Chagas' disease, a protozoan infection by the kinetoplastid Trypanosoma cruzi, constitutes a major public health problem in Latin America. With the use of mouse models of both short- and long-term forms of the disease, the efficacy of D0870, a bis-triazole derivative, was tested. D0870 was able to prevent death and induced parasitological cure in 70 to 90 percent of animals, in both the short- and long-term disease. In contrast, currently used drugs such as nifurtimox or ketoconazole prolonged survival but did not induce significant curing effects. D0870 may be useful in the treatment of human long-term Chagas' disease, a condition that is currently incurable.

We have studied the in vivo activity of the new experimental triazole derivative SCH 56592 (posaconazole) against a variety of strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, in both immunocompetent and immunosuppressed murine hosts. The T. cruzi strains used in the study were previously characterized as susceptible (CL), partially resistant (Y), or highly resistant (Colombiana, SC-28, and VL-10) to the drugs currently in clinical use, nifurtimox and benznidazole. Furthermore, all strains are completely resistant to conventional antifungal azoles, such as ketoconazole. In the first study, acute infections with the CL, Y, and Colombiana strains in both normal and cyclophosphamide-immunosuppressed mice were treated orally, starting 4 days postinfection (p.i.), for 20 consecutive daily doses. The results indicated that in immunocompetent animals SCH 56592 at 20 mg/kg of body weight/day provided protection (80 to 90%) against death caused by all strains, a level comparable or superior to that provided by the optimal dose of benznidazole (100 mg/kg/day). Evaluation of parasitological cure revealed that SCH 56592 was able to cure 90 to 100% of the surviving animals infected with the CL and Y strains and 50% of those which received the benznidazole-and nifurtimox-resistant Colombiana strain. Immunosuppression markedly reduced the mean survival time of untreated mice infected with any of the strains, but this was not observed for the groups which received SCH 56592 at 20 mg/kg/day or benznidazole at 100 mg/kg/day. However, the overall cure rates were higher for animals treated with SCH 56592 than among those treated with benznidazole. The results were confirmed in a second study, using the same model but a longer (43-dose) treatment period. Finally, a model for the chronic disease in which oral treatment was started 120 days p.i. and consisted of 20 daily consecutive doses was investigated. The results showed that SCH 56592 at 20 mg/kg/day was able to induce a statistically significant increase in survival of animals infected with all strains, while benznidazole at 100 mg/kg/day was able to increase survival only in animals infected with the Colombiana strain. Moreover, the triazole was able to induce parasitological cures in 50 to 60% of surviving animals, irrespective of the infecting strain, while no cures were obtained with benznidazole. Taken together, the results demonstrate that SCH 56592 has in vivo trypanocidal activity, even against T. cruzi strains naturally resistant to nitrofurans, nitroimidazoles, and conventional antifungal azoles, and that this activity is retained to a large extent in immunosuppressed hosts.Chemotherapy of Chagas' disease (American trypanosomiasis), a parasitic disease caused by the kinetoplastid protozoan Trypanosoma (Schizotrypanum) cruzi which afflicts 16 to 18 million people in Latin America, remains an enormous scientific and social challenge, as the drugs currently available, nitrofurans (nifurtimox; Bayer) and nitroimi...

We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30-to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3␤-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14␣ demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37°C, the MIC of SCH 56592 was 0.3 nM, again 33-to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCRbased tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.

We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of Δ²⁴⁽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 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 µM, but at 10 µM 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 µM. In both cases the antiproliferative effects were potentiated by the simultaneous incubation of the epimastigotes with inhibitors of sterol C-14α-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 Vera cells at 37°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.

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A comparative study was performed between the trypanocidal efficacy of and associated immune response to benznidazole and posaconazole in a murine model of Chagas disease. Both drugs led to 100% survival, suppression of parasitaemia and reduction of specific anti-Trypanosoma cruzi antibodies following chronic infection. All posaconazole-treated animals had negative haemocultures at 54 days post infection, whilst 50% of those treated with benznidazole had positive results. Although both drugs were effective in reducing parasitism and inflammation in the heart, posaconazole-treated animals had plasma enzymatic levels of cardiac lesion that were indistinguishable from those of uninfected mice, whilst for benznidazole the enzyme levels were significantly higher than those of uninfected controls 31 days after the start of treatment. Posaconazole was more effective than benznidazole in controlling spleen enlargement and unspecific splenocyte proliferation in the early acute phase, but allowed higher levels of activation of CD4(+) and CD8(+) T-cells in the late acute phase when the adaptive immune response takes control of the infection. These results support the notion that posaconazole could be superior to benznidazole for the treatment of T. cruzi infection in humans.

We report the effects of ketoconazole and the bistriazole ICI 195,739 acting alone or in combination with the allylamine terbinafine (Lamisil) on murine models of Chagas' disease. Mice infected with 105 Trypanosoma (Schizotrypanum) cruzi blood trypomastigotes and treated orally with 30 mg of ketoconazole per kg of body weight per day for 7 days, starting at 24 h postinoculation, had 100%o survival after 35 days, while controls (untreated) or animals that received 15 mg of ketoconazole or 100 mg of terbinafine per kg/day by the same route had O0o survival after the same period of time. However, all mice receiving the combination of 15 mg of ketoconazole plus 100 mg of terbinafine per kg/day survived for 35 days after infection; it was shown that the survival of the animals treated with this combination was statistically greater than that obtained with either drug acting alone and was indistinguishable from that observed with the high doses of ketoconazole, indicating a synergistic action of the drugs in vivo. However, most animals that survived after the 7-day treatments were not cured, as indicated by a delayed but persistent parasitemia. When the treatment was extended to 14 days, 100% survival was obtained 10 weeks after inoculation for mice treated with 30 mg of ketoconazole per kg/day and the combination of 15 mg of ketoconazole per kg/day plus 100 mg of terbinafine per kg/day, while two-thirds of the mice treated with 15 mg of ketoconazole per kg/day alone were alive after the 14-day treatment; controls or animals that received 100 mg of terbinafine per kg/day did not survive after 25 days. Parasitemia in all surviving mice was negative after 55 days but parasitological cure, as assessed by subinoculation of organs in naive animals, was predominant only in animals that received the combined drug treatment. We also investigated the bistriazole ICI 195,739 and found, as reported previously, that just 1 mg of the compound per kg/day administered orally for 5 days was enough to protect most mice from death 30 days after inoculation, but no parasitological cures were observed. However, in the protocol used in the present study, the protective activity of ICI 195,739 at suboptimal doses (0.5 mg/kg/day) could be enhanced when it was used in combination with terbinafine at doses of the allylamine that by themselves induced no significant protection. Survival of the mice was inversely correlated with the levels of parasitemia in all cases. Extension of the treatment period with the triazole to 15 days at 1 mg/kg/day afforded definitive protection against death, with parasitological cure being achieved in 50%o of mice at 10 weeks postinoculation, but no enhancement of its activity at suboptimal doses was observed when it was used in combination with terbinafine during this extended observation period. Taken together, these results support the proposition that ketoconazole used in combination with terbinafine could be useful in the treatment of humans with Chagas' disease because it can promote parasitological cure wi...

We have incorporated several inhibitors of sterol biosynthesis into long-circulating polyethyleneglycol-polylactide (PEG-PLA) nanospheres in order to improve the bioavailability of these poorly soluble compounds. Mice infected with CL and Y strains of Trypanosoma cruzi and treated for 30 consecutive days with DO870-loaded nanospheres at doses of 3 mg/kg/day, by the intravenous route, showed a significant cure rate (60-90%) for both strains. The activity was dose dependent and significant activity was observed for doses > or = 0.75 mg/kg/day. No cure was observed in mice treated with unloaded nanoparticles. Ketoconazole and itraconazole failed to induce cure against the Y strain even in the entrapped form.