In Vitro and In Vivo Trypanocidal Activity of H2bdtc-Loaded Solid Lipid Nanoparticles

et al. (2014) In Vitro and In Vivo Trypanocidal Activity of H2bdtc-Loaded Solid Lipid Nanoparticles. PLoS Negl Trop Dis 8(5): e2847. doi:10.1371/journal.pntd.0002847 In Vitro and In Vivo Trypanocidal Activity of H2bdtc-Loaded Solid Lipid Nanoparticles Zumira A. Carneiro 0 Pedro I. da S. Maia 0 Renata Sesti-Costa 0 Carla D. Lopes 0 Tatiana A. Pereira 0 Cristiane M. Milanezi 0 Marcelo A. Pereira da Silva 0 Renata F. V. Lopez 0 Joa o S. Silva 0 Victor M. Deflon 0 Michael P. Pollastri, Northeastern University, United States of America 0 1 Faculdade de Ciencias Farmaceuticas de Ribeira o Preto, University of Sa o Paulo , Ribeira o Preto, Sa o Paulo , Brazil , 2 Instituto de Qu mica de Sa o Carlos, University of Sa o Paulo, Sa o Carlos, Sa o Paulo, Brazil, 3 Departamento de Bioqu mica e Imunologia, School of Medicine, University of Sa o Paulo , Ribeira o Preto, Sa o Paulo , Brazil , 4 Instituto de F sica de Sa o Carlos, University of Sa o Paulo, Sa o Carlos, Sa o Paulo, Brazil, 5 Centro Universita rio Central Paulista - UNICEP, Sa o Carlos , Sa o Paulo , Brazil The parasite Trypanosoma cruzi causes Chagas disease, which remains a serious public health concern and continues to victimize thousands of people, primarily in the poorest regions of Latin America. In the search for new therapeutic drugs against T. cruzi, here we have evaluated both the in vitro and the in vivo activity of 5-hydroxy-3-methyl-5-phenyl-pyrazoline1-(S-benzyl dithiocarbazate) (H2bdtc) as a free compound or encapsulated into solid lipid nanoparticles (SLN); we compared the results with those achieved by using the currently employed drug, benznidazole. H2bdtc encapsulated into solid lipid nanoparticles (a) effectively reduced parasitemia in mice at concentrations 100 times lower than that normally employed for benznidazole (clinically applied at a concentration of 400 mmol kg21 day21); (b) diminished inflammation and lesions of the liver and heart; and (c) resulted in 100% survival of mice infected with T. cruzi. Therefore, H2bdtc is a potent trypanocidal agent. - Funding: This work was supported by the Research Foundation of the State of Sao Paulo (FAPESP 2010/20610-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. T. cruzi parasites are transmitted by insect vectors (triatomine bugs). T.cruzi is the causative agent of Chagas disease, which is silent and can remain asymptomatic for years [1], [2], [3]. A century after its discovery, this disease remains a serious public health issueit is closely associated with human poverty and political instability as well as with little investment in drug development. According to the World Health Organization (WHO), between seven and eight million people are infected with T. cruzi worldwide, primarily in Latin America [4], [5], [6]. One in every four Chagas patients develops a fatal symptom of the disease due to lack of adequate diagnosis and treatment. Nifurtimox and benznidazole (BZN) are currently available to treat the disease [7], [8], [9], [10]. However, neurological side effects have led commercial nifurtimox production to be discontinued [11]. As for BZN, although it is mainly effective during the acute phase of the infection, it presents undesirable side effects such as rash and gastrointestinal symptoms [12], so patients often fail to comply with the treatment [8]. Long treatment periods (30, 60, or 90 days) and appropriate pediatric formulations not available (administration of the medication to children often requires tablet fractionation) also limit BZN use [9], [11], [13]. A further concern is that no effective treatment for the symptomatic chronic phase of Chagas disease exists, so the patients usually receive palliative drugs at this stage [14], [15]. Therefore, a number of researchers are making considerable efforts to find new drugs to combat this disease. Dithiocarbazates display notable biological and pharmacological properties, including anticancer [16], [17], antimicrobial [17], [18], [19], and insecticidal activities [20]. A recent study has shown that cyclic compounds derived from S-dithiocarbazate and 1,3-diketones exhibit significant trypanocidal activity [21]: in particular, 5-hydroxy-3-methyl-5-phenyl-pyrazoline-1-(S-benzyldithiocarbazate) (previously referred to as H2L2a [21]) which was renamed H2bdtc in this reference in this work (Figure 1) constitutes a potential drug lead to develop a new agent against the trypomastigote form of Tulahuen strains of T. cruzi [21]. Nevertheless, the lipophilic character of H2bdtc may limit its administration and result in low oral bioavailability [22]. Drug delivery systems can help to circumvent this problem. Because lipids have excellent physiological acceptability and can promote drug absorption as well as selective lymphatic uptake, researchers have focused on lipid-based drug release systems [23]. In particular, solid lipids constitute solid lipid nanoparticles (SLNs) at room and body temperature. Since SLNs consist of biocompatible and biodegradable lipids with low or no human toxicity, they can function as drug delivery systems [24], [25]. SLNs offer many advantages: they protect the drug against degradation, enable controlled drug release, and dismiss the use of organic solvents. Moreover, SLNs can be produced on a large scale, meeting industrial requirements [24], [26]. The protozoan parasite Trypanosoma cruzi causes Chagas disease, a condition that affects the poorest regions of Latin America mainly. The chronic phase of this disease disables thousands of patients, constituting an important public health issue. The pharmacotherapy that is currently applied to treat the disease emerged many decades ago, is ineffective in most patients, mainly during the chronic phase, and has serious side effects. In a recent study, we showed that the compound 5-hydroxy-3-methyl-5-phenylpyrazoline-1-(S-benzyldithiocarbazate) (H2bdtc) is a potential drug candidate against the in vitro trypomastigote form of Tulahuen strains of T. cruzi. Here we report that H2bdtc loaded into solid lipid nanoparticles (H2bdtc-SLNs) displays good trypanocidal activity against the trypomastigote form of the Y strain of T. cruzi both in vitro and in vivo. Our in vivo experiments revealed that H2bdtc-SLN is 100 times more active than benznidazole (BZN), the drug that is commercially available to treat Chagas disease. Surprisingly, this compound has no side effects on the T. cruzi acute phase. Hence, we propose that H2bdtc-SLNs possesses interesting anti-Trypanosoma properties. Our group has used H2bdtc in vitro experiments involving the Tulahuen strain (group I) [21]. The resistances of this group and of the Y strain (group II) have been reported to be different, based on phosphatase activities in T. cruzi homogenates [27]. Tulahuen had an optimum phosphatase a (...truncated)