Investigation of Calcium Channel Blockers as Antiprotozoal Agents and Their Interference in the Metabolism of Leishmania (L.) infantum

Leishmaniasis and Chagas disease are neglected parasitic diseases endemic in developing countries; efforts to find new therapies remain a priority. Calcium channel blockers (CCBs) are drugs in clinical use for hypertension and other heart pathologies. Based on previous reports about the antileishmanial activity of dihydropyridine-CCBs, this work aimed to investigate whether the in vitro anti-Leishmania infantum and anti-Trypanosoma cruzi activities of this therapeutic class would be shared by other non-dihydropyridine-CCBs. Except for amrinone, our results demonstrated antiprotozoal activity for fendiline, mibefradil, and lidoflazine, with IC50 values in a range between 2 and 16 μM and Selectivity Index between 4 and 10. Fendiline demonstrated depolarization of mitochondrial membrane potential, with increased reactive oxygen species production in amlodipine and fendiline treated Leishmania, but without plasma membrane disruption. Finally, in vitro combinations of amphotericin B, miltefosine, and pentamidine against L. infantum showed in isobolograms an additive interaction when these drugs were combined with fendiline, resulting in overall mean sum of fractional inhibitory concentrations between 0.99 and 1.10. These data demonstrated that non-dihydropyridine-CCBs present antiprotozoal activity and could be useful candidates for future in vivo efficacy studies against Leishmaniasis and Chagas’ disease

Antitrypanosomal Activity of a Diterpene and Lignans Isolated from Aristolochia cymbifera

Bioguided fractionation of extract from the leaves of Aristolochia cymbifera led to the isolation of the furofuran lignans fargesin, epieudesmin, and sesamin; the dibenzylbutyrolactone lignans hinokinin and kusunokinin; and an ent-labdane diterpene named copalic acid. Our data demonstrated that copalic acid and kusunokinin were the most active compounds against trypomas-tigotes of Trypanosoma cruzi. Additionally, copalic acid demonstrated the highest parasite selectivity as a result of low toxicity to mammalian cells, despite a considerable hemolytic activity at higher concentrations. Among the isolated compounds, kusunokinin could be considered the most promising candidate, as it displayed significant activity against intracellular amastigotes (IC(50) = 17 mu M) and trypomastigotes (IC(50) = 51 mu M) without hemolytic activity. Fargesin, hinokinin, epieudesmin, and sesamin were also effective against trypomastigotes, but these compounds were highly toxic to mammalian cells and no parasite selectivity could be identified. the need for novel drugs for American trypanosomiasis is evident, and these secondary metabolites from A. cymbifera represent a useful tool for drug design.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Ciencias Exatas & Terra, BR-09972270 Diadema São Paulo, BrazilInst Adolfo Lutz Registro, Dept Parasitol, Lab Toxinol Aplicada, São Paulo, BrazilInst Plantarum Estudos Flora, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, BR-09972270 Diadema São Paulo, BrazilFAPESP: 2008/11496-9CNPq: 473405/2008-3Web of Scienc

Nesterov-aided Stochastic Gradient Methods using Laplace Approximation for Bayesian Design Optimization

Finding the best setup for experiments is the primary concern for Optimal Experimental Design (OED). Here, we focus on the Bayesian experimental design problem of finding the setup that maximizes the Shannon expected information gain. We use the stochastic gradient descent and its accelerated counterpart, which employs Nesterov's method, to solve the optimization problem in OED. We adapt a restart technique, originally proposed for the acceleration in deterministic optimization, to improve stochastic optimization methods. We combine these optimization methods with three estimators of the objective function: the double-loop Monte Carlo estimator (DLMC), the Monte Carlo estimator using the Laplace approximation for the posterior distribution (MCLA) and the double-loop Monte Carlo estimator with Laplace-based importance sampling (DLMCIS). Using stochastic gradient methods and Laplace-based estimators together allows us to use expensive and complex models, such as those that require solving partial differential equations (PDEs). From a theoretical viewpoint, we derive an explicit formula to compute the gradient estimator of the Monte Carlo methods, including MCLA and DLMCIS. From a computational standpoint, we study four examples: three based on analytical functions and one using the finite element method. The last example is an electrical impedance tomography experiment based on the complete electrode model. In these examples, the accelerated stochastic gradient descent method using MCLA converges to local maxima with up to five orders of magnitude fewer model evaluations than gradient descent with DLMC.Comment: 36 pages, 14 figure

Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response

Abstract\ud \ud Background\ud \ud Apis mellifera venom, which has already been recommended as an alternative anti-inflammatory treatment, may be also considered an important source of candidate molecules for biotechnological and biomedical uses, such as the treatment of parasitic diseases.\ud \ud \ud Methods\ud Africanized honeybee venom from Apis mellifera was fractionated by RP-C18-HPLC and the obtained melittin was incubated with promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Cytotoxicity to mice peritoneal macrophages was evaluated through mitochondrial oxidative activity. The production of anti- and pro-inflammatory cytokines, NO and H2O2 by macrophages was determined.\ud \ud \ud Results\ud Promastigotes and intracellular amastigotes were susceptible to melittin (IC50 28.3 μg.mL−1 and 1.4 μg.mL−1, respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 μg.mL−1. Uninfected macrophages treated with melittin increased the production of IL-10, TNF-α, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-α, NO and H2O2.\ud \ud \ud Conclusions\ud The results showed that melittin acts in vitro against promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Furthermore, they can act indirectly on intracellular amastigotes through a macrophage immunomodulatory effect.The authors would like to thank the State of São Paulo Research\ud Foundation (FAPESP, proc. n. 2011/23236-4 and n. 2009/53846-9), the National Council for Scientific and Technological Development (CNPq, proc. n. 563582/2010-3), the Coordination for the Improvement of Higher Education Personnel (CAPES, AUXPE Toxinologia 1219/2011, proc. n. 23038.000823/2011-21 and AUXPE proc. n. 23038.005536/2012-31) and FINEP (protocol number 01.12.0450.01). DCP (306066/2011-4), RSFJr and AGT are CNPq research fellows

Evaluation of antileishmanial potential of the antidepressant escitalopram in Leishmania infantum

Neglected tropical diseases (NTDs) such as visceral leishmaniasis (VL) present a limited and toxic therapeutic arsenal, and drug repositioning represents a safe and cost-effective approach. In this work, we investigated the antileishmanial potential and the mechanism of lethal action of the antidepressant escitalopram. The efficacy of escitalopram was determined ex-vivo using the intracellular Leishmania (L.) infantum amastigote model and the mammalian cytotoxicity was determined by the colorimetric MTT assay. The cellular and molecular alterations induced by the drug were investigated using spectrofluorimetry, a luminescence assay and flow cytometry. Our data revealed that escitalopram was active and selective against L. infantum parasites, with an IC50 value of 25 µM and a 50% cytotoxic concentration (CC50) of 184 µM. By using the fluorescent probes SYTOX® Green and DiSBAC2(3), the drug showed no alterations in the plasma membrane permeability nor in the electric potential of the membrane (∆ψp); however, after a short-time incubation, the drug caused a dose-dependent up-regulation of the calcium levels, leading to the depolarization of the mitochondrial membrane potential (∆ψm) and a reduction of the ATP levels. No up-regulation of reactive oxygen (ROS) was observed. In the cell cycle analysis, escitalopram induced a dose-dependent increase of the parasites at the sub G0/G1 stage, representing fragmented DNA. Escitalopram presented a selective antileishmanial activity, with disruption of single mitochondrion and interference in the cell cycle. Approved drugs such as escitalopram may represent a promising approach for NTDs and can be considered in future animal efficacy studies.</p

Insulin-Like Growth Factor-I Induces Arginase Activity in Leishmania amazonensis Amastigote-Infected Macrophages through a Cytokine-Independent Mechanism

Leishmania (Leishmania) amazonensis exhibits peculiarities in its interactions with hosts. Because amastigotes are the primary form associated with the progression of infection, we studied the effect of insulin-like growth factor (IGF)-I on interactions between L. (L.) amazonensis amastigotes and macrophages. Upon stimulation of infected macrophages with IGF-I, we observed decreased nitric oxide production but increased arginase expression and activity, which lead to increased parasitism. However, stimulation of amastigote-infected macrophages with IGF-I did not result in altered cytokine levels compared to unstimulated controls. Because IGF-I is present in tissue fluids and also within macrophages, we examined the possible effect of this factor on phosphatidylserine (PS) exposure on amastigotes, seen previously in tissue-derived amastigotes leading to increased parasitism. Stimulation with IGF-I induced PS exposure on amastigotes but not on promastigotes. Using a PS-liposome instead of amastigotes, we observed that the PS-liposome but not the control phosphatidylcholine-liposome led to increased arginase activity in macrophages, and this process was not blocked by anti-TGF-β antibodies. Our results suggest that in L. (L.) amazonensis amastigote-infected macrophages, IGF-I induces arginase activity directly in amastigotes and in macrophages through the induction of PS exposure on amastigotes in the latter, which could lead to the alternative activation of macrophages through cytokine-independent mechanisms

Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis

Drug delivery systems are promising pharmaceutical formulations used to improve the therapeutic index of drugs. in this study, we developed a liposomal formulation of furazolidone that targets Leishmania (Leishmania) chagasi amastigotes in a hamster model. Using laser scanning confocal microscopy, it was demonstrated that the liposomal drug co-localised with L. (L.) chagasi amastigotes within macrophages. Liposomal furazolidone administered intraperitoneally at 0.5 mg/kg for 12 consecutive days reduced spleen (74%) and liver (32%) parasite burden at a 100-fold lower dose than the free drug. Free furazolidone (50 mg/kg) also effectively reduced spleen (82.5%) and liver (85%) parasites; its in vitro activity against promastigotes and intracellular amastigotes demonstrated a high degree of parasite selectivity. Thus, furazolidone, both in the free and liposome-loaded formulation, is an effective inhibitor of L. (L.) chagasi, representing a possible cost-effective drug candidate for the treatment of visceral leishmaniasis. (C) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Inst Adolfo Lutz Registro, Dept Parasitol, Lab Appl Toxinol Antiparasit Drugs, BR-01246000 São Paulo, BrazilEscola Paulista Med, UNIFESP, Dept Microbiol Imunol & Parasitol, BR-0403902 São Paulo, BrazilUniv São Paulo, Lab Protozool, Inst Trop Med, BR-05603000 São Paulo, BrazilEscola Paulista Med, UNIFESP, Dept Microbiol Imunol & Parasitol, BR-0403902 São Paulo, BrazilCNPq: 550376/2007-0FAPESP: 2008/09260-7Web of Scienc

Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis

Drug delivery systems are promising pharmaceutical formulations used to improve the therapeutic index of drugs. In this study, we developed a liposomal formulation of furazolidone that targets Leishmania (Leishmania) chagasi amastigotes in a hamster model. Using laser scanning confocal microscopy, it was demonstrated that the liposomal drug co-localised with L. (L.) chagasi amastigotes within macrophages. Liposomal furazolidone administered intraperitoneally at 0.5 mg/kg for 12 consecutive days reduced spleen (74%) and liver (32%) parasite burden at a 100-fold lower dose than the free drug. Free furazolidone (50 mg/kg) also effectively reduced spleen (82.5%) and liver (85%) parasites; its in vitro activity against promastigotes and intracellular amastigotes demonstrated a high degree of parasite selectivity. Thus, furazolidone, both in the free and liposome-loaded formulation, is an effective inhibitor of L. (L.) chagasi, representing a possible cost-effective drug candidate for the treatment of visceral leishmaniasis. (C) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.CNPq (Conselho Nacional de Pesquisa e Desenvolvimento)[550376/2007-0]FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)[2008/09260-7