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.

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.

Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection.

The lack of translation between preclinical assays and clinical trials for novel therapies for Chagas disease (CD) indicates a need for more feasible and standardized protocols and experimental models. Here, we investigated the effects of treatment with benznidazole (Bz) and with the potent experimental T. cruzi CYP51 inhibitor VNI in mouse models of Chagas disease by using different animal genders and parasite strains and employing distinct types of therapeutic schemes. Our findings confirm that female mice are less vulnerable to the infection than males, show that male models are less susceptible to treatment with both Bz and VNI, and thus suggest that male models are much more suitable for selection of the most promising antichagasic agents. Additionally, we have found that preventive protocols (compound given at 1 dpi) result in higher treatment success rates, which also should be avoided during advanced steps of in vivo trials of novel anti-T. cruzi drug candidates. Another consideration is the relevance of immunosuppression methods in order to verify the therapeutic profile of novel compounds, besides the usefulness of molecular diagnostic tools (quantitative PCR) to ascertain compound efficacy in experimental animals. Our study aims to contribute to the development of more reliable methods and decision gates for in vivo assays of novel antiparasitic compounds in order to move them from preclinical to clinical trials for CD.

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.