Evaluation of toxicity & therapeutic efficacy of a new liposomal formulation of amphotericin B in a mouse model

Abstract

Background & objectives: Current therapy for leishmaniasis is limited and unsatisfactory. Amphotericin B, a second-line treatment is gradually replacing antimonials, the first-line treatment and is used as the preferred treatments in some regions. Though, presently it is the only drug with highest cure rate, its use is severely restricted by its acute toxicity. In the present study novel lipid-amphotericin B formulations with lower toxicity than the parent drug were evaluated for the treatment of visceral leishmaniasis (VL) in a mouse model.

Methods: The toxicity and therapeutic efficacy of a new amphiphilic formulation of amphotericin B (Kalsome10) was compared to that of amphotericin B deoxycholate (Fungizone) in a mouse model of VL using quantitative real-time PCR (qRT-PCR).

Results: The toxicity of amphotericin B was significantly less with liposomal formulation as compared to the deoxycholate form, evidenced by reduced nephrotoxicity and higher tolerated dose in BALB/c mice. The therapeutic efficacy was evaluated by quantitative real time (RT) PCR using primers highly specific for the ITS region of Leishmania donovani. There was reduction in parasite load by 2 log unit after 7 days of treatment and finally resulting in complete clearance of parasite from infected mice after 30 days of treatment with Kalsome10.

Interpretation & conclusions: This new formulation showed a favourable safety profile and better efficacy when compared to conventional amphotericin B. If production cost is kept low, it may prove to be a feasible alternative to conventional amphotericin B.

Fig. 1

Flowchart summarizing the experimental details of the study

Fig. 2

Amphotericin B loaded liposomal microspheres (Kalsome™10) analyzed by scanning electron microscopy. (A) sonicated (A) nonsonicated preparation of Kalsome™10.

Fig. 3

Single dose drug toxicity study. Values are expressed as mean of three seperate experiments ± SEM.

Fig. 4

PCR detection of Leishmania infection in mice. BALB/c mice were infected with ~1×107 promastigotes/ml of L. donovani. After 3 wk post-infection liver and spleen samples were taken out under sterile conditions, DNA was isolated and subjected to PCR analysis for confirmation of infection. Mice injected with normal saline were served as negative control. Lane 1 - Positive control (KE-16 strain DNA), lane 2 - PCR product from liver tissue sample of saline infected mice (healthy mice), lane 3 - PCR product from liver tissue sample of Leishmania infected mice (challenged mice), lane 4 - PCR product from spleen tissue sample of Leishmania infected mice (challenged mice), lane 5 - Blank (distilled water), and lane 6 - 1 Kb molecular weight marker

Fig. 5

Sensitivity of qRT-PCR. A plot of mean cycle threshold (C_T) value against the logarithmic concentration of parasite DNA (10-1000 parasites per reaction).