Looking for combination of benznidazole and Trypanosoma cruzi-triosephosphate isomerase inhibitors for Chagas disease treatment

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 113(3): 153-160, March 2018 153 Looking for combination of benznidazole and Trypanosoma cruzitriosephosphate isomerase inhibitors for Chagas disease treatment Elena Aguilera1, Javier Varela1, Elva Serna2, Susana Torres2, Gloria Yaluff2, Ninfa Vera de Bilbao2, Hugo Cerecetto1,3, Guzmán Alvarez1,4, Mercedes González1/+ Universidad de la República, Facultad de Ciencias, Grupo de Química Medicinal, Montevideo, Uruguay Universidad Nacional de Asunción, Instituto de Investigaciones en Ciencias de la Salud, Departamento de Medicina Tropical, Asunción, Paraguay 3 Universidad de la República, Facultad de Ciencias, Centro de Investigaciones Nucleares, Área de Radiofarmacia, Montevideo, Uruguay 4 Universidad de la República, Centro Universitario Regional Litoral Norte, Laboratorio de Moléculas Bioactivas, Paysandú, Uruguay 1 2 BACKGROUND The current chemotherapy for Chagas disease is based on monopharmacology with low efficacy and drug tolerance. Polypharmacology is one of the strategies to overcome these limitations. OBJECTIVES Study the anti-Trypanosoma cruzi activity of associations of benznidazole (Bnz) with three new synthetic T. cruzi- triosephosphate isomerase inhibitors, 2, 3, and 4, in order to potentiate their actions. METHODS The in vitro effect of the drug combinations were determined constructing the corresponding isobolograms. In vivo activities were assessed using an acute murine model of Chagas disease evaluating parasitaemias, mortalities and IgG anti-T. cruzi antibodies. FINDINGS The effect of Bnz combined with each of these compounds, on the growth of epimastigotes, indicated an additive action or a synergic action, when combining it with 2 or 3, respectively, and an antagonic action when combining it with 4. In vivo studies, for the two chosen combinations, 2 or 3 plus one fifth equivalent of Bnz, showed that Bnz can also potentiate the in vivo therapeutic effects. For both combinations a decrease in the number of trypomastigote and lower levels of anti-T. cruzi IgGantibodies were detected, as well clear protection against death. MAIN CONCLUSIONS These results suggest the studied combinations could be used in the treatment of Chagas disease. Key words: Chagas disease - synergism - isobologram - in vivo studies Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), represents a health threat for about 10-20 million people, being the second highest burden of disease among tropical diseases in the Americas (Nouvellet et al. 2015). The current chemotherapy is based on monopharmacology using nifurtimox (Nfx) or benznidazole (Bnz). They have limited efficacy and severe side effects (Castro et al. 2006). Some strategies to overcome the treatment limitations have included the development of new drugs, polypharmacologies and drug repositioning (Bahia et al. 2014). In the first approach, to identify new drugs, hundreds of compounds, from synthetic and natural sources, have been tested against T. cruzi (Cerecetto & González 2010, González & Cerecetto 2011). We recently described new compounds, belonging to different chemotypes, which were able to act in vivo decreasing the animal parasitaemia, i.e. compounds 1-4 (Fig. 1), surpassing the “hit-to-lead” drug discovery stage. They were designed as T. cruzi triosephosphate isomerase (TcTIM) inhibitors (Álvarez et al. 2015a, b, Aguilera et al. 2016) finding in some cases, i.e. derivatives 3 and 4, the doi: 10.1590/0074-02760170267 Financial support: Comisión Sectorial de Investigación Científica (CSIC)Universidad de la República (CSIC No 661). EA was supported by Agencia Nacional de Investigación e Innovación (ANII, POS_NAC_2014_1_102490, Uruguay). + Corresponding author: Received 7 July 2017 Accepted 25 October 2017 best results against this biological target. Although they displayed excellent in vivo behaviour some limitations were observed. For example, derivative 2 (Álvarez et al. 2015b), unlike derivative 1 at similar doses and administration regime (Álvarez et al. 2015a), showed limited survival rate of animals. On the other hand, derivatives 3 and 4, unlike derivatives 1 and 2, produced an increment of parasitaemia after the end of the treatment and limited survival rate of animals (Aguilera et al. 2016). Concerning Chagas disease, evidences have grown in favour of the use of drugs combinations to enhance treatment efficacy and tolerance. These studies focused in the combination of different chemotypes with different parasitic point of actions trying to produce complete cure, reduce drug doses or diminish duration of the treatments. Some relevant examples are the drug repositioning approach using: anti-fungal agents combined with benznidazole (Araújo et al. 2000, da Silva et al. 2012, Diniz et al. 2013, Martins et al. 2015), combination of different anti-fungals (Urbina et al. 1988), anti-fungals combined with the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase lovastatin (Urbina et al. 1993), an anti-fungal agent combined with the antiarrhythmic amiodarone (Benaim et al. 2006), an antifungal agent combined with an anti-tuberculosis drug (Veiga-Santos et al. 2015), suramin combined with Bnz (Santos et al. 2015), anti-inflammatory agents aspirin or simvastatin combined with Bnz or Nfx (López-Muñoz et al. 2010, Campos-Estrada et al. 2015), the glutathionylcysteine inhibitor L-buthionine (S,R)-sulfoximine combined with Nfx (Faúndez et al. 2008), and other combionline | memorias.ioc.fiocruz.br 154 Polypharmacology for Chagas disease • Elena Aguilera et al. Fig. 1: nifurtimox (Nfx), benznidazole (Bnz) and the Trypanosoma cruzi triosephosphate isomerase (TcTIM) inhibitors with in vivo anti-T. cruzi activity described previously by our group (Álvarez et al. 2015a, b, Aguilera et al. 2016). nations (Cencig et al. 2012). Although to a lesser extent, the use of new drugs, from synthetic or natural sources, combined with Bnz was also studied in the polypharmacology approaches (Pelizzaro-Rocha et al. 2010, Valdez et al. 2012, Rodrigues et al. 2014). As part of our ongoing program (Cerecetto & González 2008) in the search of new molecules which could provide new lead compounds for Chagas disease treatment we found excellent in vivo prototypes, such as 2, 3, and 4 (Fig. 1), that require more study from a pharmacological point of view. In this sense, herein we describe the study of these compounds combined with Bnz as potential candidates for the treatment of Chagas disease. MATERIALS AND METHODS Compounds - All chemicals were from Sigma (USA) or Merck (Germany). Compounds 2, 3, and 4 were synthesised as previously (Álvarez et al. 2015a, b, Aguilera et al. 2016). Bnz was purchased from LAFEPE (Pernambuco, Brazil). In vitro drug combination assay - To verify the effect of the combination of thiadiazole 2 and Bnz or 3 and Bnz on epimastigotes we applied method previously described (Hallander et al. 1982, Urbina et al. 1988, 1993, Veiga-Santos et (...truncated)