Rodrigo L.O.R. Cunha

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4 associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas disease.

In vitro and in vivo activity of a palladacycle complex on Leishmania (Leishmania) amazonensis.

Background Antitumor cyclopalladated complexes with low toxicity to laboratory animals have shown leishmanicidal effect. These findings stimulated us to test the leishmanicidal property of one palladacycle compound called DPPE 1.2 on Leishmania (Leishmania) amazonensis, an agent of simple and diffuse forms of cutaneous leishmaniasis in the Amazon region, Brazil. Methodology/Principal Findings Promastigotes of L. (L.) amazonensis and infected bone marrow-derived macrophages were treated with different concentrations of DPPE 1.2. In in vivo assays foot lesions of L. (L.) amazonensis-infected BALB/c mice were injected subcutaneously with DPPE 1.2 and control animals received either Glucantime or PBS. The effect of DPPE 1.2 on cathepsin B activity of L. (L.) amazonensis amastigotes was assayed spectrofluorometrically by use of fluorogenic substrates. The main findings were: 1) axenic L. (L.) amazonensis promastigotes were destroyed by nanomolar concentrations of DPPE 1.2 (IC50u200a=u200a2.13 nM); 2) intracellular parasites were killed by DPPE 1.2 (IC50u200a=u200a128.35 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50u200a=u200a1,267 nM); 3) one month after intralesional injection of DPPE 1.2 infected BALB/c mice showed a significant decrease of foot lesion size and a reduction of 97% of parasite burdens when compared to controls that received PBS; 4) DPPE 1.2 inhibited the cysteine protease activity of L. (L.) amazonensis amastigotes and more significantly the cathepsin B activity. Conclusions/Significance The present results demonstrated that DPPE 1.2 can destroy L. (L.) amazonensis in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support the potential use of DPPE 1.2 as an alternative choice for the chemotherapy of leishmaniasis.

Cytotoxicity of phenothiazine derivatives associated with mitochondrial dysfunction: a structure-activity investigation.

Phenothiazine derivatives are neuroleptic drugs used in the treatment of schizophrenia and anxiety. Several side effects are described for these drugs, including hepatotoxicity, which may be related to their cytotoxic activity. Working with isolated rat liver mitochondria, we previously showed that phenothiazine derivatives induced the mitochondrial permeability transition associated with cytochrome c release. Since the mitochondrial permeabilization process plays a central role in cell death, the aim of this work was to evaluate the effects of five phenothiazine derivatives (chlorpromazine, fluphenazine, thioridazine, trifluoperazine, and triflupromazine) on the viability of hepatoma tissue culture (HTC) cells to establish the structural requirements for cytotoxicity. All phenothiazine derivatives decreased the viability of the HTC cells in a concentration-dependent manner and exhibited different cytotoxic potencies. The EC50 values ranged from 45 to 125 μM, with the piperidinic derivative thioridazine displaying the most cytotoxicity, followed by the piperazinic and aliphatic derivatives. The addition of the phenothiazine derivatives to cell suspensions resulted in significant morphological changes and plasma membrane permeabilization. Octanol/water partition studies revealed that these drugs partitioned preferentially to the apolar phase, even at low pH values (≤4.5). Also, structural and electronic properties were calculated employing density functional theory. Interestingly, the phenothiazine derivatives promoted an immediate dissipation of the mitochondrial transmembrane potential in HTC cells, and the EC50 values were closely correlated with those obtained in cell viability assays, as well as the EC50 for swelling in isolated mitochondria. These results significantly contribute to improving our understanding of the specific structural requirements of the phenothiazine derivatives to induce cell death and suggest the involvement of the mitochondrial permeability transition in phenothiazine-induced cytotoxicity in HTC cells.

Endovascular repair of thoracoabdominal aneurysms: results of the first 48 cases

BACKGROUNDnIn 2006, we began our experience with a novel technology for fully endovascular thoracoabdominal aneurysm repair, based on a custom-made, branched stent graft design. After 48 cases, we have learned and achieved substantial progress both in technical and in clinical skills. This paper describes the partial results of this ongoing experience.nnnMETHODSnPatients in this series were selected for the presence of thoracoabdominal aortic aneurysms, with or without dissection, which was present in one patient. The observation of extensive anatomical variations in several patients prompted changes in many of the basic stent graft configurations, which are also described.nnnRESULTSnBetween August 2006 and June 2012, 48 patients were treated consecutively with custom-made branch stent grafts. The five patients with the longest follow-up available so far are at 71, 65, 60, 54 and 51 months post-procedure. The operative mortality rate, defined as death during or within a month of surgical hospitalization, was 21% (10 patients); each case is described herein. During postoperative follow up, nine patients died from causes not directly related to aneurysmal disease, at 3, 18, 20, 22, 24, 24, 37, 44 and 46 months. The main causes of death were myocardial infarction (four cases), cancer (two cases), gastrointestinal hemorrhage (one case), ischemic stroke (one case), and sepsis (one case). Permanent paraplegia occurred in one patient.nnnCONCLUSIONSnIt is still too soon to compare the results of endovascular repair of thoracoabdominal aneurysms with those of open surgical series. Despite the active and rapid progress currently observed for the endovascular method, it is still far from reaching its state-of-the-art plateau or becoming a gold standard. Further technological and technical advances in endovascular stent grafting seem to have a clear potential to provide very satisfactory operative outcomes for thoracoabdominal aortic aneurysms.

Antitrypanosomal activity and evaluation of the mechanism of action of dehydrodieugenol isolated from Nectandra leucantha (Lauraceae) and its methylated derivative against Trypanosoma cruzi

BACKGROUNDnFrom a previous screening of Brazilian biodiversity for antiprotozoal activity, the hexane extract from leaves of Nectandra leucantha (Nees & Mart.) (Lauraceae) demonstrated activity against Trypanosoma cruzi. Chromatographic separation of this extract afforded bioactive dehydrodieugenol (1). Furthermore, methylated derivative 2 (dehydrodieugenol dimethyl ether) was prepared and also tested against T. cruzi.nnnPURPOSEnTo examine the therapeutical potential of compounds 1 and 2 against T. cruzi as well as to elucidate the mechanism of action of bioactive compound 1 against T. cruzi.nnnMETHODS/STUDY DESIGNnCrude hexane extract from leaves was subjected to chromatographic steps to afford bioactive compound 1. In order to analyze the effect of additional methyl group in the antiparasitic activity of 1, derivative 2 was prepared (both are no pan-assay interference compounds - PAINS). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and analyzed for the potential effect in host cells through the production of nitric oxide and reactive oxygen species. Finally, the plasma membrane effect of the most potent compound 1 was investigated in T. cruzi trypomastigotes.nnnRESULTSnCompounds 1 and 2 displayed activity against amastigotes of T. cruzi. Although both compounds promoted activity against intracellular amastigotes, the production of nitric oxide and reactive oxygen species of host cells were unaltered, suggesting an antiparasitic activity other than host cell activation. Considering 1 the most effective compound against T. cruzi, the interference in the plasma membrane of the trypomastigotes was investigated using the fluorescent probe SYTOX® Green. After a short-term incubation, the fluidity and integrity of the plasma membrane was completely altered, suggesting it as a primary target for compound 1 in T. cruzi.nnnCONCLUSIONnCompounds 1 and 2 selectively eliminated the intracellular parasites without host cell activation and could be important scaffolds for the search of new hit compounds.

Hypervalent organotellurium compounds as inhibitors of P. falciparum calcium-dependent cysteine proteases.

Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQS 3D7 and CQR W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC50 values were 3.7±1.0μM, 1.1±0.2μM and 0.2±0.01μM, respectively. Using an assay performed in the presence of the ER Ca(2+)-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.

Specific calpain activity evaluation in Plasmodium parasites

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.

Specific effects of reactive thiol drugs on mitochondrial bioenergetics

In this minireview, the more recent findings about the effects of peculiar reactive thiol drugs on mitochondria are presented. These include the following compounds: metallo meso-tetrakis porphyrins, palladacycles, telluranes and phenothiazines. Metallo meso-tetrakis porphyrins can exhibit both beneficial and deleterious effects on mitochodria that are modulated by the central metal, cell location, and availability of axial ligands. Therefore, these compounds have the versatility to be used for cell and mitochondria protection and death. The antioxidant activity of manganese porphyrins is related to a glutathione peroxidase-like activity. By attacking exclusively the membrane protein thiol groups without glutathione depletion, palladacycles are able to induce mitochondrial permeability transition (MPT) and cytochrome c release in the absence of oxidative stress. In hepatoma cells, the mitochondrial action of palladacycles was able to induce apoptotic death. As opposed to palladacycles, telluranes and phenothiazines are able to conjugate the capacity to promote the MPT in a dose-dependent manner in association with efficient antioxidant activity toward lipids. These studies demonstrated that the action of drugs on mitochondrial bioenergetics can be modulated by peculiar reactivity with thiol groups. Therefore, they contribute to studies of toxicity as well as the design of new drugs.

“Snare-Ride”: A Bailout Technique to Catheterize Target Vessels With Unfriendly Anatomy in Branched Endovascular Aortic Repair

Purpose: To describe a novel endovascular bailout technique for successful completion of target vessel stenting during branched stent-graft repair of thoracoabdominal aortic aneurysms (TAAA) after encountering difficulties with standard catheterization techniques. Technique: Technical difficulties when using fenestrated and branched grafts should be expected, especially in difficult anatomy or when an off-the-shelf device (eg, standard 4-branch device) is used that does not perfectly “match” the anatomy. The “snare-ride technique” facilitates antegrade transaxillary side branch catheterization and stent placement during TAAA branched grafting using a snare via a transfemoral approach. The branch of the graft is catheterized from an axillary access. The respective target vessel is then catheterized via a femoral access. An Indy snare is advanced over the transfemoral wire and positioned near the entrance of the target vessel. The transaxillary wire inside the branch of the graft is then advanced, snared, and pushed inside the target vessel with the snare. The procedure is thereafter continued with antegrade bridging of the target vessel in routine fashion. Conclusion: The snare-ride technique can be a useful maneuver to catheterize target vessels with difficult anatomy in TAAA branched stent-graft repair. Early experience shows safety and feasibility.

Biocatalysis for desymmetrization and resolution of stereocenters beyond the reactive center: How far is far enough?

The kinetic resolution of racemates and desymmetrization are the most common approaches to the preparation of enantiomerically enriched compounds. These procedures allow the access of high valuable, chiral building blocks for many purposes in academic or industrial R&D endeavors. Nevertheless, the scope of stereochemistry recognition in biotransformations usually occurs at the site of the transformation or when it is close to it (not more than 3 bonds). However, there are a growing number of enzymatic transformations which surpass the limits of stereorecognition of remote chiral (or prochiral) centers. In this account, we would like to present some aspects of biocatalyzed remote resolutions and remote desymmetrizations to call attention for these challenging transformations.