a Cellular Immunology Laboratory b Steroid and Terpenoid Chemistry Division, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India
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Abstract |
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Introduction |
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The visceral form of leishmaniasis, commonly known as kala-azar, is caused by the parasite Leishmania donovani and is often fatal. Despite tremendous progress made in understanding the biochemistry and molecular biology of Leishmaniaspp., treatment by chemotherapy has seen very little progress in recent years. The toxic pentavalent antimonials remain the mainstay of treatment for leishmaniasis. The second line drugs, pentamidine and amphotericin B, although used clinically, have serious toxic side effects. 4 Therefore, improved drug therapy for leishmaniasis remains desirable.
The indole and quinoline nuclei are prevalent in a wide variety of biologically active compounds. Some indole and quinoline derivatives have been reported to possess antileishmanial activity.5,6,7,8,9 A one-step synthesis of some novel indolylquinoline derivatives has been developed using indole as the substrate under Friedel-Crafts acylation reaction conditions. 10 We investigated the antileishmanial activity of these indolylquinoline derivatives both in vitro and in vivo; the results are described in this communication.
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Materials and methods |
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Four indolylquinoline derivatives,
2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A),
2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), 2-(2''-
acetamidobenzyl)-3-(3'-indolylquinoline) (C) and
2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D) (Figure 1),
were prepared as described.10 The reaction was carried out
with indole (34 mol) as substrate, acylchloride (1 mol), and anhydrous aluminium
chloride (AlCl3, 1.5 32 mol). The substrate was dissolved in nitrobenzene,
cooled to 1520°C, and the catalyst was added in gradual increments. The acylating
agent was then added slowly with constant stirring. The reaction mixture was kept at 25°C
for 1 h, warmed to 85°C for 4 h, and then kept overnight at room temperature. The reaction
mixture was treated with an ice-HCl (1:1) mixture, neutralized with sodium bicarbonate
(NaHCO3) solution, and extracted with chloroform (CHCl3). The extract
was evaporated under reduced pressure. The concentrated gummy mass was adsorbed with silica
gel and subjected to column chromatography. Stock solutions of indolylquinoline derivatives
were made in dimethyl sulphoxide (DMSO) at 10,000 mg/L and further dilutions were made in
medium containing 8% fetal bovine serum (FBS) immediately before use. In all
experiments, the final concentration of DMSO was 0.1% (v/v) and was nontoxic to
promastigotes and amastigotes of L. donovani, and to human peripheral blood
mononuclear cells (PBMC).
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L. donovani strain AG83 was originally obtained from an Indian kala-azar patient 11 and maintained in golden hamsters. Amastigotes were isolated from spleens of L. donovani infected golden hamsters as described. 12 The spleen was rinsed in ice cold phosphate-buffered saline (PBS)-glucose (55 mM)/EDTA (2 mM), then lightly homogenized, macroscopic particles were allowed to settle, and the turbid suspension was decanted. This suspension was centrifuged at 100g for 10 min at 4°C. The amastigote-enriched suspension was centrifuged at 800g for 10 min. The pellet was suspended in 45% Percoll (8.0 mL), and finally 25% Percoll (4.0 mL) was layered over the amastigote suspension and centrifuged at 5000g for 1 h. The band containing amastigotes was taken and washed with PBS (x3), and finally resuspended in Medium-199 (Gibco Laboratories, New York, NY, USA) supplemented with 20% FBS. Promastigotes were obtained by transforming amastigotes and were maintained in vitro in Medium-199 supplemented with 8% FBS.
Isolation of normal human peripheral blood mononuclear cells (PBMC)
PBMC were separated from heparinized whole blood of normal donors by Ficoll-Hypaque density gradient centrifugation as described.13
In-vitro growth of L. donovani promastigotes in the presence of indolylquinoline derivatives
Promastigotes (1 x 106) were incubated with or without various concentrations of indolylquinoline derivatives or standard antileishmanial drugs in Medium-199 (1.0 mL) supplemented with 8% FBS at 22°C. Growth of promastigotes was monitored by counting the number of motile promastigotes microscopically.
Cytotoxicity assays
In-vitro grown promastigotes, freshly purified amastigotes, or normal human PBMC (2
x 106 of each) were washed and resuspended in Medium-199 (1.0
mL)
containing 8% FBS. Fifty microcuries of sodium chromate-51Cr (Na
251CrO4, BARC, Bombay, India) were added to
promastigotes or purified amastigotes and incubated for 6 h at 22°C. Normal human PBMC
were incubated with Na251CrO4 for 2 h at 37°C with
gentle shaking every 15 min. Radiolabelled promastigotes, amastigotes, or normal human PBMC
were washed (x4) and resuspended in Medium-199 supplemented with 8% FBS at
a
concentration of 1.0 x 106/mL. Indicated concentrations of indolylquinoline
derivatives or sodium antimony gluconate were added to radiolabelled promastigotes or
amastigotes (0.5 x 106) in a total volume of 1.0 mL in 5.0 mL tubes, and to
radiolabelled normal human PBMC (0.2 x 105) in a total volume of 0.2 mL
in microtitre plates. After 18 h of incubation at 37°C, promastigotes or amastigotes were
centrifuged and 0.5 mL aliquots of cell-free supernatants were collected and counted in a gamma
counter. For normal human PBMC, 0.1 mL of supernatants were collected from each well
without disturbing the cell pellet. The percentage 51Cr release was calculated using
the formula:
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where CPM denotes counts per minute, the spontaneous release was that obtained from promastigotes, amastigotes, or normal PBMC incubated with medium alone, the experimental release was that obtained from these cultures incubated with indolylquinoline derivatives or sodium antimony gluconate, and the total release was that obtained from these cultures incubated with 1 N HCl. In 18 h assays, the spontaneous release never exceeded 30% of the total release from either form of the parasite or normal human PBMC.
In-vitro infection of BALB/c peritoneal macrophages
Thioglycolate-elicited peritoneal exudate was used as the source of macrophages for better recovery and easier isolation. Approximately 2.5 x 105 macrophages were allowed to adhere to glass coverslips (20 mm x 25 mm) in RPMI-1640 (Gibco Laboratories) supplemented with 10% FBS and cultured for 57 days at 37°C in 5% CO2 before in-vitro infection with L. donovani. Stationary phase L. donovani promastigotes (5.0 x 106) were added to each coverslip and incubated for 6 h at 37°C in 5% CO2.
Determination of antileishmanial activity of indolylquinoline derivatives on L. donovani infected BALB/c macrophages in vitro
Coverslips were washed (x3) with 10% FBS-supplemented RPMI-1640 to remove uningested parasites and incubated for 2 days in the presence or absence of graded concentrations of indolylquinoline derivatives, pentamidine, amphotericin B (Sigma Chemical Co., St Louis, MO, USA), and sodium antimony gluconate (Gluconate Health Ltd, Calcutta, India). Infected macrophage cultures were washed with PBS, fixed with prechilled methanol, stained with Giemsa, and examined microscopically under oil immersion. At least 400 target macrophages were examined for each coverslip. Antileishmanial activity was determined by calculating the number of amastigotes per 100 macrophages.
Determination of antileishmanial activity of indolylquinoline derivatives in vivo
BALB/c mice (4550 days old) were injected in vivo with freshly transformed promastigotes of L. donovani (2 x 107/mouse). Therapy with indolylquinoline derivative A or sodium antimony gluconate started 1 month after infection. Indolylquinoline derivative was used orally (12.5 mg per kg body weight). Each mouse received a total of 21 oral administrations every day. Sodium antimony gluconate was injected im (250 mg/kg body weight). Each mouse received a total of ten injections every alternate day. Mice in the control group received PBS by oral feeding every day for 21 days. Mice in all groups were killed 2 weeks after the last treatment. The splenic parasite load was determined from impression smears after Giemsa staining. Results are expressed as the total parasite load per spleen, using the formula:
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Statistical analysis
Statistical analyses were performed by Student's t-test with the program Tadpole III (written by T. H. Caradoc-Davies, Wakari Hospital, Dunedin, New Zealand; published and distributed by Biosoft, Cambridge, UK).
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Results |
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L. donovani promastigotes can be grown in vitro at 22°C. Growth of
the promastigotes peaked within 46 days of culture (Figure 2).
To determine whether any of the indolylquinoline derivatives had any effect on the
promastigotes, various concentrations of these derivatives (dissolved in DMSO) were added to
the promastigote cultures individually. DMSO had no effect on the growth of L. donovani promastigotes in vitro at a final concentration of 0.1% (v/v). Out of four
indolylquinoline derivatives tested, derivative
C[2-(2''-acetamidobenzyl)-3-(3'-indolylquinoline)] had no
appreciable effect on the growth of L. donovani promastigotes in vitro at
concentrations as high as 10 mg/L (Figure 2). This derivative was also
ineffective at lower concentrations, i.e. 1.0 mg/L to 5.0 mg/L. By contrast, each of the remaining
three indolylquinoline derivatives (derivatives A, B, D) was found to inhibit the growth of L.
donovanipromastigotes in vitro. The degrees of inhibition by derivatives A, B and
D were very similar. Each of these three derivatives at a concentration of 10 mg/L inhibited the
growth of L. donovani promastigotes by approximately 98% on the second,
fourth, or sixth day of culture (P < 0.0001 for each of the indicated derivatives). A
lower concentration (5.0 mg/L) of these derivatives was also growth inhibitory to L.
donovani promastigotes. This concentration of derivative A, B or D inhibited promastigote
growth by approximately 70% on both the second and fourth days of culture (P
< 0.001 for each comparison) and by 40% on the sixth day (P < 0.03 for
each comparison). However, these indolylquinoline derivatives at a final concentration 2.5
mg/L had no effect on the growth of L. donovani promastigotes (Figure 2
AD). For comparison, two additional compounds (pentamidine and
amphotericin B), usually used as second-line antileishmanial drugs, were included as positive
controls. Both pentamidine and amphotericin B at a final concentration
2.5 mg/L inhibited
the in-vitro growth of L. donovani promastigotes almost completely (Figure
2). At a final concentration of 1.0 mg/L, pentamidine inhibited promastigote growth
poorly, and the differences were not significant. However, amphotericin B at the same
concentration (1.0 mg/L) inhibited promastigote growth by 28.8% on the second day,
67.4% on the fourth day (P < 0.001), and 43.8% on the sixth day of
culture (P < 0.03).
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To examine whether indolylquinoline derivatives were cytotoxic to the parasite, 51Cr-release assays were performed with radiolabelled promastigotes and amastigotes of L. donovani in the presence of varying concentrations of these derivatives. The antileishmanial drug sodium antimony gluconate (SAG) was used for comparison and normal human PBMC were used as a control for nonspecific toxicity. As shown in Figure 3, DMSO up to a concentration of 0.1% (v/v) was nontoxic to human PBMC or L. donovani promastigotes. However, DMSO was found to be toxic to purified amastigotes of L. donovaniin a dose-dependent manner. Neither any of the indolylquinoline derivatives tested (A, B, C or D) nor SAG was toxic to human PBMC. The three derivatives other than indolylquinoline derivative C were significantly cytotoxic to both promastigotes and amastigotes of L. donovani. The cytotoxicity of derivative C was only marginal over DMSO on L. donovani amastigotes and was not tested on promastigotes. SAG was nontoxic to both promastigotes and amastigotes up to a concentration of 10 mg/L (not shown). However, higher concentrations of this drug (250500 mg/L) were cytotoxic to both forms of the parasite (Figure 3).
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The protozoan parasite L. donovani survives and multiplies within mammalian macrophages. It is therefore of interest to test the efficacy of these indolylquinoline derivatives on intracellular amastigotes. Peritoneal macrophages of BALB/c mice were infected in vitro with L. donovani and then incubated with graded concentrations of these agents. SAG, pentamidine, and amphotericin B were used as positive controls. As shown in Figure 4, SAG was effective in reducing intracellular parasite burden only at a high concentration (500 mg/L). The other two known antileishmanial compounds tested, pentamidine and amphotericin B, were both effective at lower concentrations. At a concentration as low as 1.0 mg/L, pentamidine reduced the intracellular parasite load by 70%. Amphotericin B was even more effective at this concentration (90% reduction of intracellular parasite load). Of the four indolylquinoline derivatives, derivative C had no appreciable antileishmanial activity. Derivatives A and B were almost equally effective. Each of these derivatives had no appreciable effect on intracellular amastigotes at 1.0 mg/L but higher concentrations were effective. Antileishmanial activity of derivative D was not detectable up to 2.5 mg/L; however, higher concentrations of this derivative significantly reduced the intracellular amastigote load (Figure 4). Giemsa-stained micrographs of L. donovaniinfected BALB/c peritoneal macrophages incubated with indolylquinoline derivatives or sodium antimony gluconate are shown in Figure 5.
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BALB/c mice were infected with L. donovani as described above. After 1 month, groups of four or five mice were treated with PBS, SAG, or indolylquinoline derivative A. The parasite load in the spleen was then determined as described. 14 As shown in the Table, indolylquinoline derivative A was significantly more effective than sodium antimony gluconate (P < 0.01) in reducing the splenic parasite load even at a much lower concentration. SAG therapy at a dose of 250 mg per kg body weight reduced the splenic parasite load by 56%. On the other hand, indolylquinoline derivative A reduced the parasite load by 86% at a concentration as low as 12.5 mg per kg body weight.
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Discussion |
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Of four indolylquinoline derivatives tested, three had significant antileishmanial activity on cultured promastigotes, purified amastigotes, and intracellular amastigotes of L. donovani in vitro. None of these indolylquinolines was toxic to normal human cells. These indolylquinolines were less active than pentamidine or amphotericin B but were significantly more effective antileishmanial agents than sodium antimony gluconate in vitro. Our in-vivo studies with one indolylquinoline derivative in L. donovani infected BALB/c mice are encouraging. Sodium antimony gluconate was used as a positive control for in-vivo studies, and this drug partially reduced the splenic parasite load. These results are in agreement with a previous report on Leishmania majorinfection. 15 The indolylquinoline derivative was significantly more effective than sodium antimony gluconate in reducing the splenic parasite load at a much lower concentration.
A wide variety of biologically active compounds contain indole or quinoline nuclei. Some indole or quinoline derivatives have been reported to possess antileishmanial activity. 5,6,7,8,9,16 We report here that some indolylquinoline derivatives have significant antileishmanial activity both in vitro and in vivo. A recent report indicated that indolylquinoline derivatives inhibit catalytic activities of both type I and type II topoisomerases of L. donovani and that Leishmania topoisomerases are more susceptible to these agents.17 Inhibition of Leishmania topoisomerases may explain, at least in part, the antileishmanial activity of indolylquinoline derivatives, and these compounds may be exploited as antileishmanial agents.
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Acknowledgments |
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Notes |
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References |
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Received 20 May 1998; returned 5 August 1998; revised 23 September 1998; accepted 12 October 1998