RESUMEN
OBJECTIVES: There is a clear need for new strategies of leishmaniasis treatment. This work was conducted to evaluate the efficacy of the co-administration of tamoxifen and meglumine antimoniate (SbV ) in a phase II pilot clinical trial in localised cutaneous leishmaniasis patients. METHODS: A randomised controlled pilot clinical trial was conducted to evaluate the efficacy and safety of oral (40 mg/day for 20 days) or topical tamoxifen (0.1% tamoxifen citrate for 20 days) combined with meglumine antimoniate (20 mg SbV /kg/day for 20 days) vs. a standard SbV protocol (20 mg/kg/day for 20 days) for the treatment of cutaneous leishmaniasis. Primary outcome was complete epithelisation of the lesion 6 months after the end of treatment. Secondary outcomes were lesion healing 2 months after the end of treatment and frequency and severity of adverse events. RESULTS: A total of 38 subjects were included in the trial, 15 were treated with standard SbV and 23 with the combination of tamoxifen and SbV . Of the patients treated with the co-administration scheme, 12 received tamoxifen orally and 11 were treated with topical tamoxifen. Tamoxifen administered by the oral or topical routes was well tolerated. Cure rates 6 months after the end of treatment per intention to treat were 40% in the group treated with the standard SbV scheme, and 36.4% and 58%, respectively, for groups treated with SbV plus topical or oral tamoxifen. CONCLUSIONS: In the doses and schemes used in this study, co-administration of oral tamoxifen and SbV resulted in higher cure rates in comparison with the standard scheme of treatment, although not to statistically significant levels.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmaniasis Cutánea/tratamiento farmacológico , Antimoniato de Meglumina/uso terapéutico , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Tamoxifeno/uso terapéutico , Administración Oral , Administración Tópica , Adulto , Antiprotozoarios/administración & dosificación , Quimioterapia Combinada , Femenino , Humanos , Masculino , Antimoniato de Meglumina/administración & dosificación , Persona de Mediana Edad , Proyectos Piloto , Moduladores Selectivos de los Receptores de Estrógeno/administración & dosificación , Tamoxifeno/administración & dosificación , Resultado del Tratamiento , Adulto JovenRESUMEN
Cutaneous and visceral leishmaniasis are amongst the most devastating infectious diseases of our time, affecting millions of people worldwide. The treatment of these serious diseases rely on a few chemotherapeutic agents, most of which are of parenteral use and induce severe side-effects. Furthermore, rates of treatment failure are high and have been linked to drug resistance in some areas. Here, we reviewed data on current chemotherapy practice in leishmaniasis. Drug resistance and mechanisms of resistance are described as well as the prospects for applying drug combinations for leishmaniasis chemotherapy. It is clear that efforts for discovering new drugs applicable to leishmaniasis chemotherapy are essential. The main aspects on the various steps of drug discovery in the field are discussed.
Asunto(s)
Descubrimiento de Drogas/métodos , Leishmania/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológico , Insuficiencia del Tratamiento , Animales , Antiprotozoarios/efectos adversos , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Ensayos Clínicos como Asunto , Cricetinae , Modelos Animales de Enfermedad , Resistencia a Múltiples Medicamentos/genética , Quimioterapia Combinada/métodos , Humanos , Leishmaniasis Cutánea/parasitología , Leishmaniasis Visceral/parasitología , RatonesRESUMEN
The aims of the present work were to test the effect of tamoxifen administered topically and the therapeutic efficacy of tamoxifen and pentavalent antimonial combinations in an experimental model of cutaneous leishmaniasis. BALB/c mice infected with a luciferase expressing line of Leishmania amazonensis were treated with topical tamoxifen in two different formulations (ethanol or oil-free cream) as monotherapy or in co-administration with pentavalent antimonial. Treatment efficacy was evaluated by lesion size and parasite burden, quantified through luminescence, at the end of treatment and 4 weeks later. Topical tamoxifen, formulated in ethanol or as a cream, was shown to be effective. The interaction between tamoxifen and pentavalent antimonial was additive in vitro. Treatment with combined schemes containing tamoxifen and pentavalent antimonial was effective in reducing lesion size and parasite burden. Co-administration of tamoxifen and pentavalent antimonial was superior to monotherapy with antimonial.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Antimoniato de Meglumina/uso terapéutico , Piel/efectos de los fármacos , Tamoxifeno/uso terapéutico , Administración Tópica , Animales , Antiprotozoarios/administración & dosificación , Antiprotozoarios/efectos adversos , Quimioterapia Combinada/efectos adversos , Etanol/química , Femenino , Leishmania mexicana/enzimología , Leishmania mexicana/genética , Leishmaniasis Cutánea/parasitología , Luciferasas/genética , Luminiscencia , Antimoniato de Meglumina/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Piel/parasitología , Crema para la Piel/administración & dosificación , Crema para la Piel/efectos adversos , Crema para la Piel/uso terapéutico , Tamoxifeno/administración & dosificación , Tamoxifeno/efectos adversos , Tamoxifeno/químicaRESUMEN
OBJECTIVES: The objective of this study was to characterize in vitro interactions and evaluate the antileishmanial activity of tamoxifen and miltefosine combinations. METHODS: Interactions between drugs were evaluated in vitro against Leishmania amazonensis promastigotes and intracellular amastigotes by a modified isobologram method. Four different drug ratios were used to calculate the FIC index (FICI) and the mean sum of FICI. Treatment of L. amazonensis-infected BALB/c mice was initiated 4 weeks post-infection. Mice were treated with the half-maximal effective dose (ED50) or half the ED50 of tamoxifen and miltefosine orally for 15 days. Efficacy was evaluated by lesion growth and parasite burden measured through luciferase detection at the end of treatment and 30 days later. Characterization of growth curves and stepwise increase in drug concentrations in vitro were used to measure survival and resistance selection of parasite populations submitted to combination treatment. RESULTS: No in vitro interactions between tamoxifen and miltefosine were found. In infected mice, the combination of tamoxifen and miltefosine at doses corresponding to half the ED50 was more effective than monotherapy with either tamoxifen or miltefosine. When the ED50 was employed, the efficacy of the combination was equivalent to miltefosine monotherapy. In vitro, tamoxifen was able to retard or suppress the growth of parasites treated with miltefosine. CONCLUSIONS: In vitro and in vivo studies revealed no interaction between tamoxifen and miltefosine. Tamoxifen was able to hinder the emergence of miltefosine resistance.
Asunto(s)
Antiprotozoarios/administración & dosificación , Leishmania/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Fosforilcolina/análogos & derivados , Tamoxifeno/administración & dosificación , Administración Oral , Animales , Antiprotozoarios/farmacocinética , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Quimioterapia Combinada , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Ratones Endogámicos BALB C , Carga de Parásitos , Fosforilcolina/administración & dosificación , Fosforilcolina/farmacocinética , Tamoxifeno/farmacocinética , Resultado del TratamientoRESUMEN
Leishmaniasis chemotherapy remains very challenging. The high cost of active drugs, along with the severity of their side effects and the increasing failure rate of the current therapeutic schemes, calls for the discovery of new active drugs and schemes of treatment. The use of combination therapy has gained much attention in recent years as a possible strategy for overcoming the various shortcomings in the present arsenal. We recently described the effectiveness of tamoxifen in murine models of leishmaniasis, and here, we investigated the interactions between tamoxifen and amphotericin B, one of the most potent drugs used in leishmaniasis treatment. The in vitro interactions were indifferent for the association of tamoxifen and amphotericin B. The association was also assayed in vivo in Leishmania amazonensis-infected BALB/c mice and was found to yield at least additive effects at low doses of both drugs.
Asunto(s)
Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Leishmaniasis Cutánea/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB CRESUMEN
Previous work from our group showed that tamoxifen, an oral drug that has been in use for the treatment of breast cancer for over 40 years, is active both in vitro and in vivo against several species of Leishmania, the etiological agent of leishmaniasis. Using a combination of metabolic labeling with [3H]-sphingosine and myo-[3H]-inositol, alkaline hydrolysis, HPTLC fractionations and mass spectrometry analyses, we observed a perturbation in the metabolism of inositolphosphorylceramides (IPCs) and phosphatidylinositols (PIs) after treatment of L. amazonensis promastigotes with tamoxifen, with a significant reduction in the biosynthesis of the major IPCs (composed of d16:1/18:0-IPC, t16:0/C18:0-IPC, d18:1/18:0-IPC and t16:0/20:0-IPC) and PIs (sn-1-O-(C18:0)alkyl -2-O-(C18:1)acylglycerol-3-HPO4-inositol and sn-1-O-(C18:0)acyl-2-O-(C18:1)acylglycerol-3-HPO4-inositol) species. Substrate saturation kinetics of myo-inositol uptake analyses indicated that inhibition of inositol transport or availability were not the main reasons for the reduced biosynthesis of IPC and PI observed in tamoxifen treated parasites. An in vitro enzymatic assay was used to show that tamoxifen was able to inhibit the Leishmania IPC synthase with an IC50 value of 8.48⯵M (95% CI 7.68-9.37), suggesting that this enzyme is most likely one of the targets for this compound in the parasites.
Asunto(s)
Vías Biosintéticas/efectos de los fármacos , Glicoesfingolípidos/biosíntesis , Leishmania/efectos de los fármacos , Tamoxifeno/farmacología , Glicoesfingolípidos/metabolismo , Hexosiltransferasas/efectos de los fármacos , Hexosiltransferasas/metabolismo , Concentración 50 Inhibidora , Inositol/metabolismo , Leishmania/fisiología , Leishmania mexicana/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Fosfatidilinositoles/metabolismoRESUMEN
BACKGROUND: Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine. METHODOLOGY/PRINCIPAL FINDINGS: A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine. CONCLUSIONS/SIGNIFICANCE: Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmania braziliensis/enzimología , Leishmaniasis Cutánea/patología , Leishmaniasis Cutánea/parasitología , Luciferasas/metabolismo , Fosforilcolina/análogos & derivados , Anfotericina B/uso terapéutico , Animales , Antifúngicos/uso terapéutico , Antiprotozoarios/farmacología , Cricetinae , Ácido Desoxicólico/uso terapéutico , Combinación de Medicamentos , Femenino , Regulación Enzimológica de la Expresión Génica , Leishmania braziliensis/efectos de los fármacos , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmaniasis Cutánea/tratamiento farmacológico , Luciferasas/genética , Masculino , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Organismos Modificados Genéticamente , Fosforilcolina/uso terapéutico , Factores de TiempoRESUMEN
BACKGROUND: The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters. METHODOLOGY/PRINCIPAL FINDINGS: A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival. CONCLUSIONS/SIGNIFICANCE: Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmania infantum/efectos de los fármacos , Leishmania infantum/enzimología , Leishmaniasis Visceral/tratamiento farmacológico , Carga de Parásitos/métodos , Fosforilcolina/análogos & derivados , Animales , Antimonio/uso terapéutico , Cricetinae , Humanos , India , Leishmaniasis Visceral/parasitología , Luciferasas/biosíntesis , Luciferasas/genética , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Tamoxifen, an antineoplastic agent, is active in vitro and in vivo against the parasitic protozoa Leishmania. As part of our efforts to unravel this drug's mechanisms of action against the parasite and understand how resistance could arise, we tried to select tamoxifen-resistant Leishmania amazonensis. Three different strategies to generate tamoxifen resistant mutants were used: stepwise increase in drug concentration applied to promastigote cultures, chemical mutagenesis followed by drug selection and treatment of infected mice followed by selection of amastigotes. For amastigote selection, we employed a method with direct plating of parasites recovered from lesions into semi-solid media. Tamoxifen resistant parasites were not rescued by any of these methods. Miltefosine was used as a control in selection experiments and both stepwise selection and chemical mutagenesis allowed successful isolation of miltefosine resistant mutants. These findings are consistent with a multi-target mode of action to explain tamoxifen's leishmanicidal properties. Considering that drug resistance is a major concern in anti-parasitic chemotherapy, these findings support the proposition of using tamoxifen as a partner in drug combination schemes for the treatment of leishmaniasis.
Asunto(s)
Resistencia a Medicamentos , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Tamoxifeno/uso terapéutico , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapéutico , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Tamoxifeno/administración & dosificación , Tamoxifeno/farmacologíaRESUMEN
Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.
Asunto(s)
Antiprotozoarios , Resistencia a Medicamentos , Leishmania/efectos de los fármacos , Leishmaniasis Cutánea Difusa , Fosforilcolina/análogos & derivados , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Leishmaniasis Cutánea Difusa/tratamiento farmacológico , Leishmaniasis Cutánea Difusa/parasitología , Ratones , Ratones Endogámicos BALB C , Fosforilcolina/farmacología , Fosforilcolina/uso terapéuticoRESUMEN
BACKGROUND: The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. METHODOLOGY/PRINCIPAL FINDINGS: Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis-infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. CONCLUSIONS/SIGNIFICANCE: The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death.
Asunto(s)
Moduladores de los Receptores de Estrógeno/farmacología , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea/parasitología , Clorhidrato de Raloxifeno/farmacología , Tripanocidas/farmacología , Animales , Membrana Celular/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Vacuolas/efectos de los fármacosRESUMEN
Given the lack of effective and safe alternatives to the drugs already in use, considerable efforts are being applied to the search of new therapeutic options to treat leishmaniasis. A necessary step in the discovery of antileishmanial drugs is the validation of drug candidates in mouse models. The standard methods to quantify the parasite burden in animal models, mainly culture-based, are time consuming and expensive. In recent years, in vivo imaging systems have been proposed as a tool to overcome these problems, allowing parasite detection in living organisms. Here we compared different treatment efficacy evaluation approaches. Recombinant Leishmania (L.) amazonensis lines expressing the luciferase gene (La-LUC) were obtained and characterized for biological properties as compared with the wild type (WT) parental line. Bioluminescence generated by La-LUC was shown to correlate with the number of promastigotes in vitro. La-LUC promastigotes and intracellular amastigotes were equally sensitive to amphotericin B (AmB) as the WT parasites. The clinical pattern of lesion development upon infection with the transgenic lines was similar to lesions observed after infection with the WT strain. The half maximal effective dose (ED50) of AmB was determined in La-LUC infected mice through quantification of bioluminescence in vivo and ex vivo, by limiting dilution and using clinical parameters. There was agreement in the ED50 determined by all methods. Quantification of bioluminescence in vivo and/or ex vivo was elected as the best tool for determining parasite burden to assess drug efficacy in infected mice. Furthermore, the detailed analysis of AmB effectiveness in this model generated useful data to be used in drug combination experiments.