In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis

Accepted Manuscript Title: In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis Authors: Nilberto Robson Falc˜ao do Nascimento, Francisco L´eo Nascimento de Aguiar, Cl´audia Ferreira Santos, Ang´elica Maria Luna Costa, Daiana de Jesus Hardoim, K´atia da Silva Calabrese, Fernando Almeida-Souza, Eduardo Henrique Silva de Sousa, Luiz Gonzaga de Franc¸a Lopes, Maria Jania Teixeira, Vandbergue Santos Pereira, Raimunda Sˆamia Nogueira Brilhante, Marcos F´abio Gadelha Rocha PII: DOI: Reference:

S0001-706X(18)30738-1 https://doi.org/10.1016/j.actatropica.2019.01.021 ACTROP 4908

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

10 July 2018 14 January 2019 24 January 2019

Please cite this article as: do Nascimento NRF, de Aguiar FLN, Santos CF, Luna Costa AM, de Jesus Hardoim D, da Silva Calabrese K, Almeida-Souza F, de Sousa EHS, de Franc¸a Lopes LG, Jania Teixeira M, Santos Pereira V, Nogueira Brilhante RS, Gadelha Rocha MF, In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis, Acta Tropica (2019), https://doi.org/10.1016/j.actatropica.2019.01.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Original Article – Acta Tropica (2nd version) In vitro and in vivo leishmanicidal activity of a ruthenium nitrosyl complex against Leishmania (Viannia) braziliensis

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*; Francisco Léo Nascimento de Aguiar a; Cláudia

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Nilberto Robson Falcão do Nascimento

Ferreira Santos b; Angélica Maria Luna Costa b; Daiana de Jesus Hardoim c; Kátia da Silva c,d

; Eduardo Henrique Silva de Sousa e; Luiz Gonzaga

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Calabrese c; Fernando Almeida-Souza

de França Lopes e;Maria Jania Teixeira f; Vandbergue Santos Pereira f; Raimunda Sâmia

School of Veterinary, Postgraduate Program in Veterinary Sciences, State University of Ceará,

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a

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Nogueira Brilhante f; Marcos Fábio Gadelha Rocha a, f

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Fortaleza, Ceará, Brazil.

Superior Institute of Biomedical Sciences, State University of Ceará, Fortaleza, Ceara, Brazil;

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Laboratory of Immunomodulation and Protozoology, Oswaldo Cruz Institute, Oswaldo Cruz

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b

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Foundation, Rio de Janeiro, Rio de Janeiro, Brazil. Department of Pathology, State University of Maranhão, São Luiz, Maranhão, Brazil.

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Department of Organic and Inorganic Chemistry, Bioinorganic Laboratory, Federal University

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d

f

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of Ceará, Fortaleza, Ceará, Brazil. Department of Pathology and Legal Medicine, School of Medicine, Specialized Medical

Mycology Center, Postgraduate Program in Medical Microbiology, Federal University of

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Ceará, Fortaleza, Ceará, Brazil. *Corresponding author at: Nascimento, N.R.F. School of Veterinary, State University of Ceará – Av. Dr. Silas Munguba, n. 1700, Itaperi, CEP 60.714-903. Fortaleza, Ceará, Brazil. E-mail address: [email protected] (N.R.F. Nascimento)

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Graphical abstract

Highlights

The ruthenium nitrosyl complex presented antileishmanial activity.

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The ruthenium nitrosyl complex decreased the number of infected cells in vitro

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The ruthenium nitrosyl complex reduced lesion size of footpad in hamster.

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The ruthenium nitrosyl complex eliminated 99,9% of the parasites in vivo.

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ABSTRACT

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Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. There are many complications presented by the current treatment, as high toxicity, high cost and parasite

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resistance, making the development of new therapeutic agents indispensable. The present study

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aims to evaluate the leishmanicidal potential of ruthenium nitrosyl complex cis[Ru(bpy)2(SO3)(NO)](PF6) against Leishmania (Viannia) braziliensis. The effect of this metal complex on parasite-host interaction was evaluated by in vitro efficacy test in dermal fibrobast

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cells in the presence of different concentrations (1, 10, 50 and 100 µM) and by in vivo efficacy tests performed in the presence of two different concentrations of complex (100 µg/kg/day or 300 µg/kg/day) evaluating its effect on the size of the lesion and the number of parasites present in the draining lymph nodes in hamsters. Even at the lowest concentration of 1 µM of ruthenium complex, it was observed a significant decrease of the infected cells, after 24 hours exposure in

vitro, with total reduction at 50 µM of the ruthenium complex. In the in vivo cutaneous infection model, administration of daily doses of 300 µg/kg/day of complex reduced significantly lesion size by 51% (p<0.05), with a 99.9% elimination of the parasites found in the lymph nodes (p<0.001). The results suggest a promising leishmanicidal effect by that ruthenium nitrosyl

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complex against L. (V.) braziliensis.

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Keywords: Leishmania; Ruthenium nitrosyl complex; Metallodrugs; Leishmanicidal effect.

1. INTRODUCTION Leishmaniasis, a neglected tropical disease caused by protozoan parasites distributed among > 20 species of the Leishmania genus, is transmitted to humans through the bite of infected female sandflies. This disease is considered by the World Health Organization (WHO)

related death causing 20,000 to 30,000 deaths annually (WHO, 2017).

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as one of the six most important infectious diseases and is the second leading cause of parasite-

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Based on clinical manifestations, leishmaniasis is classified into: tegumentary leishmaniasis (cutaneous/mucocutaneous) and visceral leishmaniasis (Alemayehu and Alemayehu, 2017; Gradoni, 2018). Estimates suggest there are 12 million people infected, with

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1 million new cases annually, most of which are tegumentary leishmaniasis infections (Kevric

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et al., 2015; WHO, 2017).

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The tegumentary leishmaniasis is endemic in over 85 countries, predominantly in

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tropical and subtropical regions, with over 90% of cases occuring in Americas (mainly in Bolivia, Peru and Brazil) (Gradoni, 2018; WHO, 2017). In Brazil, Leishmania (Leishmania)

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amazonensis and Leishmania (Viannia) braziliensis are the main causative species of american

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tegumentary leishmaniasis, leading to the cutaneous and mucocutaneous forms, respectively (Alemayehu and Alemayehu, 2017; Kevric et al., 2015).

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The currently available treatment for tegumentary leishmaniasis relies on pentavalent antimony as the drug of first choice, and pentamidines, amphotericin B or paromomycin as the second-choice drug. However, the drugs employed for treating leishmaniasis present several

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limitations, including high toxicity, side effects, increased resistance, high cost, a complex therapeutic scheme, among others (Blum et al., 2018). In this context, the search for new highly efficacious therapeutic approaches with lower toxicity and cost becomes essential. In this perspective, the ruthenium complexes stand out for

their use in various medical applications mainly due to low toxicity and biological activity against some types of diseases (Abid et al., 2016; Li et al., 2015; Martínez et al., 2017; Southam et al., 2017). Particularly, nitrosyl ruthenium complexes have exhibited a series of promising biological activities, including anti-parasitic (Orsini et al., 2016; Silva et al., 2010), anti-

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angiogenic (Silva Sousa et al., 2016), analgesic (Staurengo-Ferrari et al., 2013), gastroprotector (Santana et al., 2015), and brain neuroprotection (Campelo et al., 2012), which have motivated

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further studies.

Considering that self-healing patients with tegumentary leishmaniasis present a considerable increase in the production of nitric oxide that may be necessary for the cure of the

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disease (Assis Souza et al., 2013; Carneiro et al., 2016), this study describes the in vitro and in

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vivo antiparasitic activity of ruthenium nitrosyl complex [Ru(bpy)2(SO3)(NO)]+ against L. (V.)

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braziliensis.

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2. MATERIAL AND METHODS 2.1. Parasites

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The L. (V.) braziliensis strain used in this study (MHOM/BR/94/H-3227) was isolated

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from a patient with cutaneous leishmaniasis and characterized by polymerase chain reaction technique and by monoclonal antibodies (Oliveira et al., 2004). The strain was maintained in

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Schneider’s medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 2% sterile human urine and antibiotics (200 U/mL penicillin, 200 µg/mL of streptomycin (Gibco, Grand Island, NY) at 24 ºC.

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2.2. Drugs

The molecule target under study was cis-[Ru(bpy)2(SO3)(NO)](PF6) (RuNO), a

ruthenium bipyridine complex. This compound was synthesized and purified as described

previously (Silva et al., 2006). Meglumine antimoniate (Glucantime; Sanofi-Aventis, São Paulo, Brazil) was used as standard drugs. 2.3. In vitro toxicity of the RuNO complex in dermal fibroblasts Dermal fibroblasts, obtained from embryonic mouse tissue, were cultured in 96-well

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plates (100 µL/well; 104 cells/mL) in DMEM, supplememnted with 10% FBS, 100 U/mL penicillin and 100 µg/mL of streptomycin, and incubated in a 5% CO2 incubator at 37 °C. Cells

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were treated with different concentrations (1, 10, 50 and 100 µM) of RuNO complex. Wells withouth cells, were kept as blank, and wells with cells without treatment were kept as control. After 4h and 24h of treatement, cell viability was evaluated by the modified colorimetric based

on

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tetrazolium

dye

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(3-(4,5-dimethylthiazol-2-yl)-2,5-

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method

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diphenyltetrazolium bromide) (Almeida-Souza et al., 2018). MTT (Sigma, USA ; 5mg/mL)

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was added to each well in a volume equal to 10% of the total. After 3 h, supernatant was

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completely removed and 0.1 mL of DMSO was added to each well to dissolve the formazan crystals. The absorbance was read on a spectrophotometer at a wavelength of 570 nm. Data

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Abs. blank × 100.

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were normalized according to the formula: % survival =Abs. sample-Abs. blank / Abs. control-

2.4. In vitro leishmanicidal effects of the RuNO complex

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The evaluation of the antileishmanial activity, in vitro, of the RuNO complex was performed as described by Costa et al (2017), with some adaptations. Dermal fibroblasts, obtained from embryonic mouse tissue, were infected with promastigotes of L. (V.) braziliensis

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at the ratio of 10:1 (105 parasite:104 fibroblast) on glass coverslips within a 24-well plate with culture medium modified DMEM, supplememnted with 10% FBS, 100 U/mL penicillin and 100 µg/mL of streptomycin, and incubated in a 5% CO2 incubator at 37 °C for 24 h. After this time, infected fibroblasts were incubated with its respective medium alone (control) or

containing different concentrations (1, 10, 50 and 100 µM) of the RuNO complex for 4 h and 24 h at 37 °C in 5% CO2. Then, the coverslips were fixed with 2% formaldehyde, stained with modified Giemsa and analyzed by optical microscopy. The percentage of infected cells and the number of amastigotes per infected cell were determined by randomly counting of 200 cells on

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each coverslip, observing internalization, adhesion or presence of Leishmania (V) braziliensis in the extracellular medium. The experiments were performed in triplicate and two independent

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experiments were conducted.

2.5. In vivo leishmanicidal efficacy tests in an experimental cutaneous model in hamster Twenty-five male golden hamsters (Mesocricetus auratus) weighing 120 g were kept in

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the Central Animal Facility of the Department of Pathology and Legal Medicine of the Federal

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University of Ceará, in groups of six to eight per cage with free access to water and food. All

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procedures involving the uses of animals were approved by the Ethics Committee for Animal

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Research of the State University of Ceará (protocol number 12236805-3/2012). The animals were injected subcutaneously in right hind footpad with 107 stationary

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phase L. (V.) braziliensis promastigotes in 20 µL of sterile saline. After the lesion appeared, the animals were divided into four groups (five animals per group) and treated for thirty days, as

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follows: I) Infected control treated with saline solution administered by the orogastric gavage; II) Infected animals treated with 100 µg/kg/day of RuNO complex administered by orogastric gavage; III) Infected animals treated with 300 µg/kg/day of RuNO complex administered by

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orogastric gavage; IV) Infected animals treated with 60 mg/kg/day of glucantime administered by intramuscular route. The RuNO compound was diluted in NaCl 0.9% and administered daily by orogastric gavage.

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2.5.1. Measurement of injury

Observation of lesion development was made by measuring the footpad swelling, each

week, with use by a gauge caliper. Lesion size was defined as the increase in footpad thickness after subtraction of the size of the controlateral uninfected footpad (Ruiz et al., 2014). 2.5.2. Quantitative parasite burden

Parasite burden was determined using the quantitative limiting dilution assay (Titus et al., 1985). Infected and healthy groups were anesthetized with ketamine (10 mg/kg) and xylazine (90 mg/kg), intramuscularly and euthanized with saturated halothane inhalation. Briefly, popliteal lymph nodes draining the infected footpad were aseptically excised and

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homogenized with a tissue glass grinder in 2 ml of Schneider's medium (Sigma Aldrich, St. Louis, MO). The homogenates were serially diluted in Schneider's medium supplemented with

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10% heat inactivated fetal calf serum (Gibco) and 2% sterile human urine in 96-well plates, 6 plates for each dilution. The plates were subsequently incubated at 25 ºC for 3 weeks and the number of viable parasites was determined under an inverted light microscope (Carl Zeiss, Jena,

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Germany). The experiments were performed in triplicate and three independent experiments

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were conducted.

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2.6. Statistical Analysis

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Number of parasites present in the draining lymph nodes was determined by chisquare test of the minimum applied to the Poisson distribution (Taswell, 1984). The data

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relating to the parasite burden and lesion size were analyzed using the Mann-Whitney test of

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GraphPad Prism program, version 7.0 (GraphPad Software, San Diego., CA). In all tests, the minimum significance was accepted when p <0.05.

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3. RESULTS

Cytotoxicity assay with dermal fibroblasts treated with RuNO complex showed no

cytotoxic effect at any of the concentrations analyzed or at any of the treatment times. Fig. 1

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shows the result of treatment with 10 μM and 50 μM of RuNO complex, after 4 hours of incubation. A significant decrease in the number of infected cells was observed (p <0.05), with reduction of 75% and 87.5%, respectively, while 100 µM concentration of RuNO complex reduced the number of infected cells to 0%. After 24 hours, treatment with RuNO complex in

all concentrations tested reduced significantly (p < 0.05) the number of infected cells when compared with control group, with no infected cells being observed in the treatments using 50 and 100 µM of RuNO complex. In addition to that, at 10 µM and higher concentrations of the RuNO complex an increase in the number of vacuoles in the cytoplasm was observed (not

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shown).

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Fig. 1. Effect of ruthenium nitrosyl complex cis-[Ru(bpy)2(SO3)(NO)](PF6) on parasite viability in dermal fibroblast. The bar graphs show the percentages of fibroblast infected with

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L. (V.) braziliensis, after 4 hours (dark gray bars) and 24 hours (light gray bars) of treatment with 1, 10, 50 and 100 µM RuNO complex in relation to drug-free infection control (black bar).

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Data represent mean ± standard deviation of three independent experiments performed in

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triplicate. *: Statistically significant difference when compared to control (p < 0.05).

In Fig. 2, it is possible to observe that from the first week of treatment (week 5) there

was a statistically significant reduction (p <0.05) in the sizes of lesion in the animals when they were treated with the two concentrations of RuNO complex. There were no statistical differences between the concentration of 300 µg/kg/day of RuNO complex and glucantime.

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Fig 2. Lesion size of hamsters footpad infected with 107 promastigotes of L. (V.) braziliensis

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and treated with ruthenium nitrosyl complex cis-[Ru(bpy)2(SO3)(NO)](PF6) (RuNO). The

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hamsters, from the fourth week of infection, were treated with saline solution (control - line with circular dots), 100 µg/kg/day (line with triangle dots) or 300 µg/kg/day (line with diamond

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dots) of RuNO complex by orogastric gavage or 60 mg/kg/day (line with square dots) of glucantime by intramuscular route for 28 days (fourth week). The points represent the

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arithmetic mean lesion size ± standard deviation. Asterisks represent statistically significant

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difference when compared to control (p < 0.05).

The reduction in the number of parasites/lymph node is quite significant in both

treatment groups with the ruthenium complex (Fig. 3). A reduction was observed in the number of parasites in animals treated with 100 μg/kg/day (75.3%) and 300 μg/kg/day (99.9%) of

RuNO complex in relation to the control treated with saline solution. The effect of 300

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μg/kg/day of RuNO complex was statistically similar to that of glucantime.

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Fig 3. Parasite burden in draining lymph node lesions of hamsters infected with 107

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promastigotes of L. (V.) braziliensis and treated with ruthenium nitrosyl complex cis[Ru(bpy)2(SO3)(NO)](PF6) (RuNO). The hamsters, from fourth week infection, were treated

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with saline solution (control), RuNO complex (100 µg/kg/day or 300 µg/kg/day) by orogastric gavage or glucantime (60 mg/kg/day) by intramuscular route for 28 days. Data represent mean

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± standard deviation. *: Statistically significant difference when compared to control (p < 0.05). 4. DISCUSSION

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Pentavalent antimonials such as glucantime are the first-choice drugs in the treatment

of leishmaniasis, however, there is a high percentage of reported side effects such as arthralgia, myalgia, anorexia, headache, fever, vomiting, and dizziness. Furthermore, these drugs are toxic to the heart, kidneys, liver, and pancreas, and this toxicity represents an important limitation in the use of these drugs by pregnant women, the elderly, and individuals with cardiac disease,

renal disease, or liver alterations (Aronson et al., 2016). Another problem is resistance to these drugs that is already being reported in endemic areas, especially in more severe cases, such as the mucosal form fo leishmaniasis (Légaré and Ouellette, 2017). In this regard, there has been an increase in the search for new substances, including nitric oxide donors, with antileishmanial

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potential. It is well known that NO is one of the crucial molecules in the control of parasite burden

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during the development of tegumentary leishmaniasis. To evade host immunity, parasites of the

genus Leishmania modulate the response in macrophages by decreasing iNOS activity and NO production by depleting the enzyme substrate, as well as increasing the production of essential

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polyamines that are needed for the growth and differentiation of the parasites (Olekhnovitch

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and Bousso, 2015).

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In the dermal fibroblast experiments, the ruthenium complex reduced the number of

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intracellular parasites in a considerable and dose dependent way. This effect was observed in the first hours of treatment and became more pronounced as the hours passed. Other studies

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have already shown that ruthenium complexes of NO donors present antileishmanial activity

2010).

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by NO release and intracellular elimination of the parasite (Orsini et al., 2016; Pereira et al.,

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Some nitrosyl metal complexes were very effective when tested against Leishmania spp. forms such as trans-[Ru(NO)(NH3)4L](X)3, [Ru(NO)Hedta], and Na2[Fe(CN)5NO]·H2O (Khouri et al., 2009; Pereira et al., 2010; Wanasen and Soong, 2008). This strategy could

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promote an increase in pharmacological properties due to a synergistic action between the NO and the metal through multiple mechanisms of action (Gambino and Otero, 2012). Particularly, the [Ru(bpy)2(SO3)(NO)](PF6) complex used in this work also exhibited a series of promising biological activities, along with a great anti-Trypanosoma cruzi effect most likely mediated by

the release of NO (and or HNO) that might impair GAPDH enzymatic activity as a biological target (Silva Sousa et al., 2016). However, due to the reactivity of NO (and HNO) along with metal complex generated, it is likely that these metallonitrosyl complexes reveal a multi-target effect in vivo.

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Additionally, we decided to use the male gold hamster model to evaluate the activity of RuNO complex in vivo based on the fact that male hamsters are known to be highly susceptible

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to Leishmania subgenus Viannia infection (Travi et al., 2002).

In the experiments with male golden hamster infected with L. (V.) braziliensis it was observed that both concentrations (100 and 300 µg/kg/day) of RuNO complex were effective

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in the treatment of leishmaniasis. The RuNO complex was effective both in reducing the

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foodpat lesion and in the parasite burden in draining lymph node lesions.

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Experimental models have shown that the outcome of Leishmania sp. infection is

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critically dependent on the immune response (Sacks and Noben-Trauth, 2002). To avoid host immunity, parasites of the Leishmania genus modulate the defense cell response, decreasing

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iNOS activity and NO production, an important molecule for intracellular elimination of the

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parasite (Horta et al., 2012). Lee and Choy (2013), reported that the addition of exogenous nitric oxide donor was able to restore iNOS levels. Moreover, previous studies have shown that the

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RuNO complex used in this study increases the levels of NO in cell cultures, supporting the antileishmanial effect observed (Orsini et al., 2016). The result obtained for the concentration of 300 μg/kg/day of the RuNO complex was

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statistically similar to that observed in the treatment with glucantime, drug of choice for the treatment of leishmaniasis. It is worth noting that pentavalent antimonials such as glucantime have several adverse effects, restricting their use in many cases (Blum et al., 2018).

In in vivo experiments, the RuNO complex has not shown any notable toxic reactions, despite the use of high doses of RuNO complex in chronic treatment. In addition, structural modifications of this compound to generate more powerful leishmanicidal compounds with lower toxicity than conventional drugs seems to be a viable alternative (Tfouni et al., 2012),

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which may be further investigated. 5. CONCLUSION

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According to the data previously exposed, we conclude that the ruthenium nitrosyl

complex, cis-[Ru(bpy)2(SO3)(NO)](PF6), has antileishmanial activity in vitro and in vivo against L. (V.) braziliensis, reducing lesion size and parasite burden. Thus, further in vivo

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to significant results for the treatment of leishmaniasis.

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studies using this compound to understand the mechanism of action and its toxicity may lead

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FUNDING

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This study was funded by Coordination for the Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development

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(CNPq),Cearense Foundation for Scientific and Technological Development Support

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(FUNCAP), Brazil (LGF Lopes 470054/2011-5, award 303732/2014-8, PRONEX PR2-010100030.01.00/15 SPU No: 3265612/2015; EHS Sousa award 312030/2015-0, Universal 01/2016

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403866/2016-2), and Foundation for Research and Scientific and Technological Development of Maranhão (F Almeida-Souza DCR03438-16).

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DECLARATIONS OF INTEREST None.

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