Antiparasitic activity of a triphenyl tin complex against Leishmania donovani

Acta Tropica 95 (2005) 1–8

Antiparasitic activity of a triphenyl tin complex against Leishmania donovani Bikramjit Raychaudhury a , Shouvik Banerjee a , Shreedhara Gupta a , Ran Vir Singh b , Salil C. Datta a,∗ a Department of Biological Chemistry, Infectious Diseases Group, Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata 700032, India b Department of Chemistry, University of Rajasthan, Jaipur 302004, India

Received 1 November 2004; received in revised form 28 February 2005; accepted 29 March 2005 Available online 17 May 2005

Abstract Visceral leishmaniasis is a life-threatening human disease commonly known as kala-azar. Leishmania donovani is the causative agent of this parasitic disease transmitted by the sand fly vector to infect hosts. Triphenyl tin salicylanilide thiosemicarbazone [Ph3 Sn(OSal·TSCZH)] (TTST) which is an organometallic complex of triphenyl tin has been evaluated to explore possibility to develop a potent chemotherapeutic agent against visceral leishmaniasis. Effect of triphenyl tin complex on growth inhibition and host–parasite interaction were examined both in vitro and in vivo. Release of toxic superoxide radical was measured spectrophotometrically through the formation of blue formazan derived from reduced nitrobluetetrazolium. To understand mode of action of Ph3 Sn(OSal·TSCZH), superoxide dismutase activity was determined spectrophotometrically by measuring ability of this enzyme to inhibit pyrogallol autoxidation and also by activity staining of the non-denaturing polyacrylamide gels after separating superoxide dismutase. Antileishmanial activity of triphenyl tin complex were found to be effective both in vitro and in vivo at lower concentrations compared to the existing toxic drugs available. IC50 of Ph3 Sn(OSal·TSCZH) was calculated as 0.05 ± 0.01 mg/L. Intracellular survival of the parasite in host macrophages was inhibited by TTST in a dose dependent manner. Parasite burden in spleen was reduced to 87% under TTST treatment (10 mg/kg body weight) and under sodium antimony gluconate (20 mg/kg body weight) reduced nearly to 65%. Its action as a chemotherapeutic agent is found to be mediated through inhibition of superoxide dismutase and simultaneous release of toxic superoxide radical. We propose that Ph3 Sn(OSal·TSCZH) may be considered as a prospective candidate to establish a better line of therapeutic process against experimental visceral leishmaniasis. © 2005 Elsevier B.V. All rights reserved. Keywords: Leishmania; Parasite; Organometallic complex; Superoxide dismutase

∗

Corresponding author. Tel.: +91 33 24736793; fax: +91 33 24723967. E-mail address: salil [email protected] (S.C. Datta).

0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.03.008

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1. Introduction Leishmaniasis has been defined by the World Health Organization as a group of diseases that severely affects 12 million people residing in the warm areas of the world (Desjeux, 2001). In most of the cases, patients cannot survive if proper treatment is not provided during development of this sand fly mediated parasitic disease. Several antileishmanial agents have already been reported (Murray, 2001; Marty and Rosenthal, 2002) but none of these proved to be the ultimate choice of drug due to varying degrees of efficacy and toxicity. Among these, pentavalent antimonials although are recognized to be the most useful drug for treatment of visceral leishmaniasis caused by Leishmania donovani (Herwaldt and Berman, 1992), discovery of antimony salt resistant pathogenic strains has made the situation worse to treat the patients against these parasites (Sundar, 2001). Miltefosine, an orally active phosphocholine analogue, also appeared to be effective in the treatment of the disease (Croft and Coombs, 2003). However, still there is a need to identify new chemotherapeutic agents for effective therapy of the visceral form of leishmaniasis, commonly abbreviated as kala-azar. Triphenyl tin salicylanilide thiosemicarbazone (TTST), an organometallic complex of tin, having chemical formula Ph3 Sn(OSal·TSCZH), has been reported to be the best in terms of efficacy to control ergot which is one of the major diseases responsible to destroy important crop like Bajra (Saini et al., 1997). Its mode of action is not known but the fungicide is essentially active without affecting the yield and quality. Moreover, toxic effect of TTST did not hamper quality of grain adversely when used as an anti-ergot agent (Jain et al., 2004), and thereby, justifies the reason to assess its leishmanicidal activtity for developing prospective new drugs against leishmaniasis. Here, we report the inhibitory effect of Ph3 Sn (OSal·TSCZH) on the growth of L. donovani and host–parasite interaction both in vitro and in vivo.

2. Materials and methods All the reagents except fetal calf serum (FCS), medium-199 and sodium antimony gluconate were ob-

Fig. 1. Structure of triphenyl tin salicylanilide thiosemicarbazone (TTST).

tained from Sigma Chemicals, USA. Fetal calf serum and medium-199 were purchased from Gibco BRL, USA. Sodium antimony gluconate was procured from GlaxoSmithKline, UK. 2.1. Preparation of triphenyl tin complex Ph3 Sn(OSal·TSCZH) (Fig. 1) which is active against crop disease like ergot was synthesized as described for similar compounds (Belwal et al., 1997). The purity of the synthesized material was tested through spectral studies and thin layer chromatography (data not shown). Purity of TTST was >90%. 2.2. Parasites L. donovani strain MHOM/IN/AG/83 was obtained from Indian kala-azar patient (Ghosh et al., 1985) and maintained by intracardial passage every 8 weeks in Syrian golden hamsters. Promastigotes were obtained by transforming amastigotes isolated from infected spleen (Jaffe et al., 1984) and maintained in medium199 supplemented with 10% fetal calf serum in vitro. 2.3. Macrophages Animal experiments conducted in this study were duly approved by the local body of our Iinstitute’s Animal Ethics Committee. Syrian golden hamsters were injected intraperitoneally with 2 ml of sterile 4% thioglycollate. Fluid was withdrawn from the peritoneal cavity after 72 h and centrifuged at 400 × g for

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10 min at 4 ◦ C. After washing, cell suspension at a density of 5 × 105 /0.3 ml in RPMI 1640 media supplemented with 10% fetal calf serum was layered on 20 × 25 mm2 sterile cover slips placed in sterile disposable petridishes and incubated for 24 h at 37 ◦ C in a 5% CO2 incubator. Non-adherent cells were removed by washing with the same media. 2.4. Cytotoxicity assay in vitro L. donovani promastigotes (Jiang et al., 1994) were cultured in medium-199 supplemented with 10% FCS after incubating with or without TTST and sodium antimony gluconate (SAG) at 22 ◦ C. Number of viable parasites were counted under microscope to monitor growth status in absence and presence of testing materials. Antileishmanial activity of TTST on amastigotes was conducted with peritoneal exudates obtained after thioglycollate elicitation. Macrophages adhered on cover slips, were challenged with promastigotes suspended in RPMI 1640 media containing 10% FCS at the ratio of 1:10 and incubated for 5 h at 37 ◦ C in 5% CO2 . After incubation, cover slips were washed with RPMI 1640 medium containing 10% FCS to remove excess promastigotes. TTST and standard drugs were then added at different dosages. Infected macrophages were washed after 48 h, fixed with cold methanol and stained with Giemsa to examine intracellular parasite load under microscope. In a separate experiment, cellular viability of hamster macrophages was determined by using 3-(4,5dimethylthiazole-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay to detect living cells which have the ability to reduce yellow MTT to a blue formazan product (Mosmann, 1983). Briefly, cells were maintained in 96-well plates. After appropriate treatment for 30 min at 37 ◦ C with varying concentration of experimental agent, MTT (0.5 mg/ml) was added and plates incubated for 4 h. 1N hydrogen chloride–isopropanol (1:24, v/v) was added, left for 15 min at room temparature and optical density was then read at 570 nm on an ELISA reader. 2.5. Effect of TTST on Leishmania infected hamster spleen in vivo Freshly transformed promastigotes (2.5 × 107 ) were injected individually to Syrian golden hamsters

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through cardiac route. After 30 days infection, treatment with TTST (10 mg/kg body weight) or sodium antimony gluconate (40 mg/kg body weight) were started. Each hamster received intramuscular injections of respective compounds (dissolved in DMSO) twice a week for one month. Animals of each group were sacrificed one month after administration of last injection to estimate spleenic parasite burden from Giemsa stained impression smears (Stauber et al., 1958). 2.6. SOD assay Superoxide dismutase (SOD) activity was determined by measuring the inhibition of pyrogallol autoxidation rate (Marklund and Marklund, 1974). The assay mixture contained 0.2 mM pyrogallol in air equilibrated 50 mM Tris–cacodylic acid buffer, pH 8.2, and 1 mM ethylenediaminetetraacetic acid. Rate of autoxidation was obtained by monitoring the increase in absorbance at 420 nm in a Hitachi spectrophotometer, No U2000. SOD has the ability to inhibit autoxidation and the extent of inhibition is taken as the measure of enzymic activity. In another experiment, 10% non-denaturing polyacrylamide gels (Laemmli, 1970) were prepared without sodium dodecyl sulfate (SDS) to separate leishmanial SOD for activity staining (Beauchami and Fridovich, 1971). Gels were incubated in solution A (20 mg nitroblue tetrazolium in 10 ml glass distilled water and soaked for 20 min), then in solution B (4 mg riboflavin, 0.4 g potassium phosphate and 600 ␮l TEMED in 50 ml glass distilled water soaked for 20 min) and finally illuminated until white bands appeared on a blue background. To determine the effect of TTST on SOD activity, promastigote lysates were incubated with different amounts of TTST for 30 min at room temperature and then subjected to native PAGE and SDS-PAGE in 10% polyacrylamide gel separately for activity staining (Beauchami and Fridovich, 1971) and coomassie blue staining, respectively (Laemmli, 1970). 2.7. Determination of superoxide radical release Promastigotes were preincubated separately for 1 h at room temperature with different concentrations of TTST. Release of superoxide radical was then mea-

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sured spectrophotometrically through the formation of blue formazan (Yasuka, 1978). Release of superoxide radical in the TTST treated infected macrophages was also determined after incubating for 1 h at 37 ◦ C. 2.8. Determination of spleenic parasite burden Parasite load in infected hamster spleens were determined before and after administration of drug from impression smears after staining with Giemsa. Total parasite burden was calculated from the following formula—organ weight (mg) × number of amastigotes per cell nucleus × (2 × 105 ) (Stauber et al., 1958).

Fig. 2. Effect of TTST on growth rate of Leishmania promastigotes in vitro. Promastigotes (2 × 106 cells/ml) were cultured for three days in media medium-199 supplemented with 10% FCS. Data are in mean ± S.D. for three individual experiments.

2.9. Assessment of TTST toxicity

3. Results

To assess toxicity of TTST, liver function test was performed using kits from Dr. Reddy’s Laboratories, Hyderabad, India. Conventional procedures were followed as described for serum alkaline phosphatase (ALP, Kind and King, 1954), serum glutamic pyruvic transaminase and serum glutamic oxaloacetic transaminase (SGPT and SGOT, Reitman and Frankel, 1957). Tests were conducted for uninfected as well as DMSO and TTST treated infected hamsters. DMSO treated infected animals were used as control.

3.1. Effect of TTST on growth of Leishmania promastigotes and intracellular amastigotes

2.10. Statistical analysis Statistical analyses were conducted through Student’s t-test as described (Mishra and Mishra, 1983).

L. donovani promastigotes were cultured in vitro up to 3 days at 22 ◦ C in presence of different concentrations of TTST dissolved in DMSO. Final concentration of DMSO was maintained at 0.1% (v/v). At this concentration DMSO had no effect on the growth of L. donovani promastigotes. Fig. 2 indicates that at a concentration of 1 mg/L TTST parasite growth was inhibited by 100%. However, at a lower concentration of 0.2–0.06 mg/L, promastigote growth was inhibited by 60–80%. IC50 of TTST was found to be 0.05 ± 0.01 mg/L compared to an established antileishmanial agent SAG whose IC50 was determined as 12 ± 2.2 mg/L (data not shown).

Table 1 Dose-dependent response of TTST and SAG on intracellular parasite burden within macrophages during leishmanial infection Concentration of TTST (mg/L)

Viable amastigotes/100 macrophage

Concentration of SAG (mg/L)

Viable amastigotes/100 macrophages

0 2 4 6 8 10

425 ± 28 370 ± 20 294 ± 19 213 ± 14 115 ± 9 N.D.

0 100 200 300 400 500

450 ± 33 401 ± 28 306 ± 12 239 ± 10 189 ± 9 135 ± 8

Data represent mean ± S.D. of three individual experiments.

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Fig. 3. Activity staining of TTST treated leishmanial SOD after separating by native PAGE in a 10% gel. Sixty micrograms of protein was loaded in each lane. Gel were incubated in nitroblue tetrazolium and riboflavin solutions and finally illuminated. SOD activity which inhibits the formation of blue formazan, was visualized as white bands on a blue background. (A) Without TTST; (B) 5 mg/L TTST; (C) 10 mg/L TTST; (D) 20 mg/L TTST; (E) 40 mg/L TTST.

Efficacy of TTST was further tested on intracellular survival of L. donovani in host macrophages. Table 1 shows that parasite burden in Leishmania infected hamster macrophages were significantly inhibited in a dose dependent manner. Intracellular amastigote in the L. donovani infected hamster macrophages could not be detected at the concentration of 10 mg/L TTST whereas 500 mg/L SAG reduced nearly 72% of intracellular parasite load.

Fig. 4. SDS-PAGE of SOD (E. coli) treated with increasing concentrations of TTST. Ten micrograms of protein sample was loaded in each lane to run on a 10% polyacrylamide gel. The gels were stained with 0.1% coomassie blue in 50% methanol. (A) Without TTST; (B) 5 mg/L TTST; (C) 10 mg/L TTST; (D) 15 mg/L TTST; (E) 20 mg/L TTST.

3.2. Impact of TTST on SOD activity and superoxide radical release

3.3. Treatment of Leishmania infected hamsters with TTST

Activity staining of native polyacrylamide gels clearly showed that SOD activity of Leishmania promastigotes were found to be inhibited in a dose dependent manner when treated with different concentrations of TTST (Fig. 3). At higher concentration (40 mg/L), SOD activity was totally lost. When pure Fe-SOD was treated with various concentrations of TTST and then subjected to SDS-PAGE, intensity of the protein bands gradually decreased. However, protein band for SOD could not be detected with 20 mg/L TTST (Fig. 4). SOD activity was inhibited upto 35.5% when promastigote lysate and TTST (10 ␮g/L) were added simultaneously in the assay mixture. 68.5% inhibition was observed when promastigote lysate was preincubated with TTST for 30 min at 22 ◦ C prior to SOD assay at the same concentration (Fig. 5). Rate of superoxide radical release was enhanced due to TTST treatment of Leishmania pathogen compared to normal parasite. Fig. 6 shows that at 10 mg/L of TTST, basal superoxide radical decay in parasites increased nearly two fold. On the other hand, release of superoxide radicals in 10 mg/L TTST treated infected

Fig. 7 indicates that compared to sodium antimony gluconate treated hamsters, parasite burden were significantly lower in the spleen of TTST treated animals. Parasite load in the sodium antimony gluconate (20 mg/kg body weight) treated spleen was found to be nearly 65%. On the contrary, treatment with TTST

macrophages were 3.5-fold higher than the untreated parasite containing macrophages.

Fig. 5. Status of Leishmania SOD activity when promastigote lysate and TTST were added simultaneously in the reaction mixture (䊉) or promastigote lysate and TTST were preincubated for 30 min at room temparature before adding to reaction mixture (
).

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Fig. 8. Viability of hamster macrophages. Cells were incubated in the absence and presence of TTST. The MTT dye reduction assay was conducted as described in Section 2. Results are in mean ± S.D. for three different experiments. Fig. 6. Rate of superoxide radical release. Leishmania promastigotes () before and () after TTST treatment for 1 h at room temparature. Infected macrophages before (
) and after () TTST treatment for 1 h at 37 ◦ C. Formation of blue formazan derived from reduced nitroblue tetrazolium in the presence of superoxide radical was measured spectrophotometrically.

Fig. 7. Effect of TTST on visceral leishmaniasis in vivo to compare its efficacy with sodium antimony gluconate (SAG). Group of five hamsters were used in each individual treatment separately. a,b,d P < 0.001 against PBS control; c,e P < 0.001 against SAG.

(10 mg/kg body weight) reduced parasite burden in spleen by 87% at a lower concentration compared to sodium antimony gluconate. When TTST was administered in to infected hamsters at a dose of 20 mg/kg body weight, spleen parasite load was reduced by 96%.

3.4. Determination of TTST toxicity Data in Table 2 indicated that ALP level in infected hamsters was higher compared to uninfected animals (P < 0.001). Significant decrease in ALP level (P < 0.001) was observed when infected hamsters were treated with TTST. On the other hand, levels of SGOT and SGPT after Leishmania infection, decreased significantly (P < 0.001) than in uninfected control but increased (P < 0.0 for SGPT and P < 0.001 for SGOT) after TTST administration. In all three cases results were found to approach normal values determined for uninfected hamsters. ED50 value (Fig. 8) for a parallel cytotoxicity evaluation with hamster macrophages was found to be 0.12 ± 0.03 mg/L. 4. Discussions Present report describes that TTST may be considered as a prospective candidate to act as a potent chemotherapeutic agent against visceral leishmaniasis. Its ability to kill intracellular parasites both in vitro and in vivo seems to be more effective in lower concentration compared to toxic antimonial drug tested.

Table 2 Effect of TTST on serum alkaline phosphatase (ALP), serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) levels of Leishmania infected hamsters Serum

ALP (KAU/dl)

SGOT (U/ml)

SGPT (U/ml)

Uninfected DMSO (after infection) After TTST treatment

22 ± 50 ± 3.6b 30 ± 2.6g

96 ± 71 ± 4.3d 85 ± 3.8h

65 ± 3.5e 39 ± 2.6f 60 ± 3.3k

2.9a

3.3c

TTST was administered to the infected hosts at a dose of 20 mg/kg body weight. (a, b, c, d, e, f, b, g, f, k) P < 0.001 and (d, h) P < 0.01. Data represents mean ± S.D. for five animals for each group. 1 KAU/dl is equivalent to 7.1 U/l.

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SOD which is one of the key enzymes of oxygen defence system is known to be an essential factor in mediating normal cellular functions (Fattman et al., 2003). As a result, the enzyme has been found to be targeted for the treatment of several diseases (Yassien et al., 2001; Briedbach et al., 2002; Mohan et al., 2000). SOD also plays a vital role during host–parasite interaction. Its activity is elevated when Leishmania parasite infects host cells (Dey et al., 1995). In our study, it has been found that SOD activity is inhibited to down regulate degree of parasite infection during TTST treatment to a great extent. At the same time release of superoxide radical which is a microbicidal mechanism of macrophages (Sies and de Groot, 1992) were found to be elevated in drug treated cells. Due to deficiency in SOD activity which is responsible to detoxify released superoxide radicals, toxic free radicals cannot be scavenged up to the maximum limit. In a recent report SOD has been demonstrated to be a key enzyme to play a vital role in the survival of intracellular parasites (Ghosh et al., 2003). Importance of this enzyme in the host–parasite interaction was established by generating SOD-deficient L. donovani. It is proposed that the mode of action of TTST is monitored through both inhibition of SOD and release of toxic oxygen metabolites to destroy internalized amastigotes responsible for Leishmania infection. It is known that the Leishmania SOD which is abundant in cytosol (Dey and Datta, 1994) is yet to be purified. The enzyme activity is lost as shown by activity staining of non-denaturing gels. Appearance of pure SOD available from a heterologous source (E. coli) was found to be diminished during SDS-PAGE analysis in a dose dependent manner. It is presumed that inhibition of enzymic activity takes place due to protein degradation caused by TTST. It is observed that apparent susceptibilities to TTST differed substantially for promastigotes and amastigotes. This is relevant, as the assay of compound conducted on infected macrophages was initiated only after 5 h of promastigote infection. Much higher time is needed for transformation of promastigotes into amstigotes. As the coverslip is read after 48 h, the results obtained are due to the transformed amastigotes derived from survived intracellular promatigotes. Basic mammalian toxicity of a compound may usually be judged by conducting liver function test through estimation of ALP, SGOT and SGPT (Manna

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et al., 2004). We observed that TTST was non-toxic up to the therapeutic dose of 20 mg/kg body weight that was administered to the host against experimental visceral leishmaniasis. None of the enzymes that were markers for liver toxicity were elevated when assayed in drug treated sera. This finding points to the lack of toxicity towards function of liver. In a parallel experiment, in vitro studies showed that TTST had a moderate cytotoxic activity against hamster macrophages. This observation may provide one of the essential clues to establish candidacy of TTST as an antileishmanial agent with better efficacy. Metal complexes have been tried to establish their antiparasitic activities (Loiseau et al., 2000; Goldberg et al., 1997) but up to this time none of them has been truly identified as the specific drug for any parasitic disease including visceral leishmaniasis. In this paper we have reported that TTST may be a better choice to act as an antileishmanial agent compared to SAG or other recently used standard drug like Amphotericin B which is not only toxic but much more expensive (Maes et al., 2004).

5. Conclusion Antileishmania activity of TTST is found to be mediated through superoxide radical production inhibiting superoxide dismutase. Data reveal that TTST may be considered as a better chemotherapeutic agent against visceral leishmaniasis and seems to be more effective compared to the existing drugs which are known to be more toxic. Acknowledgements Authors are thankful to Prof. Samir Bhattacharya, Director of the Indian Institute of Chemical Biology, Kolkata, for his keen interest in this study. Financial support from Department of Science and Technology, New Delhi, India, is gratefully acknowledged. References Beauchami, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287.

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