Miltefosine Affects Lipid Metabolism in Leishmania donovani Promastigotes

c Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14␣-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 halfknockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

Section: Resultssupporting

confidence: 78%

Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

Pandharkar

Zhu

Mathur

et al. 2014

“…These findings and the fact that promastigotes are the vector stage, differing considerably from the intracellular mammalian amastigote target, provide a convincing argument for excluding them for drug-screening purposes ( Table 2). On the other hand, susceptibility to amphotericin B and miltefosine, which are known to affect membrane integrity directly or indirectly and which are able to exert antileishmanial action independently of cell-mediated parasiticidal mechanisms, has been clearly demonstrated (20,26,40). It is therefore not too speculative to propose that promastigotes could indeed be used for determination of the resistance of field isolates to amphotericin B and miltefosine (18,25).…”

Section: Discussionmentioning

confidence: 99%

In Vitro Susceptibilities of Leishmania donovani Promastigote and Amastigote Stages to Antileishmanial Reference Drugs: Practical Relevance of Stage-Specific Differences

Vermeersch¹,

Luz²,

Toté³

et al. 2009

205

166

The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC 50 ], 0.6 to 0.7 M), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC 50 , 0.1 to 0.4 M). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC 50 , 0.4 to 3.8 M) and intracellular (IC 50 , 0.9 to 4.3 M) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC 50 , >64 g Sb V /ml) but showed good efficacy against intracellular amastigotes (IC 50 , 22 to 28 g Sb V /ml); the crystalline substance was about two to three times more potent (IC 50 , 9 to 11 g Sb V /ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC 50 , 0.1 g/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.Current first-line chemotherapy of leishmaniasis relies on a rather limited arsenal of drugs including sodium stibogluconate, meglumine antimoniate, amphotericin B, and miltefosine, but these entail either problems of emerging resistance, severe side effects, or high costs (5). Since vaccines are not yet on the horizon (23), maintenance and improvement of existing treatment regimens, combined with new drug discovery initiatives, appear to be the only ways to guarantee continued control of this important tropical disease (3,11,27).Both for drug screening and for determination of the susceptibility of field strains, different laboratory methods are being used that focus on the promastigote, the axenic amastigote, or the intracellular amastigote stage. However, it remains unclear how these models cross-validate each other. The differences in environmental conditions between promastigotes and amastigotes in vivo are reflected in their needs for in vitro cultivation. While promastigotes are easily cultured in suspension (8), amastigotes are more difficult to maintain in vitro, since they require macrophages as host cells to meet the highly acidic intracellular environment (15). Cultivat...

“…It has been suggested that miltefosine acts in Leishmania mexicana by inhibiting the alkyl-specific acyl coenzyme A-acyltransferase involved in the synthesis de novo of phosphatidylcholine (9), whereas in Trypanosoma cruzi and Leishmania donovani, miltefosine acts by inhibiting the Bremer-Greenberg pathway, specifically at level of phosphatidylethanolamine Nmethyl-transferase (8,17). In contrast, this drug inhibits phosphatidylcholine biosynthesis in humans by blocking phosphocholine citidyltransferase (24).…”

mentioning

confidence: 99%

Amiodarone and Miltefosine Act Synergistically against Leishmania mexicana and Can Induce Parasitological Cure in a Murine Model of Cutaneous Leishmaniasis

Serrano-Martín

Payares

Lucca

et al. 2009

Leishmaniasis is parasitic disease that is an important problem of public health worldwide. Intramuscularly administered glucantime and pentostam are the most common drugs used for treatment of this disease, but they have significant limitations due to toxicity and increasing resistance. A recent breakthrough has been the introduction of orally administered miltefosine for the treatment of visceral, cutaneous, and mucocutaneous leishmaniasis, but the relative high cost and concerns about teratogenicity have limited the use of this drug. Searching for alternative drugs, we previously demonstrated that the antiarrhythmic drug amiodarone is active against Leishmania mexicana promastigotes and intracellular amastigotes, acting via disruption of intracellular Ca 2؉ homeostasis (specifically at the mitochondrion and the acidocalcisomes of these parasites) and through inhibition of the parasite's de novo sterol biosynthesis (X. Serrano-Martín, Y. García-Marchan, A. Fernandez, N. Rodriguez, H. Rojas, G. Visbal, and G. Benaim, Antimicrob. Agents Chemother. 53:1403-1410, 2009). In the present work, we found that miltefosine also disrupts the parasite's intracellular Ca 2؉ homeostasis, in this case by inducing a large increase in intracellular Ca 2؉ levels, probably through the activation of a plasma membrane Ca 2؉ channel. We also investigated the in vitro and in vivo activities of amiodarone and miltefosine, used alone or in combination, on L. mexicana. It was found that the drug combination had synergistic effects on the proliferation of intracellular amastigotes growing inside macrophages and led 90% of parasitological cures in a murine model of leishmaniasis, as revealed by a PCR assay using a novel DNA sequence specific for L. mexicana.