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Francisco Gamarro - One of the best experts on this subject based on the ideXlab platform.
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the 8 aminoquinoline analogue Sitamaquine causes oxidative stress in leishmania donovani promastigotes by targeting succinate dehydrogenase
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Luis Alberto Vieira De Carvalho, Santiago Castanys, Juan Roman Luqueortega, Carmen Lopezmartin, Luis Rivas, Francisco GamarroAbstract:The 8-aminoquinoline analogue Sitamaquine (SQ) is an oral antileishmanial drug currently undergoing phase 2b clinical trials for the treatment of visceral leishmaniasis. In the present study, we investigated the mechanism of action of this drug in Leishmania donovani promastigotes. SQ causes a dose-dependent inhibition of complex II (succinate dehydrogenase) of the respiratory chain in digitonin-permeabilized promastigotes, together with a drop in intracellular ATP levels and a decrease of the mitochondrial electrochemical potential. This is associated with increases of reactive oxygen species and intracellular Ca2+ levels, a higher percentage of the population with sub-G1 DNA content, and exposure of phosphatidylserine. Taken together, these results support a lethal mechanism for SQ that involves inhibition of the respiratory chain complex II, which in turn triggers oxidative stress and finally leads to an apoptosis-like death of Leishmania parasites.
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Sitamaquine Overcomes ABC-Mediated Resistance to Miltefosine and Antimony in Leishmania
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: José M. Pérez-victoria, Carmen López-martín, Santiago Castanys, Boris I. Bavchvarov, Ivan R. Torrecillas, Marta Martínez-garcía, Mercedes Campillo, Francisco GamarroAbstract:Although oral miltefosine represented an important therapeutic advance in the treatment of leishmaniasis, the appearance of resistance remains a serious threat. LMDR1/LABCB4, a P-glycoprotein-like transporter included in the Leishmania ABC (ATP-binding cassette) family, was the first molecule shown to be involved in experimental miltefosine resistance. LMDR1 pumps drugs out of the parasite, thereby decreasing their intracellular accumulation. Sitamaquine, another promising oral drug for leishmaniasis, is currently in phase 2b clinical trials. The physicochemical features of this drug suggested to us that it could be considered for use as an LMDR1 inhibitor. Indeed, we report herein that nonleishmanicidal concentrations of Sitamaquine reverse miltefosine resistance in a multidrug resistance Leishmania tropica line that overexpresses LMDR1. This reversal effect is due to modulation of the LMDR1-mediated efflux of miltefosine. In addition, Sitamaquine is not a substrate of LMDR1, as this transporter does not affect Sitamaquine accumulation or sensitivity in the parasite. Likewise, we show that ketoconazole, another oral leishmanicidal drug known to interact with ABC transporters, is also able to reverse LMDR1-mediated miltefosine resistance, although with a lower efficiency than Sitamaquine. Molecular docking on a three-dimensional homology model of LMDR1 showed different preferential binding sites for each substrate-inhibitor pair, thus explaining this different behavior. Finally, we show that Sitamaquine is also able to modulate the antimony resistance mediated by MRPA/LABCC3, another ABC transporter involved in experimental and clinical antimony resistance in this parasite. Taken together, these data suggest that the combination of Sitamaquine with miltefosine or antimony could avoid the appearance of resistance mediated by these membrane transporters in Leishmania.
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Antileishmanial and antitrypanosomal activities of the 8-aminoquinoline tafenoquine.
Antimicrobial agents and chemotherapy, 2010Co-Authors: Vanessa Yardley, Francisco Gamarro, Simon L CroftAbstract:The neglected tropical diseases leishmaniasis, Chagas' disease, and human African trypanosomiasis (HAT), caused by trypanosomatid parasites, have a limited number of drugs for treatment and control, all with limitations of toxicity, variable efficacy, long dosing regimens, and/or parenteral administration. Recent reviews have outlined the advances made in the chemotherapy of these diseases over the past decade for visceral leishmaniasis (VL) (1), cutaneous leishmaniasis (CL) (18), Chagas' disease (22), and human African trypanosomiasis (2). The search for new treatments for these diseases has adopted various strategies, including rational design of drugs (7, 15), screening libraries of synthetic and natural products (11), and therapeutic switching. The more rapid development of a new treatment by the latter approach has been recently demonstrated for Chagas' disease with ergosterol biosynthesis inhibitors (22) and for leishmaniasis with miltefosine and paromomycin (8, 20). The 8-aminoquinolines (Fig. (Fig.1)1) have a long history as antiprotozoal drugs, in particular as antimalarials. Since the 1950s, several have also been reported as being active against Leishmania and Trypanosoma parasites (13, 21). Interest in the activity of this class of compounds for these diseases has been kept in focus by the clinical trials of Sitamaquine (WR6026) for VL (12, 23). Sitamaquine also has anti-Trypanosoma cruzi activity (6). Research on another 8-aminoquinoline, NPC1161, has identified an enantiomer with significant antileishmanial activity and a lower toxicity profile (17). Tafenoquine (TFQ) (WR238605), developed, like many agents of this class, by the Walter Reed Army Institute of Research (WRAIR), is now in clinical trials for the radical cure of Plasmodium vivax by GlaxoSmithKline (GSK) and the Medicines for Malaria Venture (MMV) (16). We present here the results of studies of the in vitro and in vivo activities of TFQ against Leishmania donovani and Trypanosoma cruzi. Studies on the mechanism of action of TFQ against Leishmania and activity against Trypanosoma brucei subsp. will be reported elsewhere. FIG. 1. Structures of tafenoquine, Sitamaquine, and primaquine. Early tests of TFQ against the promastigotes of different Leishmania species demonstrated 50% inhibitory concentrations (IC50s) below 3 μM (data not shown). Of more clinical relevance, TFQ (GSK, United Kingdom) activity was evaluated, in vitro, against intracellular amastigotes of L. donovani MHOM/ET/67/HU3 (from East Africa), L. donovani MHOM/IN/82/DD8 (from India), and L. donovani BHU1 and BHU3 (antimony-resistant strains from India generously donated by Shyam Sundar). Infected murine peritoneal macrophages were exposed to the drug as previously described (24). The percent infection was calculated, and the IC50s were derived (Prism). Subsequently, TFQ was further evaluated in the BALB/c mouse-L. donovani model of infection (9). Eight-week-old, female mice (Charles River, United Kingdom) were infected with amastigotes harvested from a donor animal. After 7 days, the mice were treated with TFQ formulated in 10% Tween 80-ethanol (EtOH) 70:30 double-distilled water (ddH2O), at 5 mg/kg, by the oral route, for 5 consecutive days. On day 14, the mice were euthanized and liver impression smears were made at necropsy. The amastigote burden was calculated (Leishman-Donovan units [LDUs]) (4), the percent inhibition was derived, and 50% effective dose (ED50) values were calculated. TFQ hydrochloride (racemate batch R146390, positive enantiomer batch R206420, and negative enantiomer batch R206422) and Sitamaquine tosylate (batch SLV3L004) were donated by GSK. Miltefosine was donated by Astra Zeneca, United Kingdom, and amphotericin B deoxycholate (Fungizone) was purchased from a commercial supplier. All in vivo experiments were carried out under license at the London School of Hygiene & Tropical Medicine (LSHTM) according to UK Home Office regulations. The efficacy of TFQ against T. cruzi (Tulahuen-LacZ strain) (5) was tested against amastigotes in vitro. Peritoneal macrophages were infected with T. cruzi harvested from feeder cell layers and exposed to TFQ. β-Galactosidase activity was measured by the addition of Nonidet P-40 (detergent) and chlorophenol red β-d-thiogalactopyranoside (CPRG; developer). Ninety-six-well assay plates were read at 570 λ, and IC50s were calculated. Benznidazole (Roche, Switzerland) was used as a positive control. Both the racemate and positive and negative enantiomers of TFQ were active against intracellular amastigotes of all of the L. donovani strains tested (see Table Table11 for IC50s) and compared favorably with the standard drugs tested alongside. In the BALB/c mouse model, TFQ was equally active against both antimony-sensitive and antimony-resistant strains (BHU1 and BHU3), with no difference seen between the racemate and enantiomers. At 5 mg/kg, TFQ achieved 99% inhibition against all L. donovani species, with the enantiomers performing similarly. In a subsequent dose-response experiment, the ED50 values ranged from 1.01 to 3.5 mg/kg (Table (Table2)2) . TABLE 1. Activity of TFQ enantiomers against L. donovani SbVa-sensitive strains by the amastigote-peritoneal exudate macrophage model TABLE 2. In vivo activities of TFQ, Sitamaquine, and SbVa in L. donovani-BALB/c mouse models We have shown that TFQ, an 8-aminoquinoline in development for the treatment of malaria (21) has, like other drugs of the same class, potential as an oral antileishmanial agent. In both in vitro and rodent models of Leishmania infection, TFQ had similar potency to Sitamaquine, the drug currently in clinical development for VL, and NPC111B, which is in preclinical development (21). The limitation of this class has been toxicity, which is of special concern for glucose-6-phosphate dehydrogenase (G6PD)-deficient patients. The extensive antimalarial safety data for TFQ, along with clinical data on Sitamaquine for VL, could support the design of appropriate treatment regimes for VL with TFQ. TFQ might also be an oral partner of interest in combination therapies for the treatment of VL (10, 19). The activities of several series of 8-aminoquinolines against the causative pathogen of Chagas' disease have been published (13). Some have undergone preclinical development (23): for example, moxipraquine for Chagas' disease (3). Sitamaquine showed potential for prevention of T. cruzi transmission through blood transfusion, with activity against trypomastigotes at 4°C (6). We did not find TFQ to be as active in vitro against T. cruzi as other 8-aminoquinolines (Table (Table3),3), as others have previously reported (3, 13, 14). TABLE 3. In vitro activity of TFQ versus T. cruzi
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Sitamaquine Sensitivity in Leishmania Species Is Not Mediated by Drug Accumulation in Acidocalcisomes
Antimicrobial agents and chemotherapy, 2008Co-Authors: Carmen López-martín, José M. Pérez-victoria, Luis Alberto Vieira De Carvalho, Santiago Castanys, Francisco GamarroAbstract:Sitamaquine (WR6026), an 8-aminoquinoline derivative, is a new antileishmanial oral drug. As a lipophilic weak base, it rapidly accumulates in acidic compartments, represented mainly by acidocalcisomes. In this work, we show that the antileishmanial action of Sitamaquine is unrelated to its level of accumulation in these acidic vesicles. We have observed significant differences in Sitamaquine sensitivity and accumulation between Leishmania species and strains, and interestingly, there is no correlation between them. However, there is a relationship between the levels of accumulation of Sitamaquine and acidotropic probes, acidocalcisomes size, and polyphosphate levels. The Leishmania major AP3δ-null mutant line, in which acidocalcisomes are devoid of their usual polyphosphate and proton content, is unable to accumulate Sitamaquine; however, both the parental strain and the AP3δ-null mutants showed similar sensitivities to Sitamaquine. Our findings provide clear evidence that the antileishmanial action of Sitamaquine is unrelated to its accumulation in acidocalcisomes.
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Characterization of an ABCG-like transporter from the protozoan parasite Leishmania with a role in drug resistance and transbilayer lipid movement
Antimicrobial agents and chemotherapy, 2008Co-Authors: Esther Castanys-muñoz, José M. Pérez-victoria, Francisco Gamarro, Santiago CastanysAbstract:Leishmaniasis treatment is hampered by the increased appearance of treatment failure. ATP-binding cassette (ABC) transporters are usually involved in drug resistance both in tumor cells and in microorganisms. Here we report the characterization of an ABCG-like transporter, LiABCG6, localized mainly at the plasma membrane in Leishmania protozoan parasites. When overexpressed, this half-transporter confers significant resistance to the leishmanicidal agents miltefosine and Sitamaquine. This resistance phenotype is mediated by a reduction in intracellular drug accumulation. LiABCG6 also reduces the accumulation of short-chain fluorescent phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. As a whole, these results suggest that LiABCG6 could be implicated in phospholipid trafficking and drug resistance.
Simon L Croft - One of the best experts on this subject based on the ideXlab platform.
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in vitro interactions between Sitamaquine and amphotericin b sodium stibogluconate miltefosine paromomycin and pentamidine against leishmania donovani
Journal of Antimicrobial Chemotherapy, 2011Co-Authors: Karin Seifert, Jane C Munday, Tahmina Syeda, Simon L CroftAbstract:OBJECTIVES: To evaluate in vitro interactions between Sitamaquine and the current antileishmanial drugs amphotericin B, sodium stibogluconate, miltefosine, paromomycin and pentamidine against intracellular Leishmania donovani amastigotes in peritoneal mouse macrophages. A second objective was to evaluate the susceptibility of antimony-resistant L. donovani isolates to Sitamaquine. METHODS: Mouse peritoneal macrophages were infected with L. donovani amastigotes. Drug susceptibility was assessed in a standard 5 day assay and drug interactions with a modified fixed ratio isobologram method. Fractional inhibitory concentrations (FICs), sum FICs (∑FICs) and an overall mean ∑FIC were calculated for each combination. The nature of interaction was classified on the basis of the mean ∑FIC as follows: synergy as mean ∑FIC≤0.5, indifference as mean ∑FIC between >0.5 and ≤4 and antagonism as mean ∑FIC>4. RESULTS: Interactions between Sitamaquine and amphotericin B, sodium stibogluconate, paromomycin and miltefosine were classified as indifferent at the 50% and 90% effective concentration (EC50 and EC90, respectively) levels. The Sitamaquine/pentamidine combination was synergistic, with overall mean ∑FICs from 0.5 to 0.6 at the EC50 level and from 0.3 to 0.7 at the EC90 level. Sitamaquine displayed in vitro activity against L. donovani isolates resistant to sodium stibogluconate. CONCLUSIONS: This study expands the preclinical data on drug combinations and provides the basis for further studies as antileishmanial chemotherapy is moving towards multidrug treatment regimens.
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Antileishmanial and antitrypanosomal activities of the 8-aminoquinoline tafenoquine.
Antimicrobial agents and chemotherapy, 2010Co-Authors: Vanessa Yardley, Francisco Gamarro, Simon L CroftAbstract:The neglected tropical diseases leishmaniasis, Chagas' disease, and human African trypanosomiasis (HAT), caused by trypanosomatid parasites, have a limited number of drugs for treatment and control, all with limitations of toxicity, variable efficacy, long dosing regimens, and/or parenteral administration. Recent reviews have outlined the advances made in the chemotherapy of these diseases over the past decade for visceral leishmaniasis (VL) (1), cutaneous leishmaniasis (CL) (18), Chagas' disease (22), and human African trypanosomiasis (2). The search for new treatments for these diseases has adopted various strategies, including rational design of drugs (7, 15), screening libraries of synthetic and natural products (11), and therapeutic switching. The more rapid development of a new treatment by the latter approach has been recently demonstrated for Chagas' disease with ergosterol biosynthesis inhibitors (22) and for leishmaniasis with miltefosine and paromomycin (8, 20). The 8-aminoquinolines (Fig. (Fig.1)1) have a long history as antiprotozoal drugs, in particular as antimalarials. Since the 1950s, several have also been reported as being active against Leishmania and Trypanosoma parasites (13, 21). Interest in the activity of this class of compounds for these diseases has been kept in focus by the clinical trials of Sitamaquine (WR6026) for VL (12, 23). Sitamaquine also has anti-Trypanosoma cruzi activity (6). Research on another 8-aminoquinoline, NPC1161, has identified an enantiomer with significant antileishmanial activity and a lower toxicity profile (17). Tafenoquine (TFQ) (WR238605), developed, like many agents of this class, by the Walter Reed Army Institute of Research (WRAIR), is now in clinical trials for the radical cure of Plasmodium vivax by GlaxoSmithKline (GSK) and the Medicines for Malaria Venture (MMV) (16). We present here the results of studies of the in vitro and in vivo activities of TFQ against Leishmania donovani and Trypanosoma cruzi. Studies on the mechanism of action of TFQ against Leishmania and activity against Trypanosoma brucei subsp. will be reported elsewhere. FIG. 1. Structures of tafenoquine, Sitamaquine, and primaquine. Early tests of TFQ against the promastigotes of different Leishmania species demonstrated 50% inhibitory concentrations (IC50s) below 3 μM (data not shown). Of more clinical relevance, TFQ (GSK, United Kingdom) activity was evaluated, in vitro, against intracellular amastigotes of L. donovani MHOM/ET/67/HU3 (from East Africa), L. donovani MHOM/IN/82/DD8 (from India), and L. donovani BHU1 and BHU3 (antimony-resistant strains from India generously donated by Shyam Sundar). Infected murine peritoneal macrophages were exposed to the drug as previously described (24). The percent infection was calculated, and the IC50s were derived (Prism). Subsequently, TFQ was further evaluated in the BALB/c mouse-L. donovani model of infection (9). Eight-week-old, female mice (Charles River, United Kingdom) were infected with amastigotes harvested from a donor animal. After 7 days, the mice were treated with TFQ formulated in 10% Tween 80-ethanol (EtOH) 70:30 double-distilled water (ddH2O), at 5 mg/kg, by the oral route, for 5 consecutive days. On day 14, the mice were euthanized and liver impression smears were made at necropsy. The amastigote burden was calculated (Leishman-Donovan units [LDUs]) (4), the percent inhibition was derived, and 50% effective dose (ED50) values were calculated. TFQ hydrochloride (racemate batch R146390, positive enantiomer batch R206420, and negative enantiomer batch R206422) and Sitamaquine tosylate (batch SLV3L004) were donated by GSK. Miltefosine was donated by Astra Zeneca, United Kingdom, and amphotericin B deoxycholate (Fungizone) was purchased from a commercial supplier. All in vivo experiments were carried out under license at the London School of Hygiene & Tropical Medicine (LSHTM) according to UK Home Office regulations. The efficacy of TFQ against T. cruzi (Tulahuen-LacZ strain) (5) was tested against amastigotes in vitro. Peritoneal macrophages were infected with T. cruzi harvested from feeder cell layers and exposed to TFQ. β-Galactosidase activity was measured by the addition of Nonidet P-40 (detergent) and chlorophenol red β-d-thiogalactopyranoside (CPRG; developer). Ninety-six-well assay plates were read at 570 λ, and IC50s were calculated. Benznidazole (Roche, Switzerland) was used as a positive control. Both the racemate and positive and negative enantiomers of TFQ were active against intracellular amastigotes of all of the L. donovani strains tested (see Table Table11 for IC50s) and compared favorably with the standard drugs tested alongside. In the BALB/c mouse model, TFQ was equally active against both antimony-sensitive and antimony-resistant strains (BHU1 and BHU3), with no difference seen between the racemate and enantiomers. At 5 mg/kg, TFQ achieved 99% inhibition against all L. donovani species, with the enantiomers performing similarly. In a subsequent dose-response experiment, the ED50 values ranged from 1.01 to 3.5 mg/kg (Table (Table2)2) . TABLE 1. Activity of TFQ enantiomers against L. donovani SbVa-sensitive strains by the amastigote-peritoneal exudate macrophage model TABLE 2. In vivo activities of TFQ, Sitamaquine, and SbVa in L. donovani-BALB/c mouse models We have shown that TFQ, an 8-aminoquinoline in development for the treatment of malaria (21) has, like other drugs of the same class, potential as an oral antileishmanial agent. In both in vitro and rodent models of Leishmania infection, TFQ had similar potency to Sitamaquine, the drug currently in clinical development for VL, and NPC111B, which is in preclinical development (21). The limitation of this class has been toxicity, which is of special concern for glucose-6-phosphate dehydrogenase (G6PD)-deficient patients. The extensive antimalarial safety data for TFQ, along with clinical data on Sitamaquine for VL, could support the design of appropriate treatment regimes for VL with TFQ. TFQ might also be an oral partner of interest in combination therapies for the treatment of VL (10, 19). The activities of several series of 8-aminoquinolines against the causative pathogen of Chagas' disease have been published (13). Some have undergone preclinical development (23): for example, moxipraquine for Chagas' disease (3). Sitamaquine showed potential for prevention of T. cruzi transmission through blood transfusion, with activity against trypomastigotes at 4°C (6). We did not find TFQ to be as active in vitro against T. cruzi as other 8-aminoquinolines (Table (Table3),3), as others have previously reported (3, 13, 14). TABLE 3. In vitro activity of TFQ versus T. cruzi
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In Vitro and In Vivo Interactions between Miltefosine and Other Antileishmanial Drugs
Antimicrobial Agents and Chemotherapy, 2006Co-Authors: Karin Seifert, Simon L CroftAbstract:The interaction of miltefosine with amphotericin B, sodium stibogluconate, paromomycin, and Sitamaquine was assessed in vitro and additionally for the first three combinations in vivo. In vitro interactions were indifferent for miltefosine combined with amphotericin B (mean sums of fractional inhibitory concentrations [mean summation operatorFICs] ranging from 1.22 to 1.51 at the 50% effective concentration [EC50] level and 1.08 to 1.38 at the EC90 level), Sitamaquine (mean summation operatorFICs from 1.33 to 1.38 and 1.0 to 1.02, respectively), and paromomycin (mean summation operatorFICs from 0.79 to 0.93 at the EC50 and 0.77 to 1.35 at the EC90 level). Some synergy was observed for miltefosine combined with sodium stibogluconate (mean summation operatorFICs from 0.61 to 0.75 at EC50 and 0.49 to 0.97 at EC90). Different interactions were found in vivo, where the highest potentiation of miltefosine activity was achieved with amphotericin B (activity enhancement index [AEI] of up to 11.3). No significant interaction was observed when miltefosine was combined with sodium stibogluconate (AEI of up to 2.38). The potentiation of miltefosine in vivo was also achieved with the combination of miltefosine and paromomycin (AEI of up to 7.22).
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In-vitro and in-vivo studies on a topical formulation of Sitamaquine dihydrochloride for cutaneous leishmaniasis.
The Journal of pharmacy and pharmacology, 2006Co-Authors: Tracy Garnier, Marc B. Brown, M. Jayne Lawrence, Simon L CroftAbstract:The efficacy of topical formulations of the 8-aminoquinoline, Sitamaquine dihydrochloride, in both in-vitro and in in-vivo models of cutaneous leishmaniasis is reported. In-vitro parasite assays confirmed that Sitamaquine dihydrochloride was active against a range of Leishmania species that cause either cutaneous or visceral leishmaniasis, with ED50 values against amastigotes over the range of 2.9 to 19.0 microM. A range of topical Sitamaquine dihydrochloride formulations (anhydrous gel, emulsions) were developed for studies on experimental cutaneous leishmaniasis using only topically acceptable excipients or those currently undergoing regulatory approval. An uptake study into murine skin confirmed in-vitro skin penetration and retention. Several formulations were tested in-vivo against Leishmania major cutaneous lesions in BALB/c mice. None of the Sitamaquine dihydrochloride formulations tested appeared to either slow lesion progression or reduce parasite burden.
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visceral leishmaniasis current status of control diagnosis and treatment and a proposed research and development agenda
Lancet Infectious Diseases, 2002Co-Authors: Simon L Croft, Shyam Sundar, Monique Wasunna, Piero Olliaro, Philippe J Guerin, Marleen Boelaert, Philippe Desjeux, Anthony BrycesonAbstract:494 Visceral leishmaniasis is common in less developed countries, with an estimated 500 000 new cases each year. Because of the diversity of epidemiological situations, no single diagnosis, treatment, or control will be suitable for all. Control measures through case finding, treatment, and vector control are seldom used, even where they could be useful. There is a place for a vaccine, and new imaginative approaches are needed. HIV coinfection is changing the epidemiology and presents problems for diagnosis and case management. Field diagnosis is difficult; simpler, less invasive tests are needed. Current treatments require long courses and parenteral administration, and most are expensive. Resistance is making the mainstay of treatment, agents based on pentavalent antimony, useless in northeastern India, where disease incidence is highest. Second-line drugs (pentamidine and amphotericin B) are limited by toxicity and availability, and newer formulations of amphotericin B are not affordable. The first effective oral drug, miltefosine, has been licensed in India, but the development of other drugs in clinical phases (paromomycin and Sitamaquine) is slow. No novel compound is in the pipeline. Drug combinations must be developed to prevent drug resistance. Despite these urgent needs, research and development has been neglected, because a disease that mainly affects the poor ranks as a low priority in the private sector, and the public sector currently struggles to undertake the development of drugs and diagnostics in the absence of adequate funds and infrastructure. This article reviews the current situation and perspectives for diagnosis, treatment, and control of visceral leishmaniasis, and lists some priorities for research and development.
Santiago Castanys - One of the best experts on this subject based on the ideXlab platform.
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the 8 aminoquinoline analogue Sitamaquine causes oxidative stress in leishmania donovani promastigotes by targeting succinate dehydrogenase
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Luis Alberto Vieira De Carvalho, Santiago Castanys, Juan Roman Luqueortega, Carmen Lopezmartin, Luis Rivas, Francisco GamarroAbstract:The 8-aminoquinoline analogue Sitamaquine (SQ) is an oral antileishmanial drug currently undergoing phase 2b clinical trials for the treatment of visceral leishmaniasis. In the present study, we investigated the mechanism of action of this drug in Leishmania donovani promastigotes. SQ causes a dose-dependent inhibition of complex II (succinate dehydrogenase) of the respiratory chain in digitonin-permeabilized promastigotes, together with a drop in intracellular ATP levels and a decrease of the mitochondrial electrochemical potential. This is associated with increases of reactive oxygen species and intracellular Ca2+ levels, a higher percentage of the population with sub-G1 DNA content, and exposure of phosphatidylserine. Taken together, these results support a lethal mechanism for SQ that involves inhibition of the respiratory chain complex II, which in turn triggers oxidative stress and finally leads to an apoptosis-like death of Leishmania parasites.
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Sitamaquine Overcomes ABC-Mediated Resistance to Miltefosine and Antimony in Leishmania
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: José M. Pérez-victoria, Carmen López-martín, Santiago Castanys, Boris I. Bavchvarov, Ivan R. Torrecillas, Marta Martínez-garcía, Mercedes Campillo, Francisco GamarroAbstract:Although oral miltefosine represented an important therapeutic advance in the treatment of leishmaniasis, the appearance of resistance remains a serious threat. LMDR1/LABCB4, a P-glycoprotein-like transporter included in the Leishmania ABC (ATP-binding cassette) family, was the first molecule shown to be involved in experimental miltefosine resistance. LMDR1 pumps drugs out of the parasite, thereby decreasing their intracellular accumulation. Sitamaquine, another promising oral drug for leishmaniasis, is currently in phase 2b clinical trials. The physicochemical features of this drug suggested to us that it could be considered for use as an LMDR1 inhibitor. Indeed, we report herein that nonleishmanicidal concentrations of Sitamaquine reverse miltefosine resistance in a multidrug resistance Leishmania tropica line that overexpresses LMDR1. This reversal effect is due to modulation of the LMDR1-mediated efflux of miltefosine. In addition, Sitamaquine is not a substrate of LMDR1, as this transporter does not affect Sitamaquine accumulation or sensitivity in the parasite. Likewise, we show that ketoconazole, another oral leishmanicidal drug known to interact with ABC transporters, is also able to reverse LMDR1-mediated miltefosine resistance, although with a lower efficiency than Sitamaquine. Molecular docking on a three-dimensional homology model of LMDR1 showed different preferential binding sites for each substrate-inhibitor pair, thus explaining this different behavior. Finally, we show that Sitamaquine is also able to modulate the antimony resistance mediated by MRPA/LABCC3, another ABC transporter involved in experimental and clinical antimony resistance in this parasite. Taken together, these data suggest that the combination of Sitamaquine with miltefosine or antimony could avoid the appearance of resistance mediated by these membrane transporters in Leishmania.
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Sitamaquine Sensitivity in Leishmania Species Is Not Mediated by Drug Accumulation in Acidocalcisomes
Antimicrobial agents and chemotherapy, 2008Co-Authors: Carmen López-martín, José M. Pérez-victoria, Luis Alberto Vieira De Carvalho, Santiago Castanys, Francisco GamarroAbstract:Sitamaquine (WR6026), an 8-aminoquinoline derivative, is a new antileishmanial oral drug. As a lipophilic weak base, it rapidly accumulates in acidic compartments, represented mainly by acidocalcisomes. In this work, we show that the antileishmanial action of Sitamaquine is unrelated to its level of accumulation in these acidic vesicles. We have observed significant differences in Sitamaquine sensitivity and accumulation between Leishmania species and strains, and interestingly, there is no correlation between them. However, there is a relationship between the levels of accumulation of Sitamaquine and acidotropic probes, acidocalcisomes size, and polyphosphate levels. The Leishmania major AP3δ-null mutant line, in which acidocalcisomes are devoid of their usual polyphosphate and proton content, is unable to accumulate Sitamaquine; however, both the parental strain and the AP3δ-null mutants showed similar sensitivities to Sitamaquine. Our findings provide clear evidence that the antileishmanial action of Sitamaquine is unrelated to its accumulation in acidocalcisomes.
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Characterization of an ABCG-like transporter from the protozoan parasite Leishmania with a role in drug resistance and transbilayer lipid movement
Antimicrobial agents and chemotherapy, 2008Co-Authors: Esther Castanys-muñoz, José M. Pérez-victoria, Francisco Gamarro, Santiago CastanysAbstract:Leishmaniasis treatment is hampered by the increased appearance of treatment failure. ATP-binding cassette (ABC) transporters are usually involved in drug resistance both in tumor cells and in microorganisms. Here we report the characterization of an ABCG-like transporter, LiABCG6, localized mainly at the plasma membrane in Leishmania protozoan parasites. When overexpressed, this half-transporter confers significant resistance to the leishmanicidal agents miltefosine and Sitamaquine. This resistance phenotype is mediated by a reduction in intracellular drug accumulation. LiABCG6 also reduces the accumulation of short-chain fluorescent phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. As a whole, these results suggest that LiABCG6 could be implicated in phospholipid trafficking and drug resistance.
Philippe M. Loiseau - One of the best experts on this subject based on the ideXlab platform.
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Sitamaquine-resistance in Leishmania donovani affects drug accumulation and lipid metabolism
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2014Co-Authors: Laurent Imbert, Sandrine Cojean, D. Libong, Pierre Chaminade, Philippe M. LoiseauAbstract: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.
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Sitamaquine as a putative antileishmanial drug candidate: from the mechanism of action to the risk of drug resistance
Parasite (Paris France), 2011Co-Authors: Philippe M. Loiseau, Sandrine Cojean, J. SchrévelAbstract: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.
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In Vitro Activities of New 2-Substituted Quinolines against Leishmania donovani
Antimicrobial agents and chemotherapy, 2011Co-Authors: Philippe M. Loiseau, Suman Gupta, Aditya Verma, Saumya Srivastava, Surendra Puri, Faten Sliman, Marie Normand-bayle, Didier DesmaeleAbstract:A series of 9 quinolines and 18 styrylquinolines was evaluated for the drugs’ in vitro antileishmanial activities and cytotoxicities. The 7-aroylstyrylquinoline scaffold appeared to be the most promising one, with the most interesting compound, no. 35, exhibiting a 50% inhibitory concentration (IC 50 )o f 1.2M and a selectivity index value of 121.5. Compound 35 was 10-fold and 8-fold more active than miltefosine and Sitamaquine, the reference compounds, with selectivity indexes 607-fold and 60-fold higher, respectively. Leishmaniasis is a family of parasitic diseases that affect about 12 million people in tropical and subtropical areas in the form of three clinical expressions: visceral leishmaniasis, which is fatal in the absence of treatment; muco-cutaneous leishmaniasis; and cutaneous leishmaniasis, which is often selfcuring. Classical drugs such as antimonials (Pentostam and Glucantime) are toxic, and drug resistance is increasing dangerously in the field (3). A liposomal amphotericin B formulation (AmBisome) less toxic than amphotericin B deoxycholate is gradually becoming the first-line therapy, especially in immunocompromised patients, but this drug must be administered by a parenteral route (11). Miltefosine (Impavido) was the first drug registered against visceral leishmaniasis in the last decade; however, its toxicity and the appearance of drug resistance justify the search for new chemical
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Mechanism of interaction of Sitamaquine with Leishmania donovani
The Journal of antimicrobial chemotherapy, 2010Co-Authors: Elaine Soares Coimbra, Sandrine Cojean, D. Libong, Pierre Chaminade, M. Saint-pierre-chazalet, A. Solgadi, L. Le Moyec, A. M. Duenas-romero, Philippe M. LoiseauAbstract: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.
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Selection of the most promising 2-substituted quinoline as antileishmanial candidate for clinical trials
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2008Co-Authors: Nashira Campos Vieira, Christine Herrenknecht, Joël Vacus, Alain Fournet, Christian Bories, Bruno Figadère, Laila S. Espindola, Philippe M. LoiseauAbstract:The antileishmanial evaluation of more than one hundred 2-substituted quinolines led us to identify three compounds for further studies: compound 1 (2-n-propylquinoline), compound 2 (2-(2methoxyethenyl)quinoline) and compound 3 (2-(2-hydroxyprop-2-enyl)quinoline). The final selection of a potential drug candidate was mainly based on chemical stability and acute oral toxicity as discriminating criteria. The most stable compound in various conditions was 2-n-propylquinoline (compound 1). Only reversible toxicity signs were observed for compound 1 at 1000 mg/kg after a treatment by oral route at a single dose and no sign was detected at 100 mg/kg. Interestingly, 2-substituted quinolines were active on a Leishmania donovani line, resistant to Sitamaquine, a 8-aminoquinoline, suggesting that 2-substituted quinolines and 8-aminoquinoline probably affect a different target in L. donovani.
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Sitamaquine glaxosmithkline walter reed army institute
Current opinion in investigational drugs, 2002Co-Authors: Clive YeatesAbstract:Sitamaquine (WR-6026) is an orally active 8-aminoquinoline analog in development by the Walter Reed Army Institute, in collaboration with GlaxoSmithKline (formerly SmithKline Beecham), for the potential treatment of visceral leishmaniasis. Phase III trials for the treatment of visceral leishmaniasis had been initiated by March 2002, at which time GlaxoSmithKline hoped to file an MAA in 2003. By 1999, the compound had also undergone phase I trials in HIV-infected individuals for the treatment of Pneumocystis carinii infection. Preclinical studies have been conducted in primates and rodents for the potential treatment of Babesia microti infection.
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Sitamaquine (GlaxoSmithKline/Walter Reed Army Institute).
Current opinion in investigational drugs (London England : 2000), 2002Co-Authors: Clive YeatesAbstract:Sitamaquine (WR-6026) is an orally active 8-aminoquinoline analog in development by the Walter Reed Army Institute, in collaboration with GlaxoSmithKline (formerly SmithKline Beecham), for the potential treatment of visceral leishmaniasis. Phase III trials for the treatment of visceral leishmaniasis had been initiated by March 2002, at which time GlaxoSmithKline hoped to file an MAA in 2003. By 1999, the compound had also undergone phase I trials in HIV-infected individuals for the treatment of Pneumocystis carinii infection. Preclinical studies have been conducted in primates and rodents for the potential treatment of Babesia microti infection.