Avalia??o da associa??o de benzonidazol e itraconazol no tratamento de camundongos infectados com clones de Trypanossoma cruzi de grupos gen?ticos distintos : efic?cia terap?utica e farmacocin?tica.

Programa de P?s-Gradua??o em Ci?ncias Farmac?uticas. CIPHARMA, Escola de Farm?cia, Universidade Federal de Ouro Preto.O perfil de resist?ncia de clones de Trypanosoma cruzi pertencentes a grupos gen?ticos distintos em rela??o ao benzonidazol (BNZ) e itraconazol (ITC) foi anteriormente demonstrado em camundongos. Considerando que a associa??o de f?rmacos tem sido mais eficiente no tratamento de diversas infec??es, o objetivo deste estudo foi avaliar a associa??o BNZ + ITC na quimioterapia experimental da doen?a de Chagas, considerando a variabilidade gen?tica do parasito e sua correla??o com a resist?ncia ao tratamento etiol?gico. Foram utilizados camundongos Swiss infectados via IP com 10x103 tripomastigotas sangu?neos de tr?s estoques clonais pertencentes aos grupos gen?ticos TcI (P209 Cl1), TcV (BUG2149 Cl10) e TcII (IVV Cl4). Dez dias p?s-inocula??o e confirma??o da infec??o, os animais foram tratados via oral com 100 mg/Kg de cada composto durante 20 e 60 dias para o BNZ e ITC respectivamente, inclusive na associa??o dos f?rmacos. A avalia??o da efic?cia terap?utica foi feita por at? 110 dias p?stratamento por exame de sangue a fresco (ESF), hemocultura (HC), PCR e ELISA. Foram considerados curados os animais que apresentaram todos os exames negativos. O tratamento com BNZ + ITC reduziu significativamente a parasitemia al?m de outros par?metros a ela relacionados nos tr?s grupos experimentais. Os ?ndices de cura entre os grupos tratados com BNZ, ITC e BNZ + ITC foram comparados. O tratamento com BNZ + ITC resultou em efic?cia terap?utica significativamente maior em rela??o ao BNZ e ITC isoladamente nos animais infectados pelos tr?s clones estudados. Os ?ndices de cura seguiram a mesma tend?ncia dos tratamentos isolados e apresentaram correla??o com a dist?ncia filogen?tica entre os clones de T. cruzi estudados. Um m?todo bioanal?tico empregando CLAE com detec??o UV precedido de extra??o l?quido-l?quido do BNZ e ITC contidos em amostras plasm?ticas de animais tratados foi desenvolvido pela primeira vez. O m?todo foi validado de acordo com as especifica??es do FDA e ANVISA, mostrando-se adequado para ser aplicado ao monitoramento farmacocin?tico no modelo murino. Os perfis plasm?ticos dos f?rmacos administrados simultaneamente em regime de dose ?nica revelaram significativa diminui??o da concentra??o plasm?tica m?xima (Cmax) do BNZ e significativo aumento do seu tempo de meia-vida de elimina??o (t??) em compara??o ? monoterapia. Estes resultados sugerem que o aumento da efic?cia do BNZ + ITC n?o se limita apenas aos distintos mecanismos de a??o dos f?rmacos, mas tamb?m ? intera??o farmacocin?tica entre eles, na qual o ITC potencializa o efeito do BNZ por prolongar sua meiavida e consequentemente aumentar sua biodisponibilidade para melhor a??o terap?utica.The resistance profiles of Trypanosoma cruzi clonal stocks from different genetic groups in relation to benznidazole (BNZ) and itraconazole (ITC) were previously demonstrated in mice. Taking into account that the drug combination has been more effective on treatment of various infections, the aim of this study was to evaluate the association BNZ + ITC in experimental chemotherapy of Chagas disease, considering the genetic variability of the parasite and its correlation with the therapeutic resistance to etiological treatment. Swiss mice infected via IP with 10,000 blood trypomastigotes of three clonal stocks from the genetic groups TcI (P209 Cl1) TcV (BUG2149 CL10) and TcII (IVV CL4) were used. Ten days after inoculation the infected animals were orally treated with 100 mg/kg of each compound for 20 and 60 days for the BNZ and ITC, respectively, including the scheme of drug association. The evaluation of treatment efficacy was carried out until 110 days post-treatment by fresh examination (ESF), hemoculture (HC), PCR and ELISA. Animals that showed all negative examinations were considered cured. The treatment with BNZ + ITC reduced significantly the parasitemia and other related parameters of the three experimental groups. The index of cure obtained with BNZ, ITC and BNZ + ITC treatments were compared. The treatment with BNZ + ITC resulted in significant higher therapeutic efficacy in relation to BNZ and ITC administered separately in the groups infected with the three clones studied. The index of cure followed the same tendency of the isolated treatments and showed correlation with the phylogenetic distance between the clones of T. cruzi studied. A bioanalytical method using HPLC-UV with liquid-liquid extraction of the drugs in the plasmatic samples from animals treated was first developed. This method was validated according to the specifications of FDA and ANVISA and was suitable for its application in pre-clinical monitoring in murine model. The pharmacokinetic profiles of the drugs simulneously administered in single dose regimen showed significant decrease of maximum plasma concentration (Cmax) of BNZ and its significant increase of half-life of elimination (t?? ) in relation to monotherapy. These results suggest that the increase of the effectiveness of the associated chemotherapy (BNZ + ITC) is not limited only to the distinct mechanisms of action of the drugs involved, but also due to the pharmacokinetic interaction between them, in which the ITC enhances the effect of BNZ and its half-life time and thereby increasing its bioavailability for better therapeutic action