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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.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-β in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-β in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Pentavalent antimonials are first-line drugs for treatment of the neglected tropical disease leishmaniasis. However, the use of these old drugs is limited by the need for daily parenteral administration, their severe side effects, and treatment failures. In light of these limitations, the World Health Organization strongly recommends research into new drugs against leishmaniasis. In this context, several structurally different nanocarriers have been investigated for the targeted delivery of antimonial drugs to infection sites of visceral leishmaniasis, as well as for the oral and topical delivery of antimony in visceral and cutaneous leishmaniasis. This chapter covers the progress achieved toward pharmaceutically acceptable nanostructured formulations for the improved delivery of antimonial drugs. Special emphasis is given to innovative nanostructures based on the unique physicochemical properties of antimony(V). The most promising nanosystems comprise liposomes for visceral leishmaniasis and micelle-like nanostructures for the oral delivery of antimony.

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.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.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.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.

Whether ultradeformable vesicles pass intact through the stratum corneum and can promote the transdermal absorption of any substance remain open questions. This paper presents different experimental approaches, based on the use of calcein as hydrophilic fluorescent marker, to probe the physicochemical and pharmacokinetic characteristics of these vesicles. Ultradeformable membranes made from natural phosphatidylcholine and sodium cholate were found to be highly permeable to calcein, as a result of the permeabilizing effects of sodium cholate and ethanol. In vitro skin permeation and in vivo transdermal (percutaneous) absorption studies were performed using hairless mice. Both studies indicated that deformable vesicles reduce the transdermal flux of calcein, when compared to a solution containing or not sodium cholate and ethanol. The data support the model that the transdermal absorption of calcein from deformable vesicles is controlled by the release of the drug from the formulation deposited onto the skin surface. Importantly, fluorescence measurements of the receptor fluid of the Franz diffusion cell after addition of Co(2+) quencher revealed that permeated calcein exists essentially under the non-encapsulated form. In conclusion, our results argue against the model that deformable vesicles would carry hydrophilic drugs across the skin and act as a sustained release system in deep tissues.

Actinic Kerarosis (AK) are hyperqueratotic cutaneous lesions, in which an abnormal proliferation of keratinocytes of the epidermis is present [1]. Since 1960s, topical 5-Fluorouracil (5-FU), a hydrophilic antimetabolite drug, has been widely used as an effective treatment for many pre-cancerous conditions and certain malignant tumors. However, the severe inflammatory reaction, clinically characterized by erosion and ulceration, which occurs in the skin following topical therapy, is still a major treatment disadvantage. Topical delivery of hydrophilic drugs through intact skin usually creates problems due to the inability of the drugs to penetrate into stratum corneum. Liposomal formulations have demonstrated capacity to increase penetration across and into skin of these drugs when compared to conventional formulations [2]. Besides, when applied on the stripped skin, in absence of barrier for drug permeation, liposomes were found to provide targeted and sustained topical delivery [3]. Thus, we hypothesized that liposomal formulation containing entrapped 5-FU could be an interesting alternative for topical treatment of AK. 5-FU encapsulation efficiency (EE) and retention in liposomes are usually low (EE less than 10%). 5-FU does not associate with the lipid bilayer [4] and EE depends mainly on the internal aqueous volume of vesicles [5]. Therefore, EE has been greater in large unilamellar vesicles (LUV) when compared to multilamellar vesicles (MLV) and the physical state of the bilayer determines the retention capacity of the vesicles [6]. Recently, MLV liposomes containing 5-FU were prepared with high EE, but with an average vesicle diameter of 5 m [7].

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.

The achievement of parasitological cure of dogs with visceral Leishmaniasis (VL) remains a great challenge, since dogs act as main reservoir for transmission of Leishmania infantum to humans and respond poorly to conventional drugs including pentavalent antimonials. Liposome-encapsulated antimonials are hundreds of times more effective than the free drugs against VL based on parasite suppression in the liver. However, complete parasite elimination in dogs seems to depend on the ability of liposomes to reach less accessible infection sites such as the bone marrow and the skin. Recently, the reduction of liposome size from 1200- to 400-nm diameter was found to improve the targeting of Sb to the bone marrow of dogs with VL. In the present work, the influence of further reduction of vesicle diameter from 400- to 175-nm on the pharmacokinetics of Sb in dogs with VL and on the distribution of Sb in the liver, spleen and bone marrow were investigated. For this purpose, two liposome formulations of meglumine antimoniate with the same lipid composition but different mean hydrodynamic diameters were prepared. The formulations were given to mongrel dogs with VL as a single intravenous bolus injection and Sb concentrations were determined by graphite furnace atomic absorption spectroscopy. Surprisingly, much more prolonged blood levels of Sb were achieved from small size (175 nm) than medium size (400 nm) liposomes. Small size vesicles were also less effective than medium size ones in targeting Sb to the liver. On the other hand, similar Sb concentrations were achieved in both spleen and bone marrow. In conclusion, the prolonged blood circulation time of liposomes with 175-nm diameter makes this nanosystem suitable for passive drug targeting to the less accessible infection sites in dogs with VL.