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 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.

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We investigated the in-vitro antiproliferative effects and mechanism of action of both enantiomers of the bis-triazole derivative ICI 195,739 against epimastigotes and amastigotes of Trypanosoma cruzi, the aetiological agent of Chagas' disease. It has recently been shown that the R(+) enantiomer, D0870, can induce radical parasitological cure in murine models of the acute and chronic forms of the disease. D0870 dose-dependently affected the growth rate of the epimastigote form (IC50 = 0.1 microM; MIC = 1-3 microM). The S(-) enantiomer was much less active (IC50 = 3 microM). Growth arrest and cell lysis induced by D0870 coincided with the complete depletion of endogenous 4,14-desmethyl sterols and their replacement by 4,14-trimethyl and dimethyl sterols. The S(-) enantiomer produced qualitatively similar changes but to a lesser extent. D0870 inhibited the incorporation of radioactivity from [2-14C]acetate into the epimastigote's 4,14-desmethyl sterols with an IC50 of 50 nM while the corresponding concentration for the S(-) enantiomer was 3 microM. D0870 eradicated the intracellular (amastigote) form of the parasite from cultured Vero cells at 10 nM; a 100-fold higher concentration of the S(-) enantiomer was required to produce a similar effect, and deleterious effects of the host cells were observed at > 100 nM. At the MIC of D0870 the endogenous amastigote sterols (ergosta-7-en-3beta-ol, 24-ethyl-cholesta-7-en-3beta-ol and ergosta-7, 24(24[1])-dien-3beta-ol) were also largely replaced by lanosterol and 24-methyl-dihydrolanosterol. Combinations of D0870 and inhibitors of sterol delta24(25) sterol methyltransferase (such as 22,26-azasterol and 24(R,S),25-epiminolanosterol) acted synergically against the intracellular forms. Taken together these results indicate that, although both enantiomers have anti-T. cruzi activity, the specific activity of the R(+) enantiomer (D0870) is nearly two orders of magnitude higher than that of its S(-) analogue. However, as the in-vitro activity of D0870 is comparable to that of standard azoles, such as ketoconazole, its remarkable in-vivo antiparasitic activity may only be explained by its particular pharmacokinetic properties.

Leishmania braziliensis (isolate 2903) was naturally resistant to ketoconazole or the bis-triazole D0870, inhibitors of sterol C-14 demethylase, which produced only moderate effects on the proliferation of promastigotes at 10 microM. In contrast, Leishmania mexicana (isolate NR) was extremely susceptible to the azoles, as complete growth arrest and cell lysis were induced by incubation of the parasites with 0.05 microM concentrations of the drugs for 72 h. The opposite response was observed with terbinafine, an inhibitor of squalene epoxidase: L. braziliensis 2903 was three times more susceptible to the drug than L. mexicana NR (MICs of 5 and 15 microM, respectively). However, when the L. braziliensis stock was grown in the presence of 1 microM terbinafine, which by itself produced only marginal (< 10%) effects on growth, it became highly susceptible to the azoles, with an MIC of 0.03 microM. Analysis of cellular free sterols by high-resolution capillary gas chromatography coupled to mass spectrometry showed that 14-methyl sterols can support normal growth of L. braziliensis 2903 but not of L. mexicana NR. On the other hand, the higher susceptibility of the L. braziliensis isolate to terbinafine was correlated with a massive accumulation of squalene in the presence of the allylamine while no significant effects on L. mexicana sterol composition were observed at drug concentrations up to 1 microM. Thus, the > 300-fold increase in the susceptibility of L. braziliensis promastigotes to azoles in the presence of terbinafine was attributed to the combined effect of squalene and the methylated sterol precursors on the physical properties of the cell's membranes, leading to the loss of cell viability. Combination therapy with azoles and terbinafine in the treatment of human L. braziliensis infections deserves further study.