[Leptospirosis: A growing health threat to fish farming and recreational activities]. / La leptospirose : une menace sanitaire croissante pour les activités piscicoles et récréatives.

Leptospirosis is a cosmopolitan infectious disease caused by the bacteria Leptospirainterrogans, which is worldwide increasing as the result of climate changes favoring the reproduction of asymptomatic reservoir rodents and also flooding that brings mammals and humans into contact with contaminated water. This disease affects many mammals among wild, domestic or farmed animals. In humans, it is manifested through frustrated to severe forms that can lead to liver and kidney failure and death in about one in ten severe cases. Diagnostic methods and treatments are satisfactory. Prophylaxis is based on individual protective measures including vaccination. The single human vaccine that is available is expensive and it protects 97% against about a third of human pathogenic serovars. Moreover, this vaccine is not covered by health insurance. Better epidemiological knowledge, particularly at European level, could stimulate research in this area in order to obtain a vaccine that would protect more comprehensively against serovars pathogenic to humans.

Synthesis and biological evaluation against Leishmania donovani of novel hybrid molecules containing indazole-based 2-pyrone scaffolds.

A series of novel indazole-pyrone hybrids were synthesized by a one pot reaction between N-alkyl-6(5)-nitroindazoles and 2-pyrone (4-hydroxy-6-methyl-2H-pyran-2-one) using indium or stannous chloride as the reducing system in the presence of acetic acid in tetrahydrofuran. The hybrid molecules were obtained in good to excellent yields (72-92%) and characterized by NMR and single crystal X-ray diffraction. Nineteen compounds were tested in vitro against both Leishmania donovani (MHOM/ET/67/HU3, also called LV9) axenic and intramacrophage amastigotes. Among all, five compounds showed anti-leishmanial activity against intracellular L. donovani with an IC50 in the range of 2.25 to 62.56 µM. 3-(1-(3-Chloro-2-ethyl-2H-indazol-6-ylamino)ethylidene)-6-methyl-3H-pyran-2,4-dione 6f was found to be the most active compound for axenic amastigotes and intramacrophage amastigotes of L. donovani with IC50 values of 2.48 ± 1.02 µM and 2.25 ± 1.89 µM, respectively. However, the cytotoxicity of the most promising compound justifies further pharmacomodulations.

New heterocyclic compounds: Synthesis and antitrypanosomal properties.

Three new series of quinoline, quinolone, and benzimidazole derivatives were synthesized and evaluated in vitro against Trypanosoma brucei gambiense. In the quinoline series, the metallo antimalarial drug candidate (ferroquine, FQ) and its ruthenium analogue (ruthenoquine, RQ, compound 13) showed the highest in vitro activities with IC50 values around 0.1 µM. Unfortunately, both compounds failed to cure Trypanosoma brucei brucei infected mice in vivo. The other heterocyclic compounds were active in vitro with IC50 values varying from 0.8 to 34 µM. One of the most interesting results was a fluoroquinolone derivative (compound 2) that was able to offer a survival time of 8 days after a treatment at the single dose of 100 µmol/kg by intraperitoneal route. Although no clear-cut structure-activity relationships emerged, further pharmacomodulations are worth to be developed in this series.

Sitamaquine-resistance in Leishmania donovani affects drug accumulation and lipid metabolism.

This study focuses on the mechanism of sitamaquine-resistance in Leishmania donovani. Sitamaquine accumulated 10 and 1.4 fold more in cytosol than in membranes of wild-type (WT) and of sitamaquine-resistant (Sita-R160) L. donovani promastigotes, respectively. The sitamaquine accumulation was a concentration-dependent process in WT whereas a saturation occurred in Sita-R160 suggesting a reduced uptake or an increase of the sitamaquine efflux. Membrane negative phospholipids being the main target for sitamaquine uptake, a lipidomic analysis showed that sitamaquine-resistance did not rely on a decrease of membrane negative phospholipid rate in Sita-R160, discarding the hypothesis of reduced uptake. However, sterol and phospholipid metabolisms were strongly affected in Sita-R160 suggesting that sitamaquine-resistance could be related to an alteration of phosphatidylethanolamine-N-methyl-transferase and choline kinase activities and to a decrease in cholesterol uptake and of ergosterol biosynthesis. Preliminary data of proteomics analysis exhibited different protein profiles between WT and Sita-160R remaining to be characterized.

Development of antileishmanial lipid nanocomplexes.

Visceral leishmaniasis is a life-threatening disease that affects nearly a million people every year. The emergence of Leishmania strains resistant to existing drugs complicates its treatment. The purpose of this study was to develop a new lipid formulation based on nanocochleates combining two active drugs: Amphotericin B (AmB) and Miltefosine (HePC). Nanocochleates composed of dioleoylphosphatidylserine (DOPS) and Cholesterol (Cho) and Ca(2+), in which HePC and AmB were incorporated, were prepared. Properties such as particle size, zeta potential, drug payload, in-vitro drug release and storage stability were investigated. Moreover, in-vitro stability in gastrointestinal fluid was performed in view of an oral administration. AmB-HePC-loaded nanocochleates with a mean particle size of 250 ± 2 nm were obtained. The particles displayed a narrow size distribution and a drug payload of 29.9 ± 0.5 mg/g for AmB, and 14.0 ± 0.9 mg/g for HePC. Drug release occurred preferentially in intestinal medium containing bile salts. Therefore, AmB-HePC-loaded nanocochleates could be a promising oral delivery system for the treatment of visceral leishmaniasis.

Strategies for the design of orally bioavailable antileishmanial treatments.

Leishmaniasis is one of the six major tropical diseases targeted by the World Health Organization. The most serious, life-threatening form is visceral leishmaniasis (VL). No vaccine is yet available for human use and chemotherapy is the main mean of dealing with this disease. This review focuses on the development of drug delivery systems (DDS) for treatment of leishmaniasis. After an overview of the significance of leishmaniasis in 2013, current chemotherapy and its limitations are considered, leading to possible strategies to improve the treatment of VL: new drugs, combinations of existing drugs and DDS, particularly for oral administration. Nanostructured biomaterials such as lipid-based or polymeric nanoparticles have unique physicochemical properties, ultra-small and controllable size, large surface area to mass ratio and the possibility of surface modification which can be used to advantage for the oral administration of antileishmanial drugs. They can improve the rate of dissolution of poorly water-soluble drugs, increase intestinal residence time by bioadhesion and, especially when lipid additives are used, influence the route and efficiency of absorption. These recent advances in this very active field should lead to better management of this serious disease.

Interactions of antileishmanial drugs with monolayers of lipids used in the development of amphotericin B-miltefosine-loaded nanocochleates.

The emergence of strains of Leishmania resistant to existing drugs complicates the treatment of life-threatening visceral leishmaniasis. The development of new lipid formulation (nanocochleates), containing two active drugs: amphotericin B (AmB) and miltefosine (hexadecylphosphocholine, HePC), could increase effectiveness, decrease toxicity and reduce the risk of appearance of resistance. Nanocochleates are cigar-shaped structures of rolled negatively charged lipid bilayers bridged by calcium, prepared from dioleoylphosphatidylserine (DOPS) and cholesterol (Cho) and able to accommodate drugs. To determine the interaction, the orientation and the stability of the amphiphilic drugs in the lipid mixture and the optimal drugs/lipids ratio, the Langmuir film balance and BAM (Brewster angle microscopy) were used. The drugs were mixed with the lipids (DOPS or 9DOPS/1Cho) and spread at the air-water interface. A stability study showed that DOPS maintained HePC at the interface at low molar fraction of HePC; this effect became more marked in the presence of Cho. The fact that HePC can be stably associated with the monolayer at low molar fraction (below 10%) suggests that in the nanocochleates HePC is inserted between the lipid molecules rather than between the bilayers. Phase diagrams and BAM images showed that, even at low pressure, DOPS maintains AmB at low molar fraction (below 10%) in the "erect" rather than the horizontal form at the interface and that the presence of Cho reinforces this effect. These results allowed us to predict the organization and the orientation of these drugs in the nanocochleates and to determine the optimal drugs/lipids ratio.

Antiprotozoal activity of ferroquine.

Ferroquine (FQ, SSR97193) is a synthetic compound currently in development for the treatment of malaria. The use of a single compound to treat several parasitoses would be very convenient for multi-infected patients and also for financial considerations. In this work, the activity of FQ was investigated on three other Protista parasites: Kinetoplastidae (Leishmania and Trypanosoma) and the cosmopolite parasite Trichomonas vaginalis. FQ exhibited a significant in vitro activity on Trypanosoma brucei brucei and Trypanosoma brucei gambiense, the agents of African trypanosomiasis in a range from 0.2 to 3.1 µM. In vivo, intraperitoneally administered FQ demonstrated a weak but significant trypanocidal activity at 100 µmol/kg, which is however higher than the maximum tolerated dose. The drop of the parasitemia of the treated mice was significantly related to the amount of injected FQ. Furthermore, this organometallic compound was responsible for a delay in the appearance of bloodstream parasites at 50 µmol/kg. However, it was not able to cure infected mice. Although no synergy was identified in vitro between FQ and pentamidine, these results justify further investigations by evaluating analogues in this chemical series.

In silico analysis of a therapeutic target in Leishmania infantum: the guanosine-diphospho-D-mannose pyrophosphorylase.

Leishmaniases are tropical and sub-tropical diseases for which classical drugs (i.e. antimonials) exhibit toxicity and drug resistance. Such a situation requires to find new chemical series with antileishmanial activity. This work consists in analyzing the structure of a validated target in Leishmania: the GDP-mannose pyrophosphorylase (GDP-MP), an enzyme involved in glycosylation and essential for amastigote survival. By comparing both human and L. infantum GDP-MP 3D homology models, we identified (i) a common motif of amino acids that binds to the mannose moiety of the substrate and, interestingly, (ii) a motif that is specific to the catalytic site of the parasite enzyme. This motif could then be used to design compounds that specifically inhibit the leishmanial GDP-MP, without any effect on the human homolog.

In vitro antileishmanial and antitrypanosomal activities of five medicinal plants from Burkina Faso.

After ethnobotanical surveys in central and western regions of Burkina Faso, five plants namely Lantana ukambensis (Verbenaceae), Xeoderris sthulmannii (Fabaceae), Parinari curatellifollia (Chrysobalanaceae), Ozoroa insignis (Anacardiaceae), and Ficus platyphylla (Moraceae) were selected for their traditional use in the treatment of parasitic diseases and cancer. Our previous studies have focused on the phytochemical, genotoxicity, antioxidant, and antiproliferative activities of these plants. In this study, the methanol extract of each plant was tested to reveal probable antileishmanial and antitrypanosomal activities. Colorimetric and spectrophotometric methods were used for the detection of antileishmanial and antitrypanosomal activities. Leishmania donovani (LV9 WT) and Trypanosoma brucei brucei GVR 35 were used to test the antileishmanial and antitrypanosomal activities, respectively. All extracts of tested plants showed a significant antitrypanosomal activity with minimum lethal concentrations between 1.5 and 25 µg/ml, the L. ukambensis extract being the most active. In the antileishmanial test, only the extract from L. ukambensis showed significant activity with an inhibitory concentration (IC(50)) of 6.9 µg/ml. The results of this study contribute to the promotion of traditional medicine products and are preliminary for the isolation of new natural molecules for the treatment of leishmaniasis and trypanosomiasis.

Identification of phospholipid species affected by miltefosine action in Leishmania donovani cultures using LC-ELSD, LC-ESI/MS, and multivariate data analysis.

Leishmaniasis is a widespread parasitic disease principally treated by intravenous drugs. Hexadecylphosphocholine (miltefosine) has recently proved its efficacy by oral route. Although its mechanism of action has been investigated, and principally relies on perturbations of the metabolism of lipids and especially phospholipids, further studies need to be conducted to detect precisely which metabolic pathways are impacted. For this purpose, the present work proposes a complete lipidomic study focused on membrane phospholipids of clones of Leishmania donovani non-treated (NT), treated (T) and resistant (R) to miltefosine. Firstly, a separation of phospholipids in normal phase high-performance liquid chromatography (NP-HPLC) was coupled to a mass spectrometer (MS) equipped with an electrospray (ESI) ion source, and response was compared to evaporative light scattering detection (ELSD). Secondly, a quantification of phospholipid classes was performed using NP-HPLC/ESI/MS on NT, T and R clones of L. donovani. Thirdly, full-scan acquisitions of analyzed samples were compared using orthogonal signal correction-partial least square-discriminant analysis (OSC-PLS-DA) to highlight phospholipid molecular species of interest between the three types of clones. Structural determination of the most relevant species has finally been performed using tandem mass spectrometry. A first hypothesis on the effect of miltefosine on lipid metabolic pathways is then proposed.

Antileishmanial activity of a formulation of 2-n-propylquinoline by oral route in mice model.

2-n-propylquinoline is presently a drug-candidate for the treatment of visceral leishmaniosis in pre-clinical development. As this compound is in an oily state, it needs to be formulated and the objectives of this study are: to prepare a formulation; to demonstrate that the new salted formulation did not alter the activity of the active ingredient; and finally, that this activity was quite good compared to the reference oral drug, miltefosine. Therefore, a 2-n-propylquinoline formulation, as camphorsulfonic salt, was prepared and characterised. On the Leishmania donovani / Balb/c mice model, a treatment by oral route at 60 mmoles/kg/day for ten consecutive days with this formulation was compared to 2-n-propylquinoline alone and to miltefosine, the oral reference drug. The salt formulation did not alter the activity of the 2-n-propylquinoline. The formulation reduced the parasite burden of 76% compared to 89% for miltefosine (not significant). The characteristics of this formulation results in a suitable drugability of 2-n-propylquinoline for further studies.

Sitamaquine as a putative antileishmanial drug candidate: from the mechanism of action to the risk of drug resistance.

Sitamaquine is a 8-aminoquinoline in development for the treatment of visceral leishmaniasis by oral route, no activity being observed on the experimental cutaneous leishmaniasis experimental models. Recent data explain how sitamaquine accumulate in Leishmania parasites, however its molecular targets remain to be identified. An advantage of sitamaquine is its short elimination half-life, preventing a rapid resistance emergence. The antileishmanial action of its metabolites is not known. The selection of a sitamaquine-resistant clone of L. donovani in laboratory and the phase II clinical trials pointing out some adverse effects such as methemoglobinemia and nephrotoxicity are considered for a further development decision.

Cationic nanoemulsion as a delivery system for oligonucleotides targeting malarial topoisomerase II.

A promising strategy based on the antisense oligonucleotides against the Plasmodium falciparum topoisomerase II has been considered using cationic nanoemulsion as oligonucleotide delivery system. Phosphodiester and chemically modified phosphorothioate oligonucleotides bearing negative charges were adsorbed on positively charged emulsion composed of medium chain triglycerides, egg lecithin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and water, at different +/- charge ratios (positive charges from cationic lipid/negative charges from oligonucleotide): +0.5/-, +2/-, +4/- and +6/-. The physicochemical properties of the complexes were determined, as well as their stability in culture medium. Their interaction with erythrocytes through hemolysis, binding experiments and confocal microscopy were also evaluated. Finally, the in vitro evaluation of parasite growth and reinfection capacity was performed. The overall results showed that antisense oligonucleotides against P. falciparum topoisomerase II gene can be efficiently adsorbed onto a cationic nanoemulsion forming complexes. Whereas unloaded nanoemulsion displayed an hemolytic effect due to the presence of the cationic lipid, this was not the case of loaded nanoemulsion at low +/- ratios. Oligonucleotide-loaded nanoemulsions were found to be located inside the infected erythrocytes, inhibiting efficiently parasite growth (until 80%) and causing a delay in P. falciparum life cycle.

Mechanism of interaction of sitamaquine with Leishmania donovani.

OBJECTIVES: This study focuses on the mechanism of interaction of sitamaquine with Leishmania donovani membranes, and its accumulation within the parasites. METHODS: A biomimetic model of the outer layer of a Leishmania plasma membrane was used to examine the interactions of sitamaquine with lipids. The plasma membranes of L. donovani promastigotes were depleted of sterol using cholesterol oxidase, in order to assess the importance of sterols in drug-membrane interactions. Sterols were quantified and sitamaquine susceptibility was assessed using the MTT test. Kinetics of sitamaquine accumulation and efflux were measured under different conditions. RESULTS: Sitamaquine interacts first with phospholipid anionic polar head groups and then with phospholipid acyl chains to insert within biological membranes and accumulates rapidly in the Leishmania cytosol according to a sterol-independent process. The rapid sitamaquine efflux observed was related to an energy-dependent mechanism since the intracellular amount of sitamaquine was enhanced three times in the absence of glucose and the efflux was inhibited in energy-depleted conditions. (1)H NMR analysis of motile lipid showed that sitamaquine did not affect lipid trafficking in Leishmania. CONCLUSIONS: We propose that sitamaquine rapidly accumulates in Leishmania by diffusion along an electrical gradient and is concentrated in the cytosol by an energy- and sterol-independent process. The affinity of sitamaquine for membranes was transitory and an energy-dependent efflux was demonstrated, suggesting the presence of an as yet uncharacterized transporter.

Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes.

OBJECTIVES: This study focuses on the importance of sterols in the action of miltefosine (hexadecylphosphocholine, HePC) against Leishmania donovani. METHODS: Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-beta-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC-lipid interactions. RESULTS: CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased. CONCLUSIONS: Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, (1)H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.

Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids.

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.

Antifungal canthin-6-one series accumulate in lipid droplets and affect fatty acid metabolism in Saccharomyces cerevisiae.

The mechanism of action of antifungal canthin-6-one series was investigated in Saccharomyces cerevisiae. After a rapid uptake, a preferential accumulation of the drug within lipid droplets was observed. The antifungal action of canthin-6-one was found as reversible. Canthin-6-one did not exhibit affinity for sterols, and membrane ergosterol was not necessary for the antifungal activity since the MICs were similar on an ergosterol-deleted and the wild-type S. cerevisiae clones. Relative amount of unsaturated alkyl chain fatty acids was significantly enhanced suggesting a stimulation of desaturase enzyme systems. No synergistic effect was observed between canthin-6-one and amphotericin B, ketoconazole and caspofungine. Canthin-6-one should now be evaluated in vivo against fungal pathogens.

Amphotericin B-gum arabic conjugates: synthesis, toxicity, bioavailability, and activities against Leishmania and fungi.

PURPOSE: Gum arabic, a branched polysaccharide consisting of more than 90% arabinogalactan having a molecular weight around 250,000 Da is the oldest and best known of all natural gums. The objective of the present investigation was to examine whether amphotericin B (AmB), the polyene antibiotic when conjugated to periodate oxidized gum arabic still retained its anti-fungal and anti-leishmanial activity and to evaluate its toxicity and bioavailability. METHODS: AmB conjugated to the oxidized polysaccharide through Schiff's linkages in the unreduced (imine) and reduced (amine) forms were characterized for the drug content, hemolytic potential, molecular mass, in vitro release and were examined for anti-fungal activity against Candida albicans and Cryptococcus neoformans and for anti-leishmanial activity against promastigotes of Leishmania donovani in culture. Toxicity and bioavailability were evaluated by intravenous (i.v) injections of the conjugates in mice and rabbits respectively. RESULTS: The conjugates were found to be non-hemolytic and mice withstood a dosage of 20 mg (AmB)/kg body weight of both conjugates. Histological examination of the internal organs of mice showed no lesions in kidney, brain, heart or liver. Estimation of the residual drug in the internal organs 7 days post injection showed that the spleen still retained 8.4 +/- 0.53 microg/g of tissue. AmB was found to be released from both conjugates in vitro although the release from the imine conjugate was much faster than from the amine conjugate. The concentrations inhibiting parasite growth by 50% (IC(50)) values for the imine conjugate against promastigotes of L. donovani LV9 and DD8 strains were 0.37 +/- 0.04 and 1.44 +/- 0.18 microM respectively. The IC(50) values for the amine conjugates were much higher. The minimum inhibitory concentration (MIC) against C. albicans and C. neoformans was in the range of 0.5-0.9 microg/mL for both imino and amino conjugates. The bioavailability of the conjugate in rabbits showed that the imine conjugate maintained a plasma concentration in the range of 20 to 5 microg/mL while for the amine conjugate it was in the range of 17 to 3 microg/mL over 24 h. CONCLUSIONS: The drug conjugates were stable, non-hemolytic and non-toxic to the internal organs of the animal and showed good anti-fungal and anti-leishmanial activity in vitro. In spite of the large molecular weight of the polysaccharide, AmB from the conjugates showed bioavailability after i.v injection. Since the highest concentration of AmB was found in the spleen after a single injection, these conjugates may have potential in anti-leishmanial therapy.

In vitro and in vivo antileishmanial efficacy of a new nitrilquinoline against Leishmania donovani.

The in vitro activity of a new analogue of 2-alkenylquinoline (2-nitrilquinoline or NQ) against Leishmania donovani was compared to oral reference drug miltefosine (HePC). IC(50) of NQ was found at 38.6 microM against promastigotes and 2.4 microM against intramacrophage amastigotes. In vivo evaluation in the L. donovani Balb/c mice model indicated that oral treatments at 12.5 and 25 mg/kg for 10 consecutive days significantly reduced the parasite burden in the liver by 68.9 and 68.5%, respectively. This activity was similar to those of HePC at 7.5 mg/kg for 10 days which reduced the parasite burden in liver by 72.5%. The present study shows the positive contribution of a nitril substitute being added into the alkenyl chain branched at the 2-position of the quinoline ring to the antileishmanial activity. In addition, any apparent toxicological disorder was observed during the experiments.