**3. Alkylphospholipids and leishmaniasis**

Miltefosine treatment leads to high cure rates in visceral leishmaniasis in India (*L. donovani*; 94% cure) [13]. However, different trials regarding the efficacy of miltefosine against cutaneous leishmaniasis in Colombia led to distinct outcomes ranging from a 90% cure [14] to an unsatisfactory cure rate of 69.8% [15]. Sensitivity to miltefosine is known to vary between *Leishmania* species [16]. In this regard, *L. braziliensis* seems to be somewhat refractory to miltefosine as shown in a number of clinical studies [14, 17-19]. Miltefosine efficacy against leishmaniasis lesions caused by *L. braziliensis*, which comprise more than 60% of cutaneous leishmaniasis in Colombia, fell to 49% [15], and was only 33% in Guatemala [14]. Additional clinical trials in Brazil showed a miltefosine cure rate of 75% and 71% for the treatment of cutaneous leishmaniasis caused by *L. braziliensis*[20] and *L. guyanensis*[21], respectively. Thus, these trials have challenged the therapeutical potential of miltefosine for the treatment of American cutaneous leishmaniasis. Miltefosine treatment has also led to approximately 70% cure rate for mucosal leishmaniasis due to *L. braziliensis* in Bolivia [18, 22], 53% for cutaneous leishmaniasis (33% for *L. braziliensis* infection, and 60% for *L. mexicana* infection) in Guatemala [14, 17, 23], and 63% for *L. tropica* infection in Afghanistan [23]. The above cure rates contrast with those reported for the treatment of visceral leishmaniasis (kala-azar) in India [12, 24] and Bangladesh [25] that were higher than 82%. These data highlight the great variability in the clinical outcome depending on the geographical area for reasons that are not well understood.

444 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

The main toxicity for miltefosine involves gastrointestinal organs in both animal and human studies. Thus, miltefosine frequently induces gastrointestinal side effects, such as anorexia, nausea, vomiting and diarrhea, that sometimes lead to drop out from treatment [2, 3, 24]. The testis and retina have been identified as target organs in rats, but the expected and corre‐ sponding effects and symptoms based on these observations have not been detected in clinical studies in humans [26]. Miltefosine distributes widely in body organs and is not metabolized by cytochrome P450 enzymes *in vitro*. Miltefosine has been found to be embryotoxic and fetotoxic in rats and rabbits, and teratogenic in rats, but not in rabbits [26]. Thus, miltefosine is potentially teratogenic, being contraindicated for use during pregnancy, and adequate contraception is required during treatment and for up to 3 months afterwards in women of child-bearing age [2, 3, 26]. An additional concern is the rapid *in vitro* generation of resistance

Miltefosine is a member of a family of structurally-related compounds collectively known as synthetic alkylphospholipids (APLs), that target cell membranes and show pleiotropic actions with multiple biomedical applications in addition to their antitumor effect, which have been widely studied [31-35]. The advent of miltefosine as a new antileishmanial drug introduces APLs as putative novel drugs for the treatment of leishmaniasis. In addition, because of the numerous studies reported on the antitumor action of these compounds, it could be envisaged that the insight acquired for their antitumor action might be of use in the treatment of leish‐

to miltefosine [27-30] that could limit its clinical use.

maniasis.

As stated above miltefosine is a member of a series of synthetic lipids structurally related and known collectively as APLs, which in turn can be classified in two major categories [31]: a) the alkyl ether phospholipids (AEPs), widely referred to collectively as alkyl-lysophospholipid analogs (ALPs), containing ether bonds in the glycerol backbone of the phospholipid, as exemplified by 1-*O*-octadecyl-2-*O*-methyl-*rac*-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) (Figure 1); b) and the alkylphosphocholines (APCs), lacking the glycerol backbone and formed by a simple long-chain alcohol esterified to a phosphobase, as exemplified by hexadecylphosphocholine (HPC; miltefosine) (Figure 1). All of these APLs show low rates of metabolism both *in vitro* and *in vivo*. APLs were initially shown to elicit actions on the immune system and to kill cancer cells [31, 33, 35]. The first studies of the antiprotozoal activity of APLs in the 1980s against *Tetrahymena pyriformis* [36] and *L. donovani* [37, 38] were reported soon after their development as anticancer drugs [39-41]. APLs have been reported to be effective against different *Leishmania* species, including *L. donovani, L. infantum* (Old World)*/chagasi* (New World)*, L. major, L. braziliensis, L. amazonensis, L. mexicana, L. panamensis, L. guyanensis, L. tropica, L. aethiopica* and *L. lainsoni* [16, 42-44].

In 1987, Croft *et al.* [38] reported the antiparasitic action of miltefosine against *L. donovani* amastigotes. Subsequently, *in vivo* studies showed the antileishmanial activity of miltefosine (10-20 mg/kg of body weight/day, orally administered) in mice infected with *L. donovani* and *L. infantum* [45]. Later on, following eight successful clinical trials [12], miltefosine (Impavi‐ do®) became the first oral treatment of visceral leishmaniasis and is the most recent drug to come to the market for the treatment of this disease.

Within APLs, edelfosine has been considered as the long-standing prototype of these com‐ pounds, and recent evidences have unveiled part of its mechanism of action as a promising antitumor drug. Another APL named perifosine, a miltefosine analog, is currently in clinical trials for different types of cancer. Thus, the insights gained in the last years on both the mechanism of action of APLs as antitumor agents, especially edelfosine, and the clinical experience acquired with some of these compounds, particularly miltefosine and perifosine, constitute an invaluable source of information for the putative use of APLs as antiparasitic drugs, as well as for the development of novel APL-related compounds as antileishmanial drugs. Very recently, edelfosine has been shown to be effective against different types of *Leishmania* parasites in *in vitro* and *in vivo* studies, displaying a better profile than miltefosine on the generation of drug resistance [30]. In addition, edelfosine exerts a potent anti-inflam‐ matory effect [46], which is of importance as several of the clinical signs of leishmaniasis are due to an inflammatory response. In this regard, it is also worth mentioning that current evidence suggests that *Leishmania* parasites are initially engulfed by neutrophils, a major inflammatory cell type, and delivered to cytoplasmic neutrophil granules [47], where they survive and retain infectivity, thus using neutrophils as transport vehicles before they enter safely and silently their ultimate host cell, the macrophage [48, 49]. Increasing evidence suggests that APLs are new promising oral leishmanicidal drugs that could act either as single drugs or as combination therapy.

The antileishmanial efficacy of miltefosine in T cell-deficient athymic mice infected with *L. donovani* (25 mg/kg of body weight dissolved in a volume of 0.3 ml tap water, orally adminis‐ tered) was similar to that found in normal mice [50, 51]. These observations were extended in T and B-cell deficient severe combined immunodeficiency (SCID) mice, where miltefosine showed a similar effect to that detected in normal BALB/c mice [52]. These results raise the possibility that miltefosine may be of interest as an initial oral treatment approach to the growing problem of AIDS-associated visceral leishmaniasis in CD4 cell-depleted patients [53].

Currently, miltefosine is the only APL in the market for the treatment of leishmaniasis, but edelfosine and perifosine show promising and potent anti-*Leishmania* activity both *in vitro* and *in vivo* [30, 54, 55], warranting further studies for their putative clinical use in the future. The mechanism of action of miltefosine is not properly understood, but a clear correlation between the accumulation of the drug within the parasite and its toxic effects has already been described [27], this notion being identical to that previously advanced for edelfosine in its antitumor activity [32, 56-58], and therefore the ability of APLs to kill different *Leishmania* species and cancer cells is critically dependent on the drug uptake.

#### **4. Mechanism of action of miltefosine as a leishmanicidal drug**

The studies directed to unravel the underlying mechanism of miltefosine as an antiparasitic drug have followed to great extent the same trends previously reported and advanced in the analysis of the molecular and cellular pathways involved in the antitumor action of APLs. Thus, the insights generated from the antitumor action of APLs on cancer cells are being extrapolated to the APL killing action on *Leishmania* parasites. In this regard, a stark example lies in the direct induction of cell death by APLs in both tumor cells and *Leishmania* parasites. Following the studies reported simultaneously in 1993 by Diomede's group in Milan [59] and Mollinedo's group in Madrid [60], showing that the APL edelfosine induced apoptosis in a wide variety of tumor cells, subsequent studies extended this proapoptotic activity against cancer cells to other APLs, including miltefosine and perifosine [61, 62]. Later on in the new millennium, a number of studies have started to unveil the underlying mechanisms, signaling pathways and subcellular structures involved in the antitumor activity of APLs, including reorganization of lipid raft membrane domains, death receptors and mitochondria [32, 34, 63, 64]. These studies have provided a conceptual framework for a better understanding of the processes involved in the anti-*Leishmania* activity of APLs. Thus, two reports came out in 2004 showing that miltefosine induced a cell death process showing several features of metazoan apoptosis in *L. donovani* promastigotes and amastigotes [65, 66], including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. Then, subsequent studies have reported the miltefosine-mediated induction of an apoptosislike cell death in promastigotes from different species of *Leishmania* promastigotes, including *L. amazonensis* [67], *L. infantum* [68], *L. tropica* [69], *L. major* [69], *L. panamensis* and many others [30]. The fact that an apoptosis-related process seems to be involved in the death of *Leishma‐ nia* parasites upon APL addition is further supported by the recent finding that tolerance to undergo apoptosis-like cell death in *Leishmania* is linked to multidrug resistance within the parasite *in vitro* [70]. This raises the concern that cross-tolerance to drug-induced apoptosislike cell death, not only against a particular selective drug promoting resistance, but also against additional drugs sharing a similar mode of killing, might lead to a facilitated emergence of cross-resistance against other drugs that have different cellular targets but analogous ways of killing [70]. However the mechanism by which APLs induce an apoptosis-like cell death in *Leishmania* parasites remains to be fully elucidated.

The antileishmanial efficacy of miltefosine in T cell-deficient athymic mice infected with *L. donovani* (25 mg/kg of body weight dissolved in a volume of 0.3 ml tap water, orally adminis‐ tered) was similar to that found in normal mice [50, 51]. These observations were extended in T and B-cell deficient severe combined immunodeficiency (SCID) mice, where miltefosine showed a similar effect to that detected in normal BALB/c mice [52]. These results raise the possibility that miltefosine may be of interest as an initial oral treatment approach to the growing problem of AIDS-associated visceral leishmaniasis in CD4 cell-depleted patients [53]. Currently, miltefosine is the only APL in the market for the treatment of leishmaniasis, but edelfosine and perifosine show promising and potent anti-*Leishmania* activity both *in vitro* and *in vivo* [30, 54, 55], warranting further studies for their putative clinical use in the future. The mechanism of action of miltefosine is not properly understood, but a clear correlation between the accumulation of the drug within the parasite and its toxic effects has already been described [27], this notion being identical to that previously advanced for edelfosine in its antitumor activity [32, 56-58], and therefore the ability of APLs to kill different *Leishmania* species and

cancer cells is critically dependent on the drug uptake.

446 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

**4. Mechanism of action of miltefosine as a leishmanicidal drug**

The studies directed to unravel the underlying mechanism of miltefosine as an antiparasitic drug have followed to great extent the same trends previously reported and advanced in the analysis of the molecular and cellular pathways involved in the antitumor action of APLs. Thus, the insights generated from the antitumor action of APLs on cancer cells are being extrapolated to the APL killing action on *Leishmania* parasites. In this regard, a stark example lies in the direct induction of cell death by APLs in both tumor cells and *Leishmania* parasites. Following the studies reported simultaneously in 1993 by Diomede's group in Milan [59] and Mollinedo's group in Madrid [60], showing that the APL edelfosine induced apoptosis in a wide variety of tumor cells, subsequent studies extended this proapoptotic activity against cancer cells to other APLs, including miltefosine and perifosine [61, 62]. Later on in the new millennium, a number of studies have started to unveil the underlying mechanisms, signaling pathways and subcellular structures involved in the antitumor activity of APLs, including reorganization of lipid raft membrane domains, death receptors and mitochondria [32, 34, 63, 64]. These studies have provided a conceptual framework for a better understanding of the processes involved in the anti-*Leishmania* activity of APLs. Thus, two reports came out in 2004 showing that miltefosine induced a cell death process showing several features of metazoan apoptosis in *L. donovani* promastigotes and amastigotes [65, 66], including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. Then, subsequent studies have reported the miltefosine-mediated induction of an apoptosislike cell death in promastigotes from different species of *Leishmania* promastigotes, including *L. amazonensis* [67], *L. infantum* [68], *L. tropica* [69], *L. major* [69], *L. panamensis* and many others [30]. The fact that an apoptosis-related process seems to be involved in the death of *Leishma‐ nia* parasites upon APL addition is further supported by the recent finding that tolerance to undergo apoptosis-like cell death in *Leishmania* is linked to multidrug resistance within the

One critical organelle that seems to be involved in the killing process in *Leishmania* is the mitochondrion. Miltefosine treatment has been found to lead to loss of mitochondrial mem‐ brane potential and the release of cytochrome *c* with consequent activation of cellular proteases in *L. donovani* promastigotes, even in arsenite-resistant L. *donovani* promastigotes displaying a multidrug resistance phenotype and overexpressing Pgp-like protein [71]. The finding that edelfosine-induced cell death in *L. infantum* promastigotes can be regulated by the ectopic expression of the antiapoptotic and proapoptotic members of the Bcl-2 family of proteins Bcl-XL and Hrk, which affect mitochondria-related processes, suggests that this process shows certain similarities to apoptosis in eukaryotic cells and that mitochondria are involved in the killing process [72]. Furthermore, miltefosine inhibits mitochondrial cytochrome *c* oxidase in *L. donovani* promastigotes, and this enzyme was suggested to act as a target for this APL [73]. In this regard, cytochrome *c* oxidase has also been identified as a potential target of miltefosine from a genomic library screen of the model yeast *Saccharomyces cerevisiae* [74]. Miltefosine inhibited cytochrome *c* oxidase activity in a dose-dependent manner, and this inhibition most likely contributed to the miltefosine-induced apoptosis-like cell death in *S. cerevisiae* [74]. Figure 2 depicts a schematic view for the antileishmanial activity of miltefosine based on current data.

**Figure 2.** Schematic view of the antileishmanial mechanism of action of miltefosine. See text for further details.
