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.

Improved glycemic control by acarbose therapy in hypertensive diabetic patients: effects on blood pressure and hormonal parameters.

A double-blind, randomized, placebo-controlled study was carried out on 44 hypertensive type 2 diabetic subjects previously treated by diet associated or not with sulfonylurea to assess the effects of acarbose-induced glycemic control on blood pressure (BP) and hormonal parameters. Before randomization and after a 22-week treatment period (100 to 300 mg/day), the subjects were submitted to a standard meal test and to 24-h ambulatory BP monitoring (ABPM) and had plasma glucose, glycosylated hemoglobin, lipid profile, insulin, proinsulin and leptin levels determined. Weight loss was found only in the acarbose-treated group (75.1 +/- 11.6 to 73.1 +/- 11.6 kg, P<0.01). Glycosylated hemoglobin decreased only in the acarbose group (6.4 +/- 1.7 to 5.6 +/- 1.9%, P<0.05). Fasting proinsulin decreased only in the acarbose group (23.4 +/- 19.3 to 14.3 +/- 13.6 pmol/l, P<0.05), while leptin decreased in both (placebo group: 26.3 +/- 6.1 to 23.3 +/- 9.4 and acarbose group: 25.0 +/- 5.5 to 22.7 +/- 7.9 ng/ml, P<0.05). When the subset of acarbose-treated patients who improved glycemic control was considered, significant reductions in diurnal systolic, diastolic and mean BP (102.3 +/- 6.0 to 99.0 +/- 6.6 mmHg, P<0.05) were found. Acarbose monotherapy or combined with sulfonylurea was effective in improving glycemic control in hypertensive diabetic patients. Acarbose-induced improvement in metabolic control may reduce BP in these patients. Our data did not suggest a direct action of acarbose on insulin resistance or leptin levels.

Improved glycemic control by acarbose therapy in hypertensive diabetic patients: effects on blood pressure and hormonal parameters

A double-blind, randomized, placebo-controlled study was carried out on 44 hypertensive type 2 diabetic subjects previously treated by diet associated or not with sulfonylurea to assess the effects of acarbose-induced glycemic control on blood pressure (BP) and hormonal parameters. Before randomization and after a 22-week treatment period (100 to 300 mg/day), the subjects were submitted to a standard meal test and to 24-h ambulatory BP monitoring (ABPM) and had plasma glucose, glycosylated hemoglobin, lipid profile, insulin, proinsulin and leptin levels determined. Weight loss was found only in the acarbose-treated group (75.1 ± 11.6 to 73.1 ± 11.6 kg, P<0.01). Glycosylated hemoglobin decreased only in the acarbose group (6.4 ± 1.7 to 5.6 ± 1.9 percent, P<0.05). Fasting proinsulin decreased only in the acarbose group (23.4 ± 19.3 to 14.3 ± 13.6 pmol/l, P<0.05), while leptin decreased in both (placebo group: 26.3 ± 6.1 to 23.3 ± 9.4 and acarbose group: 25.0 ± 5.5 to 22.7 ± 7.9 ng/ml, P<0.05). When the subset of acarbose-treated patients who improved glycemic control was considered, significant reductions in diurnal systolic, diastolic and mean BP (102.3 ± 6.0 to 99.0 ± 6.6 mmHg, P<0.05) were found. Acarbose monotherapy or combined with sulfonylurea was effective in improving glycemic control in hypertensive diabetic patients. Acarbose-induced improvement in metabolic control may reduce BP in these patients. Our data did not suggest a direct action of acarbose on insulin resistance or leptin levels

[Lovastatin in the treatment of hypercholesterolemia in non-insulin-dependent diabetes mellitus patients]. / A lovastatina no tratamento da hipercolesterolemia em portadores de diabete não dependente de insulina.

PURPOSE: To evaluate the effects of lovastatin as an hypocholesterolemic agent in non-insulin dependent diabetic (NIDDM) patients with high cholesterol plasma levels. METHODS: Twenty NIDDM patients were included in this study. Hypercholesterolemia was defined as LDL-cholesterol plasma levels above 160mg/dl in female patients and above 130mg/dl in male patients or in women presenting any other risk factor for cardiovascular disease. From the 20 patients included, 18 had also high levels of arterial blood pressure. They were evaluated for admission in the study after they have substituted the antihypertensive medication for at least 6 weeks, from beta-blockers or diuretics to angiotensin converting enzyme inhibitors or calcium channel blockers. Lovastatin was administered in a initial daily dose of 20mg to all patients for 6 weeks. After this period this dose was increased to 40mg in 11 patients with LDL-cholesterol levels above 130mg/dl. All patients were treated for a total period of 24 weeks. RESULTS: Lovastatin therapy for 24 weeks reduced LDL-cholesterol and total cholesterol plasma levels in 30% and 21%, respectively, while no changes in HDL-cholesterol or triglycerides plasma levels were observed. The medication was well tolerated and no changes in bilirrubins or transaminases plasma levels were detected. In 9 patients the serum levels of alkaline phosphatase showed an elevation and the mean level of all group increased from 109 +/- 59 to 188 +/- 60m mu/ml (p < 0.05). This was an isolated abnormality without any other clinical manifestation. CONCLUSION: Lovastatin in NIDDM showed to be an efficient agent to reduce high levels of LDL-cholesterol and total cholesterol. However, the importance of the abnormality observed in serum alkaline phosphatase levels deserves further investigation. In this condition we recommend discontinuation of lovastatin therapy.

Radioimmunoassay of carboxyl and amino terminal fragments of parathyroid hormone for the evaluation of secondary hyperparathyroidism in chronic renal failure.

1. Some parameters of calcium and phosphorus metabolism and the radioimmunoassay of plasma concentrations of both the carboxyl (COOH) (residues 53-84) and amino (NH2) terminal (residues 1-34) fragments of parathyroid hormone (PTH) were measured to evaluate secondary hyperparathyroidism in 68 patients with chronic renal failure (CRF), 34 of whom were on hemodialysis therapy. 2. The upper limits of the normal values for serum PTH-NH2 and PTH-COOH concentrations were 28 and 146 pmol/l, respectively. Patients with mild CRF (plasma creatinine (CRp) 1.2-2 mg/dl) had normal mean serum total calcium, low mean serum phosphorus, undetectable plasma levels of PTH-NH2 (less than 10 pmol/l), slightly elevated mean plasma PTH-COOH concentration and normal fractional excretion of phosphorus (FEP). Patients with moderate CRF (CRp 2.1-4 mg/dl) had normal mean serum concentrations of both total calcium and phosphorus, and elevated mean levels of both plasma PTH-COOH and PTH-NH2 associated with increased FEP. Patients with end-stage CRF (CRp greater than 4 mg/dl) and those on hemodialysis had elevated mean serum phosphorus levels and decreased mean serum total calcium concentrations compared with those with mild and moderate CRF, and more pronounced increases in both mean plasma PTH-COOH and PTH-NH2. 3. The logarithm of plasma PTH-NH2, but not PTH-COOH, concentration correlated positively with FEP and serum phosphorus concentration and negatively with total serum calcium concentration, while the logarithms of both PTH-NH2 and PTH-COOH levels correlated positively with CRp.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay of carboxyl and amino terminal fragments of parathyroid hormone for the evaluation of secondary hyperparathyroidism in chronic renal failure

1. Some parameters of calcium and phosphorus metabolism and the radioimmunoassay of plasma concentrations of both the carboxyl (COOH) (residues 53-84) and amino (NH2) terminal (residues 1-34) fragments of parathyroid hormone (PTH) were measured to evaluate secondary hyperparathyroidism in 68 patients with chronic renal falure (CRF), 34 of whon were on hemodialysis therapy. 2. The upper limits of the normal values for serum PTH-NH2 and PTH-COOH concentrations were 28 and 146 pmol/l, respectively. Patients with mild CRF (plasma creatinine (CRp) 1.2-2 mg/dl) hadh normal mean serum total calcium, low mean phosphorus, undetectable plasma levels of PTH-COOH concentration and normal fractional excretion of phosphorus (FEP). Patients with moderate CRF (CRp2.1-4 mg/dl) had normal mean serum concentrations of both total calcium and phosphorus, and elevated mean levels of both plasma PTH-COOH and PTH-NH2 associated with increased FEP. Patients with end-stage CRF (CRp > 4mg/dl) and those on hemodialysis had elevated mean serum phosphorus levels and decreased mean serum total calcium concentrations compared with those with mild and modetate CRF, and more pronounced, increases in both mean plasma PTH-COOH and PTH-NH2. 3. The logarithm of plasma PTH-NH2, but not PTH-COOH, concentration correlated positively with FEP and serum phosphorus concentration and negatively with total serum calcium concentration, while the logarithms of both PTH-NH2 and PTH-COOH levels correlated positively with CRp. 4. Calcium infusion (2 mg Kg-1 h-1 for 90 min) in eight patients with high plasma levels of PTH-NH2, and PTH-COOH resulted in a significant decrease of plasma PTH-NHL but not of plasma PTH-COOH concentration. 5. These data demonstrate increased plasma PTH levels in moderate renal failure and suggest that the assay of plasma PTH-NH2 rather than PTH-COLL is more appropriate for the evaluation of secondary hyperparathyroidism in chronic renal failure

Hypertension and diabetes. Clinical problems.

The choice of an appropriate antihypertensive agent and the hazards of postural hypotension are common problems faced in the treatment of diabetic hypertensive patients. The results of 3 studies addressing these problems are described in this report. In the first study, indoramin, an alpha-blocking agent, was administered to patients with non-insulin-dependent diabetes and mild to moderate hypertension. Blood pressure control was achieved in 57% of patients with mild, and in none with moderate hypertension. The blood glucose and insulin responses to an oral 50g glucose loading, as well as the blood concentrations of HbA1 did not change during therapy. Seven patients were excluded because of side effects. In 4 of them postural hypotension was observed. In the second study, the effects of angiotensin-converting enzyme (ACE) inhibitors, administered to patients with non-insulin-dependent diabetes and mild to moderate hypertension, were evaluated. Blood pressure control was achieved in 78% of the patients on captopril (n = 14) and in 74% of patients on enalapril therapy (n = 23). Symptomatic postural hypotension (n = 2) and hyperkalaemia (n = 2) were observed with both drugs. Significant reductions in 24-hour urinary protein or albumin excretion were detected in 12 patients on enalapril therapy. No changes in 2-hour postprandial blood glucose and HbA1 levels were observed during therapy with ACE inhibitors. In the third study, dopaminergic antagonist agents were evaluated in diabetic patients with orthostatic hypotension. In 7 patients metoclopramide (20mg intravenously) reduced the fall in mean arterial pressure induced by upright tilt.(ABSTRACT TRUNCATED AT 250 WORDS)