Repurposing strategies for Chagas disease therapy: the effect of imatinib and derivatives against Trypanosoma cruzi.

Chagas disease (CD) is a neglected parasitic condition endemic in the Americas caused by Trypanosoma cruzi. Patients present an acute phase that may or not be symptomatic, followed by lifelong chronic stage, mostly indeterminate, or with cardiac and/or digestive progressive lesions. Benznidazole (BZ) and nifurtimox are the only drugs approved for treatment but not effective in the late chronic phase and many strains of the parasite are naturally resistant. New alternative therapy is required to address this serious public health issue. Repositioning and combination represent faster, and cheaper trial strategies encouraged for neglected diseases. The effect of imatinib (IMB), a tyrosine kinase inhibitor designed for use in neoplasias, was assessed in vitro on T. cruzi and mammalian host cells. In comparison with BZ, IMB was moderately active against different strains and forms of the parasite. The combination IMB + BZ in fixed-ratio proportions was additive. Novel 14 derivatives of IMB were screened and a 3,2-difluoro-2-phenylacetamide (3e) was as potent as BZ on T. cruzi but had low selectivity index. The results demonstrate the importance of phenotypic assays, encourage the improvement of IMB derivatives to reach selectivity and testify to the use of repurposing and combination in drug screening for CD.

Repurposing Strategy of Atorvastatin against Trypanosoma cruzi: In Vitro Monotherapy and Combined Therapy with Benznidazole Exhibit Synergistic Trypanocidal Activity.

Statins are inhibitors of cholesterol synthesis, but other biological properties, such as antimicrobial effects, have also been assigned to them, leading to their designation as pleiotropic agents. Our goal was to investigate the activity and selectivity of atorvastatin (AVA) against Trypanosoma cruzi by using in vitro models, aiming for more effective and safer therapeutic options through drug repurposing proposals for monotherapy and therapy in combination with benznidazole (BZ). Phenotypic screening was performed with different strains (Tulahuen [discrete typing unit {DTU} VI] and Y [DTU II]) and forms (intracellular forms, bloodstream trypomastigotes, and tissue-derived trypomastigotes) of the parasite. On assay of the Tulahuen strain, AVA was more active against intracellular amastigotes (selectivity index [SI] = 3). Also, against a parasite of another DTU (Y strain), this statin was more active (2.1-fold) and selective (2.4-fold) against bloodstream trypomastigotes (SI = 51) than against the intracellular forms (SI = 20). A cytomorphological approach using phalloidin-rhodamine permitted us to verify that AVA did not induced cell density reduction and that cardiac cells (CC) maintained their typical cytoarchitecture. Combinatory approaches using fixed-ratio methods showed that AVA and BZ gave synergistic interactions against both trypomastigotes and intracellular forms (mean sums of fractional inhibitory concentration indexes [∑FICIs] of 0.46 ± 0.12 and 0.48 ± 0.03, respectively). Thus, the repurposing strategy for AVA, especially in combination with BZ, which leads to a synergistic effect, is encouraging for future studies to identify novel therapeutic protocols for Chagas disease treatment.

In Vitro and In Vivo Studies of the Trypanocidal Effect of Novel Quinolines.

Therapies for human African trypanosomiasis and Chagas disease, caused by Trypanosoma brucei and Trypanosoma cruzi, respectively, are limited, providing minimal therapeutic options for the millions of individuals living in very poor communities. Here the effects of 10 novel quinolines are evaluated in silico and by phenotypic studies using in vitro and in vivo models. Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties revealed that most molecules did not infringe on Lipinski's rules, which is a prediction of good oral absorption. These quinolines showed high probabilities of Caco2 permeability and human intestinal absorption and low probabilities of mutagenicity and of hERG1 inhibition. In vitro screens against bloodstream forms of T. cruzi demonstrated that all quinolines were more active than the reference drug (benznidazole [Bz]), except for DB2171 and DB2192, with five (DB2187, DB2131, DB2186, DB2191, and DB2217) displaying 50% effective concentrations (EC50s) of <3 µM (4-fold lower than that of Bz). Nine quinolines were more effective than Bz (2.7 µM) against amastigotes, showing EC50s ranging from 0.6 to 0.1 µM. All quinolines were also highly active in vitro against African trypanosomes, showing EC50s of ≤0.25 µM. The most potent and highly selective candidates for each parasite species were tested in in vivo models. Results for DB2186 were promising in mice with T. cruzi and T. brucei infections, reaching a 70% reduction of the parasitemia load for T. cruzi, and it cured 2 out of 4 mice infected with T. brucei DB2217 was also active in vivo and cured all 4 mice (100% cure rate) with T. brucei infection.

Drug repurposing strategy against Trypanosoma cruzi infection: In vitro and in vivo assessment of the activity of metronidazole in mono- and combined therapy.

Metronidazole (Mtz) is a commercial broad-spectrum nitroimidazolic derivative with relevant antimicrobial activity and relative safety profile. Therefore, it is fair to consider Mtz a candidate for drug repurposing for other neglected conditions such as Chagas disease (CD), a parasitic pathology caused by Trypanosoma cruzi. CD is treated only with benznidazole (Bz) and nifurtimox, both introduced in clinics decades ago despite important limitations, including low efficacy on the later disease stage (chronic form) and severe side effects. New cheap and fast alternative treatments for CD are needed, thus the repurposing of Mtz was assessed in vitro and in vivo in mono- and combined therapy. In vitro assays demonstrated EC50>200µM for Mtz, while for Bz the values ranged from 2.51µM (intracellular forms) to 11.5µM (bloodstream trypomastigotes). When both drugs were combined in fixed-ratio proportions, Mtz promoted Bz potency (lower EC50 values). In vivo toxicity assays for Mtz in mice showed no adverse effects neither histopathological alterations up to 2000mg/kg. Regarding experimental T. cruzi infection, Bz 100mg/kg suppressed parasitemia while Mtz (up to 1000mg/kg) in monotherapy did not, but prolonged animal survival at 250 and 500 regimen doses. The combination of both drugs (Bz 10+Mtz 250) prevented mortality (70%) besides protected against electric cardiac alterations triggered by the parasite infection. Although not able to reduce parasite load, the combination therapy prevented animal mortality; this was possibly due to a protection of the electric cardiac physiology that is normally altered in experimental infection of T. cruzi. It also suggested that the interaction with Mtz could have improved the pharmacokinetics of Bz. Our study emphasizes the importance of drug repurposing and combined therapy for CD to contribute to alternative therapies for this neglected and silent pathology.

Antitrypanosomal Activity of Sterol 14α-Demethylase (CYP51) Inhibitors VNI and VFV in the Swiss Mouse Models of Chagas Disease Induced by the Trypanosoma cruzi Y Strain.

Chagas disease is a life-threatening infection caused by a variety of genetically diverse strains of the protozoan parasite Trypanosoma cruzi The current treatment (benznidazole and nifurtimox) is unsatisfactory, and potential alternatives include inhibitors of sterol 14α-demethylase (CYP51), the cytochrome P450 enzyme essential for the biosynthesis of sterols in eukaryotes and the major target of clinical and agricultural antifungals. Here we performed a comparative investigation of two protozoon-specific CYP51 inhibitors, VNI and its CYP51 structure-based derivative VFV, in the murine models of infection caused by the Y strain of T. cruzi The effects of different treatment regimens and drug delivery vehicles were evaluated in animals of both genders, with benznidazole serving as the reference drug. Regardless of the treatment scheme or delivery vehicle, VFV was more potent in both genders, causing a >99.7% peak parasitemia reduction, while the VNI values varied from 91 to 100%. Treatments with VNI and VFV resulted in 100% animal survival and 0% natural relapse after the end of therapy, though, except for the 120-day treatment schemes with VFV, relapses after three cycles of immunosuppression were observed in each animal group, and quantitative PCR analysis revealed a very light parasite load in the blood samples (sometimes below or near the detection limit, which was 1.5 parasite equivalents/ml). Our studies support further investigations of this class of compounds, including their testing against other T. cruzi strains and in combination with other drugs.

Phenotypic Screening In Vitro of Novel Aromatic Amidines against Trypanosoma cruzi.

The current treatment of Chagas disease (CD), based on nifurtimox and benznidazole (Bz), is unsatisfactory. In this context, we performed the phenotypic in vitro screening of novel mono- and diamidines and drug interaction assays with selected compounds. Ten novel amidines were tested for their activities against bloodstream trypomastigote (BT) and amastigote forms of Trypanosoma cruzi (Y and Tulahuen strains) and their toxicities for mammalian host cells (L929 cells and cardiac cells). Seven of 10 molecules were more active than Bz against BT, with the most active compound being the diamidine DB2267 (50% effective concentration [EC50] = 0.23 µM; selectivity index = 417), which was 28-fold more active and about 3 times more selective than the standard drug. Five of the six monoamidines were also more active than Bz. The combination of DB2267 and DB2236 in fixed-ratio proportions showed an additive effect (sum of fractional inhibitory concentrations < 4) on BT. Interestingly, when intracellular forms were exposed to DB2267, its activity was dependent on the parasite strain, being effective (EC50 = 0.87 ± 0.05 µM) against a discrete typing unit (DTU) II strain (strain Y) but not against a representative DTU VI strain (strain Tulahuen) even when different vehicles (ß-cyclodextrin and dimethyl sulfoxide) were used. The intrinsic fluorescence of several diamidines allowed their uptake to be studied. Testing of the uptake of DB2236 (inactive) and DB2267 (active) by amastigotes of the Y strain showed that the two compounds were localized intracellularly in different compartments: DB2236 in the cytoplasm and DB2267 in the nucleus. Our present data encourage further studies regarding the activities of amidines and provide information which will help with the identification of novel agents for the treatment of CD.

In Vitro and In Vivo Trypanosomicidal Action of Novel Arylimidamides against Trypanosoma cruzi.

Arylimidamides (AIAs) have been shown to have considerable biological activity against intracellular pathogens, includingTrypanosoma cruzi, which causes Chagas disease. In the present study, the activities of 12 novel bis-AIAs and 2 mono-AIAs against different strains ofT. cruziin vitroandin vivowere analyzed. The most active wasm-terphenyl bis-AIA (35DAP073), which had a 50% effective concentration (EC50) of 0.5 µM for trypomastigotes (Y strain), which made it 26-fold more effective than benznidazole (Bz; 13 µM). It was also active against the Colombiana strain (EC50= 3.8 µM). Analysis of the activity against intracellular forms of the Tulahuen strain showed that this bis-AIA (EC50= 0.04 µM) was about 100-fold more active than Bz (2 µM). The trypanocidal effect was dissociated from the ability to trigger intracellular lipid bodies within host cells, detected by oil red labeling. Both an active compound (35DAP073) and an inactive compound (26SMB060) displayed similar activation profiles. Due to their high selectivity indexes, two AIAs (35DAP073 and 35DAP081) were moved toin vivostudies, but because of the results of acute toxicity assays, 35DAP081 was excluded from the subsequent tests. The findings obtained with 35DAP073 treatment of infections caused by the Y strain revealed that 2 days of therapy induced a dose-dependent action, leading to 96 to 46% reductions in the level of parasitemia. However, the administration of 10 daily doses in animals infected with the Colombiana strain resulted in toxicity, preventing longer periods of treatment. The activity of the combination of 0.5 mg/kg of body weight/day 35DAP073 with 100 mg/kg/day Bz for 10 consecutive days was then assayed. Treatment with the combination resulted in the suppression of parasitemia, the elimination of neurological toxic effects, and survival of 100% of the animals. Quantitative PCR showed a considerable reduction in the parasite load (60%) compared to that achieved with Bz or the amidine alone. Our results support further investigations of this class with the aim of developing novel alternatives for the treatment of Chagas disease.

In vitro investigation of the efficacy of novel diamidines against Trypanosoma cruzi.

Chagas' disease is a neglected tropical disease caused by Trypanosoma cruzi and constitutes a serious public health problem for Latin America. Its unsatisfactory chemotherapy stimulates the search for novel antiparasitic compounds. Amidines and related compounds exhibit well-known activity towards different microbes including T. cruzi. In this vein, our present aim was to evaluate the biological effect of 10 novel structurally related amidines in vitro against bloodstream and intracellular forms of the parasite as well as their potential toxicity on cardiac cell cultures. Our results show that although active against the extracellular forms, with some of them like DB2247 being 6-fold more effective than benznidazole and displaying very low toxicity (>96 µm), none presented superior trypanocidal effect against intracellular forms as compared with the reference drug. These results may be due to differences in susceptibility profiles related to distinct uptake/extrusion mechanisms and cellular targets between bloodstream and amastigote forms. The present study adds to the knowledge base for the future design of novel amidines that may provide promising activity against T. cruzi.

In vitro and in vivo studies of the biological activity of novel arylimidamides against Trypanosoma cruzi.

Fifteen novel arylimidamides (AIAs) (6 bis-amidino and 9 mono-amidino analogues) were assayed against Trypanosoma cruzi in vitro and in vivo. All the bis-AIAs were more effective than the mono-AIAs, and two analogues, DB1967 and DB1989, were further evaluated in vivo. Although both of them reduced parasitemia, protection against mortality was not achieved. Our results show that the number of amidino-terminal units affects the efficacy of arylimidamides against T. cruzi.

Novel amidines and analogues as promising agents against intracellular parasites: a systematic review.

Parasitic protozoa comprise diverse aetiological agents responsible for important diseases in humans and animals including sleeping sickness, Chagas disease, leishmaniasis, malaria, toxoplasmosis and others. They are major causes of mortality and morbidity in tropical and subtropical countries, and are also responsible for important economic losses. However, up to now, for most of these parasitic diseases, effective vaccines are lacking and the approved chemotherapeutic compounds present high toxicity, increasing resistance, limited efficacy and require long periods of treatment. Many of these parasitic illnesses predominantly affect low-income populations of developing countries for which new pharmaceutical alternatives are urgently needed. Thus, very low research funding is available. Amidine-containing compounds such as pentamidine are DNA minor groove binders with a broad spectrum of activities against human and veterinary pathogens. Due to their promising microbicidal activity but their rather poor bioavailability and high toxicity, many analogues and derivatives, including pro-drugs, have been synthesized and screened in vitro and in vivo in order to improve their selectivity and pharmacological properties. This review summarizes the knowledge on amidines and analogues with respect to their synthesis, pharmacological profile, mechanistic and biological effects upon a range of intracellular protozoan parasites. The bulk of these data may contribute to the future design and structure optimization of new aromatic dicationic compounds as novel antiparasitic drug candidates.

The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases - part I

Infections with protozoan parasites are a major cause of disease and mortality in many tropical countries of the world. Diseases caused by species of the genera Trypanosoma (Human African Trypanosomiasis and Chagas Disease) and Leishmania (various forms of Leishmaniasis) are among the seventeen "Neglected Tropical Diseases" (NTDs) defined as such by WHO due to the neglect of financial investment into research and development of new drugs by a large part of pharmaceutical industry and neglect of public awareness in high income countries. Another major tropical protozoan disease is malaria (caused by various Plasmodium species), which -although not mentioned currently by the WHO as a neglected disease- still represents a major problem, especially to people living under poor circumstances in tropical countries. Malaria causes by far the highest number of deaths of all protozoan infections and is often (as in this review) included in the NTDs. The mentioned diseases threaten many millions of lives world-wide and they are mostly associated with poor socioeconomic and hygienic environment. Existing therapies suffer from various shortcomings, namely, a high degree of toxicity and unwanted effects, lack of availability and/or problematic application under the life conditions of affected populations. Development of new, safe and affordable drugs is therefore an urgent need. Nature has provided an innumerable number of drugs for the treatment of many serious diseases. Among the natural sources for new bioactive chemicals, plants are still predominant. Their secondary metabolism yields an immeasurable wealth of chemical structures which has been and will continue to be a source of new drugs, directly in their native form and after optimization by synthetic medicinal chemistry. The current review, published in two parts, attempts to give an overview on the potential of such plant-derived natural products as antiprotozoal leads and/or drugs in the fight against NTDs.

The trypanocidal activity of amidine compounds does not correlate with their binding affinity to Trypanosoma cruzi kinetoplast DNA.

Due to limited efficacy and considerable toxicity, the therapy for Chagas' disease is far from being ideal, and thus new compounds are desirable. Diamidines and related compounds such as arylimidamides have promising trypanocidal activity against Trypanosoma cruzi. To better understand the mechanism of action of these heterocyclic cations, we investigated the kinetoplast DNA (kDNA) binding properties and trypanocidal efficacy against T. cruzi of 13 compounds. Four diamidines (DB75, DB569, DB1345, and DB829), eight arylimidamides (DB766, DB749, DB889, DB709, DB613, DB1831, DB1852, and DB2002), and one guanylhydrazone (DB1080) were assayed in thermal denaturation (T(m)) and circular dichroism (CD) studies using whole purified T. cruzi kDNA and a conserved synthetic parasite sequence. The overall CD spectra using the whole kDNA were similar to those found for the conserved sequence and were indicative of minor groove binding. Our findings showed that some of the compounds that exhibited the highest trypanocidal activities (e.g., DB766) caused low or no change in the T(m) measurements. However, while some active compounds, such as DB766, induced profound alterations of kDNA topology, others, like DB1831, although effective, did not result in altered T(m) and CD measurements. Our data suggest that the strong affinity of amidines with kDNA per se is not sufficient to generate and trigger their trypanocidal activity. Cell uptake differences and possibly distinct cellular targets need to be considered in the final evaluation of the mechanisms of action of these compounds.

Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12-14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD(50) values, good selectivity index, and good activity at 4 °C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection.

Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro.

Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.

Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy.

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

Biological, ultrastructural effect and subcellular localization of aromatic diamidines in Trypanosoma cruzi.

No vaccines or safe chemotherapy are available for Chagas disease. Pentamidine and related di-cations are DNA minor groove-binders with broad-spectrum anti-protozoal activity. Therefore our aim was to evaluate the in vitro efficacy of di-cationic compounds - DB1645, DB1582, DB1651, DB1646, DB1670 and DB1627 - against bloodstream trypomastigotes (BT) and intracellular forms of Trypanosoma cruzi. Cellular targets of these compounds in treated parasites were also analysed by fluorescence and transmission electron microscopy (TEM). DB1645, DB1582 and DB1651 were the most active against BT showing IC50 values ranging between 0.15 and 6.9 microm. All compounds displayed low toxicity towards mammalian cells and DB1645, DB1582 and DB1651 were also the most effective against intracellular parasites, with IC50 values ranging between 7.3 and 13.3 microm. All compounds localized in parasite nuclei and kDNA (with greater intensity in the latter structure), and DB1582 and DB1651 also concentrated in non-DNA-containing cytoplasmic organelles possibly acidocalcisomes. TEM revealed alterations in mitochondria and kinetoplasts, as well as important disorganization of microtubules. Our data provide further information regarding the activity of this class of compounds upon T. cruzi which should aid future design and synthesis of agents that could be used for Chagas disease therapy.

Diamidine activity against trypanosomes: the state of the art.

Aromatic diamidines and related compounds are DNA minor groove binders that have been screened against a variety of pathogenic microorganisms such as bacteria, fungi and protozoa and show promising results. Parasitic infections are widespread in developing countries and are major contributors to human mortality and morbidity, causing considerable economic hardship. Trypanosomes are unicellular protozoan organisms that cause serious public health problems in developing countries: African trypanosomiasis (sleeping sickness) in Africa, and Chagas' disease, in Latin America. Sleeping sickness, caused by sub-species of Trypanosome brucei (T. brucei gambiense and T. brucei rhodesiense), is a fatal disease if left untreated, with about 60 million people currently at risk. Trypanosoma cruzi is the etiological agent of Chagas' disease, an important parasitic illness that affects nearly 17 million individuals in endemic areas. The fact that the available clinical drugs are expensive, toxic, require long treatment periods, frequently exhibit reduced activity towards certain parasite strains and evolutive stages, and are beginning to show development of resistance, demonstrates the urgent need for the development of new drugs for both pathologies. For some time much attention has been focused on the effect of diamidines (and related compounds) on African trypanosomes. However more recent studies have pointed to their potential activity against T.cruzi. In this review the current therapeutic state of the art of aromatic diamidines and related compounds used against T.brucei and T.cruzi is reviewed with a focus on their potential use as antiparasitic drugs for the treatment of both these important neglected diseases.

Trypanosoma cruzi: alpha-2-macroglobulin regulates host cell apoptosis induced by the parasite infection in vitro.

A2M is a broad spectrum proteinase inhibitor and cytokine carrier, besides presenting anti-apoptotic activity through the binding to its receptor, LRP. During Trypanosoma cruzi infection, apoptosis of host cells and intracellular parasites is commonly observed both in vivo and in vitro. Since plasma as well as tissue A2M levels are increased in both murine and human acute T. cruzi infection, we evaluated the possible role of A2M (its methylamine transformed Fast form-A2M-F) in regulating apoptotic events in peritoneal macrophages and cardiomyocytes during in vitro interaction with the parasite. Our data showed that DNA fragmentation (a hallmark of apoptosis) of both host cells and parasites was inhibited by A2M-F. Impaired apoptosis was also noted when A2M-F was added to the cultures maintained under serum deprivation. In addition, macrophages from C57/BL6 mice, known to display higher LRP levels as compared to those of C3H lineage, displayed higher reduction in the apoptotic levels during the A2M-F treatment.

Cellular effects of reversed amidines on Trypanosoma cruzi.

Aromatic diamidines represent a class of DNA minor groove-binding ligands that exhibit high levels of antiparasitic activity. Since the chemotherapy for Chagas' disease is still an unsolved problem and previous reports on diamidines and related analogues show that they have high levels of activity against Trypanosoma cruzi infection both in vitro and in vivo, our present aim was to evaluate the cellular effects in vitro of three reversed amidines (DB889, DB702, and DB786) and one diguanidine (DB711) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas' disease. Our data show that the reversed amidines have higher levels of activity than the diguanidine, with the order of trypanocidal activities being as follows: DB889 > DB702 > DB786 > DB711. Transmission electron microscopy analysis showed that the reversed amidines induced many alterations in the nuclear morphology, swelling of the endoplasmic reticulum and Golgi structures, and consistent damage in the mitochondria and kinetoplasts of the parasites. Interestingly, in trypomastigotes treated with the reversed amidine DB889, multiple axoneme structures (flagellar microtubules) were noted. Flow cytometry analysis confirmed that the treated parasites presented an important loss of the mitochondrial membrane potential, as revealed by a decrease in rhodamine 123 fluorescence. Our results show that the reversed amidines have promising activities against the relevant mammalian forms of T. cruzi and display high trypanocidal effects at very low doses. This is especially the case for DB889, which merits further in vivo evaluation.

Antiparasitic activity of aromatic diamidines is related to apoptosis-like death in Trypanosoma cruzi.

Two aromatic diamidines, furamidine (DB75) and its phenyl-substituted analogue (DB569), which exhibit trypanocidal activity, were assayed against Trypanosoma cruzi and were found to induce apoptosis-like death characteristics such as nuclear DNA condensation and fragmentation, decreased mitochondrial membrane potential and phosphatidylserine exposure. DB569 displays superior trypanocidal activity compared to furamidine and also had higher ability to induce apoptosis-like death in treated parasites. The present results showing apoptosis-like death in T. cruzi after treatment with both DB75 and DB569 make important contributions to the understanding of the mechanisms of the aromatic diamidines, which represent promising trypanocidal compounds.