www.who.int/emc/diseases/leish/index.html). The clinical manifestations of ..... abolism, GPI anchor biosynthesis, and signal transduction are putative targets for ...
C Basic & Clinical Pharmacology & Toxicology 2005, 96, 60–65. Printed in Denmark . All rights reserved Copyright C ISSN 1742-7835
Leishmanicidal Activity of Edelfosine, Miltefosine and Ilmofosine Samira Azzouz, Mimoun Maache, Ramon Gil Garcia and Antonio Osuna Institute of Biotechnology, Department of Parasitology, Faculty of Sciences Campus, Fuentenueva C.P: 18071, Granada, Spain (Received August 16, 2004; Accepted September 8, 2004) Abstract: The anti-proliferative action of three alkyl-lysophospholipid derivatives, edelfosine (ET-OCH), miltefosine (Hexadecylphosphocholine), and ilmofosine (BM 14.440) has been studied on the promastigotes and amastigotes of Leishmania donovani. The effect of the three drugs has previously been studied, but the action mode was not clearly elucidated. In this study the effect on the intracellular amastigote forms was evaluated by two different methods: the traditional method, counting the amastigotes within the macrophages stained with Giemsa; and by a new method, staining the nuclear macrophages and amastigotes with ethidium bromide and counting the different population by flow cytometry. This new method, based on the flow cytometry, shows an advantage for evaluating the anti-proliferative effects in intracellular parasites. The ED50 were calculated for the drug activity after 72 hr, and for the three alkyl-lysophospholipid derivatives it were in the range of 26.73–33.31 mM against promastigotes and in the range of 16.46–23.16 against amastigotes. Also, studying the effect against macrophages J774A1, the ED50 were in the range of 24.28–26.38 mM. The effect of the alkyl-lysophospholipids in the macromolecular biosynthesis of the Leishmania donovani, was studied comparing the incorporation of labelled analogues ([3H] thymidine, [3H] uridine and [3H] leucine), respectively, in the DNA, RNA, and proteins of the flagellates treated. Miltefosine was the most active of the alkyl-lysophospholipids, especially in the inhibition of the RNA synthesis. The three compounds studied show high in vitro activity against L. donovani promastigotes and amastigotes.
The World Health Organization considers leishmaniasis one of the most serious diseases caused by protozoan parasites, with approximately 12 million cases in the world (http:// www.who.int/emc/diseases/leish/index.html). The clinical manifestations of this parasitosis vary depending on the aetiological agent (Herwaldt 1999). Visceral leishmaniasis, caused by L. donovani, L. infantum and L. chagasi, with the most characteristic symptoms, hepato- and splenomegalies, can cause 90% mortality when untreated At present, this disease is treated with pentavalent antimony and/or pentamidine salts (Manson-Bahr 1982), as well as with the antifungal drug amphotericin B, in patients who show resistance to antimonial treatments. However, although these treatments can be highly effective against the disease, the drawbacks include high toxicity and administration difficulties (Pearson & Sousa 1985). One major challenge of current research in the field of leishmaniasis is the development of new, effective molecules for chemotherapy. A new compound, derived from alkyl-lysophospholipids, has demonstrated its efficacy against leishmaniasis (Achterberg & Gerchen 1987; Croft et al. 1987, 1993 & 1996). Miltefosine was recently registered for the oral treatment of visceral leishmaniasis in India after successful clinical trials
(Sundar et al. 2000) and has also been used in treating cutaneous leishmaniasis (Soto et al. 2001). The present work provides an analysis of the action mode and the activity of three alkyl-lysophospholipid derivatives: edelfosine, and ilmofosine analogues to ether-lipids (Herrman & Bicher 1988; Kuhlencord et al. 1992), and the alkylphosphocholine derivative miltefosine (Daniel 1993), against Leishmania donovani. Materials and Methods Parasite. The strain of L. donovani used in this study was LCR-133 (Leishmania Reference Center, Jerusalem, Israel), isolated in 1967 from a human case of Kala-azar in Behenber (Ethiopia), and maintained in our laboratory since 1982 by successive passages in cultures of NNN medium, modified with a liquid phase in minimal essential medium (MEM) plus 10% of inactivated foetal bovine serum, kept in a moist air atmosphere at 28 æ. Compounds tested. The compounds studied in the present work were three ether lipids, alkyl-lysophospholipid derivatives, with effective use in cancer therapy. The edelfosine and miltefosine were from Sigma Chemical Co., ilmofosine are from Dr. Simon Croft (Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK). The chemical formulas and compositional structures of the three compounds are: 1- 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine.
Author for correspondence: S. Azzouz, Department of Parasitology (Pr. Petavy), Faculty of Pharmacy, University Claude Bernard-Lyon I, 8, avenue Rockefeller, 69373 Lyon Cedex 08, France (fax π33 (0)4 78 77 71 58, e-mail sazzouz/ugr.es).
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ALKYL-LYSOPHOSPHOLIPIDS AND LEISHMANIA DONOVANI
fected as described above but without the alkyl-lysophospholipid derivatives added.
2- Hexadecylphosphocholine
3- 1-S-hexadecyl-2-methoxymehyl-rac-glycero-3-phosphocholine.
Promastigotes assays. For the chemotherapeutic assays, the parasites were cultivated in TC-199 medium (Gibco) supplemented with 20% inactivated foetal bovine serum, inactivated at 56 æ for 30 min., in Roux flasks 75 cm2 surface area (Costar, Cambridge, MA, USA). At the end of the exponential growth phase of the promastigote, the liquid medium was centrifuged (1500¿g, 15 min.) and the number of flagellates counted and distributed into aliquots of 1¿106 parasites/ml in a 96 multi-well plate at 1.9, 19 and 190 mM concentrations. The assays were carried out in triplicate at each concentration, for 72 hr at 28 æ. Inhibition of promastigote growth was determined microscopically by counting parasite numbers in a Neubauer haemocytometer. Inhibition of growth was determined by comparison to untreated controls. Cell culture and cytotoxicity tests. The macrophagic cell line, J774A.1 (ATCC, tibb 67), was used for cytotoxicity assays. The macrophages were kept by successive subcultures in RPMI 1640 medium plus 20% inactivated foetal bovine serum at 37 æ in a moist atmosphere enriched with 5% CO2. When the macrophages had developed a monolayer, the cells were treated with the different products at 1.9, 19, and 190 mM concentrations. Cell viability was determined using the vital stain Trypan Blue (Aldrich) at 0.1% concentration in phosphate buffer (MesaValle et al. 1996). The number of dead cells was recorded, and percentage of viability was calculated versus the control culture. Amastigote assay. By optic microscope study. For the evaluation of the alkyl-lysophospholipid action in the intracellular amastigote forms of Leishmania, J-774A.1 cells were cultivated in a flat multi-well plate. Each well was covered by a borosilicate coverslip for microscopic study of the infected monolayer. After the cells adhered to the glass the medium was removed and replaced with fresh medium with the Leishmania promastigote forms in suspension. The infection rate was 1:1 flagellate forms/cell. After 12 hr at 37 æ the infected cultures were washed and the medium with the different alkyl-lysophospholipid were added at concentrations of 1.9, 19 and 95 mM, for 4 hr. Afterwards, the cultures were washed with fresh medium, and at 24, 48, and 72 hr the infected cultures were fixed in methanol and stained with Giemsa. The parasitism percentage and the parasite index (number of amastigote per cell) were calculated. The minimum number of cells counted in each replicate was 500. Control cultures were in-
Flow-cytometry study. Cultures were made in a 12 multi-well plates (Costar, Cambridge, MA, USA). After the cells adhered and developed a monolayer, the medium was removed and replaced with fresh medium with the Leishmania promastigote forms in suspension. The infection rate was 1:1 flagellate forms/cell. After 12 hr at 37 æ the infected cultures were washed and the medium with the different alkyl-lysophospholipid was added at concentrations of 1.9, 19 and 95 mM, for 4 hr. Afterwards, the cultures were washed with fresh medium and returned to RPMI 1640 medium plus 20% IFCS at 37 æ in a moist atmosphere enriched with 5% CO2. At 24, 48, and 72 hr, the treated infected cells and the control infected cells were washed 3 times with PBS and then submitted to a hypotonic solution composed of 100 mg sodium citrate, 100 ml H2O, 5 mg ethidium bromide, and 30 ml Nonidet P-40. Incubation was carried out for 15–20 min. at room temperature in the dark. The hypotonic solution is used for solubilizing membrane proteins of the cells by the detergent Nonidet P-40. Sodium citrate stabilize the cells and parasite nuclei, and keep them from sticking together. Ethidium bromide is a DNA-binding fluorochrome which, like PI, has no base preference. The cells, detached with a rubber policeman, were fixed under constant stirring in a 10% formaldehyde solution and measured in a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA, USA). The nuclei were analysed using a dual laser (488 nm visible and 395 nm ultraviolet). The laser beam was regulated to 488 nm with a power of 65 mW, to acquire the data at life size in the FSC photodetector with a 488/ 10 BP on a linear scale. The fluorescence was measured in an FL1 photodetector regulated to 460 (log scale) with 530/30 BP for fluorescein. The ethidium bromide fluorescence data were acquired in an FL2 photodetector regulated to 540 nm (log scale) with a 585/40 BP filter. A 10,000 events were recorded over a period of 350 sec. Results were statistically analysed with the Lysys II Program from Becton Dickinson. We used as controls a total lysate of infected cells and also the purified nuclei from an uninfected cell culture from which we were able to identify their peak. Effect of alkyl-lysophospholipids on DNA, RNA and protein synthesis. To measure the effect of the alkyllysophospholipid derivatives on the biosynthesis of nucleic acids and proteins, incorporation of three radiolabelled precursors, 6-[3H]-thymidine (specific activity 26–30 Ci/mmol), 5-[3H]-uridine (specific activity 25–30 Ci/ mmol) and 4.5-[3H]-leucine (specific activity 35–70 Ci/mmol) (Du Pont) was studied. The synthesis of DNA and RNA was measured by studying incorporation of thymidine and uridine, respectively, and the protein synthesis was measured by studying leucine incorporation. For this, cultures with 5¿106 parasites/ml were centrifuged, the supernatant was eliminated, and the pellet was resuspended in 1 ml of culture medium plus 95 mM of the compound. After an incubation period of 30 min., at 28 æ, the culture medium was eliminated by centrifugation. The parasites were resuspended in culture medium with the
Table 1. Activities of edelfosine, miltefosine and ilmofosine against Leishmania donovani promastigotes and amastigotes, and against macrophages. Edelfosine Macrophages Promastigotes Amastigotes
24.28 32.31 23.16a 19.47b
(21.25–27.32) (29.11–35.51) (20.09–26.23)a (18.33–20.61)b
Miltefosine 26.38 26.73 17.47a 16.46b
(23.63–29.14) (23.28–30.19) (15.82–19.12)a (14.66–18.27)b
ED50 values in micromolar with P95 confidence limits. a Values obtained by flow cytometry. b Values obtained by optic microscope. Values of two independent experiments are given with 95% confidence intervals in brackets.
Ilmofosine 26.32 31.31 21.39a 17.67b
(22.42–30.23) (27.42–35.21) (19.33–23.46)a (16.76–18.59)b
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SAMIRA AZZOUZ ET AL.
Fig. 1. Effect of edelfosine on the incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine by L. donovani promastigotes. ***P⬍0.001, Bonferroni test; **P⬍0.01, Bonferroni test; *P⬍0.05, Bonferroni test. radioactive analogue at a 5 mCi/ml dose. At 0, 45, 75 and 145 min., the cultures were centrifuged, and the pellet, after three washes with cold medium, was precipitated with 10% trichloroacetic acid (TCA, Probus) for 2 hr at 4 æ. Precipitates were filtered through Whatman GF/c filters, and washed first with TCA at 5%, and then with ethanol at 70%, and dried at 60 æ under infrared lamps. Once dried, the filters were submerged in scintillation cocktail (2,5-diphenyloxazole [PPO], 4g; 1,4-bis(5-phenyloxazolyl)benzene [POPOP] 0.1 g; toluene 1,000 ml). The radioactivity incorporated was measured in a Liquid Scintillation System model LS 6000TA (Beckman). Radioactivity contained in the precipitable material was noted as a reflection of the radioactivity present in and incorporated by the precipitable material of the parasites. Inhibition of synthesis was determined by comparison to untreated controls. Statistical analysis. ED50 values were calculated by a nonlinear regression to sigmoidal curves with the use of the Prism program (Graph Pad Software, Berkeley, CA, USA). Values are expressed as the mean∫S.D. The significance of the difference from the respective controls for each experimental test condition was assayed by using One-way Analysis of Variance (ANOVA), and Bonferroni post test was employed. Statistical differences having p⬍0.05 were considered to be significant.
Fig. 2. Effect of miltefosine on the incorporation of [3H]thymidine, [3H]uridine, and [3H]leucine by L. donovani promastigotes. ***P⬍0.001, Bonferroni test; **P⬍0.01, Bonferroni test; *P⬍0.05, Bonferroni test.
incubation of the flagellates treated with the radioactive analogues for 145 min. The data referring to the control cultures show, in all cases, increased numbers of CPM, revealing an active synthesis of DNA, RNA and proteins. The three compounds lowered the level of incorporation of the radiolabelled thymidine, uridine and leucine with respect to control. After 135 min. of incubation with the alkyl-lysophospholipids, the inhibition is more pronounced in RNA synthesis than in DNA and protein synthesis (table 2).
Results Antiproliferative effects. In the 72 hr assays edelfosine, miltefosine and ilmofosine were more active against intracellular amastigotes than against extracellular promastigotes. The range of ED50 values for edelfosine, miltefosine and ilmofosine against promastigotes was respectively 32.66, 27.33 and 31.33 mM. Against intracellular amastigotes, the ED50 values for edelfosine, miltefosine and ilmofosine were respectively 23, 17 and 21 mM for the flow citometry method, and 19,16.66 and 17 mM for the microscopic method (table 1). Effects of alkyl-lysophospholipids on macromolecule synthesis. Fig. 1, 2 and 3 present the data for incorporation of thymidine, uridine and leucine in the precipitable material after
Fig. 3. Effect of ilmofosine on the incorporation of [3H]thymidine (A), [3H]uridine (B), and [3H]leucine (C) by L. donovani promastigotes. ***P⬍0.001, Bonferroni test; **P⬍0.01, Bonferroni test; *P⬍0.05, Bonferroni test.
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ALKYL-LYSOPHOSPHOLIPIDS AND LEISHMANIA DONOVANI Table 2. Alkyl-lysophospholipids action on the biosynthesis of DNA, RNA and protein. Time (min.) 0 45 90 135
Edelfosine DNA
RNA
Miltefosine Protein
0.114∫0.001 0.084∫0.0331 0.009∫0.006 0.086∫0.012 16.5∫0.074 0.025∫0.002 41.75∫2.548 38.20∫2.36 8.89∫1.25 57.85∫0.695 73.22∫1.516 68.02∫0.739
Ilmofosine
DNA
RNA
Protein
0.016∫0.004 4.87∫0.043 46.6∫0.026 73.46∫0.072
43.06∫0.81 67.76∫0.036 90.30∫3.162 96.32∫0.751
0.006∫0.002 55.03∫0.03 64.28∫1.56 83.97∫0.045
DNA
RNA
Protein
28.66∫0.03 33.5∫0.143 9.19∫0.11 17.94∫0.001 24.19∫0.1 48.08∫0.07 49.34∫0.07 55.49∫0.283 42.72∫0.15 40.32∫0.155 73.72∫0.054 65.60∫0.31
The results are expressed in percentage of synthesis inhibition (%IC) calculated by the formula % ICΩ(TcªTp/Tc)¿100. Tc being the CPM calculated in the control tubes, and Tp the CPM corresponding to the different products tested. (nΩ3, ∫S.D.).
Discussion Antiproliferative effects. According to the results of our study, the three alkyl-lysophospholipids assayed show an in vitro anti-proliferative activity which was very high against promastigotes and intracellular amastigotes of L. donovani. This confirms the results reported by other authors, who have demonstrated the leishmanicidal activity of different alkyl-lysophospholipids (Croft et al. 1987, 1993 & 1996). The differences in the low activity of the alkyl-lysophospholipids against the promastigotes with respect to the amastigotes can be accounted for by the fact that the alkyllysophospholipids strengthen the cytotoxicity of the macrophages and consequently aid in the death of the parasite within the infected cell, either by the direct action of the compounds or by the secondary effect on the host cells (Berdel 1980; Andreesen et al. 1984; Daniel 1993). It has been demonstrated that ether lysophospholipids stimulate the oxidative burst of peritoneal macrophages (Brachwitz & Vollgraf 1995) and boost glucose consumption in these cells (Hartung 1983). In activated tumour macrophages, increases result in the production of reactiveoxygen metabolites, such as H2O2 and superoxide O2 (Hayashi et al. 1985). The results of ED50 are different from those found by other authors who studied the effect of alkyl-lysophospholipids against species of Leishmania. Croft et al. (1996) and Escobar et al. (2002), found values of ED50 more less, they cultivate Leishmania in M199 medium supplemented with 10% heat-inactivated foetal calf serum (IFCS),and with HEPES, adenosine and hemin, while we used the M199 medium supplemented with 20% of heat-inactivated foetal calf serum and only with NaHCO3 (0.356g/l). Alkyl-lysophospholipids are sensitive to the serum percentage present in the medium, and may be to the kind of medium and to his different components. Santa-Rita et al. (2000) showed that the alkyl-lysophospholipid activity against T. cruzi was much more pronounced when the incubations were performed in the absence of serum. In appropriate culture medium, they found that the ED50 after 4 hr for amastigote and epimastigotes was 59.6 and 41.7 mM, while, in PBS after 20 min., these values decreased to 7.5 and 25.7 mM, respectively. While the range of ED50 for trypomastigotes
after 24 hr was about 30 mM, after 30 min. at 30 mM in PBS, total lysis of the parasites occurred. Plasma protein binding is not surprising for hydrophobic compounds such as the alkyl-lysophospholipids (Santa-Rita et al. 2000). A ‘protecting effect’ of serum against the cytotoxicity induced by alkyl-lysophospholipids against T. cruzi was also reported for tumour cells (Petersen et al. 1992; Lohmeyer & Workman 1993). Alkyl-lysophospholipids show high activity against macrophages, the results of ED50 show low difference between the effect of alkyl-lysophospholipid against parasite and against macrophages. The effect of alkyl-lysophospholipids against tumour cells is largely studied, and the citotoxicity of edelfosine, miltefosine, and ilmofosie against macrophages was reported by many authors (Andreesen et al. 1984; Schreiber et al. 1994). The in vitro effect of the alkyl-lysophospholipid on the intracellular amastigotes was measured using two different methods: the traditional method of microscopic counting of infected macrophages stained with Giemsa, as well as the new technique based on counting by flow cytometry and staining of the cell and parasite DNA with ethidium bromide. The latter technique enables the differentiation of the fluorescent-particle size corresponding to the nucleus of the host cell as well as that of the amastigote. Flow cytometry has been used recently to study the effect of various agents against microbes as well as pathogenic prokaryotes and eukaryotes (Fouchet et al. 1993; Leonce & Burbridge 1993). Saul et al. (1982) successfully used PI to detect and count Plasmodium falciparum infected cells by flow cytometry. With this technique Azas et al. (1997), studied the effect of pentamidine, allopurinol, and amphotericin B on cell growth and in the parasite metabolism. Cell multiplication was studied by these authors by a dynamic analysis of DNA stained with propidium iodide. In another recent work (Di Giorgio et al. 2000), the authors applied cytometry to the detection of Leishmania amastigotes in macrophages, using an anti-leishmania monoclonal antibody to detect the parasite. The advantages of the method are diverse, because the technique has more reliability as microscopic counting while offering substantial time savings. The differences in the percentage of inhibition between the traditional microscopic method and flow cytometry may be due to human error in the former method.
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Abdullah et al. (1999) used the flow cytometry technique for the estimation of the percentage of U-937 cells infected with different species of leishmania. They used different dyes and they compared the results with those of the Giemsa staining method. Abdullah et al. (1999) were interested in the intracellular growth of leishmania within macrophages, while we are interested in detect and count the amastigotes of Leishmania donovani by flow cytometry after the infected treated cells were submitted to the hypotonic solution. Abdullah et al. founded that with the cells stained with PI, it was difficult to discriminate between infected and uninfected cells, PI like ethidium bromide are a DNA-binding fluorochromes. And the results they obtained by FCM analysis could not be reproduced by microscopy. In our case the treated and the infected treated cells, were lised and stabilized and stained before the FCM analysis. In the cytometer we count the nuclei of amastigotes and also those of cells, and we based on the size to differentiate between the cells and the amastigotes nuclei. FACS-analysis itself is a rapid, accurate and reproductible method of analysis. Therfore, it has the potential to serve as a tool in Leishmania research. Effects on the biosynthesis of macromolecules. The levels of macromolecular synthesis in L. donovani promastigotes treated with alkyl-lysophospholipids are reflected in fig. 1, 2 and 3. With respect to control, edelfosine depressed DNA synthesis by 57.85%, and RNA synthesis by 73.22%, and by 68.02% the protein synthesis. While miltefosine inhibited RNA synthesis by 96.32%, and DNA and protein synthesis by 73.46% and 83.97% respectively. Meanwhile, ilmofosine inhibited DNA synthesis by 40.32%, RNA synthesis by 73.72%, and protein synthesis by 65.60% (table 2). In the three cases RNA synthesis was first to be inhibited by the ALPs, followed by protein synthesis and lastly DNA synthesis. Thus, DNA synthesis proved the least affected, implying either that these products do not act directly on DNA replication. Or, that they need more time to exert their activity, given that the incubation time with the drugs was only 135 min. The effectiveness of alkyl-lysophospholipids depends largely on contact time with the cells. Several studies have demonstrated that they are capable of inducing apoptosis in leukaemic cells, but this requires adequate incubation time, which can last days (Herrman & Bicher 1988; Engelmann et al. 1996). On the other hand, RNA synthesis was the most strongly affected by the ether lipids, leaving protein synthesis in second place. The alkyl-lysophospholipids thus may exert indirect action on RNA, and therefore their activity can be recognized in the inhibition of many enzymes of phospholipid metabolism, and, in some proteins as well as enzymes responsible for the transduction signals, such as protein kinase C (Lux et al. 1996). The role of alkyl-lysophospholipids in transduction signals previously demonstrated (Croft et al. 1996), and the mechanism of phophorylation/dephosphorylation that acts
on these signals, could explain the synthesis of these new proteins (Daniel 1993). The results in the present paper and by other authors indicate that alkyl-lysophospholipids represent an effective alternative to the treatment of leishmaniasis, showing a complex action mechanism which may be due primarily to direct action on the parasite membranes, the other effects observed being a direct consequence of the aforementioned action. Acknowledgements This study was supported by CEE Antileishmanial and antitrypanosomal activities of alkyllysophospholipids project from the European Union. INCO-CT96-0084. and by the Spanish project number SAF 1998-0158.
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