Repurposing of existing therapeutics to combat drug-resistant malaria

In the era of drug repurposing, speedy discovery of new therapeutic options for the drug-resistant malaria is the best available tactic to reduce the financial load and time in the drug discovery process. Six anticancer drugs, three immunomodulators and four antibiotics were selected for the repositioning against experimental malaria owing to their mode of action and published literature. The efficacy of existing therapeutics was evaluated against chloroquine-resistant in vitro and in vivo strains of Plasmodium falciparum and P. yoelii, respectively. All the pre-existing FDA-approved drugs along with leptin were primarily screened against chloroquine-resistant (PfK1) and drug-sensitive (Pf3D7) strains of P. falciparum using SYBR green-based antiplasmodial assay. Cytotoxic profiling of these therapeutics was achieved on Vero and HepG2 cell lines, and human erythrocytes. Percent blood parasitemia and host survival was determined in chloroquine-resistant P. yoelii N67-infected Swiss mice using appropriate doses of these drugs/immunomodulators. Antimalarial screening together with cytotoxicity data revealed that anticancer drugs, idelalisib and 5-fluorouracil acquired superiority over their counterparts, regorafenib, and tamoxifen, respectively. ROS-inducer anticancer drugs, epirubicin and bleomycin were found toxic for the host. Immunomodulators (imiquimod, lenalidomide and leptin) were safest but less active in in vitro system, however, in P. yoelii-infected mice, they exhibited modest parasite suppression at their respective doses. Among antibiotics, moxifloxacin exhibited better antimalarial prospective than levofloxacin, roxithromycin and erythromycin. 5-Fluorouracil, imiquimod and moxifloxacin displayed 97.64, 81.18 and 91.77 % parasite inhibition in treated animals and attained superiority in their respective groups thus could be exploited further in combination with suitable antimalarials

Synthesis, SAR and biological studies of sugar amino acid-based almiramide analogues: N-methylation leads the way

Leishmaniasis, caused by the protozoan parasites of the genus Leishmania, is one of the most neglected diseases endemic in many continents posing enormous global health threats and therefore the discovery of new antileishmanial compounds is of utmost urgency. The antileishmanial activities of a library of sugar amino acid-based linear lipopeptide analogues were examined with the aim to identify potential drug candidates to treat visceral leishmaniasis. It was found that among the synthesized analogues, most of the permethylated compounds exhibited more activity in in vitro studies against intra-macrophagic amastigotes than the non-methylated analogues. SAR and NMR studies revealed that introduction of the N-methyl groups inhibited the formation of any turn structure in these molecules, which led to their improved activities

Nitroimidazo-oxazole compound DNDI-VL-2098: an orally effective preclinical drug candidate for the treatment of visceral leishmaniasis.

OBJECTIVES: The objective of this study was to identify a nitroimidazo-oxazole lead molecule for the treatment of visceral leishmaniasis (VL). METHODS: A library of 72 nitroimidazo-oxazoles was evaluated in vitro for their antileishmanial activity against luciferase-transfected DD8 amastigotes of Leishmania donovani. On the basis of their in vitro potency and pharmacokinetic properties, the promising compounds were tested in acute BALB/c mouse and chronic hamster models of VL via oral administration and efficacy was evaluated by microscopic counting of amastigotes after Giemsa staining. The best antileishmanial candidates (racemate DNDI-VL-2001) and its R enantiomer (DNDI-VL-2098) were evaluated in vitro against a range of Leishmania strains. These candidates were further studied in a hamster model using various dose regimens. Cytokine and inducible nitric oxide synthase estimations by real-time PCR and nitric oxide generation by Griess assay were also carried out for DNDI-VL-2098. RESULTS: In vitro screening of nitroimidazo-oxazole compounds identified the racemate DNDI-VL-2001 (6-nitroimidazo-oxazole derivative) and its enantiomers as candidates for further evaluation in in vivo models of VL. DNDI-VL-2098 (IC50 of 0.03 μM for the DD8 strain) showed excellent in vivo activity in both mouse and hamster models, with an ED90 value of 3.7 and <25 mg/kg, respectively, and was also found to be very effective against high-grade infection in the hamster model. Our studies revealed that, along with leishmanicidal activity, DNDI-VL-2098 was also capable of inducing host-protective immune cells to suppress Leishmania parasites in hamsters. CONCLUSIONS: These studies led to the identification of compound DNDI-VL-2098 as a preclinical candidate for further drug development as an oral treatment for VL

Combination of Liposomal CpG Oligodeoxynucleotide 2006 and Miltefosine Induces Strong Cell-Mediated Immunity during Experimental Visceral Leishmaniasis

<div><p>Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in <i>Leishmania donovani</i> infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.</p></div

Effect of different formulation/combination of CpG-ODN-2006 and miltefosine on parasite burden.

<p>(A) <i>L. donovani</i>-infected mice and (B) hamsters were treated with various combinations of CpG-ODN-2006 and miltefosine as described in materials and methods section. At day 4 and day 7 post treatment (A) hamsters and (B) mice of different experimental groups were sacrificed and the hepatic as well as the splenic parasite load was determined by stamps-smear method. Total parasite load in each organ is expressed in LDU unit. 1 LDU  =  amastigote per nucleated cell × organ weight in milligram. (C) Schematic representation of the experimental protocol for treatment of <i>Leishmania donovani</i> infected mice/hamsters with different formulation/combination of CpG-ODN-2006 and miltefosine. Data represents here are representative of three independent experiments. Each of the experiments was done a minimum of three times and data represents mean ± SD. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey's post test using graph pad Prism (version 5.0). Significance: Group II vs normal and all treated groups, group V vs VI and group V vs VII (*P<0.05, **P<0.01, ***P<0.001 and ns; non-significant).</p

Effect of combination therapy on production of <i>Leishmania</i>-specific IgG and its isotypes, IgG1 and IgG2.

<p>Animals (n = 5/group) were treated with various combinations of CpG-ODN-2006 and miltefosine as described in legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094596#pone-0094596-g001" target="_blank">Figure 1</a>. Sera collected from normal, infected and treated animals were processed by ELISA as described in materials and methods section to determine the (A) IgG, IgG1 and IgG2 production in hamsters and (B) IgG1 and IgG2a production in mice. Mean ± SD were calculated by the comparison of treated groups to its infected counterparts. The data are representative of three independent experiments. Each experiment was set in triplicates. The significance between various treated groups was calculated by one way ANOVA followed by Tukey's post test using graph pad Prism (version 5.0). Significance: Group II vs normal and all treated groups (*P<0.05, **P<0.01, ***P<0.001 and ns; non-significant). The data presented here are representative of two independent experiments.</p

Sequences of forward and reverse primers used for quantitative real time-PCR.

<p>Sequences of forward and reverse primers used for quantitative real time-PCR.</p

Effect of CpG-ODN-2006 and miltefosine combination therapy on cytokine response, iNOS expression and NO generation.

<p>Animals (mice and hamsters) were infected with <i>L. donovani</i> and treated with various combinations of CpG-ODN-2006 and miltefosine as described in figure legend 1. (A and B) RNA were isolated from isolated splenocytes on day 4 and day 7 post treatments and levels of mRNA expression of Th1 cytokines (IFN-γ, IL-12 and TNF-α) and iNOS (A) as well as Th2 cytokines (IL-10 and TGF-β) (B) were evaluated by real-time PCR. The mRNA level were normalized to HGPRT and GADPH for hamsters and Balb/c mice respectively and expressed as a fold change compared with uninfected control animals. (C) Nitrite generation by supernatant of splenocytes derived from different experimental groups of animals were stimulated with SLA (5 µg/mL for both hamsters and mice) for 72 h and assayed as described in materials and methods section. Data represents here are representative of three independent experiments. Each of the experiments was done a minimum of three times and data represents mean ± SD. The significance between different experimental groups was calculated by one way ANOVA followed by Tukey's post test using graph pad Prism (version 5.0). Significance: Group II vs normal and all treated groups, group VI vs VII and group VI vs VIII (*P<0.05, **P<0.01, ***P<0.001 and ns; non-significant).</p

Immunization with a Trypanosoma cruzi cyclophilin-19 deletion mutant protects against acute Chagas disease in mice

Abstract Human infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi. This protein in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation whereas in the intracellular amastigotes it participates in generating ROS promoting the growth of parasites. We have generated a parasite mutant with depleted expression of Cyp19 by removal of 2 of 3 genes encoding this protein using double allelic homologous recombination. The mutant parasite line failed to replicate when inoculated into host cells in vitro or in mice indicating that Cyp19 is critical for infectivity. The mutant parasite line also fails to replicate in or cause clinical disease in immuno-deficient mice further validating their lack of virulence. Repeated inoculation of mutant parasites into immuno-competent mice elicits parasite-specific trypanolytic antibodies and a Th-1 biased immune response and challenge of mutant immunized mice with virulent wild-type parasites is 100% effective at preventing death from acute disease. These results suggest that parasite Cyp19 may be candidate for small molecule drug targeting and that the mutant parasite line may warrant further immunization studies for prevention of Chagas disease