Antiprotozoal Activities of Phospholipid Analogues Simon L

Molecular & Biochemical Parasitology 126 (2003) 165–172

Review Antiprotozoal activities of phospholipid analogues Simon L. Croft a,∗, Karin Seifert a, Michael Ducheneˆ b a Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK b Division of Speciﬁc Prophylaxis and Tropical Medicine, Department of Pathophysiology, AKH, Währinger Gürtel 18-20, A-1090 Vienna, Austria Received 3 June 2002; received in revised form 8 August 2002; accepted 13 August 2002

Abstract

The antiprotozoal activity of phospholipid analogues, originally developed as anti-cancer drugs, has been determined in the past decade. The most susceptible parasites are Leishmania spp. and Trypanosoma cruzi with activity also shown against Trypanosoma brucei spp., Entamoeba histolytica and Acanthamoeba spp. Miltefosine, an alkylphosphocholine, was registered for the oral treatment of visceral leishmaniasis (VL) in India in March 2002. This review will focus on the biological activities of phospholipid analogues. Biochemical and molecular targets and mechanism(s) of action have been studied extensively in tumor cells but have not been determined in protozoa. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Phospholipid analogues; Miltefosine; Protozoa

1. Introduction hexadecylphosphocholine (HPC, miltefosine) for the topical treatment of skin metastases was approved in Au- Interest in phospholipid analogues as chemotherapeutic gust 1992. The discovery of the ether-lipid structure of agents resulted from the work of Munder and co-workers platelet-activating factor (PAF) also prompted further in- [1] following their investigation of the immunomodula- terest in the synthesis and activity of phospholipids [8,9] tory activity of lysophosphatidylcholine (LPC). As LPC (Fig. 1d). has a short half-life in biological systems, due to cleav- First reports of the antiprotozoal activity of phospholipid age by phospholipases or acyltransferases, more stable analogues in the 1980s against Tetrahymena pyriformis [10] derivatives were synthesized. Some of these derivatives and Leishmania donovani [11–14] occurred at the same time were found to inhibit the proliferation of tumor cells [2,3]. as their development as anti-cancer drugs [15]. This review Other analogues of LPC were synthesized by other re- will focus on current knowledge of the antiprotozoal activi- search groups as potential anti-tumor compounds and dif- ties of two groups of phospholipid analogues. Firstly, alkyl- ferences in lipid and phospholipid metabolism as well as glycerophosphocholines (AGPCs) that possess the glycerol lipid structure and composition were explored in normal backbone substituted in the sn-2 position as exemplified by and neoplastic cells [4,5]. Studies on the minimal struc- rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine tural requirement of ether-phospholipids for antineoplastic (ET-18-OCH3, edelfosine, see Fig. 1b), and secondly, the activity revealed essential cleavage sites for phospholi- alkylphosphocholines (APCs), in which phosphocholine is pase C and phospholipase D. Elimination of the glycerol esterified directly with long-chain alcohols without a glyc- backbone led to the synthesis and testing of alkylphos- erol moiety, as exemplified by hexadecylphosphocholine phocholines against tumor cells [6,7]. A formulation of (miltefosine, see Fig. 1a). The review will focus on biological activities of phospholipid analogues as biochemical and molecular targets and mechanism(s) of action have not Abbreviations: LPC, lysophosphatidylcholine; HPC, hexadecylphosphocholine; PAF, platelet-activating factor; AGPC, alkylglyc- been identified. erophosphocholine; AGPE, alkylglycerophosphoethanolamine; APC, alkylphosphocholine; APE, alkylphosphoethanolamine; ET-18-OCH3, rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine; ED50, 50% ef- 2. Antileishmanial and antitrypanosomal activities fective dose ∗ Corresponding author. Tel.: +44-20-7927-2345; fax: +44-20-7929-2807. Initial studies on Leishmania by Achterberg and Gercken E-mail address: [email protected] (S.L. Croft). [11,12] showed that several AGPCs were active against the

Fig. 1. Structures of the phospholipid analogues: (a) hexadecylphosphocholine, (b) 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), (c) 1-hexadecyl-mercapto-2-methoxymethyl-sn-glycero-3-phosphocholine (BM 41.440), (d) 1-O-hexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF). (a) is a representative alkylphosphocholine (APCs), (b) to (d) are representative alkylglycerophosphocholines (AGPCs). extracellular promastigote form of L. donovani while Croft rols, ester and ether lysophospholipids were active against et al. [13] showed that a series of APCs, including hexade- L. donovani promastigotes. Of these, the 1-O-AGPCs, cylphosphocholine (miltefosine), were active against the in- 1-O-alkylglycerophosphoethanolamines (AGPEs) and tracellular amastigote form of L. donovani both in vitro and 1-O-hexadecyl-sn-glycerol were the most active compounds in vivo. Subsequent studies have extended information on with <3 ␮MED50 values. In contrast, 1-stearoylglycerol the antileishmanial activity of the phospholipid analogues, was inactive suggesting that the ether linkage at the including other AGPCs edelfosine, ilmofosine (Fig. 1c) and sn-1 position in the glycerol was important for ac- SRI-62,834 that reached clinical trial for the treatment of tivity. The highest activity identiﬁed by Achterberg cancer, against L. donovani [16,17], against other Leishma- and Gercken [11] was by the anti-tumor compound nia species ([18,19]; Fiavey and Ngwenya, 1991, Meeting rac-1-O-octadecyl-2-O-methylglycero-3-phosphocholine Abstract of the 40th Annual Meeting of the Am Soc Trop (ET-18-OCH3, edelfosine) which has a methoxy residue at Med Hyg) and against Trypanosoma spp. [17]. the sn-2 position. Further studies on ET-18-OCH3 and two other AGPCs, 1-hexadecyl-mercapto-2-methoxymethyl-2- 2.1. Relationships of structure with antileishmanial activity deoxy-rac-glycero-3-phosphocholine (ilmofosine) and rac-2-(hydroxy-[tetrahydro-2-(octadecyloxy)methylfuran-2- The early studies of Herrmann and Gercken [14] and yl]methoxyl)-phosphinoxy - N, N, N-trimethylethaniminium Achterberg and Gercken [11,12] showed that 1-O-alkylglyce- hydroxide (SRI 62,834) showed that they had activity at S.L. Croft et al. / Molecular & Biochemical Parasitology 126 (2003) 165Ð172 167

<5 ␮M against L. donovani amastigotes in macrophage hexadecyl-, and octadecylphosphocholine against T. b. bru- models [16,17]. A dialkylGPC, rac-1-dodecyl-2-octanami- cei, T. b. rhodesiense and T. b. gambiense bloodstream form do-2-deoxy-glycerophosphocholine, was also active against trypomastigotes confirmed this low level of in vitro activity L. donovani promastigotes and amastigotes in similar mod- [29]. els in the same range as ET-18-OCH3 [20]. In contrast, C18 and C16 lyso and acyl analogues of PAF were poorly 2.3. Relationships of life cycle stage and susceptibility active (ED50s >30 ␮M) against L. donovani promastigotes and had no selective activity in the amastigote–macrophage Variation in the activities of phospholipid analogues model (Matu S, Croft SL, unpublished). However, at lower against the different life cycle stages has also been reported. concentrations acyl-PAF was shown to affect differentiation In a comparative study of six Leishmania species over 72 h, of other trypanosomatids [21,22]. promastigotes were more sensitive than amastigote stages, Alkylphosphocholines (APCs), with different alkyl chain for example ED50 values of HPC being 0.5 and 4.6 ␮M lengths and distances between P and N of the phospho- against L. donovani promastigotes and amastigotes, respec- choline moiety, have been more extensively studied for tively, and 13.1 and 37.2 ␮M against L. major promastig- their antileishmanial activity. Seven APCs, including hex- otes and amastigotes, respectively [18]. T. cruzi intracel- adecylphosphocholine (HPC, miltefosine), and one APE lular amastigotes in heart muscle cells or Vero cells were were reported active at <10 ␮g/ml against L. donovani more sensitive than extracellular epimastigotes to HPC, amastigotes and promastigotes [13]. This data was extended ET-18-OCH3 and ilmofosine [27,28]. All these compounds by Unger et al. [23] who tested 13 APCs and derivatives were significantly less active against the non-dividing T. against L. donovani promastigotes, of which 10 had ED50 cruzi bloodstream trypomastigotes [17,27]. A further sig- values <5 ␮g/ml, the most active being octadecylPC and nificant difference is seen in T. b. brucei where activity related compounds. Pyrrolidine or piperidine derivatives values of around 50 ␮M were reported on bloodstream form of octadecylPC had reduced activity in comparison to the trypomastigotes [17,29] but significantly lower ED50 val- parent compound. ues (1–5 ␮M) were observed for procyclic trypomastigotes (Croft SL, unpublished). 2.2. Susceptibility of different trypanosomatids In comparative studies between extracellular and intracellular stages it is important to consider (i) the effective drug The differing susceptibilities of tumor cell lines to phos- concentration in macrophages or other host cells might be pholipid derivatives have been reported [24] although the higher or lower than that outside the cells, (ii) the differing basis for this variation is still unclear [25]. Likewise, Leish- division rates of stages, and (iii) the different temperatures mania species are known to vary in their drug sensitivity used in assays. [26]. Although activities of both ET-18-OCH3 and HPC have been previously reported against promastigote stages 2.4. In vivo activities of other Leishmania species (Fiavey and Ngwenya, 1991; [19]) only recently has there been a comparative study of In the first study, HPC (miltefosine) was the most active species drug sensitivity [18]. In this latter study the sensi- of three APCs tested against L. donovani in BALB/c mice tivities of both promastigote and amastigote stages of six with an ED50 value of 13.8 mg/kg × 5 s.c. [13]. Based on species were determined to HPC and ET-18-OCH3.Inall the observation that oral treatment of rats was possible with assays L. donovani was the most sensitive species with val- little or no side-effects [30], a subsequent study in a simi- ues of 0.1–1.3 ␮M against promastigotes and 1.2–4.6 ␮M lar mouse model was carried out in which miltefosine gave against amastigotes, whereas L. major was the least sensi- >95% suppression of L. donovani and L. infantum amastig- tive with ED50 values for HPC of 4.8–13.2 ␮M against pro- otes in the liver, spleen and bone marrow when administered mastigotes and for HPC and ET-18-OCH3 of 7.5–37.1 ␮M orally at 20 mg/kg×5 [31]. This oral activity of HPC against against amastigotes. L. infantum in mice was confirmed by Le Fichoux et al. [32]. Phospholipid analogues also proved highly active The activity of antileishmanial drugs in immune-deficient against T. cruzi. The AGPCs ET-18-OCH3, ilmofosine and mice could be relevant to problems associated with treat- SRI-62,834 and the APC HPC were shown to have <2.0 ␮M ment of patients with L. infantum—HIV co-infections. Mil- ED50 values against intracellular amastigotes of T. cruzi (Y tefosine was shown to be highly active in T-cell deficient strain) in murine macrophages [17]. Subsequently, simi- nude mice at 25 mg/kg × 5 p.o. [33] as well as knockout lar high levels of activity of ET-18-OCH3, ilmofosine and mice lacking specific macrophage killing mechanisms [34]. HPC were reported against both extracellular epimastig- These observations were extended by Escobar et al. [35] otes and intracellular amastigotes in heart muscle or Vero who showed that miltefosine had similar ED50 and ED90 cells [27,28]. In contrast, these phospholipid analogues values against L. donovani in both BALB/c and C.B-17 scid showed significantly lower activity against Trypanosoma mice. In comparison the standard antileishmanial sodium brucei brucei and T. b. rhodesiense bloodstream form try- stibogluconate was inactive in the immunodeficient mouse pomastigotes [17]. A study of the activity of tetradecyl-, models. 168 S.L. Croft et al. / Molecular & Biochemical Parasitology 126 (2003) 165Ð172

Miltefosine was also active in experimental models considered for cutaneous leishmaniasis. In a phase I/II trial of cutaneous leishmaniasis. A 6% miltefosine ointment in Colombia oral miltefosine 150 mg/kg for 28 days gave a (MiltexTM), developed for treatment of breast cancer skin cure rate >90% [41]. metastases, was applied topically to established lesions of L. mexicana or L. major on either BALB/c, CBA/J or C57Bl/6 3. Mechanisms of antileishmanial and mice [36]. Lesions healed following applications over 2–5 antitrypanosomal action weeks accompanied by a corresponding reduction in the number of parasites in the spleen and the draining lymph Although many studies have been undertaken to reveal nodes. However, there was no promotion of a Th1-type the mechanism of action of phospholipid analogues against response in the miltefosine treated mice. tumor cells [24,42,43] no definitive mechanism of action has Similar high efficacy of these phospholipid drugs was not been identified [25]. In comparison few studies have been observed in in vivo studies against experimental trypanoso- made on mechanism(s) of action against trypanosomatids. miasis. Miltefosine, ET-18-OCH3 and ilmofosine had sup- pressive activity against T. cruzi Y and Tulahuen strains 3.1. Biochemical/molecular targets in cancer cells in mice at 30 mg/kg × 5 p.o. [17]. No other in vivo studies have been reported. However, the potential of combi- Evidence is considered for three possible mechanisms. nations, demonstrated in vitro with ketoconazole [28] must Firstly, studies suggested that mitogenic signal transduc- be considered. Against T. b. brucei in mice a top dose of tion and second messenger generation via inhibition of pro- 30 mg/kg × 5 p.o. miltefosine had no activity [17], whereas tein kinase C could be the target [44]. However, inhibi- in another study it prolonged survival time at 30 mg/kg× 10 tion of PKC does not necessarily match with the antipro- p.o., an activity that was improved by co-administration of liferative activity of miltefosine and PKC depleted cells phenylbutazone which displaces drugs from serum proteins are not changed in their sensitivity towards edelfosine [45]. [29]. Another proposed target is phosphatidylcholine biosynthesis, which has been shown to be inhibited by miltefosine 2.5. Clinical trials for leishmaniasis [46]. Miltefosine is considered to inhibit the translocation of CTP:phosphocholine-cytidylyltransferase, the key enzyme The antileishmanial activity of miltefosine in experimen- of PC-biosynthesis, from its inactive cytosolic form to its tal models and available pharmacological and toxicological active membrane-bound form. In addition, sphingomyelin data from anti-cancer clinical trials, led Asta Medica (Frank- biosynthesis has been shown to be inhibited by miltefos- furt, Germany) and WHO/TDR in 1996 to consider milte- ine, leading to increased levels of cellular ceramide [47]. fosine for clinical trials against visceral leishmaniasis (VL). It is suggested that this latter metabolite triggers cells to The initial phase I/II dose ranging study in Bihar State, In- undergo apoptosis [47,48]. Induction of apoptosis has also dia, involved 30 VL patients who received either daily or been shown for edelfosine in different human tumor cell alternate day oral doses of 50–250 mg for 28 days. Eight lines. Structural variation in the sn-2 and sn-3 position of months after treatment 7 out of 10 patients treated with 50 ET-18-OCH3 led to a loss of its ability to trigger apoptosis and 100 mg every other day relapsed, while 18 out of 19 and no such phenomenon was observed in normal cells [49]. (94.5%) treated with 100, 200 or 250 mg per day were cured according to parasitological and clinical criteria [37].Ina 3.2. Molecular targets in the trypanosomatids subsequent phase II study on 45 patients, including 17 cases who had failed to respond to previous treatment with an- Studies on Leishmania mexicana promastigotes sug- timonials, miltefosine at 100–200 mg per day for 28 days gested miltefosine and edelfosine might cause perturbation gave a 98% cure rate [38]. Another phase II trial on 120 VL of ether-lipid metabolism, GPI anchor biosynthesis and patients receiving 50–150 mg per day miltefosine for 28–42 leishmanial signal transduction [19]. Later Lux et al. [50] days, with clinical response assessed 6 months after treat- showed inhibition of the glycosomal located alkyl-specific ment, gave a 95% cure rate at 100 mg per day, again includ- acyl-Co-A acyltransferase in L.m., an enzyme involved in ing patients resistant to pentavalent antimony [39]. Mild to lipid-remodeling, by both miltefosine and edelfosine in a moderate gastrointestinal side-effects were reported in 62% dose-dependent manner. However, the IC50 for inhibition of the patients but no patient discontinued the treatment. In of this enzyme was 50 ␮M, making it unlikely to be the further clinical studies 54 patients treated with 50 mg milte- primary mechanism. Clearer evidence for inhibition of fosine twice daily for 14, 21 and 28 days showed cure rates PC biosynthesis in T. cruzi epimastigotes has been shown of, respectively 89, 100 and 100% [40]. Phase III trials have [28]. Edelfosine was found to inhibit the incorporation of 14 now been completed with a cure rate of 94% in a 299 patient l-[methyl- C]methionine into PC with an IC50 of 2 ␮M, study (Sinderman, 2002. Meeting Abstract of the COST-B9 close to the antiproliferative IC50 of 3 ␮M. The authors Expert Meeting on Antiprotozoal Chemotherapy). In March conclude that inhibition of the de novo synthesis through 2002 the drug was approved in India for oral therapy of Greenberg’s pathway could be a primary mechanism for visceral leishmaniasis. In addition, miltefosine is also being the selective activity in T. cruzi. Moreover, a change in the S.L. Croft et al. / Molecular & Biochemical Parasitology 126 (2003) 165Ð172 169

Fig. 2. Transmission electron micrographs of L. donovani amastigotes (a) untreated control, (b–c) treated with 2.5 ␮M edelfosine (N refers to the nucleus, K to the kinetoplast and FP to the flagellar pocket). Initial damage at the membrane of the flagellar pocket (b) leads to cytoplasmic disruption in the adjacent area (c). Arrows point to the areas of damage. Bar in panel represents 500 nm. sterol composition was observed, indicating inhibition of like edelfosine show high structural similarity to PAF, en- sterol C-22 desaturase. try via a PAF receptor was investigated. However, uptake of Possible effects on cell signalling have also been sug- these compounds is non-saturable and they are active against gested as edelfosine inhibited PI-PLC in T. cruzi epimastig- cells lacking a PAF receptor [24,49]. This does not exclude otes, with an involvement on PIP2 signaling pathways [51]. the existence of a receptor on trypanosomatids as PAF in- Although knowledge of signal transduction pathways in try- hibitors blocked the effect of PAF on parasite differentiation panosomatids has grown with genomic analysis [52], and [21,22]. apoptotic mechanisms have been demonstrated in trypanoso- Miltefosine also interferes with cellular carrier systems; matids [53], there is insufficient information available to en- inhibition of the incorporation of 14C-labeled desoxyglu- able comparisons to be made between trypanosomatids and cose, choline and methionine has been shown in cancer cells. mammalian cells. Interference with carrier proteins could lead to depletion of essential nutrients and thus lead to or contribute to growth retardation and cell death. Furthermore, Na+,K+-ATPase, 3.3. Cellular membranes as targets in cancer cells and another important enzyme in signal transduction, is in- trypanosomatids hibited by miltefosine [56]. Studies in multidrug-resistant The first site of interaction of the phospholipid analogues Leishmania tropica on the possible mechanisms of resis- is the cell membrane, in cancer cells [24] as well as in pro- tance to miltefosine and edelfosine demonstrated involve- tozoa. Ultrastructural analysis of miltefosine-, edelfosine-, ment of a P-glycoprotein-like transporter, a member of the and ilmofosine-treated T. cruzi epimastigotes revealed dam- ATP-binding cassette (ABC) superfamily [57]. age to the flagellar membrane which showed extensive blebbing [27]. In the same study an inhibitory effect of edelfosine on differentiation from the epimastigote to the 4. Activities against parasitic amoebae trypomastigote stage was observed. The primary site of damage in edelfosine-treated L. donovani amastigotes ap- Following the successful studies on Leishmania and Try- peared to be the flagellar pocket and the adjacent cytoplasm panosoma, the effect of alkylphosphocholines on two strains (Croft SL, unpublished) (Fig. 2). of E. histolytica was examined [58]. The cytotoxic effect was Uptake of miltefosine into cancer cells has been shown significantly dependent on the structure of the alkyl chain. In to be time-, concentration-, and temperature-dependent, addition the two tested strains showed different susceptibil- and non-saturable [54]. Cellular uptake and distribution is ity. The compounds with the highest activity were oleyl-PC, equal in sensitive and insensitive cell lines [25], whereas a octadecyl-PC, and nonadecenyl-PC with EC50 values for difference in lipid composition of the cell membrane has 48 h treatment between 15 and 21 ␮M for strain SFL-3 and been found with sensitive cells containing high amounts of between 73 and 98 ␮M for strain HM-1:IMSS. The activity ether-lipids [55], confirming other reports on this compound was not as high as against Leishmania but the levels obtained as well as edelfosine. It is also known that Leishmania in the liver and other tissues in animal models [30,59] are contain high levels of ether-lipids, representing another far above 100 ␮M, and therefore successful treatment may example of positive correlation between susceptibility to be possible in humans. phospholipid derivatives and ether-lipid content. Acanthamoeba spp. are causative agents of keratitis Interestingly, correlation of cellular uptake and action was [60], and in immunosuppressed individuals of granuloma- demonstrated for edelfosine in cancer cells [49]. As AGPCs tous amoebic encephalitis (GAE). No efficient and easily 170 S.L. Croft et al. / Molecular & Biochemical Parasitology 126 (2003) 165Ð172 manageable treatments are available for these conditions. Austrian Science Fund and a grant from the Hygiene Fund Therefore, the susceptibility of several Acanthamoeba from the Clinical Institute for Hygiene at the University of spp. to APCs was determined over a similar range as used Vienna. in the Entamoeba study [61]. All APCs showed amoebo- static effects and some of them were highly cytotoxic. In this study, HPC exhibited the highest cytotoxicity against References trophozoites resulting in complete cell death at 40 ␮M, and also displayed significant cysticidal activity. [1] Munder PG, Modolell M, Ferber E, Fischer H. 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