Uso da miltefosina no tratamento clínico de cães com leishmaniose visceral: revisão de literatura / Use of miltefosin in the clinical treatment of dogs with visceral leishmaniosis / Uso de miltefosina en el tratamiento clínico de perros con leishmaniosis visceral: revisión de la literatura

A leishmaniose visceral canina é uma doença de caráter zoonótico, acometendo os seres humanos e diversas espécies de animais silvestres e domésticos. Objetivou-se com o presente estudo realizar uma revisão de literatura sobre o uso da miltefosina no tratamento clínico de cães com leishmaniose visceral. Trata- se de uma revisão de literatura, a qual foi realizada por meio de consultas à periódicos e livros presentes na biblioteca do Cesmac. Foram utilizadas bases de dados como: portal Capes, SCIELO, Google Acadêmico; pesquisa em monografias, teses e dissertações. Causada pelo protozoário Leishmania chagasi, sendo o cão doméstico o principal reservatório desse protozoário. Por representar um problema grave de saúde pública e ser considerada uma doença potencialmente fatal (quando não tratada precocemente e adequadamente), faz- se importante que o clínico esteja familiarizado com os sinais clínicos, exames complementares e principais protocolos terapêuticos, em especial a utilização da miltefosina no tratamento da leishmaniose visceral em cães. Por ser uma zoonose que causa graves problemas de saúde pública e que vem crescendo cada vez mais no Brasil, cabe aos médicos veterinários assumirem o compromisso na conscientização sobre a importância do diagnóstico precoce além de promoverem o bem-estar animal e a saúde pública.(AU)

Canine visceral leishmaniasis is a zoonotic disease, affecting humans and several species of wild and domestic animals. The objective of the present study was to carry out a literature review on the use of miltefosine in the clinical treatment of dogs with visceral leishmaniasis. This is a literature review, which was carried out through consultations with periodicals and books present in the Cesmac library. Databases such as: Capes portal, SCIELO, Google Scholar; research in monographs, theses and dissertations. Caused by the protozoan Leishmania chagasi, with the domestic dog being the main reservoir of this protozoan. As it represents a serious public health problem and is considered a potentially fatal disease (when not treated early and properly), it is important that the clinician is familiar with the clinical signs, complementary exams and main therapeutic protocols, especially the use of miltefosine in the treatment of visceral leishmaniasis in dogs. As it is a zoonosis that causes serious public health problems and that has been growing more and more in Brazil, it is up to veterinarians to make a commitment to raise awareness of the importance of early diagnosis in addition to promoting animal welfare and public health.(AU)

La leishmaniosis visceral canina es una enfermedad zoonótica que afecta a los seres humanos y a varias especies de animales salvajes y domésticos. El objetivo de este estudio fue realizar una revisión bibliográfica sobre el uso de la miltefosina en el tratamiento clínico de perros con leishmaniosis visceral. Se trata de una revisión bibliográfica, que se realizó mediante consultas a publicaciones periódicas y libros presentes en la biblioteca del Cesmac. Se utilizaron bases de datos como: portal Capes, SCIELO, Google Académico; investigación en monografías, tesis y disertaciones. Causada por el protozoo Leishmania chagasi, siendo el perro doméstico el principal reservorio de este protozoo. Dado que representa un grave problema de salud pública y se considera una enfermedad potencialmente mortal (cuando no se trata de forma temprana y adecuada), es importante que el clínico esté familiarizado con los signos clínicos, las pruebas adicionales y los principales protocolos terapéuticos, especialmente el uso de miltefosina en el tratamiento de la leishmaniosis visceral en perros. Siendo una zoonosis que causa graves problemas de salud pública y que viene creciendo cada vez más en Brasil, corresponde a los veterinarios asumir el compromiso de concienciar sobre la importancia del diagnóstico precoz y promover el bienestar animal y la salud pública.(AU)

The heterologous expression of Escherichia coli MutT enzyme is involved in the protection against oxidative stress in Leishmania braziliensis

BACKGROUND Oxidative stress is responsible for generating DNA lesions and the 8-oxoguanine (8-oxoG) is the most commonly lesion found in DNA damage. When this base is incorporated during DNA replication, it could generate double-strand DNA breaks and cellular death. MutT enzyme hydrolyzes the 8-oxoG from the nucleotide pool, preventing its incorporation during DNA replication. OBJECTIVES To investigate the importance of 8-oxoG in Leishmania infantum and L. braziliensis, in this study we analysed the impact of heterologous expression of Escherichia coli MutT (EcMutT) enzyme in drug-resistance phenotype and defense against oxidative stress. METHODS Comparative analysis of L. braziliensis and L. infantum H2O2 tolerance and cell cycle profile were performed. Lines of L. braziliensis and L. infantum expressing EcMutT were generated and evaluated using susceptibility tests to H2O2 and SbIII, cell cycle analysis, γH2A western blotting, and BrdU native detection assay. FINDINGS Comparative analysis of tolerance to oxidative stress generated by H2O2 showed that L. infantum is more tolerant to exogenous H2O2 than L. braziliensis. In addition, cell cycle analysis showed that L. infantum, after treatment with H2O2, remains in G1 phase, returning to its normal growth rate after 72 h. In contrast, after treatment with H2O2, L. braziliensis parasites continue to move to the next stages of the cell cycle. Expression of the E. coli MutT gene in L. braziliensis and L. infantum does not interfere in parasite growth or in susceptibility to SbIII. Interestingly, we observed that L. braziliensis EcMutT-expressing clones were more tolerant to H2O2 treatment, presented lower activation of γH2A, a biomarker of genotoxic stress, and lower replication stress than its parental non-transfected parasites. In contrast, the EcMutT is not involved in protection against oxidative stress generated by H2O2 in L. infantum. MAIN CONCLUSIONS Our results showed that 8-oxoG clearance in L. braziliensis is important to avoid misincorporation during DNA replication after oxidative stress generated by H2O2.

Leishmania infantum induces high phagocytic capacity and intracellular nitric oxide production by human proinflammatory monocyte

BACKGROUND The mechanism of resistance to SbIII in Leishmania is complex, multifactorial and involves not only biochemical mechanisms, but also other elements, such as the immune system of the host. OBJECTIVES In this study, putative changes in the immunological profile of human monocytes infected with wild-type (WT) and antimony (SbIII)-resistant Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum lines were evaluated. METHODS Susceptibility assays WT and SbIII-resistant L. braziliensis and L. infantum were performed using lines THP-1 human monocytic lineage. Phagocytic capacity, cytokine profile, intracellular nitric oxide (NO) production and surface carbohydrate residues profile were performed in peripheral blood monocytes by flow cytometry. FINDINGS The phagocytic capacity and intracellular NO production by classical (CD14++CD16-) and proinflammatory (CD14++CD16+) monocytes were higher in the presence of L. infantum lines compared to L. braziliensis lines. The results also highlight proinflammatory monocytes as the cellular subpopulation of major relevance in a phagocytosis event and NO expression. It is important to note that L. infantum induced a proinflammatory cytokine profile characterised by higher levels of TNF-α in culture supernatant than L. braziliensis. Conversely, both Leishmania lines induce high levels of IL-6 in culture supernatant. Analysis of the expression profile of surface carbohydrates showed that L. braziliensis presents 4.3-fold higher expression of galactose(β1,4)N-acetylglucosamine than L. infantum line. Interestingly, the expression level of α-N-acetylgalactosamine residues was 2-fold lower in the SbIII-resistant L. braziliensis line than its counterpart WT line, indicating differences in surface glycoconjugates between these lines. MAIN CONCLUSIONS Our results showed that L. braziliensis and L. infantum induce different innate immune responses and a highly inflammatory profile, which is characteristic of infection by L. infantum, the species associated with visceral disease.

Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

In vitro activity of phospholipase A2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L) infantum chagasi

Background American visceral leishmaniasis is caused by the intracellular parasiteLeishmania (L.) infantum chagasi, and transmitted by the sand fly Lutzomyia longipalpis. Since treatment is based on classical chemotherapeutics with significant side effects, the search for new drugs remains the greatest global challenge. Thus, this in vitro study aimed to evaluate the leishmanicidal effect ofCrotalus durissus terrificus venom fractions on promastigote and amastigote forms of Leishmania (L.) infantum chagasi. Methods Phospholipase A 2 (PLA 2 ) and a pool of peptide fraction ( 3 kDa) were purified from Crotalusvenom. Furthermore, promastigotes and peritoneal macrophages of mice infected by amastigotes were exposed to serial dilutions of the PLA 2 and peptides at intervals varying between 1.5625 g/mL and 200 g/mL. Both showed activity against promastigotes that varied according to the tested concentration and the time of incubation (24, 48 and 72 h). Results MTT assay for promastigotes showed IC 50 of 52.07 g/mL for PLA2 and 16.98 g/mL for the peptide fraction of the venom. The cytotoxicity assessment in peritoneal macrophages showed IC50 of 98 g/mL and 16.98 g/mL for PLA 2 and peptide by MTT assay, respectively. In peritoneal macrophages infected by Leishmania (L.) infantum chagasi amastigotes, the PLA 2 stimulated growth of parasites, and at higher doses reduced growth by 23 %. The peptide fraction prevented 43 % of the intracellular parasite growth at a dose of 16.98 g/mL, demonstrating the toxicity of this dose to macrophages. Both fractions stimulated H 2 O 2 production by macrophages but only PLA 2 was able to stimulate NO production. Conclusion We have demonstrated the in vitro leishmanicidal activity of the PLA2 and peptide fraction ofCrotalus venom. The results encourage further studies to describe the metabolic pathways involved in cell death, as well as the prospecting of molecules with antiparasitic activity present in the peptide fraction of Crotalus durissus terrificus venom.

Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment / Acumulación celular y efecto anti- Leishmania de la protoporfirina IX exógena y endógena después del tratamiento fotodinámico

Introduction: Photodynamic therapy (PDT) using 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) constitutes an interesting alternative for cutaneous leishmaniasis treatment. Objective: To evaluate the production of PpIXbased on the administration of ALA and MAL and the effect of ALA-PDTat cellular level on non-infected and infected THP-1 cells using Leishmania ( Viannia ) panamensis or Leishmania ( Leishmania ) infantum (syn Leishmania chagasi ) parasites. Materials and methods: Protoporphyrin IX (PpIX) production and mitochondrial colocalization were evaluated by confocal microscopy. Cell toxicities were evaluated after treatment with the compounds, followed by light irradiation (597-752 nm) at 2.5 J/cm 2 fluency using a colorimetric MTT assay for THP-1 cells and a standard microscopic analysis of parasites. Results were expressed as compound concentration activity against 50% of cells or parasites (CC 50 or IC 50 ). Results: ALA or MAL induced an endogenous PpIX with a red fluorescence localized mainly in the mitochondria inside human cells. ALA and MAL-PDT induced a similar range of toxicities on THP-1 cells (CC 50 0.16±0.01mM and 0.33±0.019 mM, respectively) without any apparent inhibition of intracellular parasites in the infected cells as compared to untreated controls. Exogenous PpIX-PDT was toxic to THP-1 cells (CC 50 0.00032±0.00002 mM), L. (L.) infantum (IC 50 0.003±0.0001 mM) and L. (V.) panamensis (IC 50 0.024±0.0001 mM) promastigotes. Conclusions: Despite the effectiveness of exogenous PpIX on promastigotes and the production of PpIX by human infected cells, treatment with ALA or MAL before irradiation was unable to completely destroy L. (L.) infantum or L. (V.) panamensis intracellular amastigotes.

Introducción. El tratamiento fotodinámico con ácido 5-aminolevulínico como inductor de la protoporfirina IX (ALA-PpIX) constituye una alternativa interesante en el tratamiento de la leishmaniasis cutánea. Objetivo. Evaluar la producción de protoporfirina IX (PpIX) a partir de la administración de ALA o MAL y el efecto de la PDT con ALA a nivel celular en células THP-1 no infectadas e infectadas con Leishmania ( Viannia ) panamensis o Leishmania ( Leishmania ) infantum (syn. Leishmania chagasi ). Materiales y métodos. La producción de protoporfirina IX y su ‘colocalización´ mitocondrial se evaluaron mediante microscopía ‘confocal´. Se evaluó la toxicidad celular después del tratamiento con los compuestos y la aplicación de irradiación de luz (597-752 nm) en una fluencia de 2,5 J/cm 2 mediante el empleo de la prueba colorimétrica con metil-tiazol-tetrazolio (MTT) en las células, y de métodos microscópicos estándar en los parásitos. Los resultados se expresaron como la concentración del compuesto activo en el 50 % de las células o parásitos (CC 50 o CI 50 ). Resultados. El ácido aminolevulínico o el metil-5-aminolevulinato indujeron la protoporfirina IX endógena en células humanas, y se observó fluorescencia de color rojo en las mitocondrias. La actividad del ácido aminolevulínico y del metil-5-aminolevulinato utilizados con terapia fotodinámica fue similar en las células THP-1 (CC 50 0,16±0,01 mM y 0,33±0,019 mM, respectivamente) y, aparentemente, no inhibió los parásitos en las células infectadas, en comparación con los controles. El tratamiento exógeno con protoporfirina IX y terapia fotodinámica fue tóxico para las células THP-1 (CC 50 0,00032 ±0,00002 mM) y para los promastigotes de L. (L .) infantum (IC 50 0,003±0,0001 mM) y L. ( V .) panamensis (CI 50 0,024±0,0001 mM). Conclusiones. A pesar de la ‘fotoactividad´ del tratamiento con protoporfirina IX en promastigotes y de su producción después del tratamiento con ácido aminolevulínico y metil-5-aminolevulinato en las células infectadas con Leishmania , no se observó daño en los amastigotes presentes en las células de L. ( L .) infantum o L . ( V .) panamensis .

effect of Vitis vinifera L. leaves extract on Leishmania infantum

Vitis vinifera L.is a traditional Asian herb widely used for different health problems. In the present research, the ethanolic and the aqueous extracts of Vitis vinifera L. leaves collected from shrub, grown in Tunisia, were prepared and evaluated for the antileishmanial activity against Leishmani ainfantum promastigotes. The inhibitory concentration 50 [IC[50]] was determined and the results showed that the etahnolic extract is more active than the aqueous one [IC[50]= 0.108 mg/mL]. Microscopic observations showed that the ethanolic extract promoted the destruction of cytoplasmic and nuclear membranes of Leishmani ainfantum promastigotes and altered the overall shape of the cell. In order to explain the difference of antileishmanial activity between ethanolic and aqueous extracts, anthocyanins amount was determined by spectrophotometry. It was found that the ethanolic extract is richer in anthocyanins than the aqueous one which can explain the higher antileishmanial activity of the ethanolic extract

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

Miltefosine induces metacaspase and PARP genes expression in Leishmania infantum

OBJECTIVES: Apoptosis is the process of programmed cell death (PCD) that occurs in both animal and plant cells. Protozoan parasites possess metacaspase and these caspase-related proteases could be involved in the PCD pathways in these organisms. Therefore we analyzed the activities of metacaspase and PARP genes in Leishmania infantum (MCAN/IR/96/LON49) treated with miltefosine. MATERIALS AND METHODS: Anti-leishmania activity of miltefosine was studied by treatment of cultured promastigotes with various concentration of miltefosine. MTT assay and Annexin-V FLUOS staining by using FACS flow cytometry methods were used. Cytotoxic potential of HePC on the amastigots of L.infantum was evaluated in J774 cell line. In addition, metacaspase and PARP genes expression of treated L. infantum were studied. RESULTS: Miltefosine led to dose-dependent death of L. infantumwith features compatible with apoptosis. Over expression of metacaspase and PARP was seen 6 hr after treatment. CONCLUSIONS: Our study showed that miltefosine exerts cytotoxic effect on L. infantum via an apoptotic-related mechanism.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil / Atividade leishmanicida e citotoxicidade de constituintes químicos de duas espécies de Annonaceae cultivadas no nordeste do Brasil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10 percent phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

INTRODUÇÃO: A leishmaniose visceral é uma enfermidade endêmica em 88 países, com um total de 12 milhões de pessoas infectadas e 350 milhões em risco. Na procura de novos agentes com ação leishmanicida, alcalóides e acetogeninas isoladas de Annona squamosa e Annona muricata, foram testados contra as formas promastigotas e amastigotas de Leishmania chagasi. MÉTODOS: Foram preparados extratos com metanol: água (80: 20) das folhas de A. squamosa e sementes de A. muricata que foram extraídos com solução de ácido fosfórico 10 por cento e solventes orgânicos, para obter extratos ricos em alcalóides e acetogeninas. Estes extratos foram cromatografados em coluna de sílica gel sendo eluídos com solventes de diferentes polaridades para o isolamento dos constituintes, e feita a determinação estrutural por análise espectroscópica. Os constituintes isolados foram testados contra Leishmania chagasi, responsável pela leishmaniose visceral, utilizando o teste MTT. Testes de toxicidade foram realizados em todos os compostos isolados, sendo utilizadas células RAW 264.7. RESULTADOS: Um alcalóide benzilisoquinolínico, O-metilarmepavina, e uma C37-triidróxi-acetogenina com anel bistetrahidrofurânico adjacente foram isolados de A. squamosa e duas acetogeninas annonacinona e corossolona da A. muricata. O alcalóide mostrou um índice de inibição médio (IC50) de 23,3µg/mL e as acetogeninas apresentaram IC50 variando entre 25,9 a 37,6µg/mL contra promastigotas, e no ensaio de amastigotas, o IC50 valores variaram entre 13,5 a 28,7 µg/mL. A toxicidade mostrou resultados que variaram entre 43,5 a 79,9µg/mL. CONCLUSÕES: Estes resultados caracterizam A. squamosa e A. muricata como fontes potenciais de agentes leishmanicidas.

Chemical composition and antiprotozoal activities of Colombian Lippia spp essential oils and their major components

The chemical composition and biological activities of 19 essential oils and seven of their major components were tested against free and intracellular forms of Leishmania chagasi and Trypanosoma cruzi parasites as well as Vero and THP-1 mammalian cell lines. The essential oils were obtained from different species of Lippia, a widely distributed genus of Colombian plants. They were extracted by microwave radiation-assisted hydro-distillation and characterised by GC-FID and GC-MS. The major components were geranial, neral, limonene, nerol, carvacrol, p-cymene, ã-terpinene, carvone and thymol. The essential oil of Lippia alba exhibited the highest activity against T. cruzi epimastigotes and intracellular amastigotes with an IC50 of 5.5 ìg/mL and 12.2 ìg/mL, respectively. The essential oil of Lippia origanoides had an IC50 of 4.4 ìg/mL in L. chagasi promastigotes and exhibited no toxicity in mammalian cells. Thymol (IC50 3.2 ± 0.4 ìg/mL) and S-carvone (IC50 6.1 ± 2.2 ìg/mL), two of the major components of the active essential oils, were active on intracellular amastigotes of T. cruziinfected Vero cells, with a selective index greater than 10. None of the essential oils or major components tested in this study was active on amastigotes of L. chagasi infected THP-1 cells.

Two cases of visceral leishmaniasis in Colombia resistant to meglumine antimonial treatment / Dois casos de leishmaniose visceral da Colômbia, resistentes ao tratamento com antimoniato de meglumina

Visceral leishmaniasis (VL) affects over 500 000 people worldwide each year. The disease occurs in the Mediterranean basin, Central and South America and is caused by Leishmania infantum (syn L. chagasi). VL is an endemic disease in Colombia, particularly along the Caribbean coast and the Magdalena River Valley and 90% of VL cases occur in children under the age of five. The first line of treatment is chemotherapy with pentavalent antimonial compounds, including sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantime®). These compounds are the ones most used in Colombia, at a dose of 20 mg/kg/day for 28 days. Nevertheless resistance of L. infantum to pentavalent antimonials is becoming an important problem. No cases of VL resistant to pentavalent antimonial compounds have previously been reported from Colombia. This report describes the two cases of VL resistance to antimonial compounds in a girl and a boy who did not respond to previous treatment with Pentacarinat® and Glucantime® regimens but were treated successfully with liposomal amphotericin B. Based on our findings, we recommend liposomal amphotericin B as the first line of treatment for VL due to its low toxicity, shorter administration period and the low price obtained by WHO.

A leishmaniose visceral (VL) afeta aproximadamente 500000 pessoas anualmente no mundo. A doença ocorre no mediterrâneo, na América Central e na América do Sul, sendo causada por Leishmania infantum (syn. L. chagasi). Na Colômbia VL é uma doença endêmica, presente no litoral do Caribe e no Vale do rio Magdalena sendo que 90% de casos de VL ocorrem em crianças menores de cinco anos. O principal tratamento é a quimioterapia com compostos de antimoniais pentavalentes, incluindo stibogluconato de sódio (Pentostam®) e antimoniato de meglumina (Glucantime®). Estes compostos são os mais usados na Colômbia em dosagem de 20 mg/kg/dia durante 28 dias. Entretanto, a resistência de L. infantum aos antimoniais pentavalentes está se tornando problema importante. Na Colômbia não existiam relatos de casos de VL resistentes aos antimoniais pentavalentes. Este trabalho descreve os dois primeiros casos colombianos de VL resistentes aos compostos antimoniais em uma menina e um menino, que foram tratados com regime de Pentamidina e Glucantime®, e demonstra o sucesso obtido no tratamento com anfotericina B liposomal. Em conclusão, sugerimos como primeira opção de tratamento a anfotericina B liposomal porque é altamente efetiva no tratamento da VL, dada sua baixa toxicidade, curtos períodos de administração e o baixo preço obtido pela organização Médicos Sem Fronteiras.

In vitro and in vivo activity of meglumine antimoniate produced at Farmanguinhos-Fiocruz, Brazil, against Leishmania (Leishmania) amazonensis, L (L) chagasi and L (Viannia) braziliensis

The leishmanicidal activity of four batches of meglumine antimoniate, produced in Farmanguinhos-Fiocruz, Brazil (TAMs), was assessed and compared to Glucantime®-Aventis Pharma Ltda. Using the amastigote-like in vitro model, the active concentrations of Sb v varied from 10µg/ml to 300 µg/ml for L. (L.) chagasi and from 50µg/ml to 300µg/ml for L. (L.) amazonensis, with no statistically significant differences among the four batches of TAMs and Glucantime®. The inhibitory concentrations (IC50) determined by the amastigote-infected macrophage model for TAM01/03 and Glucantime® were, respectively: 26.3µg/ml and 127.6µg/ml for L. chagasi, 15.4µg /ml and 22.9µg/ml for L. amazonensis, and 12.1µg/ml and 24.2µg/ml for L. (V.) braziliensis. The activities of the four batches of TAMs were confirmed in an in vivo model by assessing, during eight weeks skin lesions caused by L. braziliensis in hamster that were treated with 20mg Sb v/Kg/day for 30 consecutive days. The meglumine antimoniate produced by Farmanguinhos was as effective as the reference drug, Glucantime®-Aventis, against three species of Leishmania that are of medical importance in Brazil.

Efficacy and tolerabiliy of loposomal amphotericin B (Ambisome) in the treatment of visceral leishmaniasis in Brazil

Thirty-two patients were enrolled in an open-label, dose/schedule ranging clinical trial to evaluate the efficacy and tolerability of loposomal amphotericin B (Ambisome) in the treatment of visceral leishmaniasis. All the patients received a dose of 2mg/kg daily for the first 4 days, followed by a single repeat dose of 2mg/kg at day 10 in 4 patients (total dose 10mg/kg); repeat doses on days 5, 6, and 10 in 13 patients (total dose 14/kg); or daily doses were continued on days 5 through 10 in 15 patients (total dose 20mg/kg). Patients had a mean age of 9 years, ranging between 3 and 26 years. Their mean weight was 25.9kg, ranging between 9.5kg and 75kg. All patients had splenomegaly,31/32 hepatomegaly, and 20 patients tested had leishmania documented on splenic aspirate. Six of the 32 patients were treated after relapse following antimony therapy. The duration of illness prior to therapy was a mean of 2 months, ranging between 2 weeks and 23 months. During and after treatment, there were significant reductions in liver and spleen size, and significant increases in body weight, hemoglobin levels and white blood cell counts. All patients showed initial cure at the 1 mouth follow-up. Seven patients relapsed between 2 and 6 months after the start of treatment. There was no dose relationship to the occurrence of relapse. The relapse rate in children 5 years of age or less was 7/15 (47 percent). Associated causes of relapse were refractory disease (i.e., previous relapses) in 2, severe malnutrition in 1, and concurrent disease (meningococcal meningitis) in 1. In the other 2 cases, no associated event was observed except young age (ages 3 and 5 years). One relapsed patient was treated successfully with 14 days of lipid amphotericin B, and the others were cured by use of antimony for 20 to 30 days. There were no dose related adverse events. The most common event was fever which occurred in 13/32 patients (41 percent); 3/4 patients in the 10mg dose group, 7/13 in the 14mg dose group, and 3/15 in the 20mg dose group. Three patients had cardiac arrhythmia, one also with myocarditis diagnosed 2 weeks after therapy was discontinued. One patient developed hepatitis after dose 3 and the drug was discontinued. We concluded that liposomal amphotericin B is effective in a daily dose of 2mk/kg given for 5-10 doses as an initial cure, but that relapse occurs in young children, particularly those with documented treatment resistant disease or concurrent malnutrition...

preliminary study on the inhibitory action of mystatin on the promastigote forms of leishmania tropica and leishmania infantum isolates of Pakistan

Leishmania is the etiological agent of a group of parasitic disease Leishmaniases. Recent reports show that the disease is on the rise in Pakistan. As no vaccine is yet available for any type of leishmaniasis, chemotherapy is the only means of controlling the disease. As a part of our project to find out new antileishmanial compounds, we have studied the antileishmanial activity of nystatin [mycostatin], a polyene compound, on the promastigote forms of L. tropica and L. infantum isolates of Pakistan. In in vitro cultivation, the EC50 values [the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value] obtained for Nystatin against the promastigote of L. tropica and L. infantum were 19.5-19.8 iu/ml. The mode of action proposed for this drug is same as for famous leishmanicidal drug Amphotericin B as both of these polyene drugs strongly and selectively binds to the sterols present on the surface of the parasite result in the formation of unusual pores / gaps on the surface that result in the leakage of the ions. This leakage finally leads to the destruction of the cell