Antileishmanial Activity of 2-Methoxy-4H-spiro-[naphthalene-1,2'-oxiran]-4-one (Epoxymethoxy-lawsone): A Promising New Drug Candidate for Leishmaniasis Treatment.

Epoxymethoxylawsone is a naphthoquinone derivative promising as drug candidate for the treatment of leishmaniases. In the present work the effectiveness of epoxymethoxylawsone, and meglumine antimoniate on Leishmania (Leishmania) amazonensis parasites and on mice paw lesions of infected BALB/c mice was assessed. In an intracellular amastigotes assay, the half-maximal inhibitory concentration (IC50) value for epoxymethoxylawsone was slightly higher (1.7-fold) than that found for meglumine antimoniate. The efficacy of both drugs became more evident after 48 h of exposure when either the oxirane compound and reference drug reached 18-fold and 7.4-fold lower IC50 values (0.40 ± 0.001 µM and 0.60 ± 0.02 µM), respectively. Promastigotes were also affected by epoxymethoxylawsone after 24 h of incubation (IC50 = 45.45 ± 5.0 µM), but with IC50 6-fold higher than those found for intracellular amastigotes. Cytotoxicity analysis revealed that epoxymethoxylawsone (CC50 = 40.05 ± µM) has 1.7-fold higher effects than meglumine antimoniate (CC50 = 24.14 ± 2.6 µM). Treatment of the paw lesion in infected BALB/c mice with epoxymethoxy-lawsone led to a significant 27% reduction (p < 0.05) of the lesion size, for all administrated doses, compared to the control group. Lesion reduction was also detected after mice treatment with meglumine antimoniate, reaching 31.0% (0.23 mg of Sb(V)/Kg/day and 2.27 mg of Sb(V)/Kg/day) and 64.0% (22.7 mg of Sb(V)/Kg/day). In addition, mice lesion ultrastructural changes were evidenced in amastigotes. The set of data gathered here indicate that epoxymethoxylawsone has pronounced effects on parasites and merits furthering to the preclinical stage.

Epoxy-α-lapachone has in vitro and in vivo anti-leishmania (Leishmania) amazonensis effects and inhibits serine proteinase activity in this parasite.

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm(2)), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm(2)) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm(2)). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite.

Evidences for leishmanicidal activity of the naphthoquinone derivative epoxy-α-lapachone.

In this work, we analyze the leishmanicidal effects of epoxy-α-lapachone on Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis. Promasigotes and amastigotes (inhabiting human macrophages) from both species were assayed to verify the compound's activity over the distinct morphological stages. The incubation with epoxy-α-lapachone led to a significant decrease in the numbers of promastigotes from both species in the cultures, in a dose-and time-dependent fashion. The survival of amastigotes inhabiting human macrophages was also drastically affected by the compound, as shown by the variations in the endocytic index. Our results indicate that the epoxy-α-lapachone has an antiparasitic effect over Leishmania in both morphological stages and may potentially affect a range of species in two distinct subgenera of this parasite.

The combination therapy of meglumine antimoniate and oxiranes (epoxy-α-lapachone and epoxymethyl-lawsone) enhance the leishmanicidal effect in mice infected by Leishmania (Leishmania) amazonensis.

Current treatment of cutaneous leishmaniasis includes pentavalent antimonials as first-line drugs, but this therapy has shown severe adverse effects. An alternative to minimize this issue is based on combination therapy scheme with other drugs. In this study we analyzed the potential of the association of meglumine antimoniate (MA) with the oxiranes epoxy-α-lapachone (LAP) or epoxymethyl-lawsone (LAW). Results demonstrated that association between these drugs enhanced leishmanicidal activity on Leishmania (Leishmania) amazonensis infection. The compounds were tested in monotherapy or in combinations (3:1; 1:1 and 1:3) and reduced intracellular parasite numbers, measured by the endocytic index, in all tested conditions. The most effective combination regimens were MA/LAP or MA/LAW in 3:1 ratio, which achieved a reduction of 98.3% and 93.6% in the endocytic index, respectively. BALB/c mice challenged with L. (L.) amazonensis showed significant reduction in lesion size and parasite load in both footpad and lymph nodes, after four weeks of treatment. Although, MA, LAP or LAW monotherapy were able to control the evolution of lesions when compared to untreated animals (30%, 40% and 40% of reduction, respectively), the combination of MA/LAP and LAW in 3:1 ratio showed better results reducing 61.7 and 54.4%, respectively. The results indicate that the association of meglumine antimoniate to oxiranes lead to an increment in the antileishmanial activity and represent a promising approach for the cutaneous leishmaniasis treatment.

Evidence for Tissue Toxicity in BALB/c Exposed to a Long-Term Treatment with Oxiranes Compared to Meglumine Antimoniate.

Leishmaniasis remains a serious public health problem in developing countries without effective control, whether by vaccination or chemotherapy. Part of the failure of leishmaniasis control is due to the lack of new less toxic and more effective drugs able to eliminate both the lesions and the parasite. Oxiranes derived from naphthoquinones now being assayed are promising drugs for the treatment of this group of diseases. The predicted pharmacokinetic properties and toxicological profiles of epoxy-α-lapachone and epoxymethoxy-lawsone have now been compared to those of meglumine antimoniate, and histological changes induced by these drugs in noninfected BALB/c mice tissues are described. Effects of these compounds on liver, kidney, lung, heart, and cerebral tissues of healthy mice were examined. The data presented show that both these oxiranes and meglumine antimoniate induce changes in all BALB/c mice tissues, with the lung, heart, and brain being the most affected. Epoxymethoxy-lawsone was the most toxic to lung tissue, while most severe damage was caused in the heart by epoxy-α-lapachone. Meglumine antimoniate caused mild-to-moderate changes in heart and lung tissues.

Interactions between 4-aminoquinoline and heme: Promising mechanism against Trypanosoma cruzi.

Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 µM), with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 µM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasite's lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies.

Metabolismo intermediário do Trypanosoma cruzi: estudo de ácidos carboxílicos e de enzimas envolvidas no metabolismo de trioses

A análise qualitativa e quantitativa de vários ácidos orgânicos intracelulares do Trypanosoma cruzi, mantido em meio BHI e durante a diferenciaçäo em TAU3AAG, foi realizada por HPLC. Esta metodologia mostrou-se eficaz na identificaçäo de ácidos orgânicos, e associada à medida da atividade de enzimas (hexoquinase, piruvato quinase e aldolase), permitiu observar que durante a diferenciaçäo em meio quimicamente definido, o ciclo de Krebs apresentou-se funcional na direçäo succinato oxaloacetato. Paralelamente, observou-se uma diminuiçäo da concentraçäo dos intermediários da via glicolítica, apesar da elevada concentraçäo de glicose extracelular. Por outro lado, o glicerato 2-fosfato, manteve-se em altas concentraçöes, durante o processo de diferenciaçäo, sugerindo um fluxo de substrato maior vindo do metabolismo das trioses-fosfato. Como se sabe que nesta fase ocorre uma mudança morfológica de reservossomos, um estudo envolvendo as enzimas TPI e GAPDH foi realizado. No primeiro caso, a enzima foi purificada e caracterizada a nível bioquímico-imunológico e no segundo foi analisado o efeito do óxido nítrico (ON) sobre a atividade enzimática. Este último estudo mostrou que o ON possui um efeito modulador sobre a atividade da GAPDH, podendo levar a morte do parasita. Este fato sugere que a oscilaçäo na concentraçäo de intermediários e enzimas metabólicas chaves, podem afetar drasticamente a viabilidade do T. cruzi, sendo a GAPDH um alvo do ON. Entretanto, como a via glicolítica apresenta-se menos intensa na forma infectante tripomastigota, questiona-se a eficácia desta enzima como alvo quimioterápico. (AU)

Metabolismo intermediário do Trypanosoma cruzi: estudo de ácidos carboxílicos e de enzimas envolvidas no metabolismo de trioses

A análise qualitativa e quantitativa de vários ácidos orgânicos intracelulares do Trypanosoma cruzi, mantido em meio BHI e durante a diferenciaçäo em TAU3AAG, foi realizada por HPLC. Esta metodologia mostrou-se eficaz na identificaçäo de ácidos orgânicos, e associada à medida da atividade de enzimas (hexoquinase, piruvato quinase e aldolase), permitiu observar que durante a diferenciaçäo em meio quimicamente definido, o ciclo de Krebs apresentou-se funcional na direçäo succinato oxaloacetato. Paralelamente, observou-se uma diminuiçäo da concentraçäo dos intermediários da via glicolítica, apesar da elevada concentraçäo de glicose extracelular. Por outro lado, o glicerato 2-fosfato, manteve-se em altas concentraçöes, durante o processo de diferenciaçäo, sugerindo um fluxo de substrato maior vindo do metabolismo das trioses-fosfato. Como se sabe que nesta fase ocorre uma mudança morfológica de reservossomos, um estudo envolvendo as enzimas TPI e GAPDH foi realizado. No primeiro caso, a enzima foi purificada e caracterizada a nível bioquímico-imunológico e no segundo foi analisado o efeito do óxido nítrico (ON) sobre a atividade enzimática. Este último estudo mostrou que o ON possui um efeito modulador sobre a atividade da GAPDH, podendo levar a morte do parasita. Este fato sugere que a oscilaçäo na concentraçäo de intermediários e enzimas metabólicas chaves, podem afetar drasticamente a viabilidade do T. cruzi, sendo a GAPDH um alvo do ON. Entretanto, como a via glicolítica apresenta-se menos intensa na forma infectante tripomastigota, questiona-se a eficácia desta enzima como alvo quimioterápico. (AU)