In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites.

Abstract. Leishmania (Viannia) braziliensis is the main causative species of tegumentary leishmaniasis in Brazil. In this study, we evaluated the susceptibility of 16 clinical isolates of L. (V.) braziliensis from different regions of Brazil to miltefosine in vitro. Half-maximal inhibitory concentrations of miltefosine varied from 22.9 to 144.2 μM against promastigotes and from 0.3 to 4.2 μM against intracellular amastigotes. No significant differences were found between isolates of different geographical origins. A clear correlation between the EC 50 against promastigotes and amastigotes within each isolate was found. These findings contribute to the evaluation of miltefosine's potential and limitations for the treatment of tegumentary leishmaniasis in Brazil.Tegumentary leishmaniasis is a disease of importance in Brazil, where it is mainly caused by Leishmania (Viannia) braziliensis. The efficacy of the first-line drug, meglumine antimoniate, for the treatment of cutaneous leishmaniasis in areas of L. (V.) braziliensis predominance in Brazil can be as low as 53%.1 New therapeutic alternatives are highly desirable. Miltefosine (MF) (hexadecylphosphatidylcholine) was approved for the treatment of visceral leishmaniasis in India in 2002, where pentavalent antimony was already considered as ineffective due to widespread parasite resistance. 2This oral drug has also been approved for the treatment of tegumentary leishmaniasis in Colombia, after the demonstration of equivalent efficacy to antimony, and in other countries in South America.3 However, the response is heterogeneous in areas of high prevalence of L. (V.) braziliensis: for example, a clinical trial showed 83% efficacy for MF in cutaneous leishmaniasis in Bolivia, whereas a 53% cure rate was observed in Guatemala. 3,4 In Brazil, 70% success rates were observed in two MF clinical trials of cutaneous leishmaniasis due to L. (V.) braziliensis and Leishmania (V.) guyanensis. 1,5The aim of this work was to characterize the MF susceptibility of L. (V.) braziliensis clinical isolates from Brazilian patients with tegumentary leishmaniasis from two geographically distinct regions.Eight clinical isolates were obtained from lesion biopsies of patients with tegumentary leishmaniasis attending the Anuar Auad Tropical Diseases Hospital, Goiânia, Goiás, Brazil (Leishbank), 6 and eight isolates were obtained through needle aspiration of skin lesions from patients attending the health post of Corte de Pedra, Bahia, Brazil. After the isolation, cultures were frozen and recovered to perform this study. The isolates were typed by polymerase chain reaction of internal transcribed spacer of ribosomal DNA and hsp70 gene followed by restriction analysis using Hae III, as described. 7,8 The M2903 reference strain and 16 clinical isolates produced the expected profile for L. (V.) braziliensis (data not shown).The activity of MF against promastigotes was evaluated by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay as previously described.9 Approximatel...

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.

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BackgroundLeishmania 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 FindingsA 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/SignificanceHere 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.