Antiproliferative Effects of △²⁴⁽²⁵⁾ Sterol Methyl Transferase Inhibitors on <i>Trypanosoma (Schizotrypanum) cruzi</i>: In vitro and in vivo Studies

Abstract

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.

Activities of the Triazole Derivative SCH 56592 (Posaconazole) against Drug-Resistant Strains of the Protozoan Parasite Trypanosoma ( Schizotrypanum ) cruzi in Immunocompetent and Immunosuppressed Murine Hosts

“…and given daily for 28 consecutive days followed by a 7-day rest and another 15 days of treatment, for a total of 43 doses. This course of treatment was used in our previous studies that first demonstrated the in vivo trypanocidal effects of both D0870 and SCH 56592 (13,16,(32)(33)(34)(35). In addition, other T. cruzi strains (SC-28 and VL-10, both nifurtimox and benznidazole resistant [10]) were included in the study.…”

“…In a second study, normal (immunocompetent) animals were infected with the same inoculum (10 4 ) and treatment was started at 4 days p.i. but given daily for 28 days, followed by a 7-day rest and another 15 days of treatment (16,29,(32)(33)(34)(35). Surviving animals were monitored for up to 113 days p.i.…”

Parasitological cure of Chagas disease: is it possible? Is it relevant?

“…Sterol biosynthesis inhibitors (SBI) have been found to be particularly useful, as endogenous sterols are essential for survival in these pathogenic organisms; these compounds are in most cases orally active, have broad spectrum of activity and very low toxicity (Ryder & Mieth 1992, Mercer 1993, Vanden Bossche 1995. T. cruzi has also an absolute requirement of specific endogenous sterols for cell viability and proliferation and is extremely sensitive to sterol biosynthesis inhibitors (SBI) in vitro (Docampo et al 1981, Beach et al 1986, Goad et al 1989, Urbina et al 1988, 1993, 1996b; thus, the sterol biosynthesis pathway in this organism is a valid chemotherapeutic target (Urbina 1997). Nevertheless, currently available SBI's, such as ketoconazole and itraconazole, have been shown to be unable to eradicate T. cruzi from experimentally infected animals or human patients (McCabe 1988, Moreira et al 1992, Brener et al 1993, although a recent report from Chile claims a high level (>50%) of parasitological cures in chronic patients treated with itraconazole (Apt et al 1998).…”

Novel Azasterols as Potential Agents for Treatment of Leishmaniasis and Trypanosomiasis

“…1), which has been shown to modify the sterol composition of T. cruzi epimastigotes (the parasite form found in the vector), acting either singly or in combination with other sterol biosynthesis inhibitors (32,37). In addition, the compound also affects amastigotes (13) (the intracellular form of the parasite) and has been shown to have antiproliferative effects in vitro and in vivo (31). Similarly, the compound is active against Leishmania amazonensis promastigotes and amastigotes (24) and Leishmania donovani promastigotes (8).…”