Therapeutic Efficacy of a Mixed Formulation of Conventional and PEGylated Liposomes Containing Meglumine Antimoniate, Combined with Allopurinol, in Dogs Naturally Infected with Leishmania infantum.

The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.

Mixed formulation of conventional and pegylated liposomes as a novel drug delivery strategy for improved treatment of visceral leishmaniasis.

OBJECTIVE: Test the hypothesis that pegylated meglumine antimoniate-containing liposomes (LMA) and their mixture with non-pegylated (conventional) LMA may be more effective than conventional LMA against visceral leishmaniasis (VL), because of wider drug distribution among different mononuclear phagocyte system (MPS) tissues. METHODS: Sb was determined in the blood and MPS tissues after administration of pegylated or conventional LMA intravenously to mongrel dogs naturally infected with Leishmania infantum and Swiss mice. Pegylated and conventional LMA as well as their mixture were evaluated for their antileishmanial efficacy in BALB/c infected with L. infantum through determination of parasite load in liver, spleen and bone marrow. RESULTS: An improved targeting of Sb to the bone marrow of dogs was clearly evidenced, as an important impact of pegylation. In accordance with this data, pegylated LMA significantly reduced parasite load in bone marrow of infected mice, in contrast to conventional LMA. The mixed formulation of conventional and pegylated LMA promoted parasite suppression to a higher extent in both spleen and bone marrow, compared to pegylated or conventional LMA. CONCLUSIONS: The present work establishes for the first time the potential of mixed formulations of conventional and pegylated liposomes as a drug delivery strategy for improved treatment of VL.

A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is a severe and fatal systemic chronic inflammatory disease. We investigated the alterations in, and potential associations among, antioxidant enzymes, trace elements and histopathology in CVL. Blood and tissue levels of Cu-Zn superoxide dismutase, catalase and glutathione peroxidase were measured in mixed-breed dogs naturally infected with Leishmania infantum chagasi, symptomatic (n = 19) and asymptomatic (n = 11). Serum levels of copper, iron, zinc, selenium and nitric oxide, and plasma lipid peroxidation were measured. Histological and morphometric analyses were conducted of lesions in liver, spleen and lymph nodes. We found lower blood catalase and glutathione peroxidase activity to be correlated with lower iron and selenium respectively. However, higher activity of Cu-Zn superoxide dismutase was not correlated with the increase in copper and decreased in zinc observed in infected animals compared to controls. Organ tissue was characterized by lower enzyme activity in infected dogs than in controls, but this was not correlated with trace elements. Lipid peroxidation was higher in symptomatic than in asymptomatic and control dogs and was associated with lesions such as chronic inflammatory reaction, congestion, haemosiderin and fibrosis. Systemic iron deposition was observed primarily in the symptomatic dogs showing a higher tissue parasite load. Dogs with symptomatic CVL displayed enhanced LPO and Fe tissue deposition associated with decreased levels of antioxidant enzymes. These results showed new points in the pathology of CVL and might open new treatment perspectives associated with antioxidants and the role of iron in the pathogenesis of CVL.

Efficacy of combined therapy with liposome-encapsulated meglumine antimoniate and allopurinol in treatment of canine visceral leishmaniasis.

An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.