Topical amphotericin B in ultradeformable vesicles: formulation, skin penetration studies, antifungal and antileishmanial activity.

- PEREZ AP, ALTUBE MJ, SCHILRREFF P, APEZTEGUIA G, INDIANI DE OLIVEIRA C, ZACCHINO S, ROMERO EL AND MORILLA MJ

Basilea

Conferencia; 8th CLINAM; 2015

Introduction: Amphotericin B (AmB) is a polyene antibiotic with potent antifungal and leishmanicidal activities. AmB is a high molecular weight (924 Da) molecule with amphoteric and amphiphilic behavior, poorly soluble in aqueous media and in most organic solvents. Micellar formulation of AmB with deoxycholate (Fungizone) and the unilamellar liposomal formulation of AmB (AmBisome) are used for parenteral treatment of invasive fungal infections such as Candida albicans or Aspergillus fumigatus (mostly in inmmunocompromised patients) and for treatment of visceral and mucocutaneous leishmaniasis. In the last 30 year the number of cases of invasive and superficial mycosis of Candida spp. has increased, mainly in inmmunocompromised patients. Almost 90% of AIDS patients develop at least one fungal infection over the course of disease, out of which 10?20% of infections prove fatal. Treating these patients with systemic AmB it is quite difficult because of the ongoing treatments with antiretroviral and other immunosuppressant drugs. On the other hand, Candida spp. infection in the context of burn wounds leads to invasive disease with a 14-70% mortality rate. AmB is the first line treatment for burn-related fungemia attributed to Candida species as well as many moulds. Topical AmB is the election treatment for fungal occular infections caused by Candida spp., which can cause blindness, and for cutaneous leishmaniasis (major tropical skin diseases caused by a variety of parasites of the Lesihmania genus). Counting on a topical formulation of AmB would be a goal to safely treat locally invasive fungal disease and leishmaniasis. Topical route enables a painless self administration, avoiding the use of injectablea and of systemic cytotoxicity caused by parenteral AmB. However the large molecular weight and amphoteric nature of AmB in Fungizone (the micellar formulation) hinders its adequate cutaneous penetration. Besides, the high cost and low skin penetration of AmBisome (the liposomal formulation), limits its clinical use in topical applications.Several attempts have been made to develop a topical formulation for AmB, namely liposomes, microemulsions, nanoemulsions, chitosan nanoparticles, lipid-based microtubes and ethosomes. Excepting the ethosomes however, the reminder AmB formulations act as skin surface depots, which do not enhance the AmB penetration within the epithelia. In this work we show that a deep access of AmB towards deep epithelial layers, with no aid of permeation enhancers, was achieved by loading AmB within ultradeformable nanoliposomes. Ultradeformable nanoliposomes (UDL) are highly deformable (elastic-/flexible) nanoliposomes made of phospholipids plus edge acti¬vators (EA, surfactants of high radius of curvature and mobility), capable of penetrating the intact skin across the stratum corneum and reach the viable epidermis. The challenge of this work was tuning the UDL bilayer composition so as to effectively achieve the loading of a quantitative amount of AmB. To that aim, we prepared and characterized diferent UDL lipid matrices containing AmB, soya phosphatidylcholine (SPC) and sodium cholate or Tween 80 as EA. The toxicity on keratinocytes and machophages by MTT and LDH leakage was determined for those formulations with the highest elasticty and AmB content. After that, the in vitro antifungal activity of UDL-AmB against albicans and non-albicans Candida ATCC strains, and against clinical isolates of C.albicans albicans were tested. The in vitro antileishmanial activity of UDL-AmB on Leishmania braziliensis promastigotes and intracellular amastigotes were also determined. Finally the in vitro skin penetration of UDL-AmB on human skin and storage stability were determined. Results: In first place, the influence of type of EA, SPC concentration, AmB concentration and SPC:EA ratio on deformability and AmB incorporation was tested. Nanoliposomal formulations contaning Tween 80 showed the highest deformability and AmB incorporation. The size of the resultant UDL-AmB was 107 ± 8 nm with polidispersity index of 0.078 and Z-potential of -3 ± 0.2 mV, encapsulation eficiency of 75%, being unilamellar vesicles as shown by transmission electron microscopy. UV-Vis spectral characterization of UDL-AmB showed that AmB fully associated to nanoliposomal bilayers in the monomeric form. Overall, both AmBisome and UDL-AmB caused more damage on plasma membrane than on mitochondrial activity, meaning that no cytotoxicity, as measured by MTT, was registered. Nonetheless, the lowest and highest toxicities of UDL-AmB ocurred on cells exhibiting lower (HaCaT) and higher (J774) endocytic uptake capability, respectively. The macrophage toxicity of UDL-AmB was higher than that of AmBisome: the UDL-AmB IC50 was < 1.25 µg/ml, vs 6 µg/ml for AmBisome. On the contrary, no differences were found on UDL-AmB and AmBisome IC50 on HaCaT cells cells, which resulted > 6 µg/ml. We also found that fungal strains were more sensitive that mammal cells to AmB liposomal formulations. UDL-AmB MICs (total inhibition of visible growth, equivalent to 99% inhibition) were equal or lower than AmBisome and free AmB MICs for the 5 Candida ATCC strains, and the clinical isolates of C.albicans albicans. UDL-AmB MICs were between 0.06 and 0.25 µg/ml and between 0.12 and 1 µg/ml for AmBisome. These values were 5-24 and 24-50 folds lower that IC50 on J774 for UDL-AmB and AmBisome, respectively. Besides, confocal fluorescence microscopies of rhodamine-labeled UDL-AmB incubated with C. albicans at concentration > MIC showed a fast transfer of the fluorescent dye within the fungus body. UDL-AmB showed comparable anti-leishmania activity than AmBisome. UDL-AmB showed 100% of anti-promastigote activity at 0.6 g/ml and 25 and 75 % anti-amastigote activity at 0.6 and 1.25 g/ml, respectively. Finally, upon 1 h of incubation on human skin, 33 ± 2 % and 7 ± 2 % of the administered dose of UDL-AmB was found in the stratum corneum and viable epidermis, respectively. In contrast, AmB was not found in the stratum corneum and less than 2 % of the administered dose was found viable epidermis when AmBisome was applied. The total accumulation of AmB in skin was 40 folds higher when applied as UDL-AmB than as AmBisome. Conclusions: the most remarkable finding of this work can be summarized as follows: altough UDL-AmB formulation showed comparable anti-fungal and anti-leishmanial activities with AmBisome, only UDL-AmB provided a considerable increased AmB skin deposition. Therefore the UDL-AmB formulation would be advantageous over other liposomal AmB formulations for topical fungicidal and leshminicidal treatments that require a targeted delivery of AmB to the viable epidermis and dermis.