Novel and safe single-dose treatment of cutaneous leishmaniasis with implantable amphotericin B-loaded microparticles

Abstract

The development of an effective amphotericin B (AmB) topical formulation to replace the systemically toxic injections currently used in cutaneous leishmaniasis (CL) treatment is challenging due to poor absorption through the skin. Aiming at an effective local chemotherapy, we designed PLGA (poly(lactide-co-glycolide acid) microparticles loaded with deoxycholate amphotericin B (d-AmB) for both macrophage intracellular targeting and sustained extracellular release. For that, d-AmB/PLGA microparticles with sizes ranging from 0.5 μm to 20 μm were synthesized and tested both in vitro and in vivo. In vitro, d-AmB/PLGA was more selectively active against intracellular amastigotes of Leishmania amazonensis than free d-AmB (selectivity index = 50 and 25, respectively). In vivo, the efficacy of a single intralesional (i.l) injection with d-AmB/PLGA was determined in early and established BALB/c mouse ear lesions. In early lesions, a single injection given on day 10 of infection was more effective in controlling parasite growth than eight i.l. injections with free d-AmB, as measured on day 120. Such d-AmB/PLGA injection was also effective in established lesions (day 30), leading to 97% parasite burden reduction, as compared with d-AmB or liposomal AmB (Ambisome®) i.l. injection containing the same AmB dose. Pharmacokinetic studies showed that following d-AmB/PLGA injection, AmB leaked slower from non-infected than infected ears, yet remaining in the ear tissue for as long as 30 days. Of interest, AmB was not detectable in the circulating plasma for at least two weeks of d-AmB/PLGA injection, contrasting with the rapid and durable (2 days) detection after free d-AmB injection. Despite the transient ear swelling and local cell infiltration, no alterations in AST, ALT and creatinine serum levels was induced by d-AmB/PLGA. For its approved components, local efficacy, and single-dose applicability, this novel and safe AmB microparticle depot formulation has strong potential as a new therapy for human CL.

Keywords: Chemotherapy; Drug delivery systems; Leishmania amazonensis; PLGA.

Graphical abstract

Fig. 1

Microparticle physical characteristics. A) PLGA and d-AmB/PLGA microparticles were analysed by Coulter LS230 for differential (Gaussian lines) and cumulative (progressive lines) volumes. B) Data for diameter distribution, zeta potential, and encapsulation efficiency (E.E.). Means ± SD (n = 3).

Fig. 2

Microparticle SEM and in vitro release kinetics. A-C) Dried d-AmB/PLGA microparticles were imaged by SEM in lower (A) and higher (B) magnification to confirm absence external AmB. Dry AmB (C) is shown as control. D) d-AmB/PLGA microparticles were incubated in duplicate at 37 °C in PBS under mild agitation. At the indicated times, supernatant samples were removed for quantification of AmB by HPLC, followed by PBS volume replacement. Means ± SD (n = 2).

Fig. 3

Efficacy of single-dose treatment with d-AmB/PLGA in early CL. Mice were infected with L. amazonensis in the ear pinna. On day 10 of infection, they were given a single intralesional injection with d-AmB/PLGA (5 μg of AmB), PLGA (500 μg) or PBS vehicle (10 μL). d-AmB (5 μg/dose) was given by the same route twice a week (total 8 doses). Lesion sizes were measured on the indicated days (A). Parasite loads were determined on day 120 post infection in the ear (B) and draining lymph nodes (C) by limiting dilution assay. Means ± SD (n = 5). ***p < 0.01 and *p < 0.5 in relation to d-AmB 8X.

Fig. 4

Efficacy of single-dose treatment with d-AmB/PLGA in established CL. Mice were infected in the ear with L. amazonensis. On day 30 of infection, they were given a single intralesional subcutaneous injections with d-AmB, AmBisome® or d-AmB/PLGA (5 μg of AmB each). Controls were given PLGA (500 μg) or PBS vehicle (10 μL). A) Lesion sizes were measured on the indicated days. B) Representative lesion pictures on day 90. C) Parasite loads in the ear, and D) draining lymph nodes, measured on day 90 by limiting dilution assay. Means ± SD (n = 5). ***p < 0.01 in relation to d-AmB 1X.

Fig. 5

Local and systemic pharmacokinetics. (A) Infected and non-infected mice were treated in the ear with a single dose of d-AmB/PLGA (5 μg of AmB). B) Non-infected mice were treated with d-AmB or d-AmB/PLGA (5 μg of AmB each). After the indicated time points, AmB was quantified in the ear or plasma by HPLC/UV at 405 nm. Means ± SD (n = 3).

Fig. 6

Systemic toxicology. Thirty-day infected mice were given a single dose of the indicated AmB formulations or controls, as for Fig. 4. After sixty days of treatment, sera were collected and individually assayed for AST (A), ALT (B), and creatinine (C). Positive control (1% of CCl₄) was given by intraperitoneal injection on day 87. Means ± SD (n = 5).

Fig. 7

Cutaneous hypersensitivity. Mice were pre-sensitized in the back by 3 s.c. injections with d-AmB, d-AmB/PLGA (5 μg of AmB) or PBS (10 μL) alone, on days −9, −7 and −5. Oxazolone was applied topically on the same days. On day 0, their ear pinnae was challenged in the same way, and the resulting swelling measured in the indicated days. The dashed line indicates sensitization threshold, that is 20% of achieved with 0.5% oxazolone. Means ± SD (n = 5).

Fig. 8

- Histopathological analysis. Mice were given a single s.c. injection in the ear pinna with d-AmB/PLGA (5 μg of AmB), or were left untreated (A). After 4 h (B), 1 day (C) or 30 days (D) post injection, longitudinal ear sections were obtained and stained with H&E. The inflammatory infiltrate is shown with arrows. Representative photographs of triplicate samples are shown. Bars = 100 μm.