In-vitro antiproliferative effects and mechanism of action of the bis- triazole D0870 and its S(-) enantiomer against Trypanosoma cruzi

Abstract

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.

Novel Azasterols as Potential Agents for Treatment of Leishmaniasis and Trypanosomiasis

“…For the analysis of the effects of drugs on the lipid composition of promastigotes, total lipids from control and drug-treated cells were extracted and fractionated into neutral and polar lipid fractions by silicic acid column chromatography and gas-liquid chromatography (12,13,27,28 nitrogen as the carrier gas at 24 ml/min and flame ionization detection in a Varian 3700 gas chromatograph). For quantitative analysis and structural assignments, the neutral lipids were separated in a capillary high-resolution column (Ultra-2 column,25 m by 0.20 mm inner diameter, 5% phenyl-methyl-siloxane, 0.33-m film thickness) in a Hewlett-Packard 6890 Plus gas chromatograph equipped with an HP5973A mass sensitive detector.…”

Quinuclidine Derivatives as Potential Antiparasitics

“…For the analysis of the effects of the drugs on the lipid compositions of the promastigotes, total lipids from control and drug-treated cells were extracted and fractionated into neutral and polar lipid fractions by silicic acid column chromatography and gas-liquid chromatography (19,20,42,43). The neutral lipid fractions were first analyzed by thin-layer chromatography (on Merck 5721 silica gel plates with heptane-isopropyl ether-glacial acetic acid [60: 40:4] as the developing solvent) and conventional gas-liquid chromatography (by isothermic separation in a 4-m glass column packed with 3% OV-1 on Chromosorb 100/200 mesh and nitrogen as the carrier gas at 24 ml/min and with flame ionization detection in a Varian 3700 gas chromatograph).…”

Update on Chagas' disease in Mexico

“…9 Their efficacy is also limited to the acute phase of the infection, and it is highly variable so that there is still controversial evidence that patients really benefit from such treatments. 10 A variety of new drugs is thus currently under development, [20][21][22][23][24] and some appear to show promise. However, additional studies are necessary and it will be some time before new treatments become available.…”