Effects of Miltefosine and Other Alkylphosphocholines on Human Intestinal Parasite Entamoeba histolytica

b Miltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmania promastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ϳ70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture. L eishmaniasis is caused by protozoan parasites of over 20 Leishmania species and is transmitted to humans via the bite of infected female sand flies. There are three main types of leishmaniasis: cutaneous (the most common form), visceral (the most severe clinical manifestation, which is fatal if untreated), and mucocutaneous. According to a recent report from the World Health Organization (WHO) (1), there are an estimated 1.3 million new cases of leishmaniasis worldwide and an estimated 20,000 to 30,000 deaths caused by these parasites annually.Miltefosine (MT) was the first oral drug approved for use for the treatment of visceral and cutaneous leishmaniasis and is currently registered for leishmaniasis treatment in India (2002, as Impavido), Germany (2004), and Colombia (2005) (2). This drug is also used clinically for the topical treatment of skin metastases associated with breast cancer and cutaneous lymphoma (3). MT has shown high cure rates in the treatment of visceral (4), cutaneous (5), and mucocutaneous (6) leishmaniasis. Although many studies have been conducted to identify the mechanisms underlying the action of MT against tumor cells (3) and parasites (7), these mechanisms have not yet been determined. However, due to its chemical structure, which provides MT with a high membrane affinity, the primary molecular targets of MT...

Section: Discussionmentioning

confidence: 99%

Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensis Membranes at Concentrations Similar to Those Needed for Cytotoxicity Activity

Moreira

Mendanha

Fernandes

et al. 2014

“…Numerous in vitro and in vivo experimental studies have shown that HePC is cytotoxic for both the promastigote and the amastigote stages of various species of Leishmania (7,8,15,16,29,31,58) as well as other protozoan parasites, including Trypanosoma cruzi (8,32,46,47), Trypanosoma brucei (8,28), Entamoeba histolytica (48), and Acanthamoeba spp. (64).…”

mentioning

confidence: 99%

Miltefosine Induces Apoptosis-Like Death in Leishmania donovani Promastigotes

Paris¹,

Loiseau²,

Bories³

et al. 2004

319

235

Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to Leishmania donovani. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of Leishmania donovani HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill L. donovani promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors, z-Val-Ala-DL-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.

“…This compound was also capable of diminishing ALA development in the hamster model. However, the effectiveness of C4 could be increased and its toxicity could be decreased if it were used in combination with silica beads or divalent cations or if it were administered in liposomal formulations (20,23,29). Also, we believe that several exposures of amoebas to C4 (such as during in vivo treatments) could diminish the effective dose of the carbamate required.…”

Section: Discussionmentioning

confidence: 99%

Antiamoebic and Toxicity Studies of a Carbamic Acid Derivative and Its Therapeutic Effect in a Hamster Model of Hepatic Amoebiasis

Ordaz-Pichardo¹,

Shibayama

Villa‐Treviño³

et al. 2005

Amoebiasis is an important public health problem in developing countries. Entamoeba histolytica, the causative agent of amoebiasis, may develop resistance to nitroimidazoles, a group of drugs considered to be the most effective against this parasitic disease. Therefore, research on new drugs for the treatment of this common infection still constitutes an important therapeutic demand. In the present study we determined the effects of a carbamate derivative, ethyl 4-chlorophenylcarbamate (C4), on trophozoites of E. histolytica strain HM-1: IMSS. C4 was subject to various toxicity tests, including the determination of mutagenicity for bacterial DNA and changes in the enzymatic activities of eukaryotic cells. Genotoxicity studies were performed by the mutagenicity Ames test (plate incorporation and preincubation methods) with Salmonella enterica serovar Typhimurium, with or without metabolic activation produced by the S9 fraction of rat liver. C4 toxicity studies were performed by measuring enzymatic activity in eukaryotic cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan test with Fischer 344 rat hepatocytes. C4 did not induce either frameshift mutations in S. enterica serovar Typhimurium TA97 or TA98 or base pair substitutions in strains TA100 and TA102. The compound was not toxic for cultured rat hepatic cells. Trophozoites treated with 100 g of C4 per ml were inhibited 97.88% at 48 h of culture; moreover, damage to the amoebae was also confirmed by electron microscopy. The antiamoebic activity of C4 was evaluated by using an in vivo model of amoebic liver abscess in hamsters. Doses of 75 and 100 mg/100 g of body weight reduced the extent of the amoebic liver abscess by 84 and 94%, respectively. These results justify further studies to clearly validate whether C4 is a new suitable antiamoebic drug.