Introduction
Leishmaniasis is a neglected disease caused by intracellular protozoans of the genus Leishmania. Depending on the parasite species and the patient's immune status, this disease may present a wide spectrum of clinical manifestations ranging from cutaneous leishmaniasis (CL), the most benign form, to life-threatening visceral leishmaniasis (VL). Although not fatal, CL is the most common form of the disease, which is endemic in 87 countries and affects almost 1 million people every year, according to World Health Organization (WHO) estimates (WHO, 2017). Uncomplicated CL is characterized by one or more skin ulcers at the sandfly bite site that evolves for weeks to months, normally leaving a permanent scar. In some individuals, the disease may progress to extremely morbid diffuse or mucosal forms (Aronson and Joya, Reference Aronson and Joya2019). Currently available treatments for CL are toxic and require intensive health services. For example, first-line therapy uses 20–30 daily intramuscular or intravenous injections with antimonials, pentamidine, or amphotericin B, which produces severe adverse reactions and poor patients' compliance. Liposomal formulations of amphotericin B have appeared as a less toxic alternative treatment, but the high cost and invasive administration have limited their widespread use (Uliana et al., Reference Uliana, Trinconi and Coelho2017). An ideal CL treatment should not only heal the cutaneous lesions but also prevent the development of the more morbid forms of the disease (Aronson and Joya, Reference Aronson and Joya2019).
Chalcones have appeared as a promising new class of antileishmanial (de Mello et al., Reference de Mello, Bitencourt, Pedroso, Aristides, Lonardoni and Silveira2014; Ortalli et al., Reference Ortalli, Ilari, Colotti, De Ionna, Battista, Bisi, Gobbi, Rampa, Di Martino, Gentilomi, Varani and Belluti2018). They are characterized by the presence of a 1,3-diphenylprop-2-en-1-one scaffold and have, in addition to their antileishmanial effects, a broad spectrum of pharmacological activities. Indeed, chalcones are known to affect bacteria (Kunthalert et al., Reference Kunthalert, Baothong, Khetkam, Chokchaisiri and Suksamrarn2014; Li et al., Reference Li, Chen, Zhang, Niu, Song, Luo, Lu, Liu, Zhao, Wang and Deng2016), fungi (Tiwari et al., Reference Tiwari, Pratapwar, Tapas, Butle and Vatkar2010; Łacka et al., Reference Łacka, Konieczny, Bulłakowska, Rzymowski and Milewski2011) and helminths (De Castro et al., Reference De Castro, Costa, Laktin, De Carvalho, Geraldo, De Moraes, Pinto, Couri, Pinto and Da Silva Filho2015), and to have immunosuppressive properties (Luo et al., Reference Luo, Song, Li, Zhang, Liu, Fu and Zhu2012). We have previously demonstrated the activity in vitro and in vivo of a natural methoxychalcone and its synthetic derivatives against Leishmania amazonensis (Torres-Santos et al., Reference Torres-Santos, Moreira, Kaplan, Meirelles and Rossi-Bergmann1999a; Boeck et al., Reference Boeck, Bandeira Falcão, Leal, Yunes, Filho, Torres-Santos and Rossi-Bergmann2006). Its synthetic analogue 3-nitro-2′-hydro-4′,6′-dimethoxychalcone, named CH8, displayed the highest in vitro selectivity index against L. amazonensis (S.I. = 318 in relation to murine macrophages). Additionally, intralesional injections with CH8 were more effective than the reference drug sodium stibogluconate in a murine model of CL caused by L. amazonensis (Boeck et al., Reference Boeck, Bandeira Falcão, Leal, Yunes, Filho, Torres-Santos and Rossi-Bergmann2006). The oral efficacy and safety of CH8 were also demonstrated in BALB/c mouse infections with L. amazonensis and L. infantum (Sousa-Batista et al., Reference Sousa-Batista, Escrivani-Oliveira, Falcão, Da Silva Philipon and Rossi-Bergmann2018a). More recently, the efficacy of a single injection with CH8 was achieved by loading the drug in biodegradable poly (lactic-co-glycolic acid) (PLGA) microparticles (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b). The drug encapsulation allows a prolonged release in the lesion and, in leishmaniasis, has the advantage of being taken up by phagocytic macrophages (Sousa-Batista and Rossi-Bergmann, Reference Sousa-Batista and Rossi-Bergmann2018).
Although CH8 is a promising drug candidate, its burdensome synthesis and purification is a pitfall in pharmaceutical development. Aiming to solve this problem, in the present work we proposed to identify active CH8 analogues with easier purification and better synthesis yield. The ultimate goal of this study was to choose the best analogue to encapsulate in PLGA microparticles and, thus, to develop an optimized delivery system for local and single-dose treatment of CL.
Methodology
Chalcones
Chalcone CH8 (3-nitro-2-hydroxy-4,6-dimethoxychalcone) was synthesized by aldol condensation as previously described (Boeck et al., Reference Boeck, Bandeira Falcão, Leal, Yunes, Filho, Torres-Santos and Rossi-Bergmann2006). All starting materials were commercially obtained (Merck, Germany). Fifty analogues were produced but only those with acceptable synthesis yield are described here. The chalcone analogue 1 (NAT 1) was prepared by a condensation reaction in which the appropriately substituted acetophenone (1 equiv) was added to an aqueous-alcoholic solution (40% v/v EtOH in water) of sodium hydroxide (3% w/v NaOH in water; 3 equiv) with stirring and with the cooling of the reaction mixture in an ice bath during the addition. The mixture was allowed to warm to room temperature (25°C), and the appropriately substituted benzaldehyde (1 equiv) was added with vigorous stirring until the product precipitation (Kumar et al., Reference Kumar, Ramaiah, Das and George1985). The analogues NAT22, NAT28, NAT31, and NAT49 were prepared by Claisen-Schmidt condensation (Amslinger et al., Reference Amslinger, Al-Rifai, Winter, Wörmann, Scholz and Wild2013) between aromatic acetophenones (1 equiv) and corresponding aldehydes (1 equiv) in methanol- Ba(OH)2 (1 equiv) at 50°C with magnetic agitation for 12–48 h. The products were purified by column chromatography or on preparative TLC plates to yield the corresponding chalcone. For the analogues NAT31 and NAT49, a further step of deprotection was done: to each chalcone in dry CH2Cl2 was added dropwise a solution of BCl3 in hexane at −78°C (Amslinger et al., Reference Amslinger, Al-Rifai, Winter, Wörmann, Scholz and Wild2013), after which the solution was stirred for 3 h at 0°C. All of the synthesized compounds have been previously described, and their spectral data corresponded to that in the literature. Individual yields of the compounds were as follows: NAT1 = 61%; NAT22 = 89%; NAT28 = 81%; NAT31 = 12% and NAT49 = 13%.
PLGAk and NAT22-PLGAk microparticles
Microparticles were prepared by an emulsion and solvent evaporation process. The polymers Poly(lactide-co-glycolide) 50:50 (PLGA – Purac 504, Purasorb, Corbion, Netherlands) and polyvinylpyrrolidone (PVP – Kolidon K17, BASF, Germany), at a final concentration of 90 and 10% were dissolved in dichloromethane (DCM – Vetec, Brazil). NAT22 (10%) was dissolved in this solution and the resulting homogenous mixture injected into an aqueous phase containing Polyvinyl alcohol (PVA – Sigma-Aldrich, EUA), 3% (w/v). The mixture was emulsified by agitation in an Ultra-Turrax T25 Basic (Ika, Germany) for 2 min at 13000 rpm on ice. The organic phase evaporation was performed under reduced pressure using a rotary evaporator (LOGEN Scientific, Brazil). After solvent full evaporation, the obtained microparticles (NAT22-PLGAk) were washed with water and centrifuged at 9000 rpm at 4°C. Finally, the microparticles were dispersed in trehalose 5% and cooled to −18°C for subsequent freeze-drying (FreeZone 1 lyophilizer, Labconco Corporation, EUA). The freeze-dried microparticles were stored at 4°C. Empty microparticles (PLGAk) were prepared following the same procedure but without added NAT22.
Average particle size and its distribution were measured by a laser light scattering analyzer (Zetasizer 3000, Malvern Instruments, Malvern). The particles were dispersed in 0.5% (w/w) Tween-20 solution and subsequently submitted to ultrasound for 20 s.
The drug present in the microparticles was determined directly by measuring the amount of NAT22 entrapped in the microparticles. Briefly, microparticles were accurately weighed and dissolved in 5 mL acetonitrile (Vetec, Brazil) by bath sonication until complete solubilization was achieved. The absorbance of appropriately diluted stock solutions was measured at 320 nm (SpectraMax M5), and the NAT22 concentration was calculated using calibration curves (correlation coefficient >0.999) over a concentration range of 10 to 60 mg/L NAT22. All samples were measured in triplicate.
The shape and surface of the microparticles were studied using a scanning electron microscope (s.e.m., JSM-5600LV, JEOL, Japan) operating at an acceleration voltage of 20 kV under nitrogen atmosphere. Before s.e.m. analysis, the dry microparticles were coated by sputtering with gold (JFC-1300, JEOL, Japan).
In vitro antileishmanial activity
Antileishmanial drug activity was determined against both the promastigote and the intracellular amastigote forms of L. amazonensis (MHOM/BR/75/Josefa strain).
For antipromastigote studies, 2 × 105 promastigotes mL−1 of medium 199 supplemented with 5% heat-inactivated fetal bovine serum (HIFBS – Cutilab, Brazil) were first incubated in triplicate with each compound (0, 0.1, 1, 10 and 100 μ m) to determine the IC50 range, and again with 2-fold dilutions for a more precise determination. All cultures including controls contained 1% dimethyl sulfoxide (DMSO – Sigma-Aldrich, EUA). After 72 h at 26°C, cell viability was measured by the MTS Cell Proliferation Colorimetric Assay Kit (Promega, EUA) using a plate-reader spectrometer (SpectraMax M5, Molecular Devices, EUA) at 490 nm. The results were expressed as the drug concentration that inhibited parasite growth by 50% (IC50, calculated as in 3.6 below).
For anti-amastigote activity, 5 × 105 mouse peritoneal macrophages were plated on glass coverslips and infected with 5 × 106 promastigotes (1:10) at 34°C for 4 h when non-internalized parasites were washed away with phosphate-buffered saline (PBS). After 24 h of incubation in RPMI supplemented with 5% HIFBS at 37°C, the amastigote-infected macrophages were treated with varying concentrations of compounds for 48 h. NAT22-PLGAk concentrations were relative to NAT22 content, while PLGAk concentrations were relative to its content in NAT22-PLGAk. Treatment time in the particle assay was increased to 96 h to allow more time for polymer degradation and drug release (Sousa-Batista et al., Reference Sousa-Batista, Arruda-Costa, Rossi-Bergmann and Ré2018c). Then, coverslips were stained with Giemsa for cell counting under a microscope (400x). Parasite loads in culture were calculated as total numbers of amastigotes/200 total macrophages.
Macrophages viability evaluation
Adherent mouse peritoneal macrophages were cultured in triplicate at 37°C with 2-fold dilutions of compounds for 48 h (analogue assay) or 96 h (microparticle assay). The release of the cytoplasmic enzyme lactate dehydrogenase (LDH) into the culture medium was measured using an assay kit (Doles, Brazil) and a plate-reader spectrometer (SpectraMax M5) at 340 nm, as a cytotoxicity indicator. Maximum and minimum release values were obtained with cells cultured with 2% Triton X-100 or medium, respectively, and LDH release was calculated as a percent of the positive control. Equation: % specific release = [(test release – spontaneous release)/(maximal release – spontaneous release)] × 100. Representative results from three independent experiments were expressed as the drug cytotoxic concentration for 50% of the cell in culture (CC50).
In vivo antileishmanial activity and ethical standards.
The CL model used was 8-week-old female BALB/c mice weighing about 23 g, as previously approved by the institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (protocol number CAUAP118). To compare the efficacy of CH8 and NAT22 against CL, five BALB/c mice/group were infected in the ear pinna with 106 promastigotes of green fluorescence protein GFP- L. amazonensis (Costa et al., Reference Costa, Golim, Rossi-Bergmann, Costa and Giorgio2011) at the stationary phase of growth. After 7 days of infection, animals were given local s.c. injections with 1.2 mg kg−1 of NAT22 (3.3 μg /injection) or 10 μL of the vehicle alone (PBS) twice a week during 4 weeks (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b). Alternatively, they received a total of 8 injections with NAT22 alone twice a week for 4 weeks. The parasite loads in the individual lesions were expressed as arbitrary fluorescence units (Demicheli et al., Reference Demicheli, Ochoa, da Silva, Falcão, Rossi-Bergmann, de Melo, Sinisterra and Frézard2004). The fluorescence intensity of contralateral uninfected ears was subtracted from that of the treated ears. Alternatively, mice were given a single s.c. injection with 3.3 μg of NAT22 alone or equivalents in NAT22-PLGAk. Controls received the same dose of empty PLGAk, or 10 μL of PBS alone. On day 32 of infection, animals were anaesthetized with isoflurane, sacrificed by cervical dislocation, the ears removed, grounded and assayed by limiting dilution assay (LDA) for determination of parasite loads (Lima et al., Reference Lima, Bleyenberg and Titus1997).
Statistical analysis
Groups were compared using unpaired Student´s t-test and considered different when P < 0.05. IC50 and CC50 values were calculated by logarithmic regression analysis from a sigmoidal dose-response curve. Data were normalized to run from 0% to 100% using a normalized dose-response equation. All analyses were conducted using GraphPad Prism 5 software.
Results
The need for CH8 analogues with better yields led us to synthesize approximately 50 molecules, however only the chalcones that could be synthesized more efficiently than CH8 will be shown in this work. Table 1 contains the synthesis efficiency of 5 analogues as well as their antileishmanial activity. The overall yield for each was superior to that obtained with CH8 (18%), with the highest yield (89%) being obtained for NAT22. The inhibitory activity of the analogues was tested against the amastigote and promastigote forms of L. amazonensis. CH8 displayed the highest antipromastigote activity (IC50 = 0.7 μ m), followed by NAT22 (IC50 = 1.9 μ m). All the compounds displayed potent inhibitory effects against the amastigote form of L. amazonensis, with NAT22 displaying the highest activity (IC50 = 0.1 μ m). None of the analogues had detectable cytotoxicity against murine macrophages, as indicated by the low specific release of LDH enzyme (CC50 > 100 μ m). As previously noted, the nitro group is important for optimal antiparasitic effect (activity/selectivity), as demonstrated by the superior performance of CH8 vs NAT49 and of NAT22 vs NAT28.
Synthesized chalcone analogues and their in vitro activity and safety
Maximum and minimum release optical densities were = 1.824 ± 0.003 and 1.014 ± 0.014. Means ± s.d. (n = 3).
a CC50 values as extrapolated from curve fitting.
Since NAT22 was the most active compound and its synthesis was simpler and more efficient than that of CH8 (85 and 18%, respectively), this compound was chosen for the evaluation of its efficacy in a murine model of CL. Intralesional treatment with NAT22 demonstrated significant efficacy in reducing parasite load when compared to untreated controls. At the same treatment regimen (8 doses of 3.3 μg each), although NAT22 was slightly more effective than CH8 in preventing parasite growth, the difference was not statistically significant (P > 0.05) (Fig. 1).
Effect of free chalcones in L. amazonensis-infected mice. BALB/c mice were infected with L. amazonensis-GFP in the ear. From day 7 of infection, they were treated twice a week with CH8 or NAT22 at a total dose of 1.2 mg kg−1 (3.3 μg dose−1 10 μL−1) or with 10 μl of the vehicle alone (PBS) for 4 weeks. Parasite loads were measured on day 32 of infection and expressed as specific fluorescence units (FU). Means ± s.d. (n = 5), *** P < 0.05 in relation to PBS.
With the goal of improving its local efficacy in CL, NAT22 was loaded into biodegradable polymeric microparticles. To this end, microparticles composed of a mixture of PLGA and PVP polymers were prepared by a solvent evaporation technique, yielding particles with 9.5% encapsulated NAT22 (NAT22-PLGAk). Empty microparticles were also prepared. The loaded and empty microparticles showed similar topography, with a round smooth surface (Fig. 2B) and diameters of 1.90 ± 0.01 μm (dispersion = 2.30 ± 0.07 μm) and 2.60 ± 0.13 μm (dispersion = 3.57 ± 2.10 μm), respectively, as measured by a light laser scanner (not shown). Figure 2A shows by s.e.m. the crystalline structures of unencapsulated NAT22. Drug crystals were not seen outside the microparticles allowing a durable effect, suggesting a high drug internalization (Fig. 2B).
Scanning electron microscopy of (A) large crystal structures of free NAT22, and (B) microparticle-entrapped NAT22 (NAT22-PLGAk).
When NAT22 was tested for inhibition of intracellular amastigotes, entrapment into microparticles did not significantly increase drug activity (P > 0.05), as shown in Table 2. None of the NAT22 presentations was cytotoxic to macrophages as measured by release of the cytoplasmic enzyme LDH (Table 2). Finally, we tested the efficacy of single-dose subcutaneous treatment with NAT22-PLGAk in CL (Fig. 3). The safety of PLGA s.c. injection was previously demonstrated in mice using PLGA-CH8 formulation (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b). Mice receiving a single intralesional injection with NAT22-PLGAk had, 25 days later, only 11% of the number of parasites present in animals receiving the PBS vehicle alone (100%). The resulting efficacy was higher than that obtained with a single or even eight doses of unencapsulated NAT22.
Efficacy of NAT22-PLGAk in L. amazonensis-infected mice. BALB/c mice were infected in the ear with L. amazonensis. On day 7 of infection, they were given a single (1X) or eight (8X within 4 weeks), intralesional injections with a total dose of 1.2 mg kg−1 of NAT22, NAT22-PLGAk, or PLGAk. Parasite loads were measured in the ears on day 32 of infection. Means ± s.d. (n = 5). ** P < 0.01 in the relation of PBS group, # P < 0.05.
Anti-amastigote activity and cytotoxicity of free and microparticulated NAT22
Maximum (2% Triton 100-X) and minimum LDH release values were = 1.824 ± 0.003 and 1.014 ± 0.014, respectively. Means ± s.d. (n = 3).
a CC50 values as extrapolated from curve fitting. No concentrations higher than 100 μ m were used due to dense particle deposits over cell monolayers.
Discussion
Previously, we have described the promising efficacy of the nitrochalcone CH8 for the intralesional treatment of CL caused by L. amazonensis (Boeck et al., Reference Boeck, Bandeira Falcão, Leal, Yunes, Filho, Torres-Santos and Rossi-Bergmann2006). However, the low synthesis yields together with difficult drug purification showed to be a bottleneck in the process scale-up due to interconversion of a percentage of resulting CH8 with a flavanone. Five CH8 analogues were then synthesized with fewer purification steps and higher yields.
Previous studies have shown that modifications in the positions of substituents in the structure of chalcones can improve their antileishmanial effects (Nielsen et al., Reference Nielsen, Christensen, Cruciani, Kharazmi and Liljefors1998; Kayser and Kiderlen, Reference Kayser and Kiderlen2001; Boeck et al., Reference Boeck, Bandeira Falcão, Leal, Yunes, Filho, Torres-Santos and Rossi-Bergmann2006). We produced five easily synthesized CH8 analogues bearing modifications in both aromatic rings and observed that the nitro group was important for their ability to inhibit L. amazonensis growth in vitro. The nitro group has also been demonstrated to be important in anti-trypanosome activity (Patterson and Wyllie, Reference Patterson and Wyllie2014). The nitrochalcone NAT22 was selected for further in vivo studies due to its high synthesis yield and better antileishmanial activity.
A single intralesional injection with NAT22 resulted in 40% reduction in local parasite load measured 3 weeks later. That effect is similar to observed with CH8 (37%), although in vitro NAT22 demonstrated an anti-amastigote activity greater than CH8 (0.1 and 1.4 μ m, respectively). The relative low water solubility and large crystals shown in Fig. 2 may have provided a slow dissolving depot in the injection site, providing a longer-lasting effect. Although an effective and short treatment for CL is highly desirable, the problem with the crystalline nature is needle clogging and difficult dose reproducibility.
To circumvent NAT22's poor water solubility and chemical stability, we attempted to entrap it in PLGA particles, as demonstrated previously with CH8 (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b). PLGA, a copolymer of lactic acid and glycolic acid, has been shown to be safe and effective for use in sustained-release drug delivery systems and has also been clinically approved for other uses, such as absorbable suture threads, orthopaedic scaffolds, and treatment of localized tumours (Ortega-Oller et al., Reference Ortega-Oller, Padial-Molina, Galindo-Moreno, O'Valle, Jódar-Reyes and Peula-García2015; Wan and Yang, Reference Wan and Yang2015). In order to increase NAT22 loading efficiency, reduce the initial burst release and to improve delivery characteristics, similar to that previously observed for CH8, PLGA was blended with the polymer polyvinylpyrrolidone (PVP) (Sousa-Batista et al., Reference Sousa-Batista, Arruda-Costa, Rossi-Bergmann and Ré2018c). This PVP effect is due to its higher solubility in organic polymer solution that results in extensive diffusion of PVP molecules into the dispersed droplets of polymer solution during microparticle preparation and its high capacity to interact with the drug by hydrogens bonds (Meeus et al., Reference Meeus, Scurr, Amssoms, Davies, Roberts and Van Den Mooter2013, Reference Meeus, Scurr, Appeltans, Amssoms, Annaert, Davies, Roberts and Van Den Mooter2015). Thus, the PLGA/PVP polymer blend (PLGAk) was used to improve NAT22-PLGA interaction, and the process was adjusted to provide particles with an average diameter of 1.9 μm, a size large enough to allow phagocytosis by macrophages and yet prevent absorption into blood circulation. Such particle size provides a potential advantage compared to both injected intralesional antimonials that require repeated injections (Oliveira-Neto et al., Reference Oliveira-Neto, Schubach, Mattos, da Costa and Pirmez1997) and smaller poly-lactide nanoparticles (Torres-Santos et al., Reference Torres-Santos, Rodrigues, Moreira, Kaplan and Rossi-Bergmann1999b) or larger PLGA microparticles (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b) that have been used to carry subcutaneous chalcone implants. Additionally, the NAT22-PLGAk formulation is a single dose treatment, meeting the DNDi Target Product Profile for CL that recommends the use of treatments with few injections, local effect and low cost. It is worth noting that despite the technology adding cost, a single dose treatment will have a positive impact in reduced hospital and mobility costs.
We found NAT22-PLGAk inhibited parasite growth inside macrophages in vitro, but this effect was not superior to unencapsulated NAT22, probably due to the fact that the time employed in the in vitro assay was not sufficient to achieve maximum chalcone release from the microparticles, as seen previously with CH8-PLGA (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b). It is conceivable that PVP has further slowed the intracellular release, and therefore less NAT22 was available to produce its effect on the parasite during the 96-h assay. Recently, we reported that amphotericin B release from PLGA microparticles is much faster in the subcutaneous ear tissue (30 days) than in saline solution in vitro (200 days), possibly because of the presence of enzymatic hydrolysis in the tissue (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Ré and Rossi-Bergmann2019). Whether or not NAT22 release from PLGAk microparticles follows the same kinetics remains to be determined. In addition to showing good anti-amastigote activity, NAT22-PLGAk did not induce cytotoxic effects against mammalian macrophages, supporting its safety and promise as a therapeutic tool in vivo. It appears that the anti-amastigote effect of NAT22-PLGAk is due to intracellular drug targeting rather than to macrophage activation of NO production, as this function was not affected in treated macrophages (data not shown). This is in accordance with the results obtained previously with free or encapsulated CH8 (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b) and with the well-known antioxidant (Sivakumar et al., Reference Sivakumar, Prabhakar and Doble2011) and anti-inflammatory (Herencia et al., Reference Herencia, Ferrándiz, Ubeda, Guillén, Dominguez, Charris, Lobo and Alcaraz1999) properties of chalcones. However, other potential microbicidal mechanisms of NAT22-PLGAk should not be discarded. Since, although to a much lesser extent than NAT22-PLGAk, empty PLGAk alone displayed some inhibitory effect on the intracellular parasites (IC50 > 10 μ m), suggestive of a macrophage stimulatory effect of PLGA microparticles (Luzardo-Alvarez et al., Reference Luzardo-Alvarez, Blarer, Peter, Romero, Reymond, Corradin and Gander2005).
In this work, the use of NAT22 plus the polymer blend (PLGA/PVP) in the microparticle composition allowed an increased drug content (9.5% w/w), in contrast to the previous CH8 plus PLGA which maximally incorporated 7.8% of drug (Sousa-Batista et al., Reference Sousa-Batista, Pacienza-Lima, Arruda-Costa, Falcão and Rossi-Bergmann2018b). More importantly, the easier and more efficient NAT22 synthesis yield (89%) as compared with CH8 (18%) will further reduce production costs. Because the currently available treatments for localized CL are invasive and produce systemic side effects, NAT22-PLGAk appears promising for single-dose local use. The mouse ear model of CL used here appears translatable to the human infection as the drug is discharged in the subcutaneous tissue, not on the skin surface where differential skin permeability would be more critical. Future studies on local drug kinetics, evaluation of efficacy over extended time periods, and use of other relevant parasite species like L. braziliensis and L. tropica will add further insight to this new mode of CL therapy.
Financial support
The authors thank the Brazilian agencies FAPERJ (E-26/202.402/2017 – BBP) and CNPq for financial support and The Royal Society London for an International Collaboration Award for Research Professors (#IC160044 to BRB and PGS).
Conflict of interest
None.
Ethical standards
The CL model using 8-week-old female BALB/c mice weighing about 23 g was previously approved by the institutional Animal Care and Use Committee of the Federal University of Rio de Janeiro (protocol number CAUAP118).
