In vitro and in vivo activity of a hypotoxic copper(I) complex against dermotropic Leishmania species

Cutaneous leishmaniasis is a disease caused by protozoa of the genus Leishmania and, currently, the treatment of first choice is meglumine antimoniate. However, due to its limited effectiveness and high toxicity, it is necessary to seek new active principles for leishmaniasis treatment. Metal complexes are gaining importance due to their effectiveness and low toxicity. In this context, the present study aimed to evaluate the in vitro and in vivo antileishmanial activity of the hypotoxic copper(I) complex [HB(pz)3]Cu(PCN). Four dermotropic species of Leishmania were tested with the metal complex and its effectiveness was determined through parasitic viability and infectivity rate, and cytotoxicity was determined using a redox dye (resazurin). For the in vivo tests, hamsters were infected and the lesions treated with a formulated ointment containing the complex, the effectiveness of which was assessed by measuring the diameter of the inoculum/snout location and determining the parasitic load. The results demonstrated moderate toxicity in murine macrophages and human monocytes and better efficacy in Leishmania (V.) braziliensis when compared to the other species tested, with a 50% reduction in the viability of promastigote and amastigote forms (in vitro). General data from daily topical treatment for up to 30 days showed low efficacy for reducing lesions, and no clinical and parasitological cure was observed in the experimental animals. Thus, the [HB(pz)3]Cu(PCN) complex proved to be promising in in vitro studies against L. (V.) braziliensis, and should be further tested in new formulations and new experimental treatment schemes.


INTRODUCTION
Cutaneous leishmaniasis (CL) is a neglected disease and endemic in more than 92 countries, with over 90% of cases occurring in the Americas (WHO 2021). Transmission is vectorial, and its etiological agents are the protozoans of the genus Leishmania (Blanco 2017). Currently, at least 20 species are known to be pathogenic to man and, among them, a total of seven dermotropic species are found in Brazil. Leishmania (Viannia) guyanensis, L. (V.) braziliensis and L. (Leishmania) amazonensis are highlighted in Brazil due to their wide distribution and the induction of different clinical manifestations (Teles et al. 2016).
In the treatment of CL, pentavalent antimonials are most commonly employed, and pentamidine and amphotericin B are the drugs of second choice. Considering that these drugs have low efficacy due to parasitic diversity and resistance, high toxicity, and require a long and painful treatment, the search for new active principles with more effective action is warranted (Bastos et al. 2016;Brasil 2017).
In this context, metal complexes are prominent candidates, as they have unique properties, such as the ability to bypass resistance mechanisms and the possibility of being used in multi-target molecule design (compounds that can reach the target organism by at least two independent mechanisms) (Ong et al. 2019). Transition metals have peculiar characteristics that favor performance in biological functions, and copper (Cu) is a biologically active transition metal (Krupanidhi et al. 2008).
Copper(I/II) compounds have been widely evaluated for their anti-tumor properties (e.g. Tisato et al. 2016;Hussain et al. 2019;Porchia et al. 2020), and have also been proposed for other therapeutic purposes, e.g. as antimalarial agents (Tapanelli et al. 2017). Several studies have investigated the activity of copper complexes in different oxidation states, and Cu(II) complexes have shown promising results in vitro against Leishmania spp. (Maffei et al. 2009;Portas et al. 2012;Arndt et al. 2017;Méndez-Arriaga et al. 2018). Copper is physiologically internalized by eukaryotic cells as copper(I), however, only Navarro et al. (2003) and Saeed et al. (2018) have shown the activity of copper(I) complex against promastigote forms.
The copper(I) complex [HB(pz) 3 ]Cu(PCN) presented in vitro and in vivo cytotoxic activity in cancer cells (Gandin et al. 2014). Considering that anticancer drugs can exert antileishmanial activity (Keighobadi et al. 2018), the aim of this study was to evaluate the in vitro and in vivo antileishmanial activity of the [HB(pz) 3 ]Cu(PCN) complex in Leishmania spp., and the effects of cytotoxicity on murine peritoneal macrophages and human monocytes.

Parasites
The strains used in this study were Leishmania (

In vitro biological assay
Murine peritoneal macrophages and human monocytes (primary culture/individual assays) were grown in 96-well plates (10 5 cells mL -1 ) in complete RPMI medium, in an incubator containing 5% CO 2 (Form Series II Water Jacket CO 2 Incubator, Thermo Scientific, USA) at 37 °C, for 24 h. The cells were treated with different concentrations of the copper(I) complex (0.16, 0.08, 0.04, 0.02 and 0.01 mM) or Glucantime ® (33.00, 16.50, 8.25, 4.12 and 2.06 mM). Wells without cells were kept blank and wells with cells though without treatment were maintained as controls. Cell viability was assessed using resazurin sodium salt (Sigma Aldrich™, USA) during 24, 48 and 72 h with the addition of 10 µL of stock solution of resazurin sodium salt (4 mg mL -1 in phosphate buffered saline) in each well and the plates incubated again for another 12 h at 37 ºC. The absorbance was read on a spectrophotometer (Elx800™, BIO-TEK ® , USA) at a wavelength of 590 nm. The data were normalized according to the following formula: % of survival = Abs. Sample-Abs. blank/bs. control-Abs. blank × 100 (Nascimento et al. 2019).

Biological tests in amastigote forms
The evaluation of in vitro antileishmanial activity of the copper(I) complex in amastigotes was performed as described by Nascimento et al. (2019), with some adaptations. Murine peritoneal macrophages and human monocytes (individual assays) were infected with promastigotes of each of the Leishmania species in a 10:1 ratio (10 6 parasites: 10 5 cells) on glass coverslips in 24-well plates containing complete RPMI culture medium, which was subsequently placed in an incubator at 37 °C and 5% CO 2 for up to 2 h. After this time, the infected cells were maintained either in complete RPMI medium (control) or RPMI medium containing different concentrations of the copper(I) complex (0.16, 0.08, 0.04 and 0.02 mM) and Glucantime ® (33.00, 16.50, 8.25, 4.12 and 2.06 mM) for 24, 48 and 72 h at 37 °C and 5% CO 2 . Then, the coverslips were fixed and stained every 24 h using the Quick Panoptic method (Laborclin ® , Brazil) and analyzed using optical microscopy. The percentage of infected cells was estimated by randomly counting 100 infected and uninfected cells on each slide (Comandolli-Wyrepkowki et al. 2017).

Preparation of topical formulation for in vivo biological assays
The ointment was prepared with 0.02% butyl hydroxytoluene, propylene glycol, 30% anhydrous lanolin and solid petroleum jelly. Then, 0.5 mg of copper(I) complex in 80 mg ointment was added. Base ointment without copper(I) complex was used as a placebo control.

In vivo biological assay
Ninety male golden hamsters (Mesocricetus auratus), weighing 120 g and aged 60 days, were kept in the Central Animal Facility at INPA, with water and food ad libitum, in rooms with controlled photoperiod and temperature (22-24 °C). The ninety animals were divided into three groups of thirty animals for the experimental infection procedure. From each group of 30 animals, 24 were infected by one of the species of Leishmania studied, and the other six animals were not infected and constituted the control group. In the hamster's snout, 0.1 mL -1 of sterile saline (0.9%) containing promastigotes (10 6 promastigotes mL -1 ) in stationary phase was injected intradermally. After the lesion appeared, at variable times, according to the species of Leishmania, the animals were randomly divided into five groups (six animals per group) and treated once daily, for 25 to 30 days, in the following manner: I) uninfected and untreated; II) infected and untreated; III) infected and treated with base ointment (placebo); IV) infected and treated with Glucantime ® 20 mg(Sb 5 ) kg day -1 intramuscularly; V) infected and treated with ointment containing 0.5 mg of the copper(I) complex. We did not perform in vivo tests with L. (V.) naiffi due to the lack of macroscopically visible skin lesions in the hamsters.
To evaluate the treatments, the total lesion volume was determined on a weekly basis by measuring the snout with a digital caliper (MTX ® ), the lesions were photo-documented (SM-G570M/DS, Samsung, South Korea) for morphological analysis (Comandolli-Wyrepkowki et al. 2017) and the animals were weighed.

Parasitological and hematological evaluation of treatment
All animals used in the experiment were euthanized through intramuscular injection of ketamine (10 mg kg -1 ) and xylazine (90 mg kg -1 ). After the euthanasia procedure, the liver, spleen and kidneys were removed and weighed on a precision scale. Skin tissue fragments from the inoculated site, as well as viscera tissue (liver, spleen and kidneys), were excised aseptically and cultured in Novy-MacNeal-Nicolle (NNN) medium for up to 20 days at 25 o C. The result was considered positive when at least one form of the parasite was isolated from the culture medium (Comandolli-Wyrepkowki et al. 2017).
Tissue samples from the lesion area on the snout of each animal were imprinted on glass slides. These were fixed and stained using the Quick Panoptic method and analyzed under optical microscopy. The percentage of infected cells was estimated by randomly counting 25 fields in each coverslip (Comandolli-Wyrepkowki et al. 2017).
Blood smears were prepared on slides and, subsequently, the biological material was fixed and stained using the Quick Panoptic method. The leukocyte cells were differentiated and quantified by counting 100 cells per slide at 400x magnification.

Statistical analysis
IC 50 and CC 50 were obtained through linear regression using the number of living cells A one-way ANOVA followed by Tukey's test was used to assess the significance of the differences between the groups of the in vivo biological assay, at the 5% significance level. All statistical analyses was performed using the GraphPad Prism program version 6.0 for Windows (GraphPad Software, San Diego, CA).

Ethical aspects
The in vitro tests were approved by the Ethics Committee on the Use of Animals of INPA (CEUA/INPA authorization # 023/2020 -01280.000455/2020-41) and by the Research VOL. 51 (3)

RESULTS
In the cytotoxicity evaluation, the copper(I) complex showed moderate toxicity for murine peritoneal macrophages and human monocytes, with cell viability above 80% and 75%, respectively. The CC 50 of the copper(I) complex for both cell types was > 0.4 mM during the incubation period. Cell viability was 80% in the group treated with Glucantime®, and 85% in the group treated with dimethyl sulfoxide (DMSO), and 86% and 93% in murine peritoneal macrophages and human monocytes, respectively.  (Table 1).
There was no significant difference in the parasitic viability of L. (L.) amazonensis promastigotes (Figure 1a), however, the concentrations of 0.16 and 0.08 mM of the copper(I) complex   (Table 2).
As observed in the promastigote forms, the copper(I) complex was more effective against L. (V.) braziliensis, and induced a 75% reduction in the infection rate of murine peritoneal macrophages (F = 303.1, p < 0.0001) over the entire period of incubation. There was no significant difference from cells treated with Glucantime ® (Figure 2c   2b) and in L. (V.) naiffi the significant reduction occurred at 24 and 72 h (F = 53.2, p < 0.0001) (Figure 2d). It is noteworthy that the inhibition of infectivity was not observed in human monocytes, as all groups had a high rate of monocyte infection by Leishmania spp.
In the experimental treatment of hamsters, the animals infected with L. (L.) amazonensis and left untreated had a higher average weight (173.33 g) than the average weight in the other groups (150.16 g) (p < 0.0001). In hamsetrs infected with L. (V.) braziliensis, the group treated with Glucantime ® (average 197.33 g) had a higher weight than in the other groups (average 173.45 g) (p < 0.0001). In the groups infected with L. (V.) guyanensis, there was no difference among groups in the weight of the animals.
Regarding the clinical evolution of leishmaniasis, there was no significant difference in the volume of the snout among hamster groups infected with L. (L.) amazonensis (Figure 3a). The treatment lasted only 25 days due to the lethargic behavior of the animals and the exacerbated evolution of the lesions.
Animals infected with L. (V.) guyanensis and L. (V.) braziliensis and treated with the copper(I) complex ointment showed a reduction of only 17% and 18% in the volume of the snout, respectively, by the end of the 30-day period. There was no significant difference among infected and untreated animals. In the Glucantime ® group, the volume of the snout was reduced by 73% (F = 75.03, p < 0.0001) in L. (V.) guyanensis and 70% (F = 11.96, p < 0.0001) in L. (V.) braziliensis, differing from the other treatment groups (Figure 3b,c).
For all Leishmania species, the treatment with either Glucantime ® or with the copper(I) complex ointment induced a reduction in the volume of the snout after the 15 th day of treatment. In all groups infected with L. (L.) amazonensis, until the second week of treatment, a gradual increase in the volume of the snout was observed due to the formation of the nodule and edema. After the 15 th day, there was a reduction in edema and the beginning of ulceration of the lesions, and the snout volume was larger during the entire experimental period when compared to the groups infected by other species, with ulcerated, exacerbated lesions and the presence of scabs. None of the groups presented clinical cure under any type of treatment ( Figure 4a  There was no significant difference in organ weight (liver, spleen and kidney) and hematological parameters among the experimental groups for any of the tested Leishmania species. VOL. 51 (3)  In our tests of antileishmanial activity in vitro, a dosedependent response was observed, in which the highest concentrations of the copper(I) complex (0.16 and 0.08 mM) were more effective in inhibiting promastigotes and amastigotes. Similar dose-dependent parasitic inhibition (0.365 mg mL -1 ) was reported by Hummadi et al. (2005. In the promastigote forms of L. (L.) amazonensis, there was no parasitic inhibition, however, the copper(I) complex was more efficient against amastigotes of this species, with a 75% reduction in the infection rate at 48 and 72 h. Likewise, Maffei et al. (2009) observed a IC 50 decline (at 48 and 72 hours) for three copper(II) complexes against L. (L.) amazonensis, suggesting a long-term activity for this species.
In the promastigote forms of L. (V.) braziliensis, the copper(I) complex inhibited more than 50% of the promastigote forms, with SI greater than 20 after 72 h and greater efficacy than Glucantime ® , while in the amastigote forms, the copper(I) complex decreased the infection rate by at least 75%, with similar efficacy to Glucantime ® . These results corroborate those of Caballero et al. (2014), who showed that copper(II) complexes had similar toxicity to Glucantime ® in the promastigote and amastigote forms of L. (V.) braziliensis,  ) braziliensis (C). Infected and untreated animals (black coluns), animals infected and treated with 20 mgSb 5 kg day -1 of Glucantime® (white columns), animals infected and treated with placebo (striped columns), and animals infected and treated with ointment containing 0,5 mg of copper(I) complex (dotted columns). Columns represent the mean and bars the standard deviation. Different letters indicate significant differences according to the Tukey test (p < 0.05). and a SI higher than 54.2 after 72 h. Other studies, such as those of Méndez-Arriaga et al. (2018) and Méndez-Arriaga et al. (2020), also reported the activity of copper(II) complexes in L. (V.) braziliensis, with IC 50 of 100.2 µM (IS 14.5) and 42.6 µM (IS 19.1), respectively.
To the best of our knowledge, there are no published studies on the antileishmanial activity of metal complexes for L. (V.) guyanensis and L. (V.) naiffi. Our results indicate that the copper(I) complex is efficacious against promastigote forms of these species. This is also the first report of the antileishmanial effects of a copper complex in a preclinical study with animals.
Currently there is no information available in the literature regarding the topical experimental treatment of leishmaniasis with metal complexes. The drugs currently used to treat cutaneous leishmaniasis are administered parenterally and have serious adverse effects. In this sense, topical administration of medications for leishmaniasis may be an interesting option, since the use of topical medications allows the concentration of the active ingredient in the site of infection. It offers a possible decrease in toxicity and, because it is not invasive, the treatment can be done outside the hospital environment (Bocxlaer et al. 2018). In addition, topical treatment can also be used together with other drugs, thus potentiating and reducing the time of treatment of the disease. Due to the lipophilic characteristics of the copper(I) complex, the use of hydrophobic ointments is an attractive alternative, since they have an emollient effect, are difficult to remove, do not become dry and, in addition, allow the drug to remain in prolonged contact with the skin and act as an occlusive dressing (Otto et al. 2018).
Another relevant factor for the development of a topical formulation is related to intoxication by metals because, although copper is an essential element for several physiological and biochemical functions, it is also related to diseases caused by the abundance and deficiency of this metal in the body. As such, it is necessary to stipulate doses for treatment that take into consideration the essential daily concentrations. In an adult man, these can vary from 0.9 to 2.2 mg (Baierle et al. 2010), although its topical use has a different effect in terms of absorption by the body, differently than if it were administered parenterally.
None of our treatments with the copper(I) complex achieved the clinical cure (total healing and reepithelization of the skin) and parasitological cure (the complete elimination of the parasite) of the animals infected with L. ( L.) amazonensis. In animals infected with L. (V.) guyanensis and L. (V.) braziliensis, the copper(I) complex ointment induced a reduction in lesion volume, but clinical cure was achieved only in 50% of the infected animals, and no parasitological cure was observed. In contrast, animals treated with Glucantime ® achieved 100% clinical cure, but also no parasitological cure, which had already been demonstrated in humans (Martínez-Valencia et al. 2017).
Although several in vitro studies with metal complexes have been developed in the search for new alternative treatments for cutaneous leishmaniasis, few have evaluated the effects in vivo. Among them, the study by Nascimento et al. (2019) suggested a promising leishmanicidal effect of the ruthenium nitrosil complex (300 µg kg day -1 through oral administration) against L. (V.) braziliensis in a model of skin infection in vivo (hamster), obtaining a 51% reduction in lesion size and 99.9% elimination of parasites. These authors did not observe toxic reactions to the metal complex, nor changes in size, weight and appearance of organs, or in the leukogram of test animals, which is corroborates in our study.
Our results encourage further studies that evaluate the effectiveness of the copper(I) complex in a new formulation and a new experimental treatment scheme, with emphasis on the effectiveness of the skin permeation issue, animal experimentation model, species of Leishmania involved, concentrations of the active principle and treatment time. Further research is recommended using different concentrations, formulations, routes of administration, and treatment time using the copper(I) complex, and it is also necessary to elucidate the therapeutic mechanism and the toxicological aspects.

CONCLUSIONS
The copper(I) complex showed promising in vitro activity against the evolutionary forms of Leishmania (V.) braziliensis.
In the pre-clinical study, there was a reduction of about 17% and 18% in the size of skin lesions in animals infected with parasites of the sub-genus Viannia, species L. (V.) guyanensis and L. (V.) braziliensis respectively, treated topically with the ointment containing the copper(I) complex. It is suggested that the ointment be used in conjunction with other drugs for parasitic infections by the subgenus Viannia.