Frédéric Frézard

Pentavalent antimonials: new perspectives for old drugs.

Pentavalent antimonials, including meglumine antimoniate and sodium stibogluconate, have been used for more than half a century in the therapy of the parasitic disease leishmaniasis. Even though antimonials are still the first-line drugs, they exhibit several limitations, including severe side effects, the need for daily parenteral administration and drug resistance. The molecular structure of antimonials, their metabolism and mechanism of action are still being investigated. Some recent studies suggest that pentavalent antimony acts as a prodrug that is converted to active and more toxic trivalent antimony. Other works support the direct involvement of pentavalent antimony. Recent data suggest that the biomolecules, thiols and ribonucleosides, may mediate the actions of these drugs. This review will summarize the progress to date on the chemistry and biochemistry of pentavalent antimony. It will also present the most recent works being done to improve antimonial chemotherapy. These works include the development of simple synthetic methods for pentavalent antimonials, liposome-based formulations for targeting the Leishmania parasites responsible for visceral leishmaniasis and cyclodextrin-based formulations to promote the oral delivery of antimony.

Glutathione-Induced Conversion of Pentavalent Antimony to Trivalent Antimony in Meglumine Antimoniate

ABSTRACT The standard treatment of human leishmaniases involves the use of pentavalent antimony [Sb(V)] compounds, including meglumine antimoniate. The mode of action of these compounds has not been fully elucidated. The possibility that Sb(III) is involved has been suggested; however, the biomolecule that may induce the conversion of Sb(V) to Sb(III) has not yet been identified. In the present study, we investigated both the ability of reduced glutathione (GSH) to promote the reduction of Sb(V) into Sb(III) in meglumine antimoniate and the effects of pH and temperature on this transformation. GSH did promote the reduction of Sb(V) into Sb(III) in a dose-dependent manner. When GSH and meglumine antimoniate were incubated together at a GSH/Sb molar ratio superior or equal to 5:1, all antimony was encountered in the reduced form, indicating a stoichiometry of 5:1 between GSH and Sb(V) in the reaction. The reaction between Sb(V) and GSH was favored at an acidic pH (pH 5) and an elevated temperature (37°C), conditions found within the phagolysosome, in which Leishmania resides. For instance, about 30% of the Sb(V) (concentration, 2mM) was converted to Sb(III) following incubation for 3 days with 10 mM GSH at pH 5 and 37°C. Our data support the hypothesis that Sb(V) would be converted by GSH, or a related thiol compound, to more toxic Sb(III) in the phagolysosome of macrophages.

Reduced Tissue Parasitic Load and Infectivity to Sand Flies in Dogs Naturally Infected by Leishmania (Leishmania) chagasi following Treatment with a Liposome Formulation of Meglumine Antimoniate

ABSTRACT The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

Oral Delivery of Meglumine Antimoniate-β-Cyclodextrin Complex for Treatment of Leishmaniasis

ABSTRACT The need for daily parenteral administration represents one of the most serious limitations in the clinical use of pentavalent antimonials against leishmaniasis. In this work, we investigated the ability of β-cyclodextrin to enhance the oral absorption of antimony and to promote the oral efficacy of meglumine antimoniate against experimental cutaneous leishmaniasis. The occurrence of interactions between β-cyclodextrin and meglumine antimoniate was demonstrated through the changes induced in the spin lattice relaxation times of protons in both compounds. When free and complexed meglumine antimoniate were given orally to Swiss mice, plasma antimony levels were found to be about three times higher for the meglumine antimoniate-β-cyclodextrin complex than for the free drug. Antileishmanial efficacy was evaluated in BALB/c mice experimentally infected with Leishmania amazonensis. Animals treated daily with the complex (32 mg of Sb/kg of body weight) by the oral route developed significantly smaller lesions than those treated with meglumine antimoniate (120 mg of Sb/kg) and control animals (treated with saline). The effectiveness of the complex given orally was equivalent to that of meglumine antimoniate given intraperitoneally at a twofold-higher antimony dose. The antileishmanial efficacy of the complex was confirmed by the significantly lower parasite load in the lesions of treated animals than in saline-treated controls. This work reports for the first time the effectiveness of an oral formulation for pentavalent antimonials.

New delivery strategies for the old pentavalent antimonial drugs

Importance of the field: Pentavalent antimonials are the first-line drugs for treatment of the major tropical disease leishmaniasis. However, their use is limited by the need for daily parenteral administration, their severe side effects and treatment failures. As leishmaniasis belongs to the group of neglected diseases, the improvement of old drugs through new delivery approaches has more support than the development of new chemical entities. Areas covered in this review: The review covers, from 1977 to the present, the progress achieved towards pharmaceutically acceptable liposome-based formulations of antimonials, identification of specific ligands for improved targeting of infected macrophages and new approaches for oral and topical delivery of antimonial drugs. What the reader will gain: Insights into the most promising delivery strategies to improve antimonial therapy and the chemical basis and future directions for achieving innovative orally and topically effective formulations. Take home message: The development of drug delivery strategies for the old pentavalent antimonials is a still growing and promising field, with expected innovations in the near future from improved knowledge of antimony chemistry.

Gene Expression Profiling and Molecular Characterization of Antimony Resistance in Leishmania amazonensis

Background Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis. Methods/Principal Findings We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated. Conclusions/Significance Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.

New insights into the mode of action of ultradeformable vesicles using calcein as hydrophilic fluorescent marker.

Whether ultradeformable vesicles pass intact through the stratum corneum and can promote the transdermal absorption of any substance remain open questions. This paper presents different experimental approaches, based on the use of calcein as hydrophilic fluorescent marker, to probe the physicochemical and pharmacokinetic characteristics of these vesicles. Ultradeformable membranes made from natural phosphatidylcholine and sodium cholate were found to be highly permeable to calcein, as a result of the permeabilizing effects of sodium cholate and ethanol. In vitro skin permeation and in vivo transdermal (percutaneous) absorption studies were performed using hairless mice. Both studies indicated that deformable vesicles reduce the transdermal flux of calcein, when compared to a solution containing or not sodium cholate and ethanol. The data support the model that the transdermal absorption of calcein from deformable vesicles is controlled by the release of the drug from the formulation deposited onto the skin surface. Importantly, fluorescence measurements of the receptor fluid of the Franz diffusion cell after addition of Co(2+) quencher revealed that permeated calcein exists essentially under the non-encapsulated form. In conclusion, our results argue against the model that deformable vesicles would carry hydrophilic drugs across the skin and act as a sustained release system in deep tissues.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Efficacy of Combined Therapy with Liposome-Encapsulated Meglumine Antimoniate and Allopurinol in Treatment of Canine Visceral Leishmaniasis

ABSTRACT An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes.

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.