Effect of Itraconazole-Ezetimibe-Miltefosine Ternary Therapy in Murine Visceral Leishmaniasis.

Drug combination therapy is an interesting approach to increase the success of drug repurposing for neglected diseases. Thus, the objective of this work was to evaluate binary and ternary therapies composed of itraconazole, ezetimibe and miltefosine for the treatment of visceral leishmaniasis. Intracellular Leishmania infantum amastigotes were incubated with the drugs alone or in combination for 72 h. For in vivo experiments, we tested a long-course (21 days, once per day) and a short-course treatment (5 days, twice per day) for the binary combination with itraconazole and ezetimibe. For the ternary therapy including miltefosine, we adopted the short-course treatment and varied the vehicle. None of the combinations were toxic to macrophages. Binary combination of itraconazole plus ezetimibe and ternary combination of itraconazole, ezetimibe and miltefosine had synergistic effects in intracellular amastigotes, in some of the proportions evaluated. Although the in vivo long-course therapy had been more effective than the short-course protocol, it showed hepatic toxicity signs. Ezetimibe has proven to be able to reduce the parasite burden alone or in combination. Both suspensions of the ternary combination were active, but when the drugs were suspended in the commercial ORA-Plus formulation instead of purified water, the parasite burden was reduced by 98% in the liver and spleen. Altogether, the results demonstrate for the first time the activity of ezetimibe in a viscerotropic species of Leishmania and indicate that ternary treatment composed of miltefosine, itraconazole, and ezetimibe at low doses is a promising therapeutic alternative for the treatment of visceral leishmaniasis.

Lopinavir, an HIV-1 peptidase inhibitor, induces alteration on the lipid metabolism of Leishmania amazonensis promastigotes.

The anti-leishmania effects of HIV peptidase inhibitors (PIs) have been widely reported; however, the biochemical target and mode of action are still a matter of controversy in Leishmania parasites. Considering the possibility that HIV-PIs induce lipid accumulation in Leishmania amazonensis, we analysed the effects of lopinavir on the lipid metabolism of L. amazonensis promastigotes. To this end, parasites were treated with lopinavir at different concentrations and analysed by fluorescence microscopy and spectrofluorimetry, using a fluorescent lipophilic marker. Then, the cellular ultrastructure of treated and control parasites was analysed by transmission electron microscopy (TEM), and the lipid composition was investigated by thin-layer chromatography (TLC). Finally, the sterol content was assayed by gas chromatography-mass spectrometry (GC/MS). TEM analysis revealed an increased number of lipid inclusions in lopinavir-treated cells, which was accompanied by an increase in the lipophilic content, in a dose-dependent manner. TLC and GC-MS analysis revealed a marked increase of cholesterol-esters and cholesterol. In conclusion, lopinavir-induced lipid accumulation and affected lipid composition in L. amazonensis in a concentration-response manner. These data contribute to a better understanding of the possible mechanisms of action of this HIV-PI in L. amazonensis promastigotes. The concerted action of lopinavir on this and other cellular processes, such as the direct inhibition of an aspartyl peptidase, may be responsible for the arrested development of the parasite.

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains.

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-ß-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

Oral and Intragastric: New Routes of Infection by Leishmania braziliensis and Leishmania infantum?

Although Leishmania transmission in nature is associated with the bite of an infected sandfly vector, other possible transmission routes are speculated to occur, such as the oral route. We evaluated the possibility of infection by this route in golden hamsters (Mesocricetus auratus) using Leishmania braziliensis (Lb) and Leishmania infantum (Li). Hamsters were exposed to experimental oral or intragastrical infection with axenic promastigotes, besides oral ingestion of a suspension of cultivated macrophages infected with amastigotes, lesion-fed Lutzomyia longipalpis, skin lesion or infective spleen fragment. The parasite's isolation, besides a positive PCR and IFAT, confirmed the intragastric infection by promastigote parasites. The oral ingestion of macrophages infected with L. braziliensis amastigotes was also infective. These results confirmed that Leishmania parasites could infect mammals by the intragastric route through the ingestion of promastigote forms (what can happen after a sandfly ingestion) and by the oral ingestion of infected macrophages (what can happen in nature in a predator-prey interaction). The better understanding of these alternative routes is essential to understand their transmission dynamics in nature. As far as we know, this is the first time that oral and intragastric Leishmania transmission has been experimentally demonstrated, constituting new infection routes, at least for L. infantum and L. braziliensis.

Naphthoquinones and Derivatives for Chemotherapy: Perspectives and Limitations of their Anti-trypanosomatids Activities.

Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited, and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo is essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

Carvacrol loaded nanostructured lipid carriers as a promising parenteral formulation for leishmaniasis treatment.

Leishmaniasis are a group of neglected infectious diseases caused by protozoa of the genus Leishmania with distinct presentations. The available leishmaniasis treatment options are either expensive and/or; cause adverse effects and some are ineffective for resistant Leishmania strains. Therefore, molecules derived from natural products as the monoterpene carvacrol, have attracted interest as promising anti-leishmania agents. However, the therapeutic use of carvacrol is limited due to its low aqueous solubility, rapid oxidation and volatilization. Thus, the development of nanostructured lipid carriers (NLCs) was proposed in the present study as a promising nanotechnology strategy to overcome these limitations and enable the use of carvacrol in leishmaniasis therapy. Carvacrol NLCs were obtained using a warm microemulsion method, and evaluated regarding the influence of lipid matrix and components concentration on the NLCs formation. NLCs were characterized by DSC and XRD as well. In addition, to the in vitro carvacrol release from NLCs, the in vitro cytotoxicity and leishmanicidal activity assays, and the in vivo pharmacokinetics evaluation of free and encapsulated carvacrol were performed. NLCs containing carvacrol were obtained successfully using a warm microemulsion dilution method. The NLCs formulation with the lowest particle size (98.42 ± 0.80 nm), narrowest size distribution (suitable for intravenous administration), and the highest encapsulation efficiency was produced by using beeswax as solid lipid (HLB=9) and 5% of lipids and surfactant. The in vitro release of carvacrol from NLCs was fitted to the Korsmeyer and Peppas, and Weibull models, demonstrating that the release mechanism is probably the Fickian diffusion type. Moreover, carvacrol encapsulation in NLCs provided a lower cytotoxicity in comparison to free carvacrol (p<0.05), increasing its in vitro leishmanicidal efficacy in the amastigote form. Finally, the in vivo pharmacokinetics of carvacrol after IV bolus administration suggests that this phenolic monoterpene undergoes enterohepatic circulation and therefore presented a long half-life (t1/2) and low clearance (Cl). In addition, C0, mean residence time (MRT) and Vdss of encapsulated carvacrol were higher than free carvacrol (p < 0.05), favoring a higher distribution of carvacrol in the target tissues. Thus, it is possible to conclude that the developed NLCs are a promising delivery system for leishmaniasis treatment.

Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex.

The polyene amphotericin B (AMB) exerts a powerful and broad antifungal activity. AMB acts by (i) binding to ergosterol, leading to pore formation at the fungal plasma membrane with subsequent ion leakage, and (ii) inducing the intracellular accumulation of reactive oxygen species (ROS). Herein, we have deciphered the AMB resistance mechanisms in clinical isolates of Candida haemulonii complex (C. haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera) in comparison to other clinically relevant non-albicans Candida species. Membrane gas chromatography-mass spectrometry analysis revealed that the vast majority of sterols were composed of ergosterol pathway intermediates, evidencing the absence of AMB target. Supporting this data, C. haemulonii species complex demonstrated poor membrane permeability after AMB treatment. Regarding the oxidative burst, AMB induced the formation of ROS in all species tested; however, this phenomenon was slightly seen in C. haemulonii complex isolates. Our results indicated that these isolates displayed altered respiratory status, as revealed by their poor growth in nonfermented carbon sources, low consumption of oxygen, and derisive mitochondrial membrane potential. The use of specific inhibitors of mitochondrial respiratory chain (complex I-IV) revealed no effects on the yeast growth, highlighting the metabolic shift to fermentative pathway in C. haemulonii strains. Also, C. haemulonii complex proved to be highly resistant to oxidative burst agents, which can be correlated with a high activity of antioxidant enzymes. Our data demonstrated primary evidence suggesting that ergosterol content, mitochondrial function, and fungal redox homeostasis are involved in AMB fungicidal effects and might explain the resistance presented in this multidrug-resistant, emergent, and opportunistic fungal complex.

Miltefosine-Lopinavir Combination Therapy Against Leishmania infantum Infection: In vitro and in vivo Approaches.

Concurrently, leishmaniasis and AIDS are global public health issues and the overlap between these diseases adds additional treats to the management of co-infected patients. Lopinavir (LPV) has a well characterized anti-HIV and leishmanicidal action, and to analyze its combined action with miltefosine (MFS) could help to envisage strategies to the management of co-infected patients. Here, we evaluate the interaction between LPV and MFS against Leishmania infantum infection by in vitro and in vivo approaches. The effect of the compounds alone or in association was assessed for 72 h in mouse peritoneal macrophages infected with L. infantum by the determination of the IC50s and FICIs. Subsequently, mice were orally treated twice daily during 5 days with the compounds alone or in association and evaluated after 30 days. The in vitro assays revealed an IC50 of 0.24 µM and 9.89 µM of MFS and LPV, respectively, and an additive effect of the compounds (FICI 1.28). The in vivo assays revealed that LPV alone reduced the parasite load in the spleen and liver by 52 and 40%, respectively. The combined treatment of infected BALB/c mice revealed that the compounds alone required at least two times higher doses than when administered in association to virtually eliminate the parasite. Mice plasma biochemical parameters assessed revealed that the combined therapy did not present any relevant hepatotoxicity. In conclusion, the association of MFS with LPV allowed a reduction in each compound concentration to achieve the same outcome in the treatment of visceral leishmaniasis. Although a pronounced synergistic effect was not evidenced, it does not discard that such combination could be useful in humans co-infected with HIV and Leishmania parasites.

Design, Synthesis and Antileishmanial Activity of Naphthotriazolyl-4- Oxoquinolines.

BACKGROUND: Leishmaniasis is a neglected public health problem caused by several protozoanspecies of the genus Leishmania. The therapeutic arsenal for treating leishmaniasis is quite limited, raising concerns about the occurrence of resistant strains. Furthermore, most of these drugs were developed more than 70 years ago and suffer from poor efficacy and safety and are not well adapted to the needs of patients. Therefore, research on novel natural or synthetic compounds with antiparasitic activity is urgently needed. In this paper, we evaluated the effect and the mechanism of action of naphthotriazolyl-4-oxoquinolines on promastigotes and intracellular amastigotes of Leishmania amazonensis. MATERIALS AND METHODS: The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields via the [3+2] cycloaddition reaction between 1,4-naphtoquinone and azido-4- oxoquinoline derivatives. HMPA at 100°C was established as the best solvent and temperature condition for this reaction. The structures of the compounds were confirmed by spectral analyses (infrared spectroscopy, one- and two-dimensional ¹H and ¹³C NMR spectroscopy, and high-resolution mass spectrometry). The compounds exhibited promising activities with IC50 values ranging from 0.7 to 2.0 µM against intracellular amastigotes of Leishmania amazonensis. The most selective compound was the Npentyl- substituted derivative, which showed a Selectivity Index (SI) of 8.6, making it less toxic than pentamidine (SI 4.5). RESULTS: Our results demonstrated that all compounds, except the N-propyl-substituted derivative, induce ROS production by parasites early in the culture. As a proof of concept, we demonstrated that the most selective compound was able to interfere with sterol biosynthesis in L. amazonensis. CONCLUSION: The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields. These conjugates have potent in vitro antileishmanial activity involving at least two different mechanisms of action, making them promising lead compounds for the development of new therapeutic alternatives for leishmaniasis.