mice
The therapeutic efficacy of N-251 was evaluated in L. donovani-infected mice. Briefly, mice were initially infected intraperitoneally with 108 stationary phase promastigotes, followed by a 4-week incubation period. During this time, parasites invaded the neutrophils and mononuclear phagocytes within the spleen and liver, and a chronic infection was established [28,36,37]. 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 treatment, LDU in the liver and spleen were 857.73 ± 104.05 and 13.94 ± 0.30, respectively. After treatment, in the vehicle control group that did not receive any drug, LDU increased by over 100% to 2214.34 ± 426.73 in the liver and 51.38 ± 16.24 in the spleen. In contrast, in N-251-treated mice, LDU decreased significantly (p = 0.012) by 85.73% and 93.98% to 316.00 ± 161.14 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.
At 4 wpi, 5 mice were euthanized, followed by the determination of parasite burden in spleen and liver by LDU. The remaining mice were placed into 3 groups (N-251, Miltefosine and vehicle (untreated) control) consisting of 5 mice per group treated orally at 12-hour intervals for 14 days. After treatment (6 wpi), mice were euthanized and parasite burden was determined in spleen and liver tissues by LDU and limiting dilution analyses. Results are represented by bar graphs and are expressed as mean ± SEM from 5 mice in each group. (A) Liver LDU (B) Spleen LDU (C) Liver (Limiting dilution analysis) (D) Spleen (Limiting dilution analysis). Blue: 4 wpi. Orange: 6 wpi. Mean values were compared with the untreated group (6 wpi) and *P ≤ 0.05, **P ≤ 0.01 were considered significant.
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 108 and 2.59 x 108 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 107 and 6.68 x 106 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.
Samples of compound-treated and untreated mice spleen and liver tissues were collected for histological examinations. Five μm thick paraffin tissue sections on glass slides were stained with Hematoxylin and Eosin (HE) analyzed under a light microscope. The results are indicated by representative images obtained from the light microscope. White circle: granuloma formation, Scale bars: 5 μm.
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 hyper-granulomas 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 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 Fe2+. 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 Fe2+ [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 [50–54]. 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 Ca2+ homeostasis via opening of the sphingosine-activated plasma membrane Ca2+ 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 orally-administered 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 [65–67]. 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.
Supporting information
S1 Fig. Chemical Structure of novel synthetic endoperoxide, 6-(1,2,6,7 -tetraoxaspiro[7.11]nonadec-4- yl)hexan-1-ol (N-251).
https://doi.org/10.1371/journal.pntd.0007235.s001
(TIF)
S2 Fig. Dose-dependent leishmanicidal effect of N-251 on intracellular amastigotes.
Representative Giemsa-stained images (Scale bar-5 μm) showing the dose-dependent effect of N-251 against L. donovani D10 amastigotes within RAW 264.7 macrophages.
https://doi.org/10.1371/journal.pntd.0007235.s002
(TIF)
S3 Fig. Dose–response curves showing the effect of N-251 on intracellular amastigotes.
https://doi.org/10.1371/journal.pntd.0007235.s003
(TIF)
S4 Fig. Dose-dependent leishmanicidal activity of N-251 against L. donovani complex promastigotes.
Dose–response curves showing the dose-dependent effect of N-251 against L. donovani complex promastigotes and macrophage cells.
https://doi.org/10.1371/journal.pntd.0007235.s004
(TIF)
Acknowledgments
The authors are grateful to the members of the Section of Environmental Parasitology at the Tokyo Medical Dental University (TMDU), as well as members of the Laboratory of Molecular Immunology at the Department of Animal Resource Sciences, University of Tokyo. The authors would also like to thank the staff of the Research Center for Medical Dental Sciences, TMDU, for technical advice.
References
- 1.
WHO. Visceral Leishmaniasis [Internet]. Programmes, Epidemiology. 2018 [cited 2018 Jan 17]. http://www.who.int/leishmaniasis/burden/en/
- 2. Mauricio IL, Scho G, Dujardin J, Soteriadou K, Dedet J, Haralambous C, et al. Evolutionary geographical history of the Leishmania donovani complex with a revision of current taxonomy. PNAS. 2007;104(22):9375–80.
- 3. Akhoundi M, Kuhls K, Cannet A, Votýpka J, Marty P. A Historical Overview of the Classification, Evolution, Dispersion of Leishmania Parasites Sflies. PLoS Negl Trop Dis [Internet]. 2016;10(3):1–40. Available from: http://dx.doi.org/10.1371/journal.pntd.0004349
- 4. Sundar S, Jha TK, Thakur CP, Engel J, Sindermann H, Fischer C, et al. Oral Miltefosine for Indian visceral Leishmaniasis. N Engl J Med. 2002;347(22):949–55.
- 5. Jha TK, Sundar S, Thakur CP, Bachmann P, Karbwang J, Fischer C, et al. Miltefosine, an oral agent, for the treatment of Indian visceral leishmaniasis. N Engl J Med. 1999;341(24):1795–800. pmid:10588964
- 6. Sundar S, Olliaro PL. Miltefosine in the treatment of leishmaniasis: Clinical evidence for informed clinical risk management. Ther Clin Risk Manag. 2007;3(5):733–40. pmid:18472998
- 7. Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, et al. Visceral leishmaniasis: what are the needs for diagnosis, treatment control? NatRevMicrobiol [Internet]. 2007;5(1740–1534 (Electronic)):873–82. Available from: c:%5CKARSTEN%5CPDFs%5CParasitologie-PDFs%5CParasit-2007%5CChappuis et al.-Visceral leishmaniasis- what are the needs for diagnosis treatment control.pdf
- 8. WHO. Control of the Leishmaniases. Tech Rep Ser. 2010;949(March):22–6.
- 9. Sundar S, Chakravarty J. Leishmaniasis: an update of current pharmacotherapy. Expert Opin Pharmacother. 2013;
- 10. Jefford CW. New developments in synthetic peroxidic drugs as artemisinin mimics. Drug Discov Today. 2007;12(11–12):487–95. pmid:17532534
- 11. O’Neill PM, Posner GH. A medicinal chemistry perspective on artemisinin related endoperoxides. J Med Chem. 2004;47(12):2945–64.
- 12. Sen R, Byopadhyay S, Dutta A, Mal G, Ganguly S, Saha P, et al. Artemisinin triggers induction of cell-cycle arrest apoptosis in Leishmania donovani promastigotes. J Med Microbiol. 2007;56(9):1213–8.
- 13. Sen R, Ganguly S, Saha P, Chatterjee M. Efficacy of artemisinin in experimental visceral leishmaniasis. Int J Antimicrob Agents [Internet]. 2010;36(1):43–9. Available from: http://dx.doi.org/10.1016/j.ijantimicag.2010.03.008 pmid:20403680
- 14. Rahaman M, Ghosh S, Chowdhury L, Chatterjee M. Evaluation of anti-leishmanial activity of artemisinin combined with amphotericin B or miltefosine in Leishmania donovani promastigotes. Int J Basic Clin Pharmacol [Internet]. 2014;3(4):1. Available from: http://www.ijbcp.com/?mno=159726
- 15. Ghaffarifar F, Esav Heydari F, Dalimi A, Hassan ZM, Delavari M, Mikaeiloo H. Evaluation of Apoptotic Antileishmanial Activities of Artemisinin on Promastigotes BALB/C Mice Infected with Leishmania major. Iran J Parasitol [Internet]. 2015;10(2):258–67. Available from: http://www.ncbi.nlm.nih.gov/pubmed/26246824%5Cnhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4522302
- 16. Want MY, Islammudin M, Chouhan G, Ozbak HA, Hemeg HA, Chattopadhyay AP, et al. Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis. Int J Nanomedicine. 2017;12:2189–204. pmid:28356736
- 17. Efferth T, R. Romero M, Rita Bilia A, Galal Osman A, ElSohly M, Wink M, et al. Exping the Therapeutic Spectrum of Artemisinin: Activity Against Infectious Diseases Beyond Malaria Novel Pharmaceutical Developments. World J Tradit Chinese Med [Internet]. 2016;2(2):1–23. Available from: http://www.wjtcm.org:8080/ch/reader/view_abstract.aspx?file_no=20160002&flag=1
- 18. Sen R, Saha P, Sarkar A, Ganguly S, Chatterjee M. Iron enhances generation of free radicals by Artemisinin causing a caspase-independent, apoptotic death in Leishmania donovani promastigotes. Free Radic Res [Internet]. 2010;44(11):1289–95. Available from: http://www.ncbi.nlm.nih.gov/pubmed/20815780%5Cnhttp://www.tfonline.com/doi/full/10.3109/10715762.2010.498475 pmid:20815780
- 19. Das NK, Biswas S, Solanki S, Mukhopadhyay CK. Leishmania donovani depletes labile iron pool to exploit iron uptake capacity of macrophage for its intracellular growth. Cell Microbiol. 2009;11(1):83–94. pmid:18823384
- 20. Kim H-S, Nagai Y, Ono K, Begum K, Wataya Y, Hamada Y, et al. Synthesis Antimalarial Activity of Novel Medium-Sized 1,2,4,5-Tetraoxacycloalkanes. J Med Chem. 2001;44(14):2357–61.
- 21. Sato A, Hiramoto A, Morita M, Matsumoto M, Komich Y, Nakase Y, et al. Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol. Parasitol Int [Internet]. 2011;60(3):270–3. Available from: http://dx.doi.org/10.1016/j.parint.2011.04.001 pmid:21501696
- 22. Xin CF, Kim HS, Sato A, Lee HJ, Lee YW, Pyo KH, et al. In vitro inhibition of Toxoplasma gondii by the anti-malarial cidate, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol. Parasitol Int. 2016;65(5):494–9.
- 23. Taniguchi T, Kumagai T, Shimogawara R, Ichinose S, Hiramoto A, Sato A, et al. Schistosomicidal antifecundity effects of oral treatment of synthetic endoperoxide N-89. Parasitol Int [Internet]. 2011;60(3):231–6. Available from: http://dx.doi.org/10.1016/j.parint.2011.02.007
- 24. Yamabe M, Kumagai T, Shimogawara R, Blay EA, Hino A, Ichimura K, et al. Novel synthetic compounds with endoperoxide structure damage juvenile stage of Schistosoma mansoni by targeting lysosome-like organelles. Parasitol Int [Internet]. 2017;66(1):917–24. Available from: http://dx.doi.org/10.1016/j.parint.2016.10.013 pmid:27771462
- 25. Blay EA, Kumagai T, Yamabe M, Hino A, Shimogawara R, Kim H-S, et al. Insights into the mode of action of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89) against adult Schistosoma mansoni worms. Parasitol Int [Internet]. 2018;67(4):403–12. Available from: http://linkinghub.elsevier.com/retrieve/pii/S1383576917304956%0Ahttp://www.ncbi.nlm.nih.gov/pubmed/29617630 pmid:29617630
- 26. Ueda Y, Gu W, Dansako H, Kim HS, Yoshizaki S, Okumura N, et al. Multiple antiviral activities of the antimalarial anti-hepatitis C drug cidates N-89 N-251. Biochem Biophys Reports [Internet]. 2018;15(November 2017):1–6. Available from: https://doi.org/10.1016/j.bbrep.2018.05.007
- 27. Ueda Y, Takeda M, Mori K, Dansako H, Wakita T, Kim HS, et al. New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication. PLoS One. 2013;8(8):1–11.
- 28. Stauber LA. Host Resistance to the Khartoum Strain of Leishmania donovani. Rice Inst Pam Rice Univ Stud [Internet]. 1958;45(1). Available from: http://hdl.hle.net/1911/6279
- 29. Wasan KM, Wasan EK, Gershkovich P, Zhu X, Tidwell RR, Werbovetz KA, et al. Highly Effective Oral Amphotericin B Formulation against Murine Visceral Leishmaniasis. J Inifectious Dis. 2009;200:357–60.
- 30. Allahverdiyev AM, Koc CR, Melahat B, Elcicek S, Baydar YS, Oztel NO, et al. A new approach for development of vaccine against visceral leishmaniasis: Lipophosphoglycan polyacrylic acid conjugates. Asian Pac J Trop Med. 2017;10(9):877–86.
- 31. Wyllie S, Thomas M, Patterson S, Crouch S, De Rycker M, Lowe R, et al. Cyclin-dependent kinase 12 is a drug target for visceral leishmaniasis. Nature [Internet]. 2018;560:192–7. Available from: http://dx.doi.org/10.1038/s41586-018-0356-z pmid:30046105
- 32. Van Den Kerkhof M, Mabille D, Chatelain E, Mowbray CE, Braillard S, Hendrickx S, et al. IJP: Drugs Drug Resistance In vitro in vivo pharmacodynamics of three novel antileishmanial lead series. Int J Parasitol Drugs Drug Resist. 2018;8(January):81–6.
- 33. Sharma M, Sehgal R, Kaur S. Evaluation of Nephroprotective Immunomodulatory Activities of Antioxidants in Combination with Cisplatin against Murine Visceral Leishmaniasis. PLoS Negl Trop Dis. 2012;6(5):e1629.
- 34. Pey K, Yanagi T, Pey BD, Mallik AK, Sherch JB, Kanbara H. Characterization of Leishmania isolates from Nepalese patients with visceral leishmaniasis. Parasitol Res. 2007;100(6):1361–9. pmid:17310397
- 35. Morimoto A, Omachi S, Osada Y, Chambers JK, Uchida K, Sanjoba C, et al. Hemophagocytosis in Experimental Visceral Leishmaniasis by Leishmania donovani. PLoS Negl Trop Dis [Internet]. 2016;10(3):1–16. Available from: http://dx.doi.org/10.1371/journal.pntd.0004505
- 36. Mbati PA, Abok K, Orago AS, Anjili C, Githure JI, Kagai J, et al. Establishment of an appropriate inoculum dose of Leishmania donovani promastigotes required to establish a visceral infection in laboratory animal rodent models. Afr J Health Sci. 1994;1(4):165–8. pmid:12153342
- 37. Silva JM, Zacarias DA, De Figueirêdo LC, Soares MRA, Ishikawa EAY, Costa DL, et al. BOne marrow parasite burden among patients with new world kala-azar is associated with disease severity. Am J Trop Med Hyg. 2014;90(4):621–6. pmid:24615127
- 38. Gutierrez Y, Maksem JA, Reiner NE. Pathologic changes in murine leishmaniasis (Leishmania donovani) with special reference to the dynamics of granuloma formation in the liver. Am J Pathol [Internet]. 1984;114(2):222–30. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1900324&tool=pmcentrez&rendertype=abstract pmid:6696043
- 39. Chávez-Fumagalli MA, Ribeiro TG, Castilho RO, Fernes SOA, Cardoso VN, Coelho CSP, et al. New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment. Rev Soc Bras Med Trop. 2015;48(3):235–42. pmid:26107999
- 40. Bryceson A. A policy for leishmaniasis with respect to the prevention control of drug resistance. Trop Med Int Heal. 2001;6(11):928–34.
- 41. Coelho AC. Miltefosine Susceptibility Resistance in Leishmania: From the Laboratory to the Field. J Trop Dis [Internet]. 2016;04(02):2–6. Available from: http://www.esciencecentral.org/journals/miltefosine-susceptibility-resistance-in-leishmania-from-the-laboratory-to-the-field-2329-891X-1000203.php?aid=69770
- 42. Murray HW. Tissue granuloma structure-function in experimental visceral leishmaniasis. Int J Exp Pathol. 2001;82(5):249–67. pmid:11703536
- 43.
Mcelrath MJ, Murray HW, Cohn ZA. THE DYNAMICS OF GRANULOMA FORMATION IN EXPERIMENTAL VISCERAL LEISHMANIASIS BY Leishmania donovani is an intracellular parasite that infects parenchymal macro- phages of susceptible animals (1). Unlike infection with L. major (2) or L. mexicana (3. 1988;167(June).
- 44. Antoine T, Fisher N, Amewu R, O’Neill PM, Ward SA, Biagini GA. Rapid kill of malaria parasites by artemisinin semi-synthetic endoperoxides involves ROS-dependent depolarization of the membrane potential. J Antimicrob Chemother. 2014;69(4):1005–16.
- 45. van Agtmael MA, Eggelte TA, van BC. Artemisinin drugs in the treatment of malaria: from medicinal herb to registered med- ication. Trends Pharmacol Sci. 1999;20(May 1999):199–205.
- 46. Morita M, Sanai H, Hiramoto A, Sato A, Hiraoka O, Sakura T, et al. Plasmodium falciparum endoplasmic reticulum-resident calcium binding protein is a possible target of synthetic antimalarial endoperoxides, n-89 N-251. J Proteome Res. 2012;11(12):5704–11.
- 47. Sato A, Kawai S, Hiramoto A, Morita M, Tanigawa N, Nakase Y, et al. Antimalarial activity of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) its carboxylic acid derivatives. Parasitol Int [Internet]. 2011;60(4):488–92. Available from: http://dx.doi.org/10.1016/j.parint.2011.08.017
- 48. GHOSH S, GOSWAMI S, ADHYA S. Role of superoxide dismutase in survival of Leishmania within the macrophage. Biochem J [Internet]. 2003;369(3):447–52. Available from: http://biochemj.org/lookup/doi/10.1042/bj20021684
- 49. Marquis JF, Gros P. Intracellular Leishmania: your iron or mine? Trends Microbiol. 2007;15(3):93–5. pmid:17257847
- 50. Bhari V, Sundar S, Dujardin JC, Salotra P. Elucidation of cellular mechanisms involved in experimental paromomycin resistance in leishmania donoVani. Antimicrob Agents Chemother. 2014;58(5):2580–5. pmid:24550335
- 51. Botero A, Keatley S, Peacock C, Thompson RCA. In vitro drug susceptibility of two strains of the wildlife trypanosome, Trypanosoma copemani: A comparison with Trypanosoma cruzi. Int J Parasitol Drugs Drug Resist [Internet]. 2017;7(1):34–41. Available from: http://linkinghub.elsevier.com/retrieve/pii/S2211320716301142
- 52. Laffitte MCN, Leprohon P, Legare D, Ouellette M. Deep-sequencing revealing mutation dynamics in the miltefosine transporter gene in Leishmania infantum selected for miltefosine resistance. Parasitol Res [Internet]. 2016;115(10):3699–703. Available from: http://dx.doi.org/10.1007/s00436-016-5195-y pmid:27457482
- 53. Pink R, Hudson A, Mouriès MA, Bendig M. Opportunities challenges in antiparasitic drug discovery. Nat Rev Drug Discov. 2005;4(9):727–40.
- 54. Plourde M, Coelho A, Keynan Y, Larios OE, Ndao M, Ruest A, et al. Genetic polymorphisms drug susceptibility in four isolates of Leishmania tropica obtained from Canadian soldiers returning from Afghanistan. PLoS Negl Trop Dis. 2012;6(1):1–7.
- 55. Yamabe M, Kumagai T, Shimogawara R, Blay EA, Hino A, Ichimura K, et al. Novel synthetic compounds with endoperoxide structure damage juvenile stage of Schistosoma mansoni by targeting lysosome-like organelles. Parasitol Int. 2017;66(1):917–24. pmid:27771462
- 56. Panic G, Duthaler U, Speich B, Keiser J. Drugs Drug Resistance Repurposing drugs for the treatment control of helminth infections. Int J Parasitol [Internet]. 2014;4(3):185–200. Available from: http://dx.doi.org/10.1016/j.ijpddr.2014.07.002
- 57. CRUMP A, OMURA S. Ivermectin, ‘Wonder drug’ from Japan: the human use perspective. Proc Japan Acad Ser B [Internet]. 2011;87(2):13–28. Available from: http://joi.jlc.jst.go.jp/JST.JSTAGE/pjab/87.13?from=CrossRef
- 58. Pinto-Martinez AK, Rodriguez-Durán J, Serrano-Martin X, Hernez-Rodriguez V, Benaim G. Mechanism of Action of Miltefosine on Leishmania donovani Involves the Impairment of Acidocalcisome Function the Activation of the Sphingosine-Dependent Plasma Membrane. Antimicrob Agents Chemother. 2018;62(1):1–10.
- 59. Coelho AC. Miltefosine Susceptibility Resistance in Leishmania: From the Laboratory to the Field. J Trop Dis [Internet]. 2016;04(02):2–6. Available from: http://www.esciencecentral.org/journals/miltefosine-susceptibility-resistance-in-leishmania-from-the-laboratory-to-the-field-2329-891X-1000203.php?aid=69770
- 60. Rijal S, Ostyn B, Uranw S, Rai K, Bhattarai NR, Dorlo TPC, et al. Increasing failure of miltefosine in the treatment of kala-azar in nepal the potential role of parasite drug resistance, reinfection, or noncompliance. Clin Infect Dis. 2013;56(11):1530–8.
- 61. Cojean S, Houzé S, Haouchine D, Huteau F, Lariven S, Hubert V, et al. Leishmania resistance to miltefosine associated with genetic marker. Emerg Infect Dis. 2012;18(4):704–6. pmid:22469394
- 62. Bhari V, Kulshrestha A, Deep DK, Stark O, Prajapati VK, Ramesh V, et al. Drug susceptibility in Leishmania isolates following Miltefosine treatment in cases of Visceral Leishmaniasis post Kala-Azar dermal Leishmaniasis. PLoS Negl Trop Dis. 2012;6(5):1–6.
- 63. Kremsner PG, Krishna S. Antimalarial combinations. Lancet. 2004;364(9430):285–94. pmid:15262108
- 64.
WHO. Research Priorities for Chagas Disease, Human African Trypanosomiasis Leishmaniasis. Tech Rep TDR Dis Ref Gr Chagas Dis Hum Trypanomiasis Leishmaniasis [Internet]. 2012;100. http://www.who.int/tdr/publications/research_priorities/en/
- 65. van Griensven J, Balasegaram M, Meheus F, Alvar J, Lynen L, Boelaert M. Combination therapy for visceral leishmaniasis. Lancet Infect Dis [Internet]. 2010;10(3):184–94. Available from: http://dx.doi.org/10.1016/S1473-3099(10)70011-6 pmid:20185097
- 66. Meheus F, Balasegaram M, Olliaro P, Sundar S, Rijal S, Faiz MA, et al. Cost-effectiveness analysis of combination therapies for visceral leishmaniasis in the Indian subcontinent. PLoS Negl Trop Dis. 2010;4(9).
- 67. den Boer M, Davidson RN. Treatment options for visceral leishmaniasis. Expert Rev Anti Inect Ther. 2011;9(5):555–70.