Introduction: Casiopeina III-ia (CasIII-ia) is a mixed chelate copper (II) compound capable of interacting with free radicals generated in the respiratory chain through redox reactions, producing toxic reactive oxygen species (ROS) that compromise the viability of cancer cells, bacteria and protozoa. Due to its remarkable effect on protozoa, this study evaluated the effect of CasIII-ia on Leishmania mexicana amastigotes and its potential use as a treatment for cutaneous leishmaniasis in the murine model. Methods: We analyzed the leishmanicidal effect of CasIII-ia on L. mexicana amastigotes and on their survival in bone marrow-derived macrophages. Furthermore, we evaluated the production of ROS in treated parasites and the efficacy of CasIII-ia in the treatment of mice infected with L. mexicana. Results: Our results show that CasIII-ia reduces parasite viability in a dose-dependent manner that correlates with increased ROS production. A decrease in the size of footpad lesions and in parasite loads was observed in infected mice treated with the intraperitoneal administration of CasIII-ia. Conclusions: We propose CasIII-ia as a potential drug for the treatment of cutaneous leishmaniasis.

Casiopeina III-ia (CasIII-ia, Fig. 1) is a copper-based compound that has a selective cytotoxic, antineoplastic, genotoxic, antibacterial, and antiparasitic effect. It belongs to a family of more than 100 compounds with a copper center called Casiopeinas®. It interacts with DNA, promoting mitochondrial toxicity and the generation of reactive oxygen species (ROS), causing cell damage, leading to apoptosis through the intrinsic pathway [1‒4].

Fig. 1.

Chemical structure of Casiopeina III-ia (CasIII-ia).

Fig. 1.

Chemical structure of Casiopeina III-ia (CasIII-ia).

Close modal

The general formula of Casiopeinas® is (Cu(N-N) (N-O)(H2O))NO3 and (Cu(N-N)(O-O)(H2O))NO3 in CasIII-ia, where N-N is a 4,4-dimethyl,2,2′-bipyridine and O-O is acetylacetonate ligands. Its design is based on an essential transition metal Cu (II) that reduces toxicity, and its ligands that provide a certain degree of hydrophobicity to favor absorption and the necessary distribution properties to reach the site of action. Copper is a metal that intervenes in a wide range of biological processes, due to its characteristic as a crucial redox active metal for growth and development [1, 5].

The interaction of copper with products of oxygen metabolism, such as hydrogen peroxide, induces the production of hydroxyl radicals (HO) and other ROS due to its high lipid oxidation mechanism that leads to a chain reaction, forming highly toxic free radicals that damage the fluidity and permeability of the lipid membrane in a process known as lipid peroxidation. Mitochondrial membranes are importantly exposed to this phenomenon since the highest rate of ROS is generated at this site. After damage of the mitochondrial membrane, ROS can be released into the cytoplasm causing severe cell damage due to their affinity for proteins, lipids, carbohydrates, and nucleic acids, ultimately leading to cell death by apoptosis [6‒8].

CasIII-ia has shown promising results in chemotherapy as well as for treatment of infectious diseases. Recent data have shown that CasIII-ia is a potential treatment against protozoa, such as Giardia intestinalis and Trypanosoma cruzi, by inducing the production of ROS and subsequently causing damage to their macromolecules, such as DNA [4, 9]. Due to these results, there is great interest in evaluating the effect of CasIII-ia on other protozoan infections such as leishmaniasis.

Leishmaniasis is a neglected tropical disease with diverse clinical forms depending on the parasite species. Cutaneous leishmaniasis (CL) is the most common clinical form with an estimated 600,000 to 1 million new cases occurring every year [10, 11].

One of the main problems in dealing with the disease is that first-line drugs, including pentavalent antimonials (Sb(V)), such as Glucantime®, can produce toxic side effects such as nausea, vomiting, kidney failure, and cardiac arrhythmias [12]. Furthermore, drug resistance to Sb(V) is increasing in geographical areas where the infection occurs [13]. Our study now aimed to evaluate the effect of CasIII-ia on L. mexicana amastigotes on the host cell, as well as its efficacy for treatment of Leishmania-infected BALB/c mice.

Experimental Animals

Female BALB/c mice (Mus musculus), aged 8–10 weeks, were provided by the bioterium of the Experimental Medicine Research Unit of the Faculty of Medicine, UNAM. They were handled following the guidelines established for laboratory animals (NOM-062-ZOO-1999), under the approval of the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) of the Faculty of Medicine, UNAM, with registration number 063-2020/022-CIC-2020.

Axenic Culture of Amastigotes and Promastigotes of L. mexicana

L. mexicana parasites, of the MHOM/MX/2011 Lacandona strain, were previously obtained [14]. Promastigotes were cultured in M199 medium (GIBCO by Life Technologies, Inc., Grand Island, NY, USA) at 26°C, pH 7.4, supplemented with 10% heat-inactivated FBS at 56°C for 30 min (GIBCO by Life Technologies) with 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) (Sigma-Aldrich). Metacyclic promastigotes were harvested at the stationary phase of their growth (day 5). Amastigotes were grown in Grace’s insect medium (Sigma-Aldrich), at 33°C, pH 5.5, supplemented with 20% heat-inactivated FBS with 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin), 2 mml-glutamine, and 0.35 g/L sodium bicarbonate, and were harvested in the stationary phase of their growth (days 6–7) [15].

Casiopeina III-ia Preparation

Casiopeina® III-ia (Cu(4,4′-dimethyl-2,2′-bipyridine)(acetylacetonate)(H2O))NO3, with a molecular weight of 444.92 g/mol, was synthesized at the Department of Inorganic and Nuclear Chemistry, Faculty of Chemistry of UNAM, Mexico, following the reported patent [16].

In vitro Effect of Casiopeina III-ia on L. mexicana Amastigotes

The effect of CasIII-ia on the viability of axenically grown L. mexicana amastigotes was evaluated. Amastigotes (1 × 106/mL) were cultured at 33°C with Grace’s insect culture medium supplemented with 10% heat-inactivated FBS in 5 mL Falcon® tubes (Corning, NY, USA). Cas III-ia was added at concentrations ranging from 100, 80, 60, 40, 20, and 4 μM and viability was evaluated microscopically after 24, 48, 72, and 96 h posttreatment, using erythrosine B as a viability marker. As a negative control, unstimulated parasites and parasites incubated with the vehicle at a concentration of 100 μM were used. For positive controls, parasites were cultured with Glucantime®, the IC50 was calculated using the GraphPad Prism 8 software for Mac OS X.

Murine Macrophage Differentiation

Macrophages were differentiated from bone marrow stem cells of BALB/c mice. Cells were isolated from the femur and tibia with cold PBS. After washing, 2 × 106 cells were cultured in RPMI-1640 medium (GIBCO by Life Technologies) with 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) for 7 days at 37°C with 5% CO2 in Petri dishes (Corning, NY, USA). Cultures were supplemented with 20% heat-inactivated FBS and 20% macrophage colony-stimulating factor obtained from the L929 cell line [17]. Adherent macrophages were harvested with cold PBS, and viability was determined using trypan blue as a viability marker in a Neubauer chamber.

Effect of Casiopeina III-ia on the Viability of Bone Marrow Macrophages

Murine macrophages (1 × 106) were incubated in 24-well plates with 0.2, 1, 2, and 10 μM CasIII-ia in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) at 37°C and 5% CO2 for 48 h. As a negative control, unstimulated cells were incubated with the vehicle at a concentration of 10 μM. Viability was assessed with DAPI at 3.6 mM as a viability marker (Sigma-Aldrich) and analyzed by flow cytometry in a FACSCanto II flow cytometer (BD, Becton Dickson, San José, CA, USA). The FlowJo program (Tree Star, Ashland, OR, USA) was used for data analysis.

Effect of Casiopeina III-ia on the Survival of Intracellular L. mexicana Amastigotes

Differentiated murine bone marrow macrophages (2.5 × 105) were plated in 5 wells of a 96-well plate in RPMI-1640 medium, supplemented with 10% heat-inactivated FBS and 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin), and infected with L. mexicana promastigotes in a 1:10 infection ratio (cell/parasites) for 2 h at 26°C. Thereafter, the parasites that had not been phagocytosed by macrophages were eliminated by washing with PBS. Supplemented RPMI-1640 medium was added, and the cells were incubated overnight. CasIII-ia was added at the concentrations of 0.2, 1, and 2 μM in supplemented RPMI-1640 medium, at 37°C with 5% CO2 for 48 h. Negative controls consisted of macrophages cultured without CasIII-ia or with the vehicle at the highest concentration (2 μM). After washing with PBS, the cells were cultured in M199 medium supplemented with 10% heat-inactivated FBS and 1% antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) at 26°C for 72 h. The release of viable parasites was monitored daily. Promastigotes were fixed with 0.1% glutaraldehyde and counted using a Neubauer chamber.

Analysis of ROS Production in L. mexicana Incubated with Casiopeina III-ia

L. mexicana amastigotes (1 × 106/mL) were placed in 5 mL tubes with supplemented M199 medium and coincubated with CasIII-ia at an IC50 of 58.1 μM for 48 h at 33°C. As negative controls, amastigotes were cultured without stimulus or with the vehicle at the same concentration as the IC50. The parasites were stained with H2CFDA (ROS marker) at a concentration of 50 μM for 30 min. After washing, the parasites were resuspended in PBS for reading in the flow cytometer. The data were analyzed using the FlowJo Software.

Apoptosis Assay

L. mexicana amastigotes were incubated with CasIII-ia IC50 for 48 h as described above. Vehicle and non-treated conditions were added as negative controls. After incubation, 2 × 106 parasites from each condition were stained with APC-Fire 750 Annexin V (Biolegend, Cat. No. 640953) and 7-AAD (Cytek, Tonbo Biosciences Cat. No. 13-6993-T200), which were diluted 1:20 in Annexin V Binding Buffer (BD Bioscience, Cat. No. 556454) at room temperature in the dark for 15 min. Samples were analyzed on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA). Data were analyzed by FlowJo™ Software.

Effect of Casiopeina III-ia on the Progression of L. mexicana Infection in BALB/C Mice

Mice were infected with 2 × 105L mexicana promastigotes in 10 μL PBS with a 31 G-gauge insulin syringe into the footpad of BALB/c mice. After 4 weeks, when lesions were beginning to be visible, mice were subjected to topical or intraperitoneal treatment with CasIII-ia (20 μg/kg). Additionally, non-treated and mice treated with the vehicle, either topically or intraperitoneally, were added as negative controls. Mice treated with Glucantime® (20 mg/kg) were used as positive controls [18]. The topical treatment was applied daily, while the intraperitoneal treatment was applied three times a week for 5 weeks. The progression of the infection was evaluated by measuring the increase of footpad size once a week with a digital vernier (resolution: ±0.01 mm) (Thomas Scientific).

Quantitation of the Parasitic Load

At the end of the treatment, the mice were euthanized in a CO2 chamber. The infected plantar pads were cut and weighed, and the tissue was macerated with 4 mL PBS through a 100 μm cell strainer (Corning, NY, USA), following the methods described by Lezama-Davila [19]. The samples were collected in 15-mL tubes and calibrated to 8 mL with PBS. Amastigotes were obtained by passing the homogenate through an insulin syringe seven times, and they were fixed with 0.1% glutaraldehyde and counted in a Neubauer chamber.

Statistical Analysis

The results were analyzed using the non-parametric Mann-Whitney U test for comparison between the experimental groups. The results of the IC50 determination were performed in the GraphPad Prism 8. All statistical analysis and figures were performed in the GraphPad Prism 8 software for Mac OS X, considering p ≤ 0.05 as significant. All data express the mean ± standard deviation of three independent experiments.

Effect of Casiopeina III-ia on the Survival of Intracellular L. mexicana amastigotes

The evaluation of the in vitro effect of CasIII-ia on the viability of L. mexicana amastigotes showed a dose-dependent reduction (Fig. 2). The significant effect (p ≤ 0.05) began at 48 h in cultures incubated with 80 and 100 μM CasIII-ia, showing a reduction in parasite viability of 84.6 and 92.7%, respectively. This effect increased over time, and after 96 h, a significant reduction (99%) in parasite viability was observed after incubation with CasIII-ia at concentrations of 80 and 100 μM (p ≤ 0.05). At this time point, a significant reduction (35%) was also observed after incubation with CasIII-ia at a concentration of 40 μM (p ≤ 0.05). The positive control with Glucantime® also led to a significant reduction (81%) of parasite viability (p ≤ 0.05) at 96 h, whereas the vehicle had no effect, showing similar values as the control group that had not received any stimulation (Fig. 2).

Fig. 2.

Effect of CasIII-ia on the growth of L. mexicana amastigotes. Growth curve of amastigotes incubated with different concentrations of CasIII-ia during 4 days is shown. As negative controls, a group without treatment or treated with the vehicle was added. The data represent the mean ± standard deviation of three independent experiments. Asterisks represent the significant difference (p ≤ 0.05) with respect to the control group without treatment 4.

Fig. 2.

Effect of CasIII-ia on the growth of L. mexicana amastigotes. Growth curve of amastigotes incubated with different concentrations of CasIII-ia during 4 days is shown. As negative controls, a group without treatment or treated with the vehicle was added. The data represent the mean ± standard deviation of three independent experiments. Asterisks represent the significant difference (p ≤ 0.05) with respect to the control group without treatment 4.

Close modal

From the results of the growth curve of amastigotes stimulated with CasIII-ia, the IC50 for each of the incubation times was established (Table 1), and the IC50 using 58.1 μM at 48 h was used for all subsequent experiments.

Table 1.

IC50 for amastigotes at different times

HoursIC50, μM
24 20.6 
4858.1
72 33.7 
96 32.8 
HoursIC50, μM
24 20.6 
4858.1
72 33.7 
96 32.8 

*IC50 used in experiments is marked in bold.

Effect of Casiopeina III-Ia on Bone Marrow-Derived Macrophages Infected with L. mexicana

The effect of CasIII-ia on noninfected macrophages showed that after 48 h posttreatment, the 10 μM concentration significantly reduced (p ≤ 0.05) 75.5% of cell viability (Fig. 3a), with regard to the non-stimulated control group. These results allowed to establish the CC50 of CasIII-ia for macrophages derived from bone marrow at 2.07 μM at 48 h. The vehicle showed no effect on the viability of the macrophages, which was similar to the non-stimulated control group.

Fig. 3.

Effect of CasIII-ia on infected and noninfected bone marrow-derived macrophages. a Viability of macrophages (non-infected) incubated with different concentrations of CasIII-ia during 48 h was measured by flow cytometry. b Analysis of parasite survival in bone marrow-derived macrophages stimulated with CasIII-ia for 48 h. The data shown are the mean ± standard deviation of three independent experiments, and the symbols indicate statistically significant differences from controls.

Fig. 3.

Effect of CasIII-ia on infected and noninfected bone marrow-derived macrophages. a Viability of macrophages (non-infected) incubated with different concentrations of CasIII-ia during 48 h was measured by flow cytometry. b Analysis of parasite survival in bone marrow-derived macrophages stimulated with CasIII-ia for 48 h. The data shown are the mean ± standard deviation of three independent experiments, and the symbols indicate statistically significant differences from controls.

Close modal

Thereafter, a survival assay of intracellular amastigotes was analyzed in macrophages incubated with CasIII-ia. Parasite survival tended to diminish with the increasing concentrations of CasIII-ia, showing that a significant reduction of parasite survival (66.5%) was observed at the concentration of 2 μM (p ≤ 0.05) (Fig. 3b). No effect was observed in controls exposed to the vehicle or in non-stimulated controls.

ROS Production in L. mexicana Amastigotes Induced by Casiopeina III-ia

Amastigotes were incubated with the previously established IC50 for CasIII-ia at 48 h (58.1 μM). The ROS produced by amastigotes after stimulating them with CasIII-ia during 48 h were quantified by flow cytometry. The mean fluorescence intensity showed a significant increase (97.46%) of ROS production, as compared to the control group without stimulus (p ≤ 0.05) (Fig. 4a), whereas the vehicle did not show any effect on ROS production.

Fig. 4.

Production of reactive oxygen species (ROS) and apoptosis in L. mexicana amastigotes. a ROS production was measured in amastigotes incubated with CasIII-ia at the IC50 (58.1 μM) or with the vehicle for 48 h. The bars show the mean fluorescence intensity (MFI). b Percentages of viable, apoptotic or necrotic amastigotes treated during 48 h with CasIII-ia IC50, as well as non-treated controls and vehicle are shown. Data show mean ± standard deviation of three independent experiments. Symbols represent significant differences (p ≤ 0.05) between groups.

Fig. 4.

Production of reactive oxygen species (ROS) and apoptosis in L. mexicana amastigotes. a ROS production was measured in amastigotes incubated with CasIII-ia at the IC50 (58.1 μM) or with the vehicle for 48 h. The bars show the mean fluorescence intensity (MFI). b Percentages of viable, apoptotic or necrotic amastigotes treated during 48 h with CasIII-ia IC50, as well as non-treated controls and vehicle are shown. Data show mean ± standard deviation of three independent experiments. Symbols represent significant differences (p ≤ 0.05) between groups.

Close modal

Casiopeina III-ia Induced Apoptosis in L. mexicana Amastigotes

Amastigotes were incubated with the IC50 CasIII-ia concentration established previously at 48 h. A 32% reduction of live amastigotes was observed compared to the control group that received no treatment (p ≤ 0.05). Furthermore, a 40% increase in apoptosis was observed in L. mexicana amastigotes compared to controls without treatment (p ≤ 0.05). It is noteworthy that the percentage of necrotic cells did not change with the treatment using CasIII-ia. Also, the vehicle had no effect on L. mexicana amastigotes (Fig. 4b).

Effect of Casiopeina III-ia on L. mexicana Infection in BALB/C Mice

The effect of the metallodrug was tested in BALB/c mice infected with L. mexicana. The intraperitoneal administration of CasIII-ia at a dose of 20 μg/kg showed a significant reduction (25.4%) of the footpad width after 4 weeks of treatment, compared to the non-treated control group (p ≤ 0.05). After 5 weeks, the reduction of the footpad width was 29%. In contrast, the topical application of CasIII-ia (20 μg/kg) showed no effect on the lesion size (Fig. 5a).

Fig. 5.

Lesion size and parasite load in L. mexicana-infected BALB/c mice treated with CasIII-ia. a Footpad increase of L. mexicana infected mice treated with 20 μg/kg CasIII-ia, either topically or intraperitoneally. Controls were non-treated or vehicle-treated mice. b The ratio of the parasite load per mg of tissue at the end of the experiment is shown. The bars show the mean ± standard deviation of four independent experiments. Asterisks represent the significant difference (p ≤ 0.05) between groups.

Fig. 5.

Lesion size and parasite load in L. mexicana-infected BALB/c mice treated with CasIII-ia. a Footpad increase of L. mexicana infected mice treated with 20 μg/kg CasIII-ia, either topically or intraperitoneally. Controls were non-treated or vehicle-treated mice. b The ratio of the parasite load per mg of tissue at the end of the experiment is shown. The bars show the mean ± standard deviation of four independent experiments. Asterisks represent the significant difference (p ≤ 0.05) between groups.

Close modal

When analyzing parasite load per mg of tissue, a significant decrease was observed (66.6%) after the intraperitoneal administration of CasIII-ia (p ≤ 0.05). In contrast, the topical application of CasIII-ia only led to a 20.9% reduction of the parasite load. The administration of the vehicle by both routes had no significant effect on parasite load (Fig. 5b).

Casiopeinas® are a group of metallodrugs that have received interest due to their effect against cancer cells and intracellular pathogens [2, 4, 20, 21]. Its antiparasitic effect against G. intestinalis and T. cruzi, mediated by the induction of free radicals, is a promising treatment against these pathogens [4, 9]. We now evaluated the effect of CasIII-ia on L. mexicana amastigotes, their survival within macrophages, and the induction of ROS in the parasites. We furthermore analyzed its therapeutic effect in Leishmania-infected BALB/c mice, observing that CasIII-ia is a potential alternative treatment for CL caused by L. mexicana.

Death of amastigotes induced by CasIII-ia was related to an increase in ROS production in the parasites. CasIII-ia related cell death reported in other protozoa, such as G. intestinalis and T. cruzi, was evidenced after exposure to an IC50 of 156 μM (24 h) and 6.9 μM (120 h), respectively. We now observed that for L. mexicana amastigotes, CasIII-ia achieves cell death at an IC50 of 58.1 μM (25.82 μg/mL) after 48 h of coincubation in an axenic culture. This concentration is below some of the IC50 values reported for conventional in vitro treatments against L. mexicana, such as Paromomycin (IC50 = 41 μg/mL), Glucantime (IC50 = 30 μg/mL), and Pentostam (IC50 = 48 μg/mL) [22]. Furthermore, in the case of L. mexicana-infected macrophages, CasIII-ia at a concentration of 2 μM significantly reduced the survival of intracellular amastigotes by 66%, showing the potent leishmanicidal efficacy of CasIII-ia against the infective stage of the parasite in the mammalian host.

The interest in the use of metallodrugs as treatment alternatives for pathogens has increased due to their selective affinity for biomolecules present in different pathogens [23]. Thus, Casiopeinas® are capable of reducing the concentration of antioxidant agents such as reduced glutathione (GSH) that are required to maintain a redox balance [20]. The redox metabolism of Leishmania is carried out by the glutathione conjugate N1,N8-bis (l-γ-glutamyl-L-hemicysti-nylglycyl) spermidine, or trypanothione (T(SH)2) [24]. The biosynthesis of T(SH)2 and GSH is regulated by γ-glutamylcysteine synthetase (γ-GCS) [25]. T(SH)2 has been shown to have protective effects on trypanosomatids through its antioxidant capacity against ROS and reactive nitrogen species produced by the host cell [24]. Mukherjee et al. observed that L. infantum amastigotes that lacked one allele of the gene encoding y-GCS were eliminated after macrophages produced ROS and reactive nitrogen species [25], indicating the importance of GHS and T (SH)2 for the survival of Leishmania in its host [24].

In the case of SK-N-SH neuroblastoma cells, Cu (II)-Casiopeina was observed to interact in a redox reaction with GHS, forming Cu (I)-Casiopeina + GSSH. ln the presence of H2O2 and in a new redox reaction called Fenton, the generation of hydroxide radicals (OH-) and hydroxyl (OH▪) is favored. Under these conditions, CasIII-ia decreases intracellular GSH and increases H2O2 by 21–34%, leading to morphological changes and apoptosis of the cell [3, 7].

The increase of ROS in amastigotes after coincubation with CasIII-ia may be due to Fenton reactions that could be increasing free radicals, thereby leading to parasite death. ROS produced by CasIII-ia has been found to induce death of G. intestinalis by apoptosis [4]. The effect of CasIII-ia in causing DNA damage through ROS production was also demonstrated in T. cruzi death [9].

These data suggest that the death of L. mexicana amastigotes exposed to CasIII-ia is possibly due to ROS causing oxidative damage to DNA and other biomolecules. ROS can bind to the nitrogenous bases of DNA, causing the rupture of the double helix and leading to cell death. Furthermore, the planar structure 4,4′-dimethyl-2,2′-bipyridine of CasIII-ia has the ability to internally intercalate in DNA, between the nitrogenous bases, or externally, in the groove between the 2 laps. This affinity, together with the redox properties of Cu (II) and Cu (I), favors the formation of ROS and cell death [26].

It is noteworthy that other studies using synthetic organic or natural compounds as treatments against leishmaniasis have proposed that the enhanced ROS production in. Leishmania promastigotes could lead to depolarization of the mitochondrial membrane, leading to an apoptosis-like cell death [27‒30].

The in vivo assay carried out in BALB/c mice infected with L. mexicana showed that the intraperitoneal administration of 20 μg/kg CasIII-ia led to a significant reduction of the lesion sizes (18–24%), as compared to the control group. It is noteworthy that the topical administration of the same dose of CasIII-ia showed no significant change compared to the group without treatment. Even though the intraperitoneal administration of CasIII-ia seems a promising treatment alternative for leishmaniasis in mice, potential side effects still need to be analyzed. The intraperitoneal administration of CasIII-ia at doses of 6.74 μmol/kg and 13.5 μmol/kg has been reported to induce a strong inhibition of energy metabolism and can lead to adhesion and inflammation on the peritoneal surface as a result of chronic irritation [1].

New treatment alternatives that replace or complement the currently available therapies for CL are needed [31]. We now show that CasIII-ia has a selective action on L. mexicana and therefore propose this metallodrug as a potential treatment against CL, taking into account that previous studies have positioned CasIII-ia as a promising drug for the resolution of neglected infectious diseases [4]. The potential use and administration routes of CasIII-ia for the treatment of CL caused by L. mexicana need to be further explored, in view of the fact that this metallodrug is currently in clinical phase 1 in Mexico for other pathologies and must now also be considered as a potential treatment alternative for this parasitic disease.

Our results show an important selective effect of CasIII-ia on the parasite L. mexicana, possibly due to the induction of ROS production that causes peroxidation of the mitochondrial membranes as a result of oxidative stress, releasing ROS that can interact with DNA or other biomolecules that compromise cell structure, function, and viability that ultimately lead to parasite death. Therefore, we propose that CasIII-ia should be taken into account as a potential treatment against CL caused by L. mexicana.

All animal studies were approved by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) of the Faculty of Medicine, UNAM, with registration number 063-2020/022-CIC-2020.

The authors declare that there are no competing interests.

This work was supported by UNAM-PAPIIT IG201221, UNAM-PAPIIT IN230020, UNAM-FQ 5000-9047, and CONACyT 6682. The authors declare that this work has not been influenced by any financial, personal, or professional interest. The funders had no role in study design, data preparation and analysis, decision to publish, or preparation of the manuscript.

José Delgado-Domínguez, Lizet Mejia Camacho, Lisset Torres-Martínez, Jaime Zamora-Chimal, and Rocely Cervantes-Sarabia participated in the conceptualization, study design, conducted all the experiments, data curation, and reviewed drafts of the manuscript. Adrián Espinoza-Guillen and Lena Ruiz-Azuara participated in Casiopeina III-ia synthesis, critically revised the manuscript, and approved of the final drafts and funding. Ingeborg Becker participated in conceptualization, supervision, visualization, and approval of the final draft, as well as funding.

The data generated during the current study are publicly available from the corresponding author (J.D.).

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