Liposomal-lupane system as alternative chemotherapy against cutaneous leishmaniasis: Macrophage as target cell

Leishmania amazonensis causes human diseases that range from self-healing to diffusion cutaneous lesions. The chemotherapy of leishmaniasis requires long-term treatment and has been based on the use of pentavalent antimonials. Liposomes have been used as antileishmanial drug carries and have adjuvant activity in vaccines against several microorganisms, representing an important option to the development of new therapeutics for the disease. In this study, we developed a liposomal formulation containing lupane [3β,6β,16β-trihydroxylup-20(29)-ene], isolated from fruits of Combretum leprosum with pharmacological properties as antinociceptive, anti-inflammatory, antiulcerogenic and antileishmanial activities. The aim of the present study was to evaluate the efficacy of liposomal-lupane in L. amazonensis-infection model. Liposomes were prepared by the extrusion method with DPPC, DPPS and cholesterol at 5:1:4 weight ratio. The lupane (2 mg/mL) was added to the lipid mixture, solubilized in chloroform and dried under nitrogen flow. The activity of liposomal-lupane was conducted in vitro with mouse peritoneal infected macrophages. Furthermore, mice were infected in the right hind footpad with 10⁵ stationary growth phase of L. amazonensis promastigotes. After 6 weeks, animals were treated with liposomal-lupane for 15 days by intraperitoneal injection. The evolution of disease was monitored weekly by measuring footpad thickness with a caliper. Three days after the treatment, peritoneal macrophages were collected, plated and production of the cytokines IL-10 and IL-12 was evaluated in supernatants of the cultures after 24 h. The results indicate that the liposomal system containing lupane achieved here is a promising tool to confer antileishmanial activity to infected macrophages.     

In vitro antileishmanial activity and cytotoxicity of essential oil from Lippia sidoides Cham

Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. There is no vaccine available for Leishmania infections and conventional treatments are very toxic to the patients. Therefore, antileishmanial drugs are urgently needed. In this study we have analyzed the effects of essential oils from Lippia sidoides (LSEO) and its major compound thymol on the growth, viability and ultrastructure of Leishmania amazonensis. The essential oil and thymol showed significant activity against promastigote forms of L. amazonensis, with IC₅₀/48 h of 44.38 and 19.47 μg/mL respectively. However, thymol showed toxicity against peritoneal macrophages and low selectivity against the promastigotes when compared with the crude LSEO. On the other hand, no cytotoxic effect was observed in macrophages treated with the crude essential oil. Incubation of L. amazonensis-infected macrophages with LSEO showed a marked reduction in amastigote survival within the macrophages. Significant morphological alterations as accumulation of large lipid droplets in the cytoplasm, disrupted membrane and wrinkled cells were usually seen in treated parasites. The LSEO's activity against both promastigote and the amstigote forms of L. amazonensis, together with its low toxicity to mammalian cells, point to LSEO as a promising agent for the treatment of cutaneous leishmaniasis.     

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.     

Antileishmanial activity of sulphonamide nanoemulsions targeting the β-carbonic anhydrase from Leishmania species

The β-carbonic anhydrase (CA, EC 4.2.1.1) from Leishmania spp. (LdcCA) is effectively inhibited by aromatic/heterocyclic sulphonamides, in the low nanomolar range, but no in vitro antileishmanial activity was detected for such compounds. We formulated some of these sulphonamides as nanoemulsions (NEs) in clove oil, and tested them in vitro against Leishmania infantum MHOM/BR/1974/PP75 and Leishmania amazonensis IFLA/BR/1967/PH8 strains. Interesting inhibitory concentrations IC₅₀ were observed for some of the sulphonamides NEs, with IC₅₀ as low as 3.90?µM (NE-3F) and 2.24?µM (NE-5B) for L. amazonensis and 3.47?µM (NE-5B) for L. infantum. Some of the investigated NEs displayed toxicity for macrophages beyond the parasites. For the same nonoemulsions, a selective index (SI) greater than for Amphotericin B. Haemolytic assay using human red blood cells indicate that the NEs were less cytotoxic than amphotericin B, a widely used antifungal agent. NEs demonstrated to be an excellent strategy for increasing the penetration of these hydrophilic drugs through membranes, with a huge increase of efficacy over the sulphonamide CA inhibitor (CAI) alone.     

Constitutive nitric oxide synthase-like enzyme in two species involved in cutaneous and mucocutaneous leishmaniasis

Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/− 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/− 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.     

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10² and 2.5 x 10⁶ promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.     

Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro

Leishmaniasis’ treatment is based mostly on pentavalent antimonials or amphotericin B long-term administration, expensive drugs associated with severe side effects. Considering these aforementioned, the search for alternative effective and safe leishmaniasis treatments is a necessity. This work evaluated a neolignan, licarin A anti-leishmanial activity chemically synthesized by our study group. It was observed that licarin A effectively inhibited Leishmania (Leishmania) major promastigotes (IC₅₀ of 9.59 ± 0.94 μg/mL) growth, by inducing in these parasites genomic DNA fragmentation in a typical death pattern by apoptosis. Additionally, the neolignan proved to be even more active against intracellular amastigotes of the parasite (EC₅₀ of 4.71 ± 0.29 μg/mL), and significantly more effective than meglumine antimoniate (EC₅₀ of 216.2 ± 76.7 μg/mL) used as reference drug. The antiamastigote activity is associated with an immunomodulatory activity, since treatment with licarin A of the infected macrophages induced a decrease in the interleukin (IL)-6 and IL-10 production. This study demonstrates for the first time the antileishmanial activity of licarin A and suggests that the compound may be a promising in the development of a new leishmanicidal agent.     

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.     

Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages

We investigated the role of autophagy in infection of macrophages by Leishmania amazonensis. Induction of autophagy by IFN-γ or starvation increased intracellular parasite load and the percentages of infected macrophages from BALB/c but not from C57BL/6 mice. In contrast, starvation did not affect the replication of either Leishmania major or Trypanosoma cruzi in BALB/c macrophages. In BALB/c macrophages, starvation resulted in increased monodansylcadaverine staining and in the appearance of double-membrane and myelin-like vesicles characteristic of autophagosomes. Increased parasite load was associated with a reduction in NO levels and was attenuated by wortmannin, an inhibitor of autophagy. In infected macrophages from BALB/c, but not from C57BL/6 mice, starvation increased the number of lipid bodies and the amounts of PGE₂ produced. Exogenous PGE₂ increased parasite load in macrophages from BALB/c, but not C57BL/6 mice. The cyclooxygenase inhibitor indomethacin prevented the increase of parasite load in starved BALB/c macrophages, and actually induced parasite killing. These results suggest that autophagy regulates the outcome of L. amazonensis infection in macrophages in a host strain specific manner.     

New salicylamide and sulfonamide derivatives of quinoxaline 1,4-di-N-oxide with antileishmanial and antimalarial activities

Continuing with our efforts to identify new active compounds against malaria and leishmaniasis, 14 new 3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivatives were synthesized and evaluated for their in vitro antimalarial and antileishmanial activity against Plasmodium falciparum Colombian FCR-3 strain and Leishmania amazonensis strain MHOM/BR/76/LTB-012A. Further computational studies were carried out in order to analyze graphic SAR and ADME properties. The results obtained indicate that compounds with one halogenous group substituted in position 6 and 7 provide an efficient approach for further development of antimalarial and antileishmanial agents. In addition, interesting ADME properties were found.     

Synthetic Oxoisoaporphine Alkaloids: In Vitro,In Vivo and In Silico Assessment of Antileishmanial Activities

Leishmaniasis is a growing health problem worldwide. As there are certain drawbacks with the drugs currently used to treat human leishmaniasis and resistance to these drugs is emerging, there is a need to develop novel antileishmanial compounds, among which isoquinoline alkaloids are promising candidates. In this study, 18 novel oxoisoaporphine derivatives were synthesized and their possible antileishmanial activity was evaluated. The in vitro activity of these derivatives against Leishmania amazonensis axenic amastigotes was first evaluated, and the selected compounds were then tested in an inhibition assay with promastigotes of L. infantum, L. braziliensis, L. amazonensis and L. guyanensis, and with intracellular amastigotes of L. infantum and L. amazonensis. Finally, the most active compounds, OXO 1 (2,3-dihydro-7H-dibenzo[de,h]quinolin-7-one) and OXO 13 (2,3,8,9,10,11-hexahydro-7H-dibenzo[de,h]quinolin-7-one), were tested in BALB/c mice infected with L. infantum. Treatment of mice at a dose of 10 mg/kg with OXO 1 yielded significant reductions (p<0.05) in parasite burden in liver and spleen (99% and 78%, respectively) whereas with OXO 13 were not significant. Although previous reports suggest that this family of molecules displays inhibitory activity against monoamine oxidase A and acetylcholinesterase, these enzymes were not confirmed as targets for antileishmanial activity on the basis of the present results. However, after development of a new bioinformatics model to analyze the Leishmania proteome, we were able to identify other putative targets for these molecules. The most promising candidates were four proteins: two putative pteridine reductase 2 (1MXF and 1MXH), one N-myristoyltransferase (2WUU) and one type I topoisomerase (2B9S).     

Amidine Derivatives and Leishmania amazonensis: an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction

Previous work has demonstrated that N-N′-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes and Trypanosoma evansi trypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infection's supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production by L. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present in Leishmania parasites for leishmanicidal drugs.     

Evaluation of antileishmanial activity of eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens

Infection with Leishmania spp. causes a disease with multifaceted clinical manifestations in humans. The treatment for leishmaniasis is dependent on a limited range of drugs. Here we investigated the antileishmanial activity of eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. We showed that eupomatenoid-5 had a dose-dependent activity during 72 h of treatment, exhibiting IC₅₀ of 9.0 µg/mL and 13.0 µg/mL for promastigote and axenic amastigote forms, respectively, and IC₅₀ of 5.0 µg/mL for intracellular amastigote forms of Leishmania amazonensis. When L. amazonensis was treated with eupomatenoid-5, it underwent considerable ultrastructural alterations, as shown by transmission electron microscopy. Among the alterations was the appearance of intense exocytic activity in the region of the flagellar pocket, myelin-like figures, and vacuoles in the cytoplasm of parasites treated with 9.0 µg/mL. Cells treated with 25.0 µg/mL showed a very large structure, apparently an extension of the endoplasmic reticulum. Also, mitochondrial swelling was detected at this concentration, indicating damage and significant change in this organelle. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa and it is not toxic to macrophages. Our studies indicated that eupomatenoid-5 might be a potential new drug for the treatment of leishmaniasis, because this compound displays interesting antileishmanial activity in vitro against promastigote, axenic amastigote, and intracellular amastigote forms of L. amazonensis.     

Morphological and physiological changes in Leishmania promastigotes induced by yangambin, a lignan obtained from Ocotea duckei

We have previously demonstrated that yangambin, a lignan obtained from Ocotea duckei Vattimo (Lauraceae), shows antileishmanial activity against promastigote forms of Leishmania chagasi and Leishmania amazonensis. The aim of this study was to determine the in vitro effects of yangambin against these parasites using electron and confocal microscopy. L. chagasi and L. amazonensis promastigotes were incubated respectively with 50 μg/mL and 65 μg/mL of pure yangambin and stained with acridine orange. Treated-parasites showed significant alterations in fluorescence emission pattern and cell morphology when compared with control cells, including the appearance of abnormal round-shaped cells, loss of cell motility, nuclear pyknosis, cytoplasm acidification and increased number of acidic vesicular organelles (AVOs), suggesting important physiological changes. Ultrastructural analysis of treated-promatigotes showed characteristics of cell death by apoptosis as well as by autophagy. The presence of parasites exhibiting multiples nuclei suggests that yangambin may also affect the microtubule dynamic in both Leishmania species. Taken together our results show that yangambin is a promissing agent against Leishmania.     

In vitro antileishmanial and cytotoxic activities of nerolidol are associated with changes in plasma membrane dynamics

The sesquiterpene nerolidol is a membrane-active compound that has demonstrated antitumor, antibacterial, antifungal and antiparasitic activities. In this study, we used electron paramagnetic resonance (EPR) spectroscopy and biophysical parameters determined via cell culture assays to study the mechanisms underlying the in vitro antileishmanial activity of nerolidol. The EPR spectra of a spin-labeled stearic acid indicated notable interactions of nerolidol with the cell membrane of Leishmania amazonensis amastigotes. The nerolidol IC₅₀ values in L. amazonensis amastigotes and promastigotes were found to depend on the cell concentration used in the assay. This dependence was described by an equation that considers various cell suspension parameters, such as the 50% inhibitory concentrations of nerolidol in the cell membrane (c_m50) and the aqueous phase (c_w50) and the membrane-water partition coefficient of nerolidol (K_M/W). Via cytotoxicity (CC₅₀) and hemolytic potential (HC₅₀) data, these parameters were also determined for nerolidol in macrophages and erythrocytes. With a c_w50 of 125 μM, macrophages were less sensitive to nerolidol than amastigotes and promastigotes, which had mean c_w50 values of 56 and 74 μM, respectively. The estimated c_m50 values of nerolidol for amastigotes and promastigotes and macrophages were between 2.6 and 3.0 M, indicating substantial accumulation of nerolidol in the cell membrane. In addition, the spin-label EPR data indicated that membrane dynamic changes occurred in L. amazonensis amastigotes at concentrations similar to the nerolidol IC₅₀ value.     

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4⁺ and CD8⁺ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.     

Pterocarpanquinone LQB-118 Induces Apoptosis in Leishmania (Viannia) braziliensis and Controls Lesions in Infected Hamsters

Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the induction of parasite apoptosis and shows promising therapeutic option by oral or local routes in leishmaniasis.     

Entomological surveys of Lutzomyia flaviscutellata and other vectors of cutaneous leishmaniasis in municipalities with records of Leishmania amazonensis within the Bragança region of Pará State,Brazil

In southeast Amazon, Lutzomyia (Nyssomyia) flaviscutellata is the incriminated vector of Leishmania (Leishmania) amazonensis, a causative agent of zoonotic cutaneous leishmaniasis (CL). The optimal methods for surveying Lu. flaviscutellata were investigated in the Bragança region, northeast Pará State, Brazil, selected for the presence of Le. amazonensis. The performances of modified Disney traps and CDC light traps were compared in four ecotopes within and around four village transects during the wet and dry seasons. The physiological age of female sand flies was estimated and natural infection by flagellates was evaluated by dissection. Disney traps were better for detecting the presence of Lu. flaviscutellata, while CDC traps performed well for detecting Lutzomyia (Nyssomyia) antunesi, suspected vector of Leishmania lindenbergi. The former was more abundant during the wet season, when female flies were naturally infected with Le. amazonensis. These findings identified the environments of local transmission. In order to improve surveys of Lu. flaviscutellata as part of integrated epidemiological surveillance of CL, our recommendations include focusing vector surveys with Disney traps on forest fragments where people work, during the seasonal peak of the vector. Further field studies are required to make model‐based predictions of seasonal variations in the vectorial capacity of vector populations.     

Chemotherapy of leishmaniasis part X: Synthesis and bioevaluation of novel terpenyl heterocycles

Some novel α and β ionone based chalcones and their dihydropyrazolidines/pyrazolidines have been synthesized and evaluated for their in vitro and in vivo antileishmanial activities against Leishmania donovani. Amongest all, one compound (4d) exhibited significant in vitro activity against intracellular amastigotes of Leishmania donovani with IC₅₀ values of 7.49 μM and was found promising as compared to reference drug, miltefosine. On the basis of good Selectivity Index (S.I.), the compound was further tested for its in vivo response against Leishmania donovani/hamster model and has shown significant inhibition of parasite multiplication (81%). The present study has helped us in identifying a new lead that could be exploited as a potential antileishmanial agent.     

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.