Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is considered a neglected tropical disease due to the scarcity of safe and effective treatments ¹ . Current therapeutic options are limited to nifurtimox (NFX) and benznidazole (BZ), both developed over 40 years ago.

Etiological treatment during the acute stage results in high rates of parasitemia clearance and serological negativity, while treatment success during the chronic phase is currently arguable. Adverse events are usual, especially in adults, forcing treatment discontinuation (Pérez-Molina and Molina, 2018).

Despite the urgent need to develop safer drugs, the pharmaceutical industry has shown limited interest in research for new chemical entities, probably due to the socioeconomic condition of most affected patients (Paulino et al., 2005).

Drug repurposing (i.e., the study of drugs already developed and tested for other diseases) (Chatelain and Ioset, 2011) and combination therapy arise as cost-effective strategies to fill the gap in the drug discovery pipeline (Keenan et al., 2013a; Andrews et al., 2014). Additionally, a positive drug combination increases drug efficacy by targeting multiple metabolic pathways, minimizing the risk of drug resistance, and reducing the frequency and intensity of dose-dependent adverse reactions (Field et al., 2017).

Initially developed for treating skin metastases in breast tumors, miltefosine (MLT), also known as hexadecylphosphocholine, is the first and to date only oral drug for visceral and cutaneous leishmaniasis (Dorlo et al., 2012). Extensive experience with MLT in leishmaniasis clinical trials suggests an excellent safety profile for patients, including pediatric patients, with low to moderate adverse events (Singh et al., 2006; Mbui et al., 2019). Still, some concerns remain regarding MLT effects on the gastrointestinal tract and potential teratogenicity (Dorlo et al., 2012).

MLT is considered a protein kinase B (PKB) inhibitor with a key role in intracellular signaling for cell viability (Dorlo et al., 2012). However, the mechanisms of action related to its anti-protozoal activity are not fully clarified yet. Some evidence suggests an inhibitory effect on phosphatidylcholine and sphingomyelin synthesis, triggering apoptosis (Maya et al., 2007; Apt, 2010) or inhibiting the mitochondrial cytochrome c oxidase and altering the organelle membrane (Dorlo et al., 2012; Ghosh et al., 2013). Similarly, it was proposed that MLT inhibits the parasite phosphatidylcholine biosynthesis through the transmethylation pathway (Lira et al., 2001).

Recently, it was proposed that MLT targeted the sphingosine-activated plasma membrane directly Ca⁺² channel, both on Leishmania donovani and T. cruzi, affecting the intracellular calcium homeostasis (Benaim et al., 2020).

Previous reports indicated that MLT exhibits both in vitro (Croft et al., 1996; Santa-Rita et al., 2000) and in vivo activity (Saraiva et al., 2002; Martinez-Peinado et al., 2021) against T. cruzi.

However, in vivo efficacy was evaluated by different monotherapy treatment schemes and cure criteria hampering the assessment of the MLT ability to eradicate T. cruzi infection. Therefore, we conducted a set of in vitro and in vivo experiments based on previous guidelines (Romanha et al., 2010) to evaluate MLT efficacy alone and in combination with BZ.

MLT is a lipophospholipid analog developed originally as an antineoplastic agent (Dorlo et al., 2012). Based on previous in vitro and in vivo studies and results from oncology clinical trials, MLT was repositioned for visceral and cutaneous leishmaniasis treatment. Since its registration in 2002, MLT remains the only oral drug available for leishmaniasis (Reimão et al., 2020).

MLT and other alkyl-lipophospholipids have proven activity on T. cruzi (Croft et al., 1996; Luna et al., 2009). Our results support the MLT effect on inhibiting amastigote development in vitro and the low efficacy on trypomastigote stage (Croft et al., 1996; Santa-Rita et al., 2000). Moreover, MLT high selectivity index suggests a specific antiparasitic mechanism of action without affecting the host cell.

To date, only three studies have assessed the in vivo efficacy of MLT (Croft et al., 1996; Saraiva et al., 2002; Martinez-Peinado et al., 2021) in mice models of acute infection. While Croft et al. observed transient parasitemia suppression without a curative effect or reduction of mortality in mice treated for 5 days at up to 30 mg/kg/day by oral route, Saraiva et al. reported parasitic sterilization in all mice treated with 25 mg/kg/day, determined by direct blood observation at 120 dpi and spleen culture for 15 days. Recently, Martinez-Peinado et al. studied MLT efficacy by giving 30 mg/kg/day for 10 days, using the Brazil strain expressing firefly luciferase, which yielded statistical differences in luciferase activity compared to infected non-treated mice.

It is well known that several experimental variables affect the infection course and the main outcomes in animal models of T. cruzi infection (Chatelain and Konar, 2015), and their heterogeneity make difficult the direct comparison between results.

In this work, we evaluated the in vivo efficacy of MLT using state-of-the-art methodology based on previous guidelines for drug screening (Romanha et al., 2010) by using qPCR and immunosuppression cycle to elucidate the possible reemergence of parasites from tissues.

The murine model of acute infection with T. cruzi was previously standardized (Gulin et al., 2018), using the VD strain (TcVI), originally isolated from a patient infected by a congenital route. This model becomes relevant since mother-to-child infection is the main transmission mode in vector-free areas within and outside Latin America (Carlier et al., 2019). Furthermore, the VD strain displays high parasitemia levels and mortality rates in infected non-treated mice and a moderate BZ and NFX effect to eradicate the infection.

Despite a temporary animal discomfort observed in MLT oral administration, the treatment was well tolerated by the animals. Also, the weight loss and ruffled fur agree with previously reported effects (Saraiva et al., 2002). The body weight loss could be explained by the gastrointestinal discomfort caused by MLT at all assayed doses, an adverse effect widely reported in clinical trials (Jha et al., 1999; Sundar et al., 2002).

As described by Saraiva et al., the parasitemia course throughout the acute phase of infection was not suppressed with 25 mg/kg/day of MLT. However, in our infection model, the days of patent parasitemia and the MPR were lowered in a dose-dependent manner by MLT, and the 100 mg/kg/day dose was able to reduce blood trypomastigotes circulation to undetectable levels by optical microscopy.

When administered alone, MLT at lower doses leads to a parasitemia reduction, and this could be explained in part due to the widespread drug distribution and the slow clearance rate. In mice, oral MLT is absorbed slowly but extensively and accumulates mainly in the kidneys, liver, and lungs (Breiser et al., 1987). Clinical pharmacokinetic studies indicate that, due to its prolonged half-life, MLT accumulates during treatment reaching a steady-state concentration in the last week of a 28-day treatment schedule (Kip et al., 2018).

Although the drug failed to produce parasitic sterilization, it was able to maintain the burden of amastigotes at low levels, which was reactivated by CYP-induced immunosuppression. Moreover, animals treated with MLT showed lower rates of amastigote nests in the myocardium and in skeletal muscle compared to NT, which could be explained by the MLT extensive distribution in these tissues, as previously documented in mice and rats (Breiser et al., 1987; Marschner et al., 1992).

Failure to reach parasitological sterilization was also detected in a percentage of the animals treated with BZ or NFX. These results coincide with the previously published characterization of the VD strain (Gulin et al., 2018), and with other T. cruzi strains from the same DTU, usually classified as BZ-sensitive (Fernández et al., 2010; Keenan et al., 2013b).

MLT effect on Leishmania spp. was also studied in combination with reference drugs or with repositioned compounds. In vitro additive synergism with sodium stibogluconate and in vivo potentiate synergism potentiation combined with paromomycin or amphotericin B were reported (Seifert and Croft, 2006). Costa et al. also reported the parasiticidal effect of efavirenz on L. infantum and a lower IC₅₀ adding MLT (Costa et al., 2016).

The combined effect of MLT with other compounds on T. cruzi viability was previously described by in vitro combination with ketoconazole, both on trypomastigotes and intracellular amastigotes (Santa-Rita et al., 2005). However, this is the first study of the anti-T cruzi effect of the MLT and BZ combination.

The analysis of the MLT+BZ in vitro combination suggests a synergistic effect on trypomastigotes viability and an additive interaction on intracellular amastigote development. This association was not evaluated previously, although similar in vitro interactions were reported when combining MLT with paromomycin (de Morais-Texeira et al., 2014) or nitazoxanide (Mesquita et al., 2014) on Leishmania spp.

We furthered the evaluation of the MLT and BZ association on a murine model of acute infection with T. cruzi. MLT+BZ combination at sub-therapeutic doses could not achieve an effect different from that obtained with the drugs separately. However, it decreased parasite loads in blood and prevented mortality in treated mice. Nevertheless, MLT+BZ at half the total dose (i.e., 50mg/kg) produced negative parasitemia and prevented the reactivation after the immunosuppression protocol.

The qPCR yielded disparate results in target organs such as skeletal muscle and myocardium in both experimental groups, with detection of parasitic DNA in low-loading or even non-quantifiable cases. These results could then question qPCR as a method to establish parasitological cure in cases where T. cruzi DNA is not detected in the blood.

Residual DNA in tissues and phagocytic cells has been reported in experimentally infected mice treated with BZ and considered cured by different methods (Guarner et al., 2001; Martins et al., 2008), which would indicate the presence of non-viable parasites or even cross-contamination with parasitic DNA.

However, recently described spontaneous dormancy in T. cruzi (Sánchez-Valdéz et al., 2018) could also explain the presence of parasitic DNA in tissues due to the non-replicative amastigote stage in the absence of bloodstream trypomastigotes, which would deserve further investigation to fully understand therapeutic failure both in preclinical studies and clinical trials.

Although our results demonstrate the inability of MLT to achieve a complete parasitological cure in an acute murine model of T. cruzi infection, the positive interaction with BZ both in vitro and in vivo opens a promising alternative in drug repurposing and combinatory therapy approach for Chagas disease treatment. MLT and other synthetic alkylphospholipids arise as drug candidates for combined treated schemes and serve as chemical scaffolds for new compound entities.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee from the Faculty of Veterinary Sciences (University of Buenos Aires) (Protocol #2014/04).

JENG: Writing - original draft, Writing -review & editing, Investigation, Validation, Formal analysis. MMCB: Writing -review & editing, Investigation, Validation, Formal analysis. DR and MES: Investigation, Validation, Formal analysis. JA: Conceptualization, Methodology, Funding acquisition, Resources, Supervision. FG-B: Conceptualization, Methodology, Writing - review & editing, Funding acquisition, Resources, Supervision. All authors contributed to the article and approved the submitted version.

This work was supported by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación, Ministerio de Ciencia y Técnica, Argentina (Grant: Proyectos de Investigación Cientı́fica y Tecnológica PICTO-GLAXO 2012-035).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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