This study was performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (COBEA) and had the approval of the Animal Ethics Committee of Oswaldo Cruz Foundation (license number L-026/2015).

The Leishmania infantum (MHOM/MA/67/ITMAP-263) used in this study is a well-established strain and was kindly provided by Instituto de Biologia Molecular e Celular, Porto University, Portugal.

Cyclobenzaprine (CBP), DTNB (Ellman's reagent), propidium iodide (PI), resazurin, lipopolysaccharide (LPS), concanavalin A (ConA), miltefosine and rhodamine 123 were purchased from Sigma–Aldrich (St Louis, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) from Molecular Probes (Eugene, USA), and Glucantime from Sanofi-Aventis (São Paulo, Brazil).

Promastigotes of L. infantum (strain MHOM/MA/67/ITMAP-263) at 1.0x10⁶ cells/mL were cultivated with cyclobenzaprine in triplicate (0–50 μM) at 26°C in Schneider’s medium (Sigma-Aldrich) supplemented with 20% heat-inactivated fetal calf serum (HIFCS), for 72 h. Inhibition of parasite growth was assessed employing resazurin [6].

For the intracellular amastigote assay, resident peritoneal macrophages were collected from BALB/c mice, plated in RPMI (Sigma-Aldrich) at 2x10⁶ cells/mL in duplicate, infected with promastigotes of L. infantum (5:1) and incubated at 37°C in 5% CO₂ for 4 h. After washing, macrophages were incubated with cyclobenzaprine for further 72 h. The slides were stained and the amastigotes were counted using light microscopy.

Dose Translation: The initial dose was calculated based on the human dose, as follows [7]: Animal dose (mg/kg) = (human K_m / animal K_m) x human dose (mg/kg), where mouse K_m = 3, human K_m = 37.

Protocol: BALB/c mice (five per group) were infected intraperitoneally with 1.0x10⁸ stationary-phase L. infantum promastigotes [8–10]. After 25 days, mice were treated with three CBP doses (12.32, 24.64 and 49.28 mg/kg) by oral gavage for five days [11]. Control groups were treated with miltefosine 20.55 mg/kg and vehicle (water). After the treatment, the animals were euthanized and spleen and liver were aseptically removed and the parasite load was estimated by limiting dilution assay (LDA) [12]. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using laboratory colorimetric diagnostic kits (Doles, Goiânia, Brazil).

The assay was adapted from Hamilton et al. [13]. Briefly, promastigotes of L. infantum were centrifuged and resuspended in buffer containing 40 mM HEPES, 1 mM EDTA and protease inhibitor cocktail (Sigma-Aldrich). The parasites were lysed and centrifuged at 17.500g/15 min. The supernatant was considered to contain the trypanothione reductase (TryR). The assay was performed in triplicate with: 40 mM HEPES (pH 7.5), 1 mM EDTA, 100 μM of DTNB (Ellman's reagent), 1 μM substrate (trypanothione trifluoroacetate, Sigma-Aldrich), 0.1 mM NADPH and different concentrations of cyclobenzaprine (0–100μM). Reading was initiated after adding 4 mg/mL soluble protein (enzyme) at 410nm using a Spectra Max M2 spectrofluorometer (Molecular Devices, Silicon Valley, CA, USA) at 10 min intervals for 1 h. Clomipramine was used as positive control (PC). This drug has well-known action on the inhibition of trypanothione reductase enzyme [14]. Apparent Ki was assumed to be the value of the 50% inhibitory concentration obtained from the dose-response curve inhibition of TryR by CBP in 60 minutes. Ki value was determined by nonlinear least-squares fitting of these data to the 4-parameter logistic plot equation, as described by Holloway GA, Charman WN, Fairlamb AH, et al, (2009): y = A+{(B–A)/[1+(C/x)^D]} [15].

Intracellular ROS levels were measured as described by Ribeiro et al [16]. Briefly, 1×10⁷ promastigotes/mL of L. infantum were incubated with CBP (0 to 50 μM) in the presence of 20 μM H₂DCFDA in Schneider’s medium with 20% HIFCS. The fluorescence was quantified at 1 h intervals for 6 h.

Briefly, macrophages obtained from peritoneal cavity were plated at 1x10⁵ cells/mL in a 24 well plate with complete RPMI 1640 medium supplemented with 10% HIFCS and infected overnight with amastigotes of L. infantum (ratio 10:1). After 18 hours of infection, macrophages were co-cultured with splenocytes obtained from BALB/c mouse at a ratio 1:6. Cells were treated with a non-toxic concentration of CBP (100 μM) for 48 h. Afterwards, the cell supernatant was analyzed for IL-6, IL-10, monocyte chemotactic protein-1 chemokine (MCP-1), IFN-γ and TNF-α levels using flow cytometry and the Cytometric Beads Array (CBA) kit (BD Bioscience, San Diego, CA, USA). Controls consisted of sham-treated macrophages infected with L. infantum, LPS (1 μg/mL) and concanavalin A (1 μg/mL), and the negative control (macrophages without the drug). The nitric oxide content was colorimetrically determined by the Griess reaction in the culture supernatants of peritoneal macrophages [17]. Cells were treated for 24 h with cyclobenzaprine at 12 μM, corresponding to the IC₅₀ value against intracellular amastigotes. Bacterial lipopolysaccharide (LPS) was used as positive control (1 μg/mL). The obtained results were extrapolated from a standard curve prepared with NaNO₂ at different concentrations (0 to 400 μM).

CBP demonstrated a concentration-dependent activity in L. infantum promastigotes growth, with an IC₅₀ value of 14.5±1.1μM and an IC₉₀ value of 74.5±1.2 μM (Fig 1A). The drug was also effective against clinically relevant forms, the intracellular amastigotes, with an IC₅₀ of 12.6±1.05 μM and an IC₉₀ of 28.7±1.3 μM (Fig 1B). The treatment with 50 μM reduced this index by 99% when compared to control (insert Fig 1B).

Cyclobenzaprine was effective in reducing the parasite load by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively (Fig 2A) and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg (Fig 2B), after a short-term treatment. We did not observe changes in hepatic transaminases levels in groups treated with CBP. However, there was a significant increase of ALT level in the group treated with 20.55 mg/kg of miltefosine.

CBP inhibited the TryR activity in a concentration-dependent manner (Fig 3A), with a Ki of 86 ± 7.7 μM. CPB also increased the ROS production in promastigotes in a time and concentration-dependent manner in the first 6 hours, consistent with TryR inhibition (Fig 3B).

Flow cytometry data demonstrated that CBP inhibited the production of IL-6 in infected macrophages, decreasing the production of this cytokine by 53% when compared to untreated macrophages (Fig 4a and 4b). MCP-1 levels of macrophages co-cultured with splenocytes were not altered by CBP treatment (Fig 4c and 4d). The control of cytokines production was confirmed in the LPS and ConA-treated groups. The treatment of macrophages with CBP resulted in unaltered levels of TNF-α, IFN-γ and IL-10 (Fig 4e–4j). Griess assay demonstrated an upregulation of nitric oxide levels of 3.2 times in macrophages incubated with CBP (Fig 5).