Abstract Background: Cutaneous leishmaniasis (CL) is a parasitic disease caused by the protozoan Leishmania spp. Pentavalent antimonial agents have been used as an effective therapy, despite their side effects and resistant cases. Their pharmacokinetics remain largely unexplored. This study aimed to investigate the pharmacokinetic profile of meglumine antimoniate in a murine model of cutaneous leishmaniasis using a radiotracer approach. Methods: Meglumine antimoniate was neutron-irradiated inside a nuclear reactor and was administered once intraperitoneally to uninfected and L. amazonensis-infected BALB/c mice. Different organs and tissues were collected and the total antimony was measured. Results: Higher antimony levels were found in infected than uninfected footpad (0.29% IA vs. 0.14% IA, p = 0.0057) and maintained the concentration. The animals accumulated and retained antimony in the liver, which cleared slowly. The kidney and intestinal uptake data support the hypothesis that antimony has two elimination pathways, first through renal excretion, followed by biliary excretion. Both processes demonstrated a biphasic elimination profile classified as fast and slow. In the blood, antimony followed a biexponential open model. Infected mice showed a lower maximum concentration (6.2% IA/mL vs. 11.8% IA/mL, p = 0.0001), a 2.5-fold smaller area under the curve, a 2.7-fold reduction in the mean residence time, and a 2.5-fold higher clearance rate when compared to the uninfected mice. Conclusions: neutron-irradiated meglumine antimoniate concentrates in infected footpad, while the infection affects antimony pharmacokinetics.

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.

Pentavalent antimonials such as meglumine antimoniate (MA) are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania) infantum chagasi-infected mice. MA (Glucantime(r)) was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

OBJETIVO: Avaliar o comportamento mecânico e os efeitos no processo de calcificação pós-implante do pericárdio bovino tratado com glutaraldeído submetido a várias doses de radiação gama. MÉTODO: Pericárdios bovinos fixados com glutaraldeído foram submetidos a radiação gama, nas doses de 0 a 10000 Gy. Seis amostras de cada grupo foram avaliadas pela microscopia óptica, determinação da temperatura de desnaturação do colágeno e ensaio mecânico de tração e implantadas subcutaneamente em ratos. Após quatro meses do implante, as amostras foram explantadas e o conteúdo de Ca2+ determinado pela espectrometria de absorção atômica. RESULTADOS: Níveis de Ca2+ (em µg/mg): 0 Gy (controle) - 194,45; 50 Gy - 154,64; 100 Gy - 169,37; 200 Gy - 163,64; 500 Gy - 199,89; 1000 Gy - 184,02; 2000 Gy - 198,95; 5000 Gy - 227,95 e 10000 Gy - 362,62. Houve alteração significativa no comportamento mecânico do tecido irradiado, quando comparado ao grupo controle, mesmo com o emprego de baixas doses de radiação. CONCLUSÃO: O emprego da radiação gama no pericárdio bovino tratado com glutaraldeído não reduziu os níveis de Ca2+ em implantes subcutâneos em ratos por quatro meses e promoveu alteração significativa no comportamento mecânico do tecido, com redução na sua resistência, quando comparados ao grupo controle.

OBJECTIVE: To evaluate the effect of gamma irradiation on glutaraldehyde-fixed bovine pericardium. METHOD: Glutaraldehyde-fixed bovine pericardium was exposed to gamma radiation (doses from 0 to 10000 Gy). Six samples from each of nine groups were evaluated by optic microscopy, and shrinking and mechanical tests and the denaturation temperature was determined. Additionally, they were subcutaneously implanted in rats and after four months they were explanted and Ca2+ levels measured by atomic absorption spectroscopy. RESULTS: The Ca2+ levels were (in µg/mg): control (0 Gy) - 194.45; 50 Gy - 154.64; 100 Gy - 169.37; 200 Gy - 163.64; 500 Gy - 199.89; 1000 Gy - 184.02; 2000 Gy - 198.95; 5000 Gy - 227.95; 10000 Gy - 362.62. Gamma irradiation caused a significant effect on the biomechanical properties of the tissue. CONCLUSION: e-fixed bovine pericardium.

OBJETIVO: Avaliar possível efeito sinérgico dos agentes anticalcificantes mais promissores investigados na literatura, por meio de tratamento seqüencial. MÉTODO: Pericárdios bovinos fixados com glutaraldeído foram tratados com: aquecimento a 50ºC, éter dietílico 80%, quitosana 0,125%, heparina, NaBH4 e formaldeído 4%. Amostras foram avaliadas pela microscopia óptica, determinação da temperatura de desnaturação do colágeno e ensaio mecânico de tração e implantadas subcutaneamente em ratos. Após quatro meses do implante, as amostras foram explantadas e o conteúdo de Ca2+ determinado pela espectrometria de absorção atômica. RESULTADOS: Níveis de Ca2+ (em µg/mg): Controle -194,45; aquecimento a 50ºC - 6,87; éter dietílico - 3,69; quitosana - 68,89; quitosana+heparina - 6,81; formaldeído - 107,34; tratamento seqüencial - 0,17. O comportamento mecânico variou de acordo com os tratamentos empregados. CONCLUSÃO: O tratamento seqüencial foi efetivo na inibição da calcificação e o tecido apresentou comportamento mecânico adequado à confecção de biopróteses.

OBJECTIVE: To evaluate a possible synergic effect of the most promising anti-calcifying agents investigated in literature by sequential treatment. METHOD: Glutaraldehyde-fixed bovine pericardium was treated by: heating to 50ºC, 80% ether, 0.125% chitosan, heparin, NaBH4 and 4% formaldehyde. Samples were evaluated by optic microscopy, shrinking and mechanical tests and implanted subcutaneously in rats. Four months later the samples were explanted and Ca2+ levels measured by atomic absorption spectroscopy. RESULTS: Ca2+ levels were (in µg/mg): control - 194.45; heating at 50ºC - 6.87; ether - 3.69; chitosan - 68.89; heparin - 6.81; formaldehyde - 107.34; sequential treatment - 0.17. The mechanical characteristics changed according to the treatments employed. CONCLUSION: Sequential treatment was effective to inhibit calcification and the tissue showed an adequate mechanical structure for the manufacture of bioprosthesis.

Os antimoniais pentavalentes, como o antimoniato de meglumina (Glucantime® ) ou estibogluconato de sódio (Pentostam® ), são o principal tratamento para a leishmaniose, um complexo de doenças causadas pelo protozoário parasita Leishmania, uma doença endêmica e negligenciada no Brasil. Apesar do seu uso clínico por mais de meio século, seu mecanismo de ação, toxicidade e dados de farmacocinética permanecem desconhecidos. Os métodos analíticos para determinação de antimônio em sistemas biológicos são complexos e apresentam baixa sensibilidade. Estudos utilizando radiotraçadores têm papel potencial no desenvolvimento farmacológico. O objetivo deste estudo foi desenvolver um radiotraçador de antimônio, com propriedades físicas e biológicas adequadas. O antimoniato de meglumina foi irradiado por nêutrons no reator nuclear IEA-R1, produzindo dois radioisótopos: 122Sb e 124Sb, com alta pureza radionuclídica e boa atividade específica. Este composto mostrou atividade antileishmania similar ao fármaco não irradiado. O uso de radiotraçadores, facilmente produzidos por irradiação por nêutrons pode ser um importante instrumento para elucidar questões sobre a farmacologia dos antimoniais.

Pentavalent antimony, as meglumine antimoniate (Glucantime® ) or sodium stibogluconate (Pentostam® ), is the main treatment for leishmaniasis, a complex of diseases caused by the protozoan Leishmania, and an endemic and neglected threat in Brazil. Despite over half a century of clinical use, their mechanism of action, toxicity and pharmacokinetic data remain unknown. The analytical methods for determination of antimony in biological systems remain complex and have low sensitivity. Radiotracer studies have a potential in pharmaceutical development. The aim of this study was to obtain a radiotracer for antimony, with suitable physical and biological properties. Meglumine antimoniate was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This compound showed the same antileishmanial activity as the native compound. The use of the radiotracers, easily created by neutron irradiation, could be an interesting tool to solve important questions in antimonial pharmacology.