Imidazo[1,2-a]pyrimidine as a New Antileishmanial Pharmacophore against Leishmania amazonensis Promastigotes and Amastigotes.

Leishmania poses a substantial threat to the human population all over the globe because of its visceral and cutaneous spread engendered by all 20 species. Unfortunately, the available drugs against leishmania are already hobbled with toxicity, prolonged treatment, and increasing instances of acquirement of resistance. Under these grave circumstances, the development of new drugs has become imperative to keep these harmful microbes at bay. To this end, a Groebke-Blackburn-Bienaymé multicomponent reaction-based library of different imidazo-fused heterocycles has been synthesized and screened against Leishmania amazonensis promastigotes and amastigotes. Among the library compounds, the imidazo-pyrimidine 24 has been found to be the most effective (inhibitory concentration of 50% (IC50) < 10 µM), with selective antileishmanial activity on amastigote forms, a stage of the parasite related to human disease. The compound 24 has exhibited an IC50 value of 6.63 µM, being ∼two times more active than miltefosine, a reference drug. Furthermore, this compound is >10 times more destructive to the intracellular parasites than host cells. The observed in vitro antileishmanial activity along with suitable in silico physicochemical and absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of compound 24 reinforce the imidazo-pyrimidine scaffold as a new antileishmanial pharmacophore and encourage further murine experimental leishmaniasis studies.

Mitracarpus frigidus reduces lipid metabolism and PGE2 levels in inflammatory cells.

OBJECTIVES: To evaluate the ability of the aqueous extract of Mitracarpus frigidus (MFAq) to inhibit lipid body formation and inflammatory mediator production in macrophages stimulated with lipopolysaccharide (LPS) and interferon gamma (IFN-γ). METHODS: MFAq was chemically characterized by ultrafast liquid chromatography/quadruple time-of-flight tandem mass spectrometry. The macrophages obtained from mice were incubated with MFAq. Cell viability and membrane integrity were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and propidium iodide assays, respectively. Moreover, NO, reactive oxygen species (ROS), transforming growth factor beta (TGF-ß), prostaglandin E2 (PGE2) levels and lipid bodies (LBs) were examined in macrophages that were stimulated with LPS and IFN-γ and treated with MFAq. Finally, molecular docking analysis was conducted to investigate the interaction of MFAq with the cyclooxygenase 2 (COX-2) enzyme. KEY FINDINGS: Chlorogenic acid, clarinoside, harounoside, rutin, kaempferol-3O-rutinoside and 2-azaanthraquinone were identified in MFAq. MFAq significantly inhibited NO, ROS and LBs, and did not affect the membrane integrity of macrophages. MFAq-treated cells showed significantly lower levels of TGF-ß and PGE2. Molecular docking demonstrated that the compounds found in MFAq are able to inhibit COX-2 by binding to important residues in the catalytic site. CONCLUSIONS: MFAq interferes with lipid metabolism in stimulated macrophages, leading to the reduction of important inflammatory mediators. Furthermore, MFAq can directly inhibit the COX-2 enzyme or inhibit its expression owing to its ability to reduce NO production.

In vitro evaluation of antileishmanial activity, mode of action and cellular response induced by vanillin synthetic derivatives against Leishmania species able to cause cutaneous and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, mainly due to their toxicity of the drugs, high cost and/or by the emergence of parasite resistant strains. In this context, new therapeutics should be searched. In this study, two novel synthetic derivatives from vanillin: [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] or 3s and [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde] or 3t, were evaluated regarding their antileishmanial activity against distinct parasite species able to cause cutaneous and visceral leishmaniasis. Results showed that compounds 3s and 3t were effective against Leishmania infantum, L. amazonensis and L. braziliensis promastigote and amastigote-like forms, showing selectivity index (SI) of 25.1, 18.2 and 22.9, respectively, when 3s was used against promastigotes, and of 45.2, 7.5 and 15.0, respectively, against amastigote-like stage. Using the compound 3t, SI values were 45.2, 53.0 and 80.0, respectively, against promastigotes, and of 35.9, 46.0 and 58.4, respectively, against amastigote-like forms. Amphotericin B (AmpB) showed SI values of 5.0, 7.5 and 15.0, respectively, against promastigotes, and of 3.8, 5.0 and 7.5, respectively, against amastigote-like stage. The treatment of infected macrophages and inhibition of the infection upon pre-incubation with the molecules showed that they were effective in reducing the infection degree and inhibiting the infection in pre-incubated parasites, respectively, as compared to data obtained using AmpB. The mechanism of action of 3s and 3t was evaluated in L. infantum, revealing that both 3s and 3t altered the parasite mitochondrial membrane potential leading to reactive oxygen species production, increase in lipid corps and changes in the cell cycle, causing the parasite' death. A preliminary assay using the cell culture supernatant from treated and infected macrophages showed that 3s and 3t induced higher IL-12 and lower IL-10 values; suggesting the development of an in vitro Th1-type response in the treated cells. In this context, data indicated that 3s and 3t could be considered therapeutic agents to be tested in future studies against leishmaniasis.

Isolation and Chemical Characterization of Antifungal, Antioxidant, and Anti-Inflammatory Compounds from Centrosema coriaceum using GC/MS, UFLC-QTOF-MS, and FACE.

In recent years, natural products with biological activities have been increasingly researched. The elucidation of phytoconstituents is necessary for the development of drugs as a natural alternative for the treatment of various diseases. The work aimed to evaluate in vitro and in silico bioactivities of hexane (CCHE) and methanol (CCME) fractions of ethanolic extract from Centrosema coriaceum Benth (Fabaceae) leaves and elucidate their phytoconstituents. CCHE and CCME showed antifungal activity for Candida glabrata (MIC of 1000 µg/mL) with fungistatic effect and action in cell envelope by sorbitol and ergosterol assays. CCHE and CCME presented promising antioxidant activity against the DPPH radical with IC50 of 13.61±0.50 and 6.31±0.40 µg/mL, respectively, and relative antioxidant activity (RAA%) of 45.77±3.61/ 28.53±2.25 % for CCHE and 82.18±2.25/51.99±3.23 % for CCME when compared to rutin and quercetin, respectively. Moreover, these fractions demonstrated promising results for the inhibition of lipid peroxidation by ß-carotene/linoleic acid assay. For anti-inflammatory and cytotoxicity activities, CCHE and CCME significantly inhibited the production of nitric oxide and TNF-α, without toxicity on murine intraperitoneal macrophages, respectively. Esters, alkanes, steroids, tocopherols, and terpenes were identified in CCHE by GC/MS. Flavonoids, phenolic acids, and disaccharides were detected in CCME by UFLC-QTOF-MS and FACE. Furthermore, rutin was purified from CCME. In silico predictions evidenced that compounds present in both fractions have high affinity to the fungal membrane besides antioxidant and anti-inflammatory activities. Based on these observations, CCHE and CCME have a noteworthy potential for the design of novel antifungal and anti-inflammatory agents that should be explored in future studies.

Effects of a Serine Protease Inhibitor N-p-Tosyl-L-phenylalanine Chloromethyl Ketone (TPCK) on Leishmania amazonensis and Leishmania infantum.

Studies have previously demonstrated the importance of serine proteases in Leishmania. A well-known serine protease inhibitor, TPCK, was used in the present study to evaluate its in vitro and in vivo antileishmanial effects and determine its mechanism of action. Despite slight toxicity against mammalian cells (CC50 = 138.8 µM), TPCK was selective for the parasite due to significant activity against L. amazonensis and L. infantum promastigote forms (IC50 = 14.6 and 31.7 µM for L. amazonensis PH8 and Josefa strains, respectively, and 11.3 µM for L. infantum) and intracellular amastigotes (IC50 values = 14.2 and 16.6 µM for PH8 and Josefa strains, respectively, and 21.7 µM for L. infantum). Leishmania parasites treated with TPCK presented mitochondrial alterations, oxidative stress, modifications in lipid content, flagellar alterations, and cytoplasmic vacuoles, all of which are factors that could be considered as contributing to the death of the parasites. Furthermore, BALB/c mice infected with L. amazonensis and treated with TPCK had a reduction in lesion size and parasite loads in the footpad and spleen. In BALB/c mice infected with L. infantum, TPCK also caused a reduction in the parasite loads in the liver and spleen. Therefore, we highlight the antileishmanial effect of the assessed serine protease inhibitor, proposing a potential therapeutic target in Leishmania as well as a possible new alternative treatment for leishmaniasis.

Pharmacological investigation of antioxidant and anti-inflammatory activities of aqueous extract from Mitracarpus frigidus (Rubiaceae).

OBJECTIVES: This study aimed to evaluate the potential of aqueous extract from Mitracarpus frigidus aerial parts (MFAq) in the treatment of inflammation and oxidative stress, as well as to characterize its chemical constituents. METHODS: Total phenolic and flavonoid contents were determined, and phytoconstituents were detected by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-QTOF-MS). The antioxidant activity was evaluated by DPPH, TAC and ß-carotene/linoleic acid assays. In-vitro anti-inflammatory activity, cell viability and cell cycle were performed in J774A.1 cell line. In-vivo anti-inflammatory activity was investigated by two ear oedema assays (croton oil and phenol). KEY FINDINGS: Chlorogenic acid, clarinoside, quercetin-hexosylpentoside, rutin, kaempferol-3-O-rutinoside, kaempferol-rhamnosylhexoside, quercetin-pentosylrhamnosylhexoside, harounoside, 2-azaanthraquinone and sucrose were identified by UFLC-QTOF-MS. MFAq showed antioxidant activity, which was positively correlated to the content of phenolic compounds. MFAq significantly inhibited the production of nitric oxide, did not decrease viability in MTT assay (all concentrations) and showed no changes in membrane permeability and cell cycle of J774A.1 cell line. Furthermore, MFAq showed a reduction in ear oedema in all tested doses. CONCLUSION: MFAq was effective in some antioxidant and inflammatory parameters, in the experimental conditions that were used in the study. This is the first report of chemical composition and bioactivities from this extract.

Toll-like receptor-7/8 agonist kill Leishmania amazonensis by acting as pro-oxidant and pro-inflammatory agent.

OBJECTIVES: Evaluation of the anti-Leishmanial activity of imidazoquinoline-based TLR7/8 agonists. METHODS: TLR7/8-active imidazoquinolines (2 and 3) were synthesized and assessed for activity against Leishmania amazonensis-intracellular amastigotes using mouse peritoneal macrophages. The production of reactive oxygen species (ROS), nitric oxide (NO) and cytokines was determined in infected and non-infected macrophages. KEY FINDINGS: The imidazoquinolines, 2 and 3, were primarily agonists of TLR7 with compound 3 also showing modest TLR8 activity. Docking studies showed them to occupy the same binding pocket on TLR7 and 8 as the known agonists, imiquimod and resiquimod. Compounds 2 and 3 inhibited the growth of L. amazonensis-intracellular amastigotes with the most potent compound (3, IC50 = 5.93 µM) having an IC50 value close to miltefosine (IC50 = 4.05 µM), a known anti-Leishmanial drug. Compound 3 induced macrophages to produce ROS, NO and inflammatory cytokines that likely explain the anti-Leishmanial effects. CONCLUSIONS: This study shows that activating TLR7 using compounds 2 or 3 induces anti-Leishmanial activity associated with induction of free radicals and inflammatory cytokines able to kill the parasites. While 2 and 3 had a very narrow cytotoxicity window for macrophages, this identifies the possibility to further develop this chemical scaffold to less cytotoxic TLR7/8 agonist for potential use as anti-Leishmanial drug.

Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.

In vitro and in vivo antileishmanial activity of ß-acetyl-digitoxin, a cardenolide of Digitalis lanata potentially useful to treat visceral leishmaniasis.

Current treatments of visceral leishmaniasis face limitations due to drug side effects and/or high cost, along with the emergence of parasite resistance. Novel and low-cost antileishmanial agents are therefore required. We report herein the antileishmanial activity of ß-acetyl-digitoxin (b-AD), a cardenolide isolated from Digitalis lanata leaves, assayed in vitro and in vivo against Leishmania infantum. Results showed direct action of b-AD against parasites, as well as efficacy for the treatment of Leishmania-infected macrophages. In vivo experiments using b-AD-containing Pluronic® F127 polymeric micelles (b-AD/Mic) to treat L. infantum-infected mice showed that this composition reduced the parasite load in distinct organs in more significant levels. It also induced the development of anti-parasite Th1-type immunity, attested by high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and specific IgG2a antibodies, in addition to low IL-4 and IL-10 contents, along with higher IFN-γ-producing CD4+ and CD8+ T-cell frequency. Furthermore, low toxicity was found in the organs of the treated animals. Comparing the therapeutic effect between the treatments, b-AD/Mic was the most effective in protecting animals against infection, when compared to the other groups including miltefosine used as a drug control. Data found 15 days after treatment were similar to those obtained one day post-therapy. In conclusion, the results obtained suggest that b-AD/Mic is a promising antileishmanial agent and deserves further studies to investigate its potential to treat visceral leishmaniasis.

TITLE: Activité antileishmaniale in vitro et in vivo de la ß-acétyl-digitoxine, un cardénolide de Digitalis lanata potentiellement utile pour traiter la leishmaniose viscérale. ABSTRACT: Les traitements actuels de la leishmaniose viscérale font face à des limitations dues aux effets secondaires des médicaments et/ou à leur coût élevé, ainsi qu'à l'émergence d'une résistance parasitaire. Des agents antileishmaniaux nouveaux et peu coûteux sont donc nécessaires. Nous rapportons ici l'activité antileishmaniale de la ß-acétyl-digitoxine (b-AD), un cardénolide isolé à partir de feuilles de Digitalis lanata, mesurée in vitro et in vivo contre Leishmania infantum. Les résultats ont montré une action directe de la b-AD contre les parasites, ainsi qu'une efficacité pour le traitement des macrophages infectés par Leishmania. Des expériences in vivo utilisant des micelles polymériques Pluronic® F127 contenant de la b-AD (b-AD/Mic) pour traiter des souris infectées par L. infantum ont montré que cette composition réduisait à des niveaux plus significatifs la charge parasitaire dans différents organes, ainsi que le développement d'une immunité antiparasitaire de type Th1, attestée par les taux élevés d'IFN-γ, d'IL-12, de TNF-α, de GM-CSF, de nitrite et d'anticorps IgG2a spécifiques, en plus des faibles taux d'IL-4 et IL-10, ainsi qu'une fréquence plus élevée des cellules T CD4+ and CD8+ productrices d' IFN-γ. De plus, une faible toxicité a été trouvée dans les organes des animaux traités. En comparant l'effet thérapeutique des traitements, b-AD/Mic était le plus efficace pour protéger les animaux contre l'infection, par rapport aux autres groupes comprenant la miltefosine utilisée comme contrôle médicamenteux. Les données trouvées 15 jours après le traitement étaient similaires à celles obtenues un jour après le traitement. En conclusion, les résultats obtenus suggèrent que b-AD/Mic est un agent antileishmanial prometteur et mérite des études supplémentaires pour étudier son potentiel à traiter la leishmaniose viscérale.

Ivermectin presents effective and selective antileishmanial activity in vitro and in vivo against Leishmania infantum and is therapeutic against visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.

Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 µg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 µg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum-infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.

In vitro and in vivo evaluation of cnicin from blessed thistle (Centaurea benedicta) and its inclusion complexes with cyclodextrins against Schistosoma mansoni.

Schistosomiasis, caused by a blood fluke of the genus Schistosoma, afflicts over 230 million people worldwide. Treatment of the disease relies on just one drug, praziquantel. Cnicin (Cn) is the sesquiterpene lactone found in blessed thistle (Centaurea benedicta) that showed antiparasitic activities but has not been evaluated against Schistosoma. However, cnicin has poor water solubility, which may limit its antiparasitic activities. To overcome these restrictions, inclusion complexes with cyclodextrins may be used. In this work, we evaluated the in vitro and in vivo antischistosomal activities of cnicin and its complexes with ß-cyclodextrin (ßCD) and 2-hydroxypropyl-ß-cyclodextrin (HPßCD) against Schistosoma mansoni. Cnicin were isolated from C. benedicta by chromatographic fractionation. Complexes formed by cnicin and ßCD (Cn/ßCD), as well as by cnicin and HPßCD (Cn/HPßCD), were prepared by coprecipitation and characterized. In vitro schistosomicidal assays were used to evaluate the effects of cnicin and its complexes on adult schistosomes, while the in vivo antischistosomal assays were evaluated by oral and intraperitoneal routes. Results showed that cnicin caused mortality and tegumental alterations in adult schistosomes in vitro, also showing in vivo efficacy after intraperitoneal administration. The oral treatment with cnicin or Cn/ßCD showed no significant worm reductions in a mouse model of schistosomiasis. In contrast, Cn/HPßCD complex, when orally or intraperitoneally administered to S. mansoni-infected mice, decreased the total worm load, and markedly reduced the number of eggs, showing high in vivo antischistosomal effectiveness. Permeability studies, using Nile red, indicated that HPßCD complex may reach the tegument of adult schistosomes in vivo. These results demonstrated the antischistosomal potential of cnicin in preparations with HPßCD.

Antischistosomal properties of aurone derivatives against juvenile and adult worms of Schistosoma mansoni.

Schistosomiasis is a neglected disease caused by helminth flatworms of the genus Schistosoma, affecting over 240 million people in more than 70 countries. The treatment relies on a single drug, praziquantel, making urgent the discovery of new compounds. Aurones are a natural type of flavonoids that display interesting pharmacological activities, particularly as chemotherapeutic agents against parasites. In pursuit of treatment alternatives, the present work conducted an in vitro and in vivo antischistosomal investigation with aurone derivatives against Schistosoma mansoni. After preparation of aurone derivatives and their in vitro evaluation on adult schistosomes, the three most active aurones were evaluated in cytotoxicity and haemolytic assays, as well as in confocal laser-scanning microscope studies, showing tegumental damage in parasites in a concentration-dependent manner with no haemolytic or cytotoxic potential toward mammalian cells. In a mouse model of schistosomiasis, at a single oral dose of 400 mg/kg, the selected aurones showed worm burden reductions of 35% to 65.0% and egg reductions of 25% to 70.0%. The most active thiophenyl aurone derivative 18, unlike PZQ, had efficacy in mice harboring juvenile S. mansoni, also showing significant inhibition of oviposition by parasites, giving support for the antiparasitic potential of aurones as lead compounds for novel antischistosomal drugs.

Antileishmanial compounds from Connarus suberosus: Metabolomics, isolation and mechanism of action.

Leishmaniasis is a disease impacting public health worldwide due to its high incidence, morbidity and mortality. Available treatments are costly, lengthy and toxic, not to mention the problem of parasite resistance. The development of alternative treatments is warranted and natural products demonstrate promising activity. This study investigated the activity of Connarus suberosus extracts and compounds against Leishmania species. Several C. suberosus extracts were tested against L. amazonensis promastigotes. Active and inactive extracts were analyzed by UHPLC-MS and data evaluated using a metabolomics platform, revealing an unknown neoflavonoid (connarin, 3), isolated together with the pterocarpans: hemileiocarpin (1) and leiocarpin (2). The aforementioned compounds (1-3), together with the benzoquinones: rapanone (4), embelin (5) and suberonone (6) previously isolated by our group from the same species, were tested against: (i) L. amazonensis and L. infantum promastigotes, and (ii) L. amazonensis intracellular amastigotes, with the most active compound (3) also tested against L. infantum amastigotes. Cytotoxicity against murine peritoneal macrophages was also investigated. Compounds 2 and 3 presented an IC50 33.8 µM and 11.4 µM for L. amazonensis promastigotes; and 44.3 µM and 13.3 µM for L. infantum promastigotes, respectively. For L. amazonensis amastigotes, the IC50 of 2 was 20.4 µM with a selectivity index (SI) of 5.7, while the IC50 of 3 was 2.9 µM with an SI of 6.3. For L. infantum amastigotes, the IC50 of 3 was 7.7 µM. Compounds 2 and 3 presented activity comparable with the miltefosine positive control, with compound 3 found to be 2-4 times more active than the positive control, depending on the Leishmania species and form. The extracts and isolated compounds showed moderate toxicity against macrophages. Compounds 2 and 3 altered the mitochondrial membrane potential (ΔΨm) and neutral lipid body accumulation, while 2 also impacted plasma membrane permeabilization, culminating in cellular disorder and parasite death. Transmission electron microscopy of L. amazonensis promastigotes treated with compound 3 confirmed the presence of lipid bodies. Leiocarpin (2) and connarin (3) demonstrated antileishmanial activity. This study provides knowledge of natural products with antileishmanial activity, paving the way for prototype development to fight this neglected tropical disease.

Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.

AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.

In vitro Assessment of Camphor Hydrazone Derivatives as an Agent Against Leishmania amazonensis.

PURPOSE: Due to serious problems with the treatment of leishmaniasis all around the world, here is an urgent need in the search for new drugs that are more effective and safer for the treatment of the various forms of leishmaniasis. Actual therapy is limited and lacks sufficient efficacy due to incomplete elimination of the parasites form of patients. In this sense, we decided to evaluate, by first-time, a series of seventeen camphor hydrazone derivatives (2a-2p) against Leishmania amazonensis. METHODS: The compounds previously synthesized from camphor, an abundant natural compound, were evaluated in vitro against the extra and intracellular forms of Leishmania amazonensis, and murine macrophages. RESULTS: The majority of compounds, fourteen, displayed activity against the intracellular form of the parasite (amastigote) with IC50 values ranging from 21.78 to 58.23 µM, being six compounds active for both forms of the parasite. The compound 2i exhibited higher activity against the amastigote form with the value of IC50 (21.78 µM) close to standard utilized miltefosine (12.74 µM) and selectivity index of at least 6.9. Six compounds displayed activity against promastigote form of Leishmania amazonensis 2g, 2j-2n (41.17-69.59 µM), with the compound 2m being the more active with IC50 = 41.17 µM, 1.9 times less active than the reference drug (IC50 = 21.39 µM). The compound 2m was the more selective to this form, with a selectivity index of at least 3.6. All the compounds were non-cytotoxic to macrophages. CONCLUSIONS: Most compounds showed activity against amastigote form of Leishmania amazonensis, being that they were not cytotoxic to macrophage at the maximum tested concentration, showing the selective property of these compounds. Since amastigotes are the parasite stages that cause the disease in humans, these results highlight the antileishmanial effect of the compounds. This study indicates the possible development of candidates to leishmanicidal drugs from an abundant natural compound of easy access.

A chloroquinoline derivate presents effective in vitro and in vivo antileishmanial activity against Leishmania species that cause tegumentary and visceral leishmaniasis.

The identification of new therapeutics to treat leishmaniasis is desirable, since available drugs are toxic and present high cost and/or poor availability. Therefore, the discovery of safer, more effective and selective pharmaceutical options is of utmost importance. Efforts towards the development of new candidates based on molecule analogs with known biological functions have been an interesting and cost-effective strategy. In this context, quinoline derivatives have proven to be effective biological activities against distinct diseases. In the present study, a new chloroquinoline derivate, AM1009, was in vitro tested against two Leishmania species that cause leishmaniasis. The present study analyzed the necessary inhibitory concentration to preclude 50% of the Leishmania promastigotes and axenic amastigotes (EC50 value), as well as the inhibitory concentrations to preclude 50% of the murine macrophages and human red blood cells (CC50 and RBC50 values, respectively). In addition, the treatment of infected macrophages and the inhibition of infection using pre-treated parasites were also investigated, as was the mechanism of action of the molecule in L. amazonensis. To investigate the in vivo therapeutic effect, BALB/c mice were infected with L. amazonensis and later treated with AM1009. Parasitological and immunological parameters were also evaluated. Clioquinol, a known antileishmanial quinoline derivate, and amphotericin B (AmpB), were used as molecule and drug controls, respectively. Results in both in vitro and in vivo experiments showed a better and more selective action of AM1009 to kill the in vitro parasites, as well as in treating infected mice, when compared to results obtained using clioquinol or AmpB. AM1009-treated animals presented significantly lower average lesion diameter and parasite burden in the infected tissue and organs evaluated in this study, as well as a more polarized antileishmanial Th1 immune response and low renal and hepatic toxicity. This result suggests that AM1009 should be considered a possible therapeutic target to be evaluated in future studies for treatment against leishmaniasis.

Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.

Insights about resveratrol analogs against trypanothione reductase of Leishmania braziliensis: Molecular modeling, computational docking and in vitro antileishmanial studies.

In this work, we combined molecular modeling, computational docking and in vitro analysis to explore the antileishmanial effect of some resveratrol analogs (ResAn), focusing on their pro-oxidant effect. The molecular target was the trypanothione reductase of Leishmania braziliensis (LbTryR), an essential component of the antioxidant defenses in trypanosomatid parasites. Three-dimensional structures of LbTryR were modeled and molecular docking studies of ResAn1-5 compounds showed the following affinity: ResAn1 > ResAn2 > ResAn4 > ResAn5 > ResAn3. Positive correlation was observed between these compounds' affinity to the LbTryR and the IC50 values against Leishmania sp (ResAn1 < ResAn2 < ResAn4), which allows for TryR being considered an important target for them. As the compound ResAn1 showed the best antileishmanial activity, and docking studies showed its high affinity for NADP binding site (NS) of TryR, plus having been able to induce ROS production in L. braziliensis promastigotes treated, ResAn1 probably occupies NS interfering in the electron transfer processes responsible for the catalytic reaction. The in silico prediction of ADMET properties suggests that ResAn1 may be a promising drug candidate with properties to cross biological membranes and high gastrointestinal absorption, not violating Lipinski's rules. Ultimately, the antileishmanial effect of ResAn can be associated with a pro-oxidant effect which, in turn, can be exploited as an antimicrobial agent. Communicated by Ramaswamy H. Sarma.

In vivo antileishmanial efficacy of a naphthoquinone derivate incorporated into a Pluronic® F127-based polymeric micelle system against Leishmania amazonensis infection.

New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania parasites, but low toxicity in mammalian hosts. Flau-A is a naphthoquinone derivative recently showed to presents an in vitro effective action against Leishmania amazonensis and L. infantum species. In the present work, the in vivo efficacy of Flau-A, which was incorporated into a Poloxamer 407-based micelle system, was evaluated in a murine model against L. amazonensis infection. Amphotericin B (AmB) and Ambisome® were used as controls. The animals were infected and later treated with the compounds. Thirty days after the treatment, parasitological and immunological parameters were evaluated. Results showed that AmB, Ambisome®, Flau-A or Flau-A/M-treated animals presented significantly lower average lesion diameter and parasite burden in tissue and organs evaluated, when compared to the control (saline and micelle) groups. Flau-A or Flau-A/M-treated mice were those presenting the most significant reductions in the parasite burden, when compared to the others. These animals developed also a more polarized antileishmanial Th1 immune response, which was based on significantly higher levels of IFN-γ, IL-12, TNF-α, GM-CSF, and parasite-specific IgG2a isotype; associated with low levels of IL-4, IL-10, and IgG1 antibody. The absence of toxicity was found in these animals, although mice receiving AmB have showed high levels of renal and hepatic damage markers. In conclusion, results suggested that the Flau-A/M compound may be considered as a possible therapeutic target to be evaluated against human leishmaniasis.