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.

Drug resistance mechanisms in clinical isolates of Leishmania donovani.

Leishmania are protozoan parasites distributed worldwide. About 1.5-2.0 million cases are reported in the world annually from this disease and the death toll is estimated to be 57,000. Along with Brazil, Sudan and Bangladesh, India contributes to 90 per cent of the global burden of visceral leishmaniasis (VL). The absence of effective vaccines and vector control programmes, makes chemotherapy the most widely used tool against leishmaniasis. Chemotherapy based on pentavalent antimonials has been used for more than 50 years and remains the mainstay for treatment of leishmaniasis. Clinical resistance to pentavalent antimonials, in the form of sodium antimony gluconate (SAG), has become a major problem in the treatment of kala-azar (visceral leishmaniasis) in India. The mechanism of resistance is unclear in these clinical isolates although a lot of work has been carried out with Leishmania mutants selected in vitro by step-wise increasing drug concentration using the antimony related metal arsenic and more recently sodium antimony gluconate. We for the first time, investigated the molecular aspect of drug resistance in clinically confirmed sodium antimony gluconate resistant field isolates and found that the parasite evaded cytotoxic effects of therapy by enhanced efflux of drugs through overexpressed membrane proteins belonging to the superfamily of ABC (ATP-binding cassette) transporters. Additionally, our study also points towards cell surface changes in resistant isolates.

Childhood visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani and transmitted by the bite of infected sandfly Phlebotomus argentipes. Nearly half of the VL cases occur in children (childhood or paediatric VL). The clinical manifestations of childhood VL are more or less same as in the adults. Prolonged fever with anorexia and loss of appetite are the major presenting features. Marked enlargement of the spleen and liver (spleen larger than liver) with moderate to severe anaemia and changes in hair take place. Bacterial infection is a common coinfection and intestinal parasitic infestations are very common in children with VL. Liver function tests, blood, urine and stool may show abnormalities. Confirmation of diagnosis is made by demonstration of parasite by microscopic examination and culture of materials obtained by bone marrow aspiration or splenic puncture. Sodium antimony gluconate (stibogluconate) has been the drug of choice for over past 50 yr. Pentamidine isothionate, though effective is relatively toxic. Amphotericin B is the most effective drug for the treatment of VL. Miltefosine is the first-ever oral drug, is highly effective. Post kala-azar dermal leishmaniasis (PKDL) in children poses a therapeutic challenge. In the absence of an ideal vaccine for VL, control measures would essentially include prevention of transmission through vector control and community awareness.

Visceral leishmaniasis - current therapeutic modalities.

Major therapeutic obstacles in the treatment of visceral leishmaniasis (VL) include the alarming increase in antimonial unresponsiveness especially in Bihar, India and relapses in HIV-Leishmania co-infected patients. The therapeutic armamentarium for VL is currently plagued with several limitations as the available drugs are toxic, majority are effective only parenterally and need to be administered for extended periods. The first orally effective drug, miltefosine has been approved for treating VL. In antimony refractory zones, pentavalent antimony has been largely replaced by amphotericin B deoxycholate, but prolonged hospitalization, toxic effects, and requirement for monitoring greatly hamper its widespread application in endemic regions. Lipid formulations of amphotericin B, a remarkable advance in amphotericin B therapy, have greatly reduced toxicity enabling large doses to be delivered over a short period. Even a single dose treatment with liposomal amphotericin B cures > 90 per cent patients; however, the stumbling block is its prohibitive cost that precludes its widespread accessibility in endemic countries. Studies using paromomycin in VL are encouraging, and judging by the preliminary results of a recently concluded phase III trial, it could be an extremely useful and affordable antileishmanial drug. Other orally effective drugs include the azoles and allopurinol but these have met with limited success owing to either poor efficacy or unacceptable toxicity. Sitamaquine has undergone limited evaluation, and the data suggest effective antileishmanial activity; its role has to be delineated for which additional developmental studies are proposed. This review highlights the progress made in the treatment of VL, including the multiple mechanisms of action of antileishmanial drugs with a view to enable the researcher to undertake the challenge of providing affordable and effective chemotherapy.

Magnitude of unresponsiveness to sodium stibogluconate in the treatment of visceral leishmaniasis in Bihar.

BACKGROUND: The Indian government proposes to eliminate kala-azar, which has been a serious public health problem in Bihar. This study aimed to assess the magnitude of unresponsiveness to sodium stibogluconate in the treatment of new cases of visceral leishmaniasis and to identify the associated factors. METHODS: Patients with clinically and parasitologically confirmed visceral leishmaniasis (n = 182) who had received no prior treatment, were enrolled for the study. The patients were treated with sodium stibogluconate (20 mg/kg body weight; upper limit 850 mg), intramuscularly for 30 days. The vital parameters and side-effects, if any, were monitored. Patients who developed toxicity during treatment were excluded from the study but were given rescue treatment with liposomal amphotericin B. All patients who completed the treatment were followed up for 6 months. RESULTS: Unresponsiveness to sodium stibogluconate at the end of treatment was 43%. It was higher in women (48%) compared to men (40%). A significant association was observed between unresponsiveness and level of endemicity (p = 0.0002), large spleen size (p = 0.04) and immune response (migration inhibition factor) (p = 0.00002). At the end of 6 months' follow up, 27% of patients relapsed, giving a total unresponsiveness rate of 58%. CONCLUSION: Unresponsiveness to sodium stibogluconate is a serious problem in the management of patients with visceral leishmaniasis. In patients with factors associated with nonresponse to sodium stibogluconate, alternative drugs such as miltefosine or amphotericin B should be considered as first-line drugs.

Studies on stibanate resistant Leishmania donovani isolates of Indian origin.

Studies with 26 clones of L. donovani promastigotes derived from three different Indian isolates indicated that wild type parasites are mixture of stibanate sensitive and resistant cells. Both forms of the parasite were resistant to the drug. Infection with resistant parasites appears to be the primary reason of high rate of pentavalent antimony unresponsiveness among Indian kala-azar patients. It was observed that the resistant parasites originated as a result of irregular and often incomplete treatment of kala-azar patients with pentavalent antimonials.

Effect of sodium stibogluconate on hepatic mixed function oxidase system and marker enzymes of golden hamsters during Leishmania donovani infection.

During L. donovani infection in golden hamsters, tremendous hepatic damage was observed as apparent from increased activities of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, succinate dehydrogenase, glucose-6-phosphatase and acid ribonuclease. The levels of cytochrome P-450 and related monooxygenases, viz. aniline hydroxylase and aminopyrine-N-demethylase registered significant decrease in infected animals. Sodium stibogluconate, a standard antileishmanial drug, though caused the removal of parasites from infected tissues, but did not help in the recovery of deranged hepatic markers. The results explain the higher mortality of stibanate treated infected animals as compared to untreated animals infected with L. donovani.

Glucantine resistant Leishmania promastigotes are sensitive to pentostam

Diferentes amostras de leishmania foram analisadas quanto à susceptibilidade in vitro ao pentostam - uma cepa de L. (V) braziliensis considerada sensível ao glucantine, três cepas (duas L. (V) braziliensis e uma L. (L) amazonensis) consideradas naturalmente resistentes ao glucantine, uma linhagem resitente (L. (V) guyanensis) selecionada in vitro pela exposiçäo em alta concentraçäo de droga. A elevada sensibilidade destas amostras em contraposiçäo à resistência observada para a glucantine sugere existir relaçäo entre a estrutura química e a atividade destes compostos. Estes dados indicam a necessidade de uma avaliaçäo comparativa de atividade clínica do pentostam e do glucantime no tratamento da leishamniose

Effect of sodium stibogluconate on the status of interleukin-I production in normal & Leishmania donovani infected BALB/c mice.

Sodium stibogluconate, did not bring about significant increase in the production of IL-1, when both specific leishmanial antigen, or non specific Staphylococcus epidermidis was used as stimulus in normal uninfected animals. However, Staph. epidermidis was found to be a better stimulus as it brought about a significant increase (P less than 0.001) in IL-1 production when compared with leishmania antigen. In BALB/c mice infected with L. donovani there was a significant reduction (P less than 0.001) in IL-1 levels on various post infection days irrespective whether Staph. epidermidis or leishmanial antigen was used as stimulus when compared with controls. IL-1 levels were significantly increased (P less than 0.05) when L. donovani infected animals were treated with SSG, after 14 days post infection, irrespective of the stimuli used.