Antiproliferative effects of Delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: In vitro and in vivo studies

We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of Delta(24(25)) sterol methyl transferase (E.C. 2.1.1.43) of yeasts and fungi on epimastigotes and amastigotes on Trypanosoma (Schizotrypanum) cruzi, the causative agents of Chagas disease, as well as its chemotherapeutic effecs in a murine model of the disease. On the epimastigote form proliferating in liver infusion tryptose medium at 28 degrees C 22,26-azasterol (AZA), a cholestanol analog with a 6-membered aza ring as a side chain produced a dose-dependent reduction of the growth rate up to 3 mu M but at 10 mu M complete growth arest and cell lysis took place after 120-144 h. For 24(R,S),25-epiminolanosterol (EIL) complete growth arrest and lysis took place with 6 mu M. In both cases the antiproliferative effects were potentiated by the simultaneous incubation of the epimastigotes with inhibitors of sterol C-14 alpha-demethylase such as ketoconazole or SDZ 89,485, as indicated by concave isobolograms and fractional inhibitory concentrations ranging from 0.11 to 0.46. Analysis of the sterol composition in control and treated cells by thin-layer and capillary gas-liquid chromatography coupled to mass spectrometry showed that growth inhibition correlated with the complete disappearance of the native endogenous sterols of the parasite (ergosterol and 24-ethyl analogs) and the accumulation of 24-desalkyl sterols. Against the clinically relevant amastigote form proliferating inside cultured Vero cells at 37 degrees C, AZA eradicated the parasite of 100 nM, while the corresponding concentration for EIL was 300 nM. Synergic effects of both inhibitors when combined with ketoconazole against this form of the parasite was demonstrated using a three-dimensional analytic method which allowed the identification of optimal drug concentrations. Finally, it was found that daily oral administration of AZA at 50 mg/kg/day for a total of 43 doses to mice infected with a lethal inoculum of T. cruzi allowed survival of all treated animals 25 days after infection, while all control (untreated) animals were dead at this point of time. Increased survival correlated with a 90% reduction in parasitemia in the treated animals. The antiparasitic effects of the azasterol were potentiated in combined treatments with ketoconazole. This is the first report of a successful application of a sterol methyl transferase inhibitor as a chemotherapeutic agent in a protozoal infection.