Avaliação da eficácia de uma nova formulação de anfotericina B poliagregada em diferentes esquemas terapêuticos no tratamento da esporotricose experimental causada por <i>Sporothrix brasiliensis</i>

Abstract

Sporotrichosis is a chronic subcutaneous mycosis caused by several thermo-dimorphic species of the <i>Sporothrix schenckii</i> complex. This mycosis presents different clinical manifestations, and the most common form is the lymphocutaneous form. Severe cases caused by <i>Sporothrix brasiliensis</i> have been recently described, which required a prolonged treatment with high toxic antifungal drugs as amphotericin B-deoxycholate or its less toxic and costly versions. The aim of this work was to test the <i>in vitro</i> and <i>in vivo</i> efficacy of a new intravenous formulation of a poli-aggregated amphotericin B (D-AmB) and to test the in vivo efficacy semi-solid version (topical AmB), using as reference drugs the itraconazole (ITC) and amphotericin B-desoxycholate (D-AmB). <i>In vitro</i> susceptibility tests with <i>S. brasiliensis</i> showed that this species is susceptible to reference antifungals and to P-AmB. For <i>in vivo</i> tests a murine model of disseminated sporotrichosis and other of subcutaneous sporotrichosis, caused by <i>S. brasiliensis</i>, were established. For the disseminated model of sporotrichosis BALB/c intravenously inoculated with <i>S. brasiliensis</i> yeast cells were treated 72 h post- infection, treated with the drugs already mentioned, under different therapeutic regimens: i) a monotherapy ITC, D-AmB or P-AmB; ii) a therapeutic combination of D-AmB and ITC and P-AmB and ITC; iii) a system of pulse with D-AmB or P-AmB. Survival (n=nine) and fungal load in internal organs (n=three at least) were evaluated, and it was observed that monotherapy with ITC and P-AmB and D-AmB was the least effective regimen. The combination therapy was more effective than the monotherapy, and the combination of D-AmB and ITC was more effective than the combination with P-AmB and ITC. The pulse regimen, using D-AmB or P-AmB, was the most effective of all since prolonged the survival of animals and reduced the fungal burden in the organs. <i>In vivo</i> toxicity tests with the different drugs showed that the ITC and D-AmB induced hepatic and renal toxicity in animals, respectively, but not P-AmB. <i>In vitro</i> cytotoxicity tests it was observed that ITC was less cytotoxic and hemolytic and most selective drugs tested, followed by P- AmB, which is less cytotoxic and more selective than D-AmB. In the subcutaneous sporotrichosis model mice of the same strain were inoculated subcutaneously with conidia of <i>S. schenckii</i> and <i>S. brasiliensis</i> (n=9/group). The animals infected with <i>S. brasiliensis</i> showed regression of primary lesions and dissemination. Using the subcutaneous murine sporotrichosis model caused by <i>S. brasiliensis</i> we tested preliminarly the topical poly-aggregated AmB, wich reduced the extent of lesions from infected animals infected. This is the first study to evaluate different dosing regimens of disseminated murine sporotrichosis caused by <i>S. brasiliensis</i> using ITC, D-AmB and a new less toxic formulation of poly-aggregated amphotericin B. This study revealed that the pulse regimen as the most effective for the formulations of intravenous AmB. Our study also established pioneering a model of subcutaneous sporotrichosis induced by <i>S. brasiliensis</i>, which has proved a useful tool to compare the virulence of the complex species of <i>S. schenckii</i> complex and to test the efficacy of new antifungal agentes.