Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC₅₀ and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (K_M/W), and the concentration of the compound in the membrane (c_m50) and in the aqueous phase (c_w50), which inhibits cell growth by 50%. The K_M/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 10⁸ parasites/mL the compounds did not show significantly different results. The c_m50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.     

Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10⁶ parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC₅₀ values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC₅₀ values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC₅₀ values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC₅₀ values. For assays with high cell concentrations (2×10⁹ parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC₅₀ values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.     

Antiproliferative Activity and Ultrastructural Changes in Promastigote and Amastigote forms of Leishmania amazonensis Caused by Limonene-Acylthiosemicarbazide Hybrids

Leishmaniasis is a tropical zoonotic disease. It is found in 98 countries, with an estimated 1.3 million people being affected annually. During the life cycle, the Leishmania parasite alternates between promastigote and amastigote forms. The first line treatment for leishmaniasis are the pentavalent antimonials, such as N-methylglucamine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). These drugs are commonly related to be associated with dangerous side effects such as cardiotoxicity, nephrotoxicity, hepatotoxicity, and pancreatitis. Considering these aspects, this work aimed to obtain a new series of limonene-acylthiosemicarbazides hybrids as an alternative for the treatment of leishmaniasis. For this, promastigotes, axenic amastigotes, and intracellular amastigotes of Leishmania amazonensis were used in the antiproliferative assay; J774-A1 macrophages for the cytotoxicity assay; and electron microscopy techniques were performed to analyze the morphology and ultrastructure of parasites. ATZ−S-04 compound showed the best result in both tests. Its IC₅₀, in promastigotes, axenic amastigotes and intracellular amastigotes was 0.35±0.08 μM, 0.49±0.06 μM, and 15.90±2.88 μM, respectively. Cytotoxicity assay determined a CC₅₀ of 16.10±1.76 μM for the same compound. By electron microscopy, it was observed that ATZ−S-04 affected mainly the Golgi complex, in addition to morphological changes in promastigote forms of L. amazonensis.     

In vitro effects of bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 on the physiology and interaction process of Leishmania amazonensis

Leishmaniasis is one of the most relevant neglected tropical diseases in the world, affecting 14 million people. Despite the high morbidity, mortality and socio-economic impact, few therapeutic options are available for this disease. To make matters worse, the available molecules have several limitations such as limited efficacy, high cost, side effects and increased resistance. In this context, our group previously synthesized new compounds with anti-leishmania potential being the bis(N-[4-(hydroxyphenyl)methyl]-2-pyridinemethamine)zinc perchlorate monohydrate 4 (complex 4) the most promising one. Therefore, this present work revealed some morphological and physiological changes promoted by complex 4 on Leishmania amazonensis promastigotes as well as it was evidenced its potential against intramacrophage amastigotes. Complex 4 promoted a progressive reduction on the promastigotes size and a remarkable increase on the granularity/complexity as judged by flow cytometry. Transmission electron microscopy (TEM) analysis revealed extensive mitochondrial and plasma membrane alterations, although plasma membrane integrity remained. The mitochondrial changes observed by TEM were accompanied by a decrease in the activity of mitochondrial dehydrogenases with increased production of reactive oxygen species. Interestingly, promastigotes also showed changes in lipid metabolism. Besides the very low toxicity to macrophages, complex 4 had a great effect on intramacrophage amastigotes, displaying an IC₅₀ of 3.91 μM. Collectively, the data presented here reinforce the potential of aminopyridyl compounds complexed to zinc against L. amazonensis. Thus, our work serves as a basis for in vivo assays to be designed or even the synthesis of more selective/effective compounds with lower cost.     

Leishmanicidal effect of 1,3,4-thiadiazolium mesoionic salts on Leishmania amazonensis in vitro

Leishmaniasis is a neglected broad clinical spectrum disease caused by protozoa of the genus Leishmania, which affect millions of people annually in the world and the treatment has severe side effects and resistant strains have been reported. Mesoionic salts are a subclass of the betaine group with extensive biological activity such as microbicide and anti-inflammatory In this work, we analyze the cytotoxic effects of mesoionic salts, 4-phenyl-5-(X-phenyl)-1,3,4-thiadiazolium-2-phenylamine chloride (X = 4 Cl; 3,4 diCl and 3,4 diF), on Leishmania amazonensis in vitro. Initially, Mesoionic salts toxicity were evaluated by XTT assay on L. amazonensis promastigotes. Our results show that the mesoionic salts MI-3,4 diCl, MI-4 Cl and MI-3,4 diF were toxic to the promastigote parasite with IC₅₀ values of 14.3, 40.1 and 61.8 μM, respectively. The amastigote survival was evaluated in treated infected-macrophages, and the results demonstrate that MI-4 Cl (IC₅₀ = 33 μM) and MI-3,4 diCl (IC₅₀ = 43 μM) have a toxic effect against these forms. None of the mesoionic compounds tested present host cell toxicity up to the tested concentration of 100 μM. The selectivity index for MI-3,4 diCl and MI-4 Cl were 3.94 and 6.97, respectively. Nitric oxide (NO) production assayed by Griess reagent, in LPS-activated macrophages or not, in the presence of the salts showed that only the MI-3,4 diCl compound reduced NO levels. Lipid profile analysis of treated-promastigotes showed no alteration of neutral lipids. Evaluation of mitochondrial membrane potential (∆Ψm) showed that the MI-4Cl compound was able to reduce (∆Ψm) by 50%. Therefore, our results suggest that the chlorinated compounds are promising biomolecules, which cause inhibition of L. amazonensis promastigotes, amastigotes, leading to mitochondrial damage.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

Benzaldehyde Thiosemicarbazone Derived from Limonene Complexed with Copper Induced Mitochondrial Dysfunction in Leishmania amazonensis

Background

Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains.

Methodology/Principal Findings

We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC₅₀ concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC₅₀ of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis.

Conclusion/Significance

Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.     

Leishmanicidal Effect of Synthetic trans-Resveratrol Analogs

Background

Stilbene-based compounds show antitumoral, antioxidant, antihistaminic, anti-inflammatory and antimicrobial activities. Here, we evaluated the effect of the trans-resveratrol analogs, pterostilbene, piceatannol, polydatin and oxyresveratrol, against Leishmania amazonensis.

Methodology/Principal Findings

Our results demonstrated a low murine macrophage cytotoxicity of all four analogs. Moreover, pterostilbene, piceatannol, polydatin and oxyresveratrol showed an anti-L. amazonensis activity with IC₅₀ values of 18 μM, 65 μM, 95 μM and 65 μM for promastigotes, respectively. For intracellular amastigotes, the IC₅₀ values of the analogs were 33.2 μM, 45 μM, 29 μM and 30.5 μM, respectively. Among the analogs assayed only piceatannol altered the cell cycle of the parasite, increasing 5-fold the cells in the Sub-G0 phase and decreasing 1.7-fold the cells in the G0-G1 phase. Piceatannol also changed the parasite mitochondrial membrane potential (ΔΨm) and increased the number of annexin-V positive promastigotes, which suggests incidental death.

Conclusion/Significance

Among the analogs tested, piceatannol, which is a metabolite of resveratrol, was the more promising candidate for future studies regarding treatment of leishmaniasis.     

Synthesis,antileishmanial and antitrypanosomal activities of N-substituted tetrahydro-β-carbolines

A series of N-substituted tetrahydro-β-carbolines were synthesized and screened for antileishmanial activity through an in vitro assay that involves promastigotes and axenic amastigotes of Leishmania donovani, the causative agent for visceral leishmaniasis. The thiophen-2-yl analogs 9b and 11f and naphthyl analog 11h were found to show significant activity against promastigotes with IC₅₀ values of 12.7, 9.1 and 22.1 μM, respectively. Analogs 9b and 11h were also effective against axenic amastigotes with IC₅₀ values of 62.8 and 87.6 μM, respectively. The antileishmanial activity of analogs was then tested in human macrophage cell line infected with L. donovani amastigotes and 2-naphthyl linked analog 11h was found to be effective with IC₅₀ value of 28.3 μM. Several analogs also displayed antitrypanosomal activity against Trypanosoma brucei, the causative agent for human African trypanosomiasis. Compounds 11e, 11f and 11h were more effective than others with IC₅₀ values of 1.0, 8.9 and 10.2 μM, respectively. All synthesized analogs were not cytotoxic towards mammalian cell lines including Vero (monkey kidney fibroblasts), HEPG2 (human hepatoma cells), LLC-PK₁ (pig kidney epithelial cells) and THP-1 (human macrophages).     

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC₅₀ values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC₅₀ = 199 µM) compared to the parent strain (IC₅₀ = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.     

Drimanes from Drimys brasiliensis with leishmanicidal and antimalarial activity

This paper evaluates CHCl₃ and CH₃OH extracts of the stem bark, branches and leaves of Drimys brasiliensis and drimane sesquiterpenes isolated from the stem bark against strains of Leishmania amazonensis and Leishmania braziliensis promastigotes and Plasmodium falciparum trophozoites. All of the extracts and compounds were tested in cell lines in comparison with reference standards and cell viability was determined by the XTT method. The CHCl₃ and CH₃OH extracts from the stem bark and branches yielded promising results against two strains of Leishmania, with 50% inhibitory concentrations (IC₅₀ ) values ranging from 39-100 µg/mL. The CHCl₃ extract of the stem bark returned IC₅₀ values of 39 and 40.6 µg/mL for L. amazonensis and L. braziliensis, respectively. The drimanes were relatively effective: 1-β-(p-coumaroyloxy)-polygodial produced IC₅₀ values of 5.55 and 2.52 µM for L. amazonensis and L. braziliensis, respectively, compared with 1-β-(p-methoxycinnamoyl)-polygodial, which produced respective IC₅₀ values of 15.85 and 17.80 µM. The CHCl₃ extract demonstrated activity (IC₅₀ of 3.0 µg/mL) against P. falciparum. The IC₅₀ values of 1-β-(p-cumaroyloxyl)-polygodial and 1-β-(p-methoxycinnamoyl)-polygodial were 1.01 and 4.87 µM, respectively, for the trophozoite strain. Therefore, the results suggest that D. brasiliensis is a promising plant from which to obtain new and effective antiparasitic agents.     

Reactive oxygen species production and mitochondrial dysfunction contribute to quercetin induced death in Leishmania amazonensis

Background

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects more than 12 million people worldwide. Quercetin has generated considerable interest as a pharmaceutical compound with a wide range of therapeutic activities. One such activity is exhibited against the bloodstream parasite Trypanosoma brucei and amastigotes of Leishmania donovani. However, the mechanism of protozoan action of quercetin has not been studied.

Methodology/Principal Findings

In the present study, we report here the mechanism for the antileishmanial activity of quercetin against Leishmania amazonensis promastigotes. Quercetin inhibited L. amazonensis promastigote growth in a dose- and time- dependent manner beginning at 48 hours of treatment and with maximum growth inhibition observed at 96 hours. The IC₅₀ for quercetin at 48 hours was 31.4 µM. Quercetin increased ROS generation in a dose-dependent manner after 48 hours of treatment. The antioxidant GSH and NAC each significantly reduced quercetin-induced cell death. In addition, quercetin caused mitochondrial dysfunction due to collapse of mitochondrial membrane potential.

Conclusions/Significance

The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. Quercetin has been described as a pro-oxidant, generating ROS which are responsible for cell death in some cancer cells. Mitochondrial membrane potential loss can be brought about by ROS added directly in vitro or induced by chemical agents. Taken together, our results demonstrate that quercetin eventually exerts its antileishmanial effect on L. amazonensis promastigotes due to the generation of ROS and disrupted parasite mitochondrial function.     

Antileishmanial activity and trypanothione reductase effects of terpenes from the Amazonian species Croton cajucara Benth (Euphorbiaceae)

BackgroundLeishmaniasis comprises several infectious diseases caused by protozoa parasites of Leishmania genus. In recent years, there has been a growing interest in the therapeutic use of natural products to treat parasitic diseases. Among them Croton cajucara Benth. (Euphorbiaceae) is a plant found in the Amazonian region with a history of safe use in folk medicine.PurposeThe purpose of this study was to investigate the effects of clerodane diterpenes, trans-dehydrocrotonin (DCTN), trans-crotonin (CTN) and acetylaleuritolic acid (AAA) obtained from powdered bark of C. cajucara against promastigotes, axenic and intracellular amastigotes of Leishmania amazonensis. Furthermore, the effects of DCTN and CTN on the trypanotiona reductase enzyme were also investigated. The extraction of the terpenes was carried out as previously reported (Maciel et al., 1998, Maciel et al., 2003).MethodsThe effect of the isolated compounds (DCTN, CTN and AAA) from the bark of C. cajucara was assessed in vitro against promastigotes, axenic amastigotes and intracellular amastigotes of L. amazonensis by counting of remaining parasites in a Neubauer chamber in comparison to pentamidine used as standard drug. The action of natural products on trypanothione reductase was assessed using soluble protein fraction of promastigotes. The assays were performed by incubation with HEPES, EDTA, NADPH and trypanothione disulfide to quantify the NAPH consumption by TryR.ResultsThe results showed very high efficacy, especially of the diterpene DCTN, against promastigotes (IC₅₀ = 6.30 ± 0.06 µg/ml) and axenic amastigotes (IC₅₀ = 19.98 ± 0.05 µg/ml) of L. amazonenesis. The cytotoxic effect of the best active natural product was evaluated on mouse peritoneal infected macrophages (IC₅₀ = 0.47 ± 0.03 µg/ml in 24 h of culture), and the treatment revealed that DCTN never reaches toxic concentrations while reducing the infection and, most importantly, with no toxicity (>100 µg/ml with 0% of macrophage kill) when compared to pentamidine (37.5 µg/ml with 100% of macrophage kill). Furthermore, all of the natural products assayed on the trypanothione reductase enzyme inhibited the enzyme activity compared to the control.ConclusionClerodane diterpenes from C. cajucara showed promising in vitro antileishmanial effects against L. amazonensis, specially the DCTN with no macrophage toxicity up to the assayed concentration. In addition, the action on trypanothione reductase enzyme revealed a possible mechanism of action.     

In vitro growth inhibitory effects of 13,28-epoxyoleanane triterpene saponins in cancer cells

Two new 13,28-epoxyoleanane triterpene saponins, magnosides A (1) and B (2), were isolated from the 95% ethanolic extract of Cybianthus magnus (Mez) Pipoly roots. Their structures were deduced by a combination of spectral analyses and chemical evidences as compared to data reported in the literature. The hemolytic activity of both compounds was measured. Compound 1 was shown to exhibit the strongest hemolytic activity with a HD₅₀ of 3.8 μM followed by 2 with a HD₅₀ of 33.5 μM. The bioactivity of compounds 1 and 2 was also evaluated in vitro against different cellular models including Mycobacterium tuberculosis, Leishmania amazonensis axenic amastigotes, mouse peritoneal macrophages and eight cancer cell lines. While neither of the tested compounds displayed any activity against M. tuberculosis, both exhibited anti-leishmanial activity against axenic amastigotes as well as in vitro growth inhibitory activity against all tested cancer cell lines with IC₅₀ growth inhibitory concentrations ranging between 4 μM and 33 μM. The compounds displayed similar growth inhibitory activity in cancer cell lines sensitive to pro-apoptotic stimuli versus those displaying various levels of resistance to such stimuli. Quantitative videomicroscopy analyses revealed that compounds 1 and 2 are cytotoxic.     

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC_{50 L. braziliensis} 30.47 ± 6.35 μM; IC_{50 L. amazonensis} 40.46 ± 14.21 μM; IC_{50 L. infantum} 65.93 ± 15.12 μM) and amastigotes (IC_{50 L. braziliensis} 68.75 ± 16.55 μM; IC_{50 L. amazonensis} 38.45 ± 12.05 μM; IC_{50 L. infantum} 64.08 ± 23.52 μM), with low cytotoxicity against mouse peritoneal macrophages (CC₅₀ 235.80 ± 36.95 μM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.     

Effect of the Synadenium carinatum latex lectin (ScLL) on Leishmania (Leishmania) amazonensis infection in murine macrophages

Antiparasitic effect of a lectin isolated from Synadenium carinatum latex (ScLL) was evaluated against Leishmania (Leishmania) amazonensis promastigotes/amastigotes. Pretreatment of murine inflammatory peritoneal macrophages with ScLL reduced by 65.5% the association index of macrophages and L. (L) amazonensis promastigotes. Expression of cytokines (IL-12, IL-1 and TNF-α) was detected in infected macrophages pretreated with ScLL (10 μg/mL). ScLL also reduced the growth of L. (L) amazonensis amastigote intracellular forms, showing no in vitro cytotoxic effects in mammalian host cells. ScLL treatment in infected murine inflammatory peritoneal macrophages did not induce nitric oxide production, suggesting that a nitric oxide independent pathway is activated to decrease the number of intracellular Leishmania.     

Antileishmanial Activity of the Estrogen Receptor Modulator Raloxifene

Background

The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis.

Methodology/Principal Findings

Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC₅₀ values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene''s mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC₂(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis – infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden.

Conclusions/Significance

The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.     

Phenotypic characterization of Leishmania mexicana pentamidine-resistant promastigotes. Modulation of the resistance during in-vitro developmental life cycle

Two clones of Leishmania mexicana resistant to 5 μM (LmR5CL2) and 20 μ M (LmR20CLl) pentamidine, derived from a parental wild-type clone (LmWTCL3) were selected in vitro using a continuons drug pressure protocol. Both resistant clones expressed a cross-resistance to diminazene aceturate. No differences in their in-vitro infectivity for mouse peritoneal macrophages between wild-type and pentamidine-resistant promastigotes were observed. During these experiments, promastigotes of LmR20CL1 derived from intramacrophagic amastigote forms reverted to the pentamidine-sensitive phenotype, unlike the lower resistant ones. In the same way, when a complete developmental sequence of L. mexicana was achieved in axenic cultures, LmR20CL1 promastigotes derived from axenically growing amastigotes expressed an IC₅₀ value close to the wild-type one, whereas resulting LmR5CL2 promastigotes remained pentamidine resistant. This modulation of the chemoresistance during the developmental life cycle could be significant in the transmission of drug-resistant strains by Phlebotominae as well as in basic research to follow drug resistance during the in-vitro and in-vivo life cycle of Leishmania.     

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 μM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 μM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.     

Synthesis and activity of novel tetrazole compounds and their pyrazole-4-carbonitrile precursors against Leishmania spp

A new series of 5-(1-aryl-3-methyl-1H-pyrazol-4-yl)-1H-tetrazole derivatives (4a–m) and their precursor 1-aryl-3-methyl-1H-pyrazole-4-carbonitriles (3a–m) were synthesized and evaluated as antileishmanials against Leishmania braziliensis and Leishmania amazonensis promastigotes in vitro. In parallel, the cytotoxicity of these compounds was evaluated on the RAW 264.7 cell line. The results showed that among the assayed compounds the substituted 3-chlorophenyl (4a) (IC₅₀/24 h = 15 ± 0.14 μM) and 3,4-dichlorophenyl tetrazoles (4d) (IC₅₀/24 h = 26 ± 0.09 μM) were the most potent against L. braziliensis promastigotes, as compared the reference drug pentamidine, which presented IC₅₀ = 13 ± 0.04 μM. In addition, 4a and 4d derivatives were less cytotoxic than pentamidine. However, these tetrazole derivatives (4) and pyrazole-4-carbonitriles precursors (3) differ against each of the tested species and were more effective against L.braziliensis than on L. amazonensis.