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.

Copper(II) complex-loaded castor oil-based nanostructured lipid carriers used against Mycobacterium tuberculosis : Development, characterisation, in vitro and in vivo biological assays.

A copper(II) complex-loaded castor oil-based nanostructured lipid carrier was evaluated to enhance the poor water solubility of antimicrobial compounds, improving their biological properties and antimicrobial activity against Mycobacterium tuberculosis. Nanostructured lipid carriers were composed of the castor oil, polyoxyethylene 40 stearate and caprylic/capric triglyceride, poloxamer 407, cetyltrimethylammonium bromide and three different copper(II) complexes. The systems were ultrasonicated at an amplitude of 8% for 20 min and an ice bath was used throughout the procedure. The blank nanostructured lipid carrier (F5) and nanostructured lipid carriers loaded with copper(II) complex 1, 2 and 3 (F5.1, F5.2 and F5.3, respectively) for 45 days presented values of mean diameter, poly dispersity index and zeta potential ranging from 186 to 199 nm, 0.14 to 0.2 and 24 to 30 mV, respectively. Atomic force microscopy indicated that the nanostructured lipid carriers were distributed at the nanoscale, corroborating the mean diameter data. Differential scanning calorimetry determined the melting points of the constituents of the nanostructured lipid carriers. The antimicrobial activity of copper(II) complexloaded F5 against M. tuberculosis H37Rv showed better anti-tuberculosis activity than the free complexes. In vivo biological assays of complex-loaded F5 demonstrated reduced toxicity. Our results suggest that nanostructured lipid carriers could be a potential nanotechnological strategy to optimise tuberculosis 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.

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.

Uric acid levels are associated with microvascular endothelial dysfunction in patients with Type 1 diabetes.

AIMS: Recent data identified uric acid as an independent risk factor for cardiovascular disease. The aim of the present study was to assess the association between uric acid and endothelial dysfunction in 57 patients with Type 1 diabetes and 53 healthy control subjects. METHODS: Microvascular endothelial function was evaluated using laser Doppler perfusion monitoring coupled with pharmacological (iontophoretic administration of acetylcholine and sodium nitroprusside) and physiological (post-occlusive reactive hyperaemia and thermal hyperaemia) stimuli. RESULTS: Uric acid was higher in subjects without diabetes than in those with diabetes (P = 0.03). Microvascular vasodilator response to acetylcholine was significantly reduced in Type 1 diabetes (P = 0.002) and was correlated to disease duration (r = -0.3, P = 0.01), triglyceride (r = -0.37, P = 0.005), insulin dose (r = -0.28, P = 0.03), fasting plasma glucose levels (r = -0.3, P = 0.02), HbA(1c) (r = -0.34, P = 0.001) and uric acid (r = -0.3, P = 0.005). On stepwise multivariate analysis, age, HbA(1c) and uric acid were the most important independent variables that were associated with the endothelium-dependent response in Type 1 diabetes (P = 0.02). CONCLUSIONS: Glycaemic control and uric acid in the normal range were the most important contributing factors to the decreasing endothelium-dependent responses associated with Type 1 diabetes. Consequently, uric acid could be a new potential marker of microvascular endothelial dysfunction in these patients. Further studies are required to explore the clinical relevance of the relationship between uric acid levels, oxidative stress and endothelial dysfunction in patients with Type 1 diabetes, as well as whether treatment with uric acid-lowering drugs for slight elevations in uric acid would benefit these patients.

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.