Innovative formulations co-incorporating amphotericin b and retinoic acid in liposomes for cutaneous leishmaniasis treatment

Abstract

Leishmaniasis is a neglected disease that primarily affects socially vulnerable populations and immunocompromised individuals, posing significant treatment challenges. This disease is caused by different species of Leishmania, leading to two primary clinical forms: cutaneous (CL) and visceral (VL). In 2022 alone, 205,986 new CL cases and 12,842 new VL cases were reported, highlighting the urgent need for effective treatment strategies. Amphotericin B (AmB), one of the most potent antileishmanial drugs, faces obstacles such as low solubility, self-aggregation, and high toxicity. Although the liposomal AmB (AmBisome®) presents an improved profile, it is expensive, and its efficacy remains limited in complicated disease cases. This work investigated the classical chemotherapy (AmB) combined with an immunomodulator to address this limitation. All-trans-retinoic acid (RA) appears promising in this context, as recent studies showed both immunomodulatory and antileishmanial effects. Thus, this thesis aimed to evaluate an innovative approach based on liposomal formulations co-incorporating AmB and RA. The specific objectives were to (i) develop and characterize a rigid PEGylated liposome for parenteral treatment (LAmB-RA) and a deformable liposome (DLAmB-RA) for topical application; (ii) evaluate the stability of AmB-RA formulations; (iii) investigate LAmB-RA hemolytic activity in vitro; (iv) perform a pharmacokinetic study of LAmB-RA in mice; and (v) assess LAmB-RA antileishmanial efficacy in murine models of CL. Rigid liposomes were prepared using a simple and innovative method to co-encapsulate both drugs, exploiting their pH-dependent solubilities and the effect of temperature on membrane fluidity. While RA and AmB are highly soluble in alkaline solutions, their solubility decreases at neutral pH, favoring their incorporation into the liposomal membrane. LAmB-RA formulation exhibited a hydrodynamic diameter of 123.3 ± 6.5 nm and a low polydispersity index (PdI) of 0.11 ± 0.02, making it suitable for parenteral administration. With a high encapsulation efficiency of 97.1 ± 4.4% for AmB and 94.7 ± 5.2% for RA, LAmB-RA remained stable in solution for 30 days at 4 ºC. Moreover, LAmB-RA demonstrated reduced hemolytic activity compared to the commercial formulation Anforicin B® (p <0.0001). Pharmacokinetic analysis by LC-MS showed greater and more prolonged plasma concentrations of AmB from the PEGylated LAmB-RA formulation compared to AmBisome® (conventional liposome). These pharmacokinetic improvements likely contributed to the significant efficacy of LAmB-RA in murine models of L. major and L. amazonensis infections. In the L. major model, LAmB-RA treatment significantly reduced lesion growth (-1.1 ± 2.1 mm versus 3.5 ± 1.4 mm) and parasite load compared to the Control group (p < 0.05). In the L. amazonensis model, LAmB-RA significantly reduced lesion growth (1.1 ± 0.7 mm) compared to both untreated (3.3 ± 1.4 mm) and AmBisome®-treated groups (2.5 ± 0.8 mm), as well as the parasite burden in the spleen (p < 0.05). Additionally, LAmB-RA treatment enhanced the IFN-γ/IL-10 ratio in splenocyte cultures stimulated with Leishmania antigens, indicating a favorable immunomodulatory response. DLAmB-RA, in turn, was achieved by RA solubilization in ethanol and AmB solubilization in sodium deoxycholate, surfactant used to obtain deformable vesicles. This liposome showed a vesicle size of 101.4 ± 1.3 nm, a PdI of 0.20 ± 0.01, and encapsulation efficiency higher than 90% for both AmB and RA, being stable at least 20 days at 25 oC. These findings support LAmB-RA and DLAmB-RA as promising alternatives for CL treatment, warranting further investigations.