In vitro activity of C 20 -diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum

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1 International Journal of Antimicrobial Agents 25 (2005) In vitro activity of C 20 -diterpenoid alkaloid derivatives in promastigotes and intracellular amastigotes of Leishmania infantum Patricia González a, Clotilde Marín a, Isabel Rodríguez-González a, Ana B. Hitos a, Maria Jose Rosales a, Matías Reina b, Jesús G. Díaz c, Azucena González-Coloma d, Manuel Sánchez-Moreno a, a Departamento de Parasitología, Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, C/ Severo Ochoa s/n, Granada, Spain b Instituto de Productos Naturales y Agrobiología, CSIC, Avda. Astrofísico F. Sánchez, 3, La Laguna, Tenerife, Spain c Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, La Laguna, Tenerife, Spain d Centro de Ciencias Medioambientales. CSIC, Serrano 115-dpdo Madrid, Spain Received 22 March 2004; accepted 6 August 2004 Abstract The in vitro anti-proliferative effects are described of several atisine-type diterpenoid alkaloids against the protozoan parasite Leishmania infantum, which causes human visceral leishmaniasis and canine leishmaniasis in the Mediterranean basin, as well as human cutaneous leishmaniasis throughout the Mediterranean region. From a total of 43 compounds tested, including C 19 - and C 20 -diterpene alkaloids from several chemical classes, only 15,22-O-diacetyl-19-oxo-dihydroatisine, azitine and isoazitine were highly active against cultures of the parasite (promastigote form) with IC 50 values within the range of the reference drug pentamidine-isothionate ( mg/l for the test compounds, mg/l for the positive control). These compounds were not toxic to the host cell. When treated with a dosage of 5 g/ml of the active compounds (half of their IC 50 ), the promastigote forms lost 80% of their infection capacity and the multiplication of extracellular forms of L. infantum was severely affected. The study showed that atisine-type C 20 -diterpenoid alkaloids exhibited promising anti-leishmanial properties with strong molecular selectivity. These might have implications for other intracellular pathogens- or phylogenetically related parasites, such as Trypanosoma spp Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Leishmania infantum; Anti-leishmanials; Norditerpenoid alkaloids; In vitro 1. Introduction Among the major worldwide health problems, especially in developing countries, are protozoan diseases of different types. Leishmaniasis, caused by several species of flagellated protozoa belonging to genus Leishmania, which are transmitted exclusively by the bite of female phlebotomine sandfly, affects some 12 million people. About 350 million are exposed to the risk of infection, with over new cases annually [1]. Over the last few years, regions not formerly considered endemic to Leishmania, registered in- Corresponding author. Tel.: ; fax: address: msanchem@ugr.es (M. Sánchez-Moreno). creases in recorded cases. In Mediterranean Europe, visceral leishmaniasis is recognized as a re-emerging disease [2]. The classic forms of leishmaniasis (e.g. visceral and cutaneous leishmaniasis) still impose specific difficulties in terms of diagnosis and treatment. Drug treatment today is restricted to a limited number of clinically useful drugs, such as pentamidine or amphotericin B. However, both drugs have serious side effects. Furthermore, especially improved formulations, such as liposome-encapsulated amphotericin B, though effective, have made general treatment unaffordable for many afflicted countries, revealing an urgent need for new, safer and cheaper drugs [3]. In Central and South America, where there is a high prevalence of these diseases, the drug of choice, commonly used /$ see front matter 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi: /j.ijantimicag

2 P. González et al. / International Journal of Antimicrobial Agents 25 (2005) in the industrialized world, is rarely available, and moreover, most affected patients belong to the poorer classes, who cannot afford these expensive medicines. Instead, following traditional medicine, these countries use plants to treat most diseases, including leishmaniasis [4]. For all these reasons, one strategy to discover new drug leads is to investigate natural products from traditional medicinal plants. Plant species of the genera Aconitum, Delphinium and Consolida are known sources of C 19 -norditerpene and C 20 -diterpene alkaloids (NDAs and DAs, respectively) of pharmacological and economic importance [5,6]. NDAs act as potent nicotinic cholinergic receptor (nacchr) agonists and antagonists in invertebrates, including insects and in vertebrates [7]. The insecticidal and anti-feedant activity of NDAs [8 10], suggest a defensive role played by these compounds. However, only a few DAs have been studied for their plant defensive and pharmacological properties, including their effects on Trypanosoma cruzi epimastigote forms [9,11 15] and their neurotoxic effects are unknown. Here, for the first time, we report the inhibitory effects of DAs on the extracellular promastigote and the intracellular amastigote stages of Leishmania infantum in comparison with their direct effects on macrophage host cells. These data suggest that DAs are a class of compounds with potential for further development for anti-protozoal therapy. 2. Materials and methods 2.1. Parasite strain L. infantum strain UCM10 (zimodeme MON1) was isolated in Madrid (Spain) and kindly supplied by Professor Alunda JM of the Faculty of Veterinarian Medicine Madrid (Spain) Reagents compounds The compounds tested here were isolated from Aconitum, Delphinium and Consolida spp. [10,15] (Fig. 1) Promastigote assay The promastigote forms used for the chemotherapy assays were cultured at 28 C in RPMI 1640 medium (Flow Laboratories, Irvine, UK) in Roux flasks (Corning, USA) of 75 cm 2 in surface area, supplemented with 10% inactivated Fig. 1. Structure of the three artisine-type diterpine alkaloids (ADs) used.

3 138 P. González et al. / International Journal of Antimicrobial Agents 25 (2005) calf serum. At the exponential-phase flagellate growth, the liquid medium was centrifuged at 1500 rpm for 10 min and the number of parasites were counted and distributed in 96 multiwell plates (Becton Dickinson, USA) to parasites/ml. The compounds were dissolved in dimethyl sulfoxide (DMSO, Panreac, Barcelona, Spain) at a concentration of 0.1%, after this had been assayed as non-toxic and without inhibitory effects on parasite growth, as previously demonstrated [16]. The compounds were dissolved in the culture medium, and the dosages used were: 100, 50, 25, 10 and 1 g/ml. The effect of each compound at these concentrations against promastigote forms, were evaluated at 24, 48 and 72 h, using a Neubauer haemocytometric chamber. The leishmanicidal effects were expressed as IC 50, that is, the concentration required to give 50% inhibition, calculated by linear regression analysis from the K c values at the concentrations used Cell culture and cytotoxicity tests Macrophage line J774A.1 (ECACC number ) was obtained from a tumour in a female BALB/c rat in Macrophages were kept in the laboratory by cryopreservation in liquid nitrogen and then by successive subcultures in RPMI medium. For the cytotoxicity test, macrophages were placed in 25 ml cone-based bottles (Sterling), and centrifuged at 750 rpm for 5 min. The culture medium was removed and Hank s solution was added to a final concentration of 10 6 cells/ml. This cell suspension was distributed in a culture tray (with 24 wells) at a rate of 100 L per well and incubated for 24 h at 37 C in a humid atmosphere enriched with 5% CO 2. The medium was removed, and the fresh medium was added together with the product to be studied (at concentrations of 100, 50, 25, 10 and 1 g/ml). The cultures were incubated for 72 h. The vital stain trypan blue (0.1% P/B in phosphate buffer) was used to determine cell viability. The number of dead cells was recorded, and the percentage of viability was calculated in comparison to that of the control culture, and the IC 50 was calculated by linear regression analysis from the K c values at the concentrations used Amastigote-macrophage assay Two experimental procedures were designed for this study, in all cases beginning with macrophages J774A.1, which were diluted to 10 6 cells/ml in RPMI medium plus 10% IFCS, plated in 24-well tissue-culture chamber slides and allowed to adhere for 24 h at 37 C in a mixture of 5% CO 2 and 95% air Experimental model no. 1 (M + Li + drug) Adherent macrophages were infected in vitro at a ratio of 10:1 with L. infantum promastigotes in the exponential growth phase. The drugs (5 g/ml concentrations) were added at the same time and the trays were incubated for 12 h at 37 Cin5%CO 2 in air. Afterwards, non-phagocytosed parasites and the drugs were removed by washing, and infected cultures were grown for 8 days in fresh RPMI medium. The medium was renewed every 48 h Experimental model no. 2 ([M Li] + drug) Adherent macrophages were infected with exponentialphase promastigotes of L. infantum at a ratio of 10:1 and maintained for 12 h at 37 C in 5% CO 2 in air. Nonphagocytosed parasites were removed by washing, and the infected cultures were incubated with the drugs (5 g/ml concentrations) for another 12 h and afterwards washed and cultured for 8 days in fresh RPMI medium. The culture medium was renewed every 48 h. In all cases, the drug activity was determined from the percentage of infected cells and number of amastigotes per infected macrophage in treated and untreated cultures in methanol-fixed and Giemsa-stained preparations. Values are means of four separate determinations Ultrastructural alterations The parasites, at a density of cells/ml, were cultured in their corresponding medium, containing the three drugs at 5 mg/ml. After 72 h, the cultures were centrifuged at 1500 g for 10 min, and the pellets washed in PBS and then fixed with 2% (v/v) p-formaldehyde-glutaraldehyde in 0.05 M cacodylate buffer (ph 7.4) for 2 h at 4 C. Pellets were prepared for transmission-electron microscopy following the technique of Luque et al. [16]. 3. Results and discussion From a total of 43 diterpene alkaloids (x NDAs and y DAs from several chemical classes) tested at 100 g/ml, only three atisine-type ADs markedly inhibited the growth of the in vitro forms of L. infantum promastigotes (data not shown). The in vitro leishmanicidal activity of the active compounds (1, 2 and 3; Fig. 1) against promastigote L. infantum are shown in Table 1. Pentamidine-isothionate was included here as the anti-leishmanial reference drug. Compound 3 exhibited the highest toxicity to the extracellular L. infantum Table 1 In vitro susceptibility of Leishmania infantum promastigotes to compounds tested Compound IC 50 (mg/l) Toxicity IC (h) 48 (h) 72 (h) (mg/l) a Pentostam > >300 IC 50, the concentration required to give 50% inhibition, calculated by linear regression analysis from the K c values at the concentrations tested (1, 10, 25, 50 and 100 g/ml). Note: average of four separate determinations. a On J774.2 macrophages at 72 h of culture.

4 P. González et al. / International Journal of Antimicrobial Agents 25 (2005) Table 2 Effects of the drugs (at 5 g/ml concentration) on the infection rate of J774A.1 macrophages and on the average number of Leishmania infantum amastigotes per infected macrophage during 8 days of culture, under different conditions Compounds (%) M s a IP/C b 48 (h) 96 (h) 144 (h) 192 (h) 48 (h) 96 (h) 144 (h) 192 (h) None (control) ± ± ± ± 1.1 M +Li+1 c ± 0.7 6,8 ± ± ± 0.7 [M Li] + 1 c ± ± ± ± 1.2 Mø+Li+2 c ± ± ± ± 0.9 [M Li] + 2 c ± ± ± ± 1.2 M +Li+3 c ± ± ± ± 1.1 [M Li] + 3 c ± ± ± ± 0.6 a Percentage macrophage parasitism. Values are mean ± standard deviations of four separate determinations. b Number of amastigotes per macrophage infected. Values are mean ± standard deviations of four separate determinations. c Details are given in Section 2. parasites (IC 50 of 13.38, 9.70 and 7.39 mg/l at 24, 48 and 72 h of culture, respectively) with IC 50 values lower than these obtained for the reference drug (IC 50 of mg/l at 72 h of culture). ADs 2 and 1 also showed pronounced effects against promastigotes (IC 50 of and mg/l, respectively, at 72 h of culture). This leishmanicidal activity was associated with a lack of toxicity to murine macrophages by compounds 3 and 2 (IC 50 = >300, >200 mg/l, respectively) and only weak toxicity by 1 (IC 50 = mg/l). Table 2 shows the effect of compounds 1 3 (at 5 mg/l) on the infection rate of J774A.1 macrophages and the average number of amastigotes per cell infected up to 8 days of culture. Two experimental models were used to discriminate the effects of the test compounds on the growth of the extracellular forms or the infection capacity and growth of the intracellular forms of L. infantum. The first experimental model (M + Li + drug) consisted of adherent macrophages infected with drug-treated promastigotes in exponential growth phase for 12 h. Afterwards, the non-phagocytosed parasites and the drugs were removed and the culture kept in fresh medium for 8 days. The percentage of parasitism was strongly inhibited ( =78%) at 48 h of culture by compound 3. This inhibition persisted and even increased with time, reaching values of 86% inhibition at 8 days of culture. The number of amastigotes per cell were greatly diminished ( =87%) when compared with the control. This effect continued and increased slightly over the 8 experimental days. Similar results were found for compound 2. Alkaloid 1 showed the least activity on infected macrophages (14% at 48 h of culture, maintaining the same percentage for 8 days) and on the number of amastigotes, although in this case a marked fall was noted in the multiplication of the intracellular forms (64% at 48 h, increasing to 81% at 8 days), indicating that 1 had a greater action on the intracellular forms than against the infective capacity of the extracellular forms of L. infantum. The results of this experimental model confirm that: (1) alkaloids 3, 2, and to a lesser extent 1, are toxic to promastigote forms of L. infantum, (2) these compounds lower the infective capacity of these forms of the parasite and (3) the three compounds assayed are very active against the extracellular forms of the parasite. When the parasites were incubated with the macrophages for 12 h before the addition of the alkaloids (Section 2.4.3), the percentage of parasitism was not significantly affected. This result is not surprising since 12 h incubation of the macrophages with the promastigotes in the absence of drugs is sufficient time to allow for maximum parasitism. Nevertheless, the number of amastigotes was significantly reduced (71 49%) by the three products tested. This inhibitory effect increased slightly over the 8 experimental days, indicating that there was a direct action on the intracellular forms and their multiplication. Fig. 2A shows a control culture of L. infantum promastigotes with the characteristic structure of kinetoplastids. The typical kinetoplast (K) is formed by maxi- and minicircles that appear as a prolongation of the mitochondria, the mitochondria (M) have well-defined mitochondrial crypts and a number of glycosomes (G), a nucleus (N) with its nucleolus (Nu) normally centred and the characteristic microtubules (MT) may be seen. L. infantum promastigotes treated with compound 3 (Fig. 2B) showed major nuclear and cytoplasmic alterations. Although the cytoplasmic membrane appeared intact, we were able to detect small dense bodies (D) associated with the microtubular structure. The nucleus (N) appeared irregular and with little electrodensity, and a double nuclear membrane was not visible. The cytoplasm showed little electrodensity and contained a large number of empty vacuoles (V) throughout, and even the flagellar sack contained small vacuoles. The mitochondria (M) were very scarce. Compound 3 (Fig. 2C) induced notable morphological changes in the parasites, which appeared with a rounded, extremely swollen appearance. The cytoplasm, with abundant vacuoles and little electrodensity, contained numerous glycosomes (G) and swollen mitochondria (M). Similar alterations were provoked by 2 (Fig. 2D). The parasites appeared very swollen with enormous empty vacuoles (V) or vacuoles composed of granular material. In some of

5 140 P. González et al. / International Journal of Antimicrobial Agents 25 (2005) Fig. 2. Ultrastructural effect of C 20 -diterpenoid alkaloid derivatives (5 g/ml) on promastigotes Leishmania infantum cultured in RPMI 1640 medium for 72 h. (A) Control, (bar = ); (B) isoazitine, (bar = ); (C) iosoazitine, (bar = ); (D) azitine, (bar = ) and (E) 15,22-O-diacetyl-19-oxo-dihydroatisine, (bar = ). V, vacuoles; K, kinetoplast; M, mitochondrion; MT, microtubules; N, nucleus; G, glycosomes and D, dense bodies. these, a large vacuole occupied almost the entire cytoplasm. Also, the mitochondria in which the crypts could be distinguished, appeared swollen. The kinetoplast appeared disorganized, without visible maxi- or minicircles. Compound 1 (Fig. 2E) proved to be the most harmful to L. infantum promastigotes. Most of the parasites were dead and many were fragmented, and thus, the culture was filled with cell fragments, and intact parasites were scarce. In some of these, the cytoplasmic membrane appeared disintegrated in some areas (arrow), leading to direct contact of the cytoplasmic organelles and the nucleus with the culture medium and resulting in fragmentation. This compound acts fundamentally at the level of the cytoplasmic membrane of the parasites, although alterations were detected also particularly in the mitochondria (M), which were extremely rare and lacked their normal morphology and appeared as uniform sacks without crypts. Compound 1 also led to the loss of the typical structure in maxi- and minicircles in the kinetoplast (K). This is the first study that has been made on the leishmanicidal activity of DAs. Previous results have shown that T. cruzi mortality increased with ADs 13-oxo-cardiopetamine (inactive against L. infantum, data not shown) and 1, while 2 and 3 were inactive which suggests species-related selectivity for the anti-parasitic action of these compounds, with L. infantum being more sensitive to DAs than T. cruzi. Furthermore, none of 43 NDAs tested on T. cruzi affected its viability as shown here for L. infantum [10,15] indicating a strong molecular selectivity for the trypanocidal and leishmanicidal effect of DAs. Our results showed that compounds 1 3 were very active in vitro against both the extracellular as well as the intracellular forms of L. infantum. These compounds are not toxic for the host cells and are effective at concentrations similar to or lower than the reference drug used in the present study. The in vitro growth rate of L. infantum was reduced, its capacity to infect cells was negatively

6 P. González et al. / International Journal of Antimicrobial Agents 25 (2005) affected and the multiplication of the amastigotes was greatly reduced. In conclusion, our study provides data that DAs 1 3 has promising anti-leishmanial properties. These could have implications for other intracellular pathogens or phylogenetically related parasites as shown for Trypanosoma spp. The potent leishmanicidal activities of the alkaloids described here represent an exciting advance in the search for new antiprotozoal agents. References [1] WHO Control of the Leishmaniases. Report of a WHO Expert Committee, Technical Report Series 793. Geneva; [2] Kayser O, Kiderlen AF, Croft SL. Antileishmanial activity of two -pyrones from Podolepsis hieracioides (Asteraceae). Acta Trop 2003;86: [3] Kayser O, Kinderlen AF, Folkens U, et al. Antileishmanial activity of aurones. Planta Med 1999;65: [4] Berger I, Passreiter CM, Cáceres A, et al. Antiprotozoal activity of Neurolaena lobata. Phytother Res 2001;15: [5] Atta-ur-Rahman M, Choudary MI. Diterpenoid and steroidal alkaloids. Nat Prod Rep 1999;16: [6] Panter KE, Manners GD, Steigelmeier BL, et al. Larkspur poisoning: toxicology and alkaloid structure activity relationships. Biochem Syst Ecol 2002;30: [7] Seitz U, Ameri A. Different effects of [ 3 H] noradrenaline uptake of the aconitum alkaloids aconitine, 3-acetylaconitine, lappaconitine, and N-desacetylla-ppaconitine in rat hippocampus. Biochem Pharmacol 1998;55: [8] Jennings KR, Brown DG, Wright DPJ. Methyllycaconitine, a naturally occuring insecticide with a high affinity for the insect cholinergic receptor. Experientia 1986;42: [9] Ulubelen A, Mericli A, Kilincer N, et al. Insect repellent activity of diterpenoid alkaloids. Phytother Res 2001;15: [10] González-Coloma A, Reina M, Guadaño A, et al. Structural diversity and defensive properties of norditerpenoid alkaloids. J Chem Ecol 2004;30: [11] Bessonova IA, Shaidkhodzaeva ShA. Hetisane-type diterpene alkaloids. Chem Nat Comp 2000;36: [12] González-Coloma A, Guadaño A, Gutiérrez C, et al. Antifeedant Delphinium diterpene alkaloids. Structure-activity relationships. Agric Food Chem 1998;46: [13] Li L, Shen Y-M, Yang X-S, et al. Antiplatelet aggregation activity of diterpene alkaloids from Spiraea japonica. Eur J Pharmacol 2002;449:23 8. [14] Li L, Shen Y-M, Yang X-S, et al. Effects of spiramine T on antioxidant enzymatic activities and nitric oxide production in cerebral ischemia-reperfusion gerbils. Brain Res 2002;944: [15] González-Coloma A, Reina M, Guadaño A, et al. Antifeedant C 20 Diterpene Alkaloids. Chem Biodivers 2004;1: [16] Luque F, Fernández-Ramos C, Entrala E, et al. In vitro evaluation of newly synthesised [1,2,4] triazolo [1,5a] pyrimidine derivatives against Trypanosoma cruzi, Leishmania donovani and Phytomonas staheli. Comp Biochem Phys C 2000;126:39 44.