Intranasal delivery of LaAg vaccine improves immunity of aged mice against visceral Leishmaniasis.

There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis.

Synthesis and evaluation of hybrid sulfonamide-chalcones with potential antileishmanial activity.

Leishmaniasis is an emerging tropical infectious disease caused by a protozoan parasite of the genus Leishmania. In this work, the molecular hybridization between a trimethoxy chalcone and a sulfonamide group was used to generate a series of sulfonamide-chalcones. A series of eight sulfonamide-chalcone hybrids were made with good yields (up to 95%). These sulfonamide-chalcones were tested against promastigotes of Leishmania amazonensis and cytotoxicity against mouse macrophages, which showed good antileishmanial activity with IC50 = 1.72-3.19 µM. Three of them (10c, 10g, and 10h) were also highly active against intracellular amastigotes and had a good selectivity index (SI > 9). Thus, those three compounds were docked in the cytosolic tryparedoxin peroxidase (cTXNPx) enzyme of the parasite, and molecular dynamics simulations were carried out. This enzyme was selected as a target protein for the sulfonamide-chalcones due to the fact of the anterior report, which identified a strong and stable interaction between the chalcone NAT22 (6) and the cTXNPx. In addition, a prediction of the drug-likeness, and the pharmacokinetic profile of all compounds were made, demonstrating a good profile of those chalcones.

Crosslinked chitosan microparticles as a safe and efficient DNA carrier for intranasal vaccination against cutaneous leishmaniasis.

Intranasal (i.n.) vaccination with adjuvant-free plasmid DNA encoding the leishmanial antigen LACK (LACK DNA) has shown to induce protective immunity against both cutaneous and visceral leishmaniasis in rodents. In the present work, we sought to evaluate the safety and effectiveness of d,l-glyceraldehyde cross-linked chitosan microparticles (CCM) as a LACK DNA non-intumescent mucoadhesive delivery system. CCM with 5 µm of diameter was prepared and adsorbed with a maximum of 2.4 % (w/w) of DNA with no volume alteration. Histological analysis of mouse nostrils instilled with LACK DNA / CCM showed microparticles to be not only mucoadherent but also mucopenetrant, inducing no local inflammation. Systemic safeness was confirmed by the observation that two nasal instillations one week apart did not alter the numbers of bronchoalveolar cells or blood eosinophils; did not alter ALT, AST and creatinine serum levels; and did not induce cutaneous hypersensitivity. When challenged in the footpad with Leishmania amazonensis, mice developed significantly lower parasite loads as compared with animals given naked LACK DNA or CCM alone. That was accompanied by increased stimulation of Th1-biased responses, as seen by the higher T-bet / GATA-3 ratio and IFN-γ levels. Together, these results demonstrate that CCM is a safe and effective mucopenetrating carrier that can increase the efficacy of i.n. LACK DNA vaccination against cutaneous leishmaniasis.

Cysteinyl-leukotrienes promote cutaneous Leishmaniasis control.

Leishmaniasis is a neglected tropical parasitic disease with few approved medications. Cutaneous leishmaniasis (CL) is the most frequent form, responsible for 0.7 - 1.0 million new cases annually worldwide. Leukotrienes are lipid mediators of inflammation produced in response to cell damage or infection. They are subdivided into leukotriene B4 (LTB4) and cysteinyl leukotrienes LTC4 and LTD4 (Cys-LTs), depending on the enzyme responsible for their production. Recently, we showed that LTB4 could be a target for purinergic signaling controlling Leishmania amazonensis infection; however, the importance of Cys-LTs in the resolution of infection remained unknown. Mice infected with L. amazonensis are a model of CL infection and drug screening. We found that Cys-LTs control L. amazonensis infection in susceptible (BALB/c) and resistant (C57BL/6) mouse strains. In vitro, Cys-LTs significantly diminished the L. amazonensis infection index in peritoneal macrophages of BALB/c and C57BL/6 mice. In vivo, intralesional treatment with Cys-LTs reduced the lesion size and parasite loads in the infected footpads of C57BL/6 mice. The anti-leishmanial role of Cys-LTs depended on the purinergic P2X7 receptor, as infected cells lacking the receptor did not produce Cys-LTs in response to ATP. These findings suggest the therapeutic potential of LTB4 and Cys-LTs for CL treatment.

Intradermal Immunization of SARS-CoV-2 Original Strain Trimeric Spike Protein Associated to CpG and AddaS03 Adjuvants, but Not MPL, Provide Strong Humoral and Cellular Response in Mice.

Despite the intramuscular route being the most used vaccination strategy against SARS-CoV-2, the intradermal route has been studied around the globe as a strong candidate for immunization against SARS-CoV-2. Adjuvants have shown to be essential vaccine components that are capable of driving robust immune responses and increasing the vaccination efficacy. In this work, our group aimed to develop a vaccination strategy for SARS-CoV-2 using a trimeric spike protein, by testing the best route with formulations containing the adjuvants AddaS03, CpG, MPL, Alum, or a combination of two of them. Our results showed that formulations that were made with AddaS03 or CpG alone or AddaS03 combined with CpG were able to induce high levels of IgG, IgG1, and IgG2a; high titers of neutralizing antibodies against SARS-CoV-2 original strain; and also induced high hypersensitivity during the challenge with Spike protein and a high level of IFN-γ producing CD4+ T-cells in mice. Altogether, those data indicate that AddaS03, CpG, or both combined may be used as adjuvants in vaccines for COVID-19.

Development of Immunological Assays Based on Leishmania donovani Antigen for Diagnosis of Canine Visceral Leishmaniasis and Their Multicenter Evaluation in Brazil and Italy.

Canine visceral leishmaniasis (CVL) due to Leishmania infantum infection is a zoonotic disease prevalent in the areas of South America and the Mediterranean. Infected dogs as reservoirs can contribute to disease transmission and can be a scourge to public health. Therefore, early diagnosis of infected dogs may play a pivotal role in circumscribing disease progression. Invasive tissue aspiration and insufficient serological methods impair a single assay for prompt CVL diagnosis. In the present study, we aimed to evaluate the potential of Leishmania donovani isolated membrane protein, LAg, for the diagnosis of CVL through immunological assays. Initially, enzyme-linked immunosorbent assay was done with Brazilian dog sera to evaluate the performance of LAg in diagnosing CVL and found sensitivity and specificity of 92.50% and 95%, respectively. The study further confirmed the diagnostic efficacy of LAg in a dipstick format. The dipstick test of canine sera from three centers in Brazil and one center in Italy collectively showed sensitivity values in the range of 53.33% to 100% in recognizing symptomatic dogs and specificity values between 75% and 100% to rule out healthy dogs. Moreover, a rapid immunochromatographic test was developed and optimized using LAg. This test was able to identify 94.73% of CVL of Brazilian origin with specificity of 97.29%. The current results highlight the reactive potential of the L. donovani antigen, LAg, for L. infantum CVL diagnosis and support our previous findings, which suggest the utility of LAg for the diagnosis of both L. donovani and L. infantum human VL in a variety of endemic regions. LAg as a diagnostic candidate may be employed to identify comprehensive CVL cases in epidemiological areas.

Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

Eugenol derivatives with 1,2,3-triazole moieties: Oral treatment of cutaneous leishmaniasis and a quantitative structure-activity relationship model for their leishmanicidal activity.

Leishmaniasis is a group of neglected vector-borne tropical diseases caused by protozoan parasites of the genus Leishmania that multiply within phagocytic cells and have a wide range of clinical manifestations. Cutaneous leishmaniasis (CL) is a serious public health that affects more than 98 countries, putting 350 million people at risk. There are no vaccines that have been proven to prevent CL, and the treatment relies on drugs that often have severe side effects, justifying the search for new antileishmanial treatments. In the present investigation, it is demonstrated that 4-(3-(4-allyl-2-methoxyphenoxy)propyl)-1-(4-methylbenzyl)-1H-1,2,3-triazole (7k) presents significant antileishmanial activity (IC50 of 7.4 µmol L-1 and 1.6 µmol L-1 for promastigote and amastigote forms, respectively), low cytotoxicity against macrophage cells (IC50 of 211.9 µmol L-1), and a selective index of 132.5. Under similar conditions, compound 7k outperformed glucantime and pentamidine, two commonly used drugs in clinics. In vivo assays on CL-infected female BALB/c mice demonstrated that compound 7k had activity similar to intralesional glucantime when administered orally, with decreased lesion and parasitic load, and a low systemic toxic effect. Given the importance of understanding the relationship between compound structure and biological activity in the research and development of new drugs, the development of a quantitative structure-activity relationship (QSAR) model for the leishmanicidal activity presented by the eugenol derivatives with 1,2,3-triazole functionalities is also described herein. This study demonstrates the therapeutic potential of orally active eugenol derivatives against CL and provides useful insights into the relationship between the chemical structures of triazolic eugenol derivatives and their biological profile.

Targeting chalcone binding sites in living Leishmania using a reversible fluorogenic benzochalcone probe.

Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.

In Vivo Safety and Efficacy of Chalcone-Loaded Microparticles with Modified Polymeric Matrix against Cutaneous Leishmaniasis.

Current chemotherapy of cutaneous leishmaniasis (CL) is based on repeated systemic or intralesional administration of drugs that often cause severe toxicity. Previously, we demonstrated the therapeutic potential of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with 8% of the nitrochalcone CH8 (CH8/PLGA) prepared by a conventional bench method. Aiming at an industrially scalable process and increased drug loading, new MPs were prepared by spray drying: CH8/PDE with PLGA matrix and CH8/PVDE with PLGA + polyvinylpyrrolidone (PVP) matrix, both with narrower size distribution and higher drug loading (18%) than CH8/PLGA. Animal studies were conducted to evaluate their clinical feasibility. Both MP types induced transient local swelling and inflammation, peaking at 1−2 days, following a single intralesional injection. Different from CH8/PDE that released 90% of the drug in the ear tissue in 60 days, CH8/PVDE achieved that in 30 days. The therapeutic efficacy of a single intralesional injection was evaluated in BALB/c mice infected with Leishmania (Leishmania) amazonensis and golden hamsters infected with L. (Viannia) braziliensis. CH8/PVDE promoted greater reduction in parasite burden than CH8/PDE or CH8/PLGA, measured at one month and two months after the treatment. Thus, addition of PVP to PLGA MP matrix accelerates drug release in vivo and increases its therapeutic effect against CL.

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis.

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

MPLA and AddaVax® Adjuvants Fail to Promote Intramuscular LaAg Vaccine Protectiveness against Experimental Cutaneous Leishmaniasis.

There is so far no vaccine approved for human leishmaniasis, mainly because of the lack of appropriate adjuvants. This study aimed to evaluate in mice the capacity of a mixture of monophosphoryl lipid A (MPLA) and AddaVax® adjuvants in enhancing the efficacy of a Leishvacin®-like vaccine comprised of Leishmania amazonensis whole antigens (LaAg). For that, mice were immunized with LaAg plus MPLA/AddaVax® by the intramuscular route (i.m.) prior to challenge with 2 × 105 and 2 × 106 living parasites. Immunization with LaAg alone reduced the lesion growth of the 2 × 105-challenged mice only in the peak of infection, but that was not accompanied by reduced parasite load, and thus not considered protective. Mice given a 2 × 106 -challenge were not protected by LaAg. The association of LaAg with MPLA/AddaVax® was able to enhance the cutaneous hypersensitivity response compared with LaAg alone. Despite this, there was no difference in proliferative cell response to antigen ex vivo. Moreover, regardless of the parasite challenge, association of LaAg with MPL/AddaVax® did not significantly enhance protection in comparison with LaAg alone. This work demonstrated that MPL/AddaVax® is not effective in improving the efficacy of i.m. LaAg vaccine against cutaneous leishmaniasis.

Antileishmanial Chemotherapy through Clemastine Fumarate Mediated Inhibition of the Leishmania Inositol Phosphorylceramide Synthase.

Current chemotherapeutics for leishmaniasis have multiple deficiencies, and there is a need for new safe, efficacious, and affordable medicines. This study describes a successful drug repurposing approach that identifies the over-the-counter antihistamine, clemastine fumarate, as a potential antileishmanial drug candidate. The screening for inhibitors of the sphingolipid synthase (inositol phosphorylceramide synthase, IPCS) afforded, following secondary screening against Leishmania major (Lmj) promastigotes, 16 active compounds. Further refinement through the dose response against LmjIPCS and intramacrophage L. major amastigotes identified clemastine fumarate with good activity and selectivity with respect to the host macrophage. On target engagement was supported by diminished sensitivity in a sphingolipid-deficient L. major mutant (ΔLmjLCB2) and altered phospholipid and sphingolipid profiles upon treatment with clemastine fumarate. The drug also induced an enhanced host cell response to infection indicative of polypharmacology. The activity was sustained across a panel of Old and New World Leishmania species, displaying an in vivo activity equivalent to the currently used drug, glucantime, in a mouse model of L. amazonensis infection. Overall, these data validate IPCS as an antileishmanial drug target and indicate that clemastine fumarate is a candidate for repurposing for the treatment of leishmaniasis.

Vitamin D increases killing of intracellular Leishmania amazonensis in vitro independently of macrophage oxidative mechanisms.

Vitamin D has been reported to activate macrophage microbicidal mechanisms by inducing the production of antimicrobial peptides and nitric oxide (NO), but conversely has been shown to contribute to a greater susceptibility to Leishmania amazonensis infection in mice. Thus, this study aimed to evaluate the role of vitamin D during intracellular infection with L. amazonensis by examining its effect on macrophage oxidative mechanisms and parasite survival in vitro. Vitamins D2 and D3 significantly inhibited promastigote and amastigote growth in vitro. Vitamin D3 was not able to induce NO and reactive oxygen species (ROS) production in uninfected macrophages or macrophages infected with L. amazonensis. In addition, vitamin D3 in combination with interferon (IFN)-γ did not enhance amastigote killing and in fact, significantly reduced NO and ROS production when compared with the effect of IFN-γ alone. In this study, we demonstrated that vitamin D directly reduces parasite growth in infected macrophages (approximately 50-60% at 50 µm) but this effect is independent of the activation of macrophage oxidative mechanisms. These findings will contribute to a better understanding of the role of vitamin D in cutaneous leishmaniasis.

Nanoparticles Loaded with a New Thiourea Derivative: Development and In vitro Evaluation Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 µM for promastigotes and an IC50 of 70 µM for amastigotes. OBJECTIVE: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems. METHODS: Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM). RESULTS: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite Selectivity Index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). CONCLUSION: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

Single-dose treatment for cutaneous leishmaniasis with an easily synthesized chalcone entrapped in polymeric microparticles.

Cutaneous leishmaniasis (CL) is a major health problem in many countries and its current treatment involves multiple parenteral injections with toxic drugs and requires intensive health services. Previously, the efficacy of a single subcutaneous injection with a slow-release formulation consisting of poly(lactide-co-glycolide) (PLGA) microparticles loaded with an antileishmanial 3-nitro-2-hydroxy-4,6-dimethoxychalcone (CH8) was demonstrated in mice model. In the search for more easily synthesized active chalcone derivatives, and improved microparticle loading, CH8 analogues were synthesized and tested for antileishmanial activity in vitro and in vivo. The 3-nitro-2',4',6'-trimethoxychalcone (NAT22) analogue was chosen for its higher selectivity against intracellular amastigotes (selectivity index = 1489, as compared with 317 for CH8) and more efficient synthesis (89% yield, as compared with 18% for CH8). NAT22 was loaded into PLGA / polyvinylpyrrolidone (PVP) polymeric blend microspheres (NAT22-PLGAk) with average diameter of 1.9 µm. Although NAT22-PLGAk showed similar activity to free NAT22 in killing intracellular parasites in vitro (IC50 ~ 0.2 µm), in vivo studies in Leishmania amazonensis - infected mice demonstrated the significant superior efficacy of NAT22-PLGAk to reduce the parasite load. A single intralesional injection with NAT22-PLGAk was more effective than eight injections with free NAT22. Together, these results show that NAT22-PLGAk is a promising alternative for single-dose localized treatment of CL.

Leishmanicidal activity of Piper marginatum Jacq. from Santarém-PA against Leishmania amazonensis.

Leishmaniasis is an infectious disease that has high endemicity and is among the six parasitic diseases of higher occurrence in the world. The current treatments are limited due to their toxicity, treatment resistance and high cost which have increased the search for new substances of natural origin for its therapy. Based on this, an in vitro biological and chemical investigation was carried out to evaluate the potential of Piper marginatum against Leishmania amazonesis. P. marginatum leaves were collected to obtain the essential oil (EO) and the ethanolic extract (CE). The chemical profile of the CE and fractions was obtained by 1H NMR. The analysis of the EO chemical composition was performed by GC-MS. EO, CE and fractions were submitted to antileishmanial and cytotoxicity assays against macrophages. The chromatographic profiles of EO, CE and fractions showed the presence of phenolic compounds and terpenoids, having 3,4-Methylenedioxypropiophenone as a major compound. All P. marginatum samples showed low toxicity to macrophages. The CE and the methanolic, hexane and ethyl acetate fractions had low cytotoxicity when compared to Pentamidine. All tested samples inhibited growth of L. amazonensis promastigotes. The antileishmanial activity of EO, CE and fractions were evaluated in macrophages infected with L. (L.) amazonensis and treated with the concentrations 1, 10 and 100 µg/mL for 48 h. All samples were active, but EO and CE showed superior activity against amastigote forms when compared to the promastigote forms of L. amazonensis. This work describes for the first time the antileishmanial activity of the species P. marginatum and its cytotoxicity against macrophages, suggesting that it can be an alternative source of natural products in the phytotherapeutic treatment of leishmaniasis.

Encapsulation in lipid-core nanocapsules improves topical treatment with the potent antileishmanial compound CH8.

Cutaneous leishmaniasis (CL) is a neglected parasitic disease conventionally treated by multiple injections with systemically toxic drugs. Aiming at a more acceptable therapy, we developed lipid-core nanocapsules (LNCs) entrapping the potent antileishmanial chalcone (CH8) for topical application. Rhodamine-labeled LNC (Rho-LNC-CH8) was produced for imaging studies. LNC-CH8 and Rho-LNC-CH8 had narrow size distributions (polydispersity index <0.10), with similar mean sizes (~180 nm) by dynamic light scattering. In vitro, Rho-LNC-CH8 was rapidly internalized by extracellular Leishmania amazonensis parasites macrophages in less than 15 min. LNC-CH8 activated macrophage oxidative mechanisms more efficiently than CH8, and was more selectively toxic against the intracellular parasites. In vivo, topically applied Rho-LNC-CH8 efficiently permeated mouse skin. In L. amazonensis-infected mice, LNC-CH8 reduced the parasite load by 86% after three weeks of daily topical treatment, while free CH8 was ineffective. In conclusion, LNC-CH8 has strong potential as a novel topical formulation for CL treatment.

Novel and safe single-dose treatment of cutaneous leishmaniasis with implantable amphotericin B-loaded microparticles.

The development of an effective amphotericin B (AmB) topical formulation to replace the systemically toxic injections currently used in cutaneous leishmaniasis (CL) treatment is challenging due to poor absorption through the skin. Aiming at an effective local chemotherapy, we designed PLGA (poly(lactide-co-glycolide acid) microparticles loaded with deoxycholate amphotericin B (d-AmB) for both macrophage intracellular targeting and sustained extracellular release. For that, d-AmB/PLGA microparticles with sizes ranging from 0.5 µm to 20 µm were synthesized and tested both in vitro and in vivo. In vitro, d-AmB/PLGA was more selectively active against intracellular amastigotes of Leishmania amazonensis than free d-AmB (selectivity index = 50 and 25, respectively). In vivo, the efficacy of a single intralesional (i.l) injection with d-AmB/PLGA was determined in early and established BALB/c mouse ear lesions. In early lesions, a single injection given on day 10 of infection was more effective in controlling parasite growth than eight i.l. injections with free d-AmB, as measured on day 120. Such d-AmB/PLGA injection was also effective in established lesions (day 30), leading to 97% parasite burden reduction, as compared with d-AmB or liposomal AmB (Ambisome®) i.l. injection containing the same AmB dose. Pharmacokinetic studies showed that following d-AmB/PLGA injection, AmB leaked slower from non-infected than infected ears, yet remaining in the ear tissue for as long as 30 days. Of interest, AmB was not detectable in the circulating plasma for at least two weeks of d-AmB/PLGA injection, contrasting with the rapid and durable (2 days) detection after free d-AmB injection. Despite the transient ear swelling and local cell infiltration, no alterations in AST, ALT and creatinine serum levels was induced by d-AmB/PLGA. For its approved components, local efficacy, and single-dose applicability, this novel and safe AmB microparticle depot formulation has strong potential as a new therapy for human CL.