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In vitro and in vivo identification of tetradentated polyamine complexes as highly efficient metallodrugs against Trypanosoma cruzi

https://doi.org/10.1016/j.exppara.2016.02.004Get rights and content

Highlights

  • Low toxicity alternative treatment against Trypanosoma cruzi in mice.

  • Tetraamine 2 in vitro was 108 times better than Bz.

  • Parasitemia reactivation of only 10.1% in mice treated with Tetraamine 2.

  • 50% of cure in the mice treated with Tetraamine 3 after chronic phase.

Abstract

In order to identify new compounds to treat Chagas disease during the acute phase with higher activity and lower toxicity than the reference drug benznidazole (Bz), a series of tetraamine-based compounds was prepared and their trypanocidal effects against Trypanosoma cruzi were evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by flow cytometry assays against Vero cells. In vivo assays were performed in BALB/c mice, in which the parasitemia levels were quantified by fresh blood examination; the assignment of a cure was determined by PCR and reactivation of blood parasitemia levels after immunosuppression. The mechanism of action was elucidated at metabolic and ultra-structural levels by 1H NMR and TEM studies. Finally, as tetraamines are potentially capable of casuing oxidative damage in the parasites, the study was completed by assessing their activity as potential iron superoxide dismutase (Fe-SOD) and trypanothione reductase (TR) inhibitors. High-selectivity indexes observed in vitro were the basis of promoting three of the tested compounds to in vivo assays. The tests on the murine model for the acute phase of Chagas disease showed better parasitemia inhibition values than those found for Bz. Tetraamines 2 and 3 induced a remarkable decrease in the reactivation of parasitemia after immunosuppression and curative rates of 33 and 50%, respectively. Tetraamine 3 turned out to be a great inhibitor of Fe-SOD and TR. The high anti-parasitic activity and low toxicity render these tetraamines appropriate molecules for the development of an affordable anti-Chagas agent.

Introduction

The World Health Organisation (WHO) lists Chagas disease as one of the most neglected tropical diseases. Chagas disease is endemic in Latin America; it is estimated that 10 million people are infected worldwide and more than 25 million are living at risk of infection (World Health Organization, 2012). The main drugs used for the treatment of Chagas disease are benznidazole (Bz, LAFEPE) and nifurtimox (Lampit®, Bayer), which present significant side effects and cure <20% of patients with chronic Chagas disease (Mckerrow et al., 2009). Thus, many researchers have combined efforts to discover drugs against new targets, looking for lower toxicity and a greater tolerance in patients, aiming not only for efficiency in the acute phase, but also in the chronic phase.

One of the self-defence strategies of Trypanosoma cruzi (the etiological agent of Chagas disease) is its highly active and exclusive iron superoxide dismutase (Fe-SOD), which differs from Cu–Zn-SOD or Mn-SOD operating in mammals. Fe-SOD is an extremely efficient enzyme for preventing any oxidative damage from the host in combination with peroxidases. The other self-defence strategy is trypanothione, a parasitic molecule involved in protozoan protection against oxidative stress, which is now considered a virulence factor of Chagas disease (Piacenza et al., 2013) Thus, the use of molecules that interfere with the enzyme trypanothione reductase (TR), which keep the trypanothione molecule in its reduced status, may lead to parasite death. In this sense, we focused on complexes capable affecting or interfering with these exclusive enzymes of T. cruzi.

In previous work, we designed iron and manganese coordination complexes containing polyamine ligands, which are capable of generating highly oxidising species (Cussó et al., 2013b, Garcia-Bosch et al., 2012, Company et al., 2011) Highly reactive Mn(IV)double bondO and Fe(IV)double bondO species embedded in these ligand scaffolds have been prepared and their oxidative reactivity studied. Furthermore, these polyamine ligands bind very strongly to iron and manganese ions, forming coordination complexes that are stable under highly acidic and oxidative conditions.

Herein, we report on the in vitro and in vivo anti-trypanosomal properties of Mn-based polyamines and polyamine complexes (Fig. 1), which represent a class of compounds that have, so far, only rarely been explored for Chagas disease chemotherapy. Finally, we analyse the possible mechanism of action over the parasite structure and function as well as the protective enzymes mentioned above.

Section snippets

Chemistry

Most of our compounds (1, 2, 4 and 5) have been previously synthesized for organic synthesis purposes (Cussó et al., 2013a, Costas and Que, 2002) On the other hand, the compound 3 has been designed as a structural variant of compound 2 and synthesized for the first time in this work. The structures are shown in Fig. 1.

Compound 3 synthesis

A suspension of Mn(OTf)2 (69.7 mg, 0.16 mmol) in anhydrous dichloromethane (1 mL) was added dropwise to a vigorously stirred solution of (R,R)-dMMBPMCN (Cussó et al., 2013a)

Chemistry

Manganese-based coordination complexes (13) were selected for their ability to generate high-oxidation-state compounds, which are potentially capable of causing oxidative damage in the parasite. Additionally, water-soluble compounds (4, 5) were studied to evaluate potential Fe-chelation in causing Fe-SOD dysfunction and inducing parasite death.

In vitro trypanosomicidal evaluation

All five polyamines showed better selectivity indexes (SI = IC50 Vero cells/IC50 extracellular and intracellular forms of T. cruzi) compared to the

Conclusion

The trypanocidal properties of a series of compound derivatives have been examined both in vivo and in vitro. The experiments allowed us to select compounds that displayed improved efficiency and lower toxicity than the reference drug. Parallel studies have been carried to establish the mechanisms of action. Compound 3 selectively inhibits the Fe-SOD and TR enzymes of the parasite. Moreover, polyamine 3 showed an in vivo cure rate of 50% at a security standard dosage of 15 mg/kg body mass.

Financial & competing interests disclosure

This work was supported by: Spanish Ministry of Economy and Competitiveness (MINECO), CONSOLIDER-INGENIO 2010 CSD2010-00065. European Research Foundation for Project ERC-2009-StG-239910, MICINN for project CTQ2009-08464. INNPLANTA project INP-2011-0059-PCT-420000-ACT1. Authors declare no conflicts of interest. No written assistance has been required for the content but the English has been corrected by the company Proof Reading Service (Devonshire Business Centre. Works Road. Letchworth Garden

Acknowledgements

We acknowledge Generalitat de Catalunya for an ICREA Academia Award (X.R. and M.C.). F.O. is grateful for a FPU Grant from the Ministry of Education of Spain (AP-2010-3562) and he also thanks Natalie Dirdjaja and Dr. Alejandro Leroux for their support with the kinetic analysis of TR.

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