Therapy with Sodium Stibogluconate in Stearylamine-Bearing Liposomes Confers Cure against SSG-Resistant Leishmania donovani in BALB/c Mice

Abstract

BackgroundResistance of Leishmania donovani to pentavalent antimonials, the first-line treatment of visceral leishmaniasis (VL), has become a critical issue worldwide. Second-line and new drugs are also not devoid of limitations. Suitable drug-delivery systems can improve the mode of administration and action of the existing antimonials, thus increasing their clinical life.Methodology/Principal FindingsWe investigated the efficacy of sodium stibogluconate (SSG) in phosphatidylcholine (PC)–stearylamine-bearing liposomes (PC-SA-SSG), PC-cholesterol liposomes (PC-Chol-SSG) and free amphotericin B (AmB) against SSG-resistant L. donovani strains in 8-wk infected BALB/c mice. Animals were sacrificed and parasites in liver, spleen and bone marrow were estimated 4-wk post-treatment by microscopic examination of stamp smears and limiting dilution assay. A set of PC-SA-SSG and AmB treated mice were further studied for protection against reinfection. Serum antibodies and cytokine profiles of ex-vivo cultured splenocytes were determined by ELISA. Uptake of free and liposomal SSG in intracellular amastigotes was determined by atomic absorption spectroscopy. Rhodamine 123 and 5-carboxyfluorescein, known substrates of Pgp and MRP transporter proteins, respectively, were used in free and liposomal forms for efflux studies to estimate intracellular drug retention. Unlike free and PC-Chol-SSG, PC-SA-SSG was effective in curing mice infected with two differentially originated SSG-unresponsive parasite strains at significantly higher levels than AmB. Successful therapy correlated with complete suppression of disease-promoting IL-10 and TGF-β, upregulation of Th1 cytokines and expression of macrophage microbicidal NO. Cure due to elevated accumulation of SSG in intracellular parasites, irrespective of SSG-resistance, occurs as a result of increased drug retention and improved therapy when administered as PC-SA-SSG versus free SSG.Conclusions/SignificanceThe design of this single-dose combination therapy with PC-SA-SSG for VL, having reduced toxicity and long-term efficacy, irrespective of SSG-sensitivity may prove promising, not only to overcome SSG-resistance in Leishmania, but also for drugs with similar resistance-related problems in other diseases.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis

“…VL infection induces the T-cell anergy, and thus, effective cell-mediated response is necessary for disease suppression. 19 This impairment in T-cell response is successfully reversed by treatment with NLA and AmB, which reveals the clearance of the intracellular parasites. 48 Progression of disease is very much dependent on the induction of Th2-related cytokines, whereas disease resolution is attributable to Th1 cytokines.…”

“…[11][12][13][14][15][16][17][18] Depending upon their physical properties, liposomes are recognized as foreign particles after in vivo administration and are easily cleared by the reticuloendothelial system that harbors Leishmania parasites. [19][20][21] Thus, the association or incorporation of chemotherapeutic agents in liposomes has immense therapeutic benefits against the intracellular infection of Leishmania. Liposomal drug delivery holds great promise for the treatment of infectious disease and is already used in clinical practice.…”

Cationic Liposomal Sodium Stibogluconate (SSG), a Potent Therapeutic Tool for Treatment of Infection by SSG-Sensitive and -Resistant Leishmania donovani

“…In addition, we reported earlier that cationic liposomes bearing egg phosphatidylcholine (PC) and stearylamine (SA) (i.e., PC-SA) had direct killing activity against various strains of Leishmania, an activity not observed with anionic or neutral liposomes (18,19). Encapsulation of a suboptimal dose of SSG within these cytolytic vesicles (i.e., PC-SA-SSG) further enhanced the therapeutic potential of either of the monotherapies, as single-dose treatments against both SSGresponsive and non-SSG-responsive established murine VL (20,21). The aim of antileishmanial therapy is to achieve maximum efficacy with minimum doses of the drug and carrier for a successful cure.…”

“…For comparative evaluation of efficacy, groups of BALB/c mice were treated with a single injection of 75 g of SSG in 5 mg of PC either as PC-DDAB-SSG or as PC-SA-SSG. At 90 days postinfection, animals were sacrificed, and liver and spleen parasite burdens were determined by examining methanol-fixed, Giemsa-stained imprints of the cut organs; results were expressed as Leishman-Donovan units (LDU), calculated as the number of amastigotes per 1,000 nucleated cells ϫ organ weight (mg) (21). In the case of bone marrow, 100 l of PBS-flushed bone marrow suspension was smeared over glass slides, fixed with methanol, and then stained with Giemsa stain to estimate the number of parasites (21).…”

“…At 90 days postinfection, animals were sacrificed, and liver and spleen parasite burdens were determined by examining methanol-fixed, Giemsa-stained imprints of the cut organs; results were expressed as Leishman-Donovan units (LDU), calculated as the number of amastigotes per 1,000 nucleated cells ϫ organ weight (mg) (21). In the case of bone marrow, 100 l of PBS-flushed bone marrow suspension was smeared over glass slides, fixed with methanol, and then stained with Giemsa stain to estimate the number of parasites (21). In selected experiments, limiting dilution assays were performed, in which homogenates of liver and spleen were serially diluted and cultured for 2 weeks.…”