Induction of apoptosis by zerumbone isolated from Zingiber zerumbet (L.) Smith in protozoan parasite Leishmania donovani due to oxidative stress

Abstract In the present context of emergence of resistance aligned with the conventional anti-leishmanial drugs and occasional treatment failure compelled us to continue the search for replaceable therapeutic leads against Leishmaniainfection. Various ginger spices of the Zingiberaceae family are widely used as spices, flavouring agents, and medicines in Southeast Asia because of their unique flavour as well as due to their medicinal properties. Zerumbone, a natural component of Zingiber zerumbet (L.) Smith, has been studied for its pharmacological potential as antiulcer, antioxidant, anticancer, and antimicrobial. In this study, we have shown that zerumbone could induce ROS mediated apoptosis in Leishmania donovani promastigotes and also found effective in reducing intracellular amastigotes in infected-macrophages. We emphasized the potential of zerumbone to be employed in the development of new therapeutic drugs against L. donovaniinfection and provided the basis for future research on the application of transitional medicinal plants.

Effect of different serine protease inhibitors in validating the 115 kDa Leishmania donovani secretory serine protease as chemotherapeutic target.

Proteases have been considered as an important group of targets for development of antiprotozoal drugs due to their essential roles in host-parasite interactions, parasite immune evasion, life cycle transition and pathogenesis of parasitic diseases. The development of potent and selective serine protease inhibitors targeting L. donovani secretory serine protease (pSP) could pave the way to the discovery of potential antileishmanial drugs. Here, we employed different classical serine protease inhibitors (SPIs), such as aprotinin, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N-tosyl-lysine chloromethyl ketone (TLCK), benzamidine (Bza) and pSP-antibody to determine the role of the protease in parasitic survival, growth and infectivity. Among the different classical SPIs, aprotinin appeared to be more potent in arresting L. donovani promastigotes growth with significant morphological alterations. Furthermore, aprotinin and anti-pSP treated parasites significantly decreased the intracellular parasites and percentage of infected macrophages. These results suggest that SPIs may reduce the infectivity by targeting the serine protease activity and may prove useful to elucidate defined molecular mechanisms of pSP, as well as for the development of novel antileishmanial drugs in future.

Antiparasitic activity of plumericin & isoplumericin isolated from Plumeria bicolor against Leishmania donovani.

Background & objectives: The severe toxicity, exorbitant cost and emerging resistance of Leishmania species against most of the currently used drugs underscores the urgent need for the alternative drugs. The present study evaluates in vitro anti-leishmanial activity of Plumeria bicolor and its isolated compounds. Methods: The in vitro anti-parasitic activity of chloroform extract of Plumeria bicolor, plumericin and isoplumericin were tested alongwith appropriate controls against promastigote and amastigote forms of Leishmania donovani using 96 well microtiter plate. The concentration used for assessing the anti-leishmanial activity of extract of Plumeria bicolor and both isolated compounds were 100 μg/ml and 15 μM, respectively. The viability of the cells was assessed by MTT assay. The cytotoxicity of these compounds was performed against J774G8 murine macrophage cells lines at the concentration of 30 μM. Results: The Plumeria bicolor extract showed activity with the IC50 of 21±2.2 and 14±1.6 μg/ml against promastigote and amastigote forms of L. donovani, respectively. Plumericin consistently showed high activity with the IC50 of 3.17±0.12 and 1.41±0.03 μM whereas isoplumericin showed the IC50 of 7.2±0.08 μM and 4.1±0.02 μM against promastigote and amastigote forms, respectively. Cytotoxic effect of the chloroform extract of P. bicolor, plumericin and isoplumericin was evaluated in murine macrophage (J774G8) model with CC50 value of 75±5.3 μg/ml, 20.6±0.5 and 24±0.7 μM, respectively. Interpretation & conclusions: Our results indicated that plumericin showed more potent activity than isoplumericin and might be a promising anti-leishmanial agent against L. donovani.

In vitro and in vivo antileishmanial efficacy of a combination therapy of diminazene and artesunate against Leishmania donovani in BALB/c mice / Estudo in vitro e in vivo da eficácia anti leishmaniótica de terapêutica combinada de Diminazene e Artesunate contra Leishmania donovani em camundongos Balb/c

The in vitro and in vivo activity of diminazene (Dim), artesunate (Art) and combination of Dim and Art (Dim-Art) against Leishmania donovani was compared to reference drug; amphotericin B. IC50 of Dim-Art was found to be 2.28 ± 0.24 µg/mL while those of Dim and Art were 9.16 ± 0.3 µg/mL and 4.64 ± 0.48 µg/mL respectively. The IC50 for Amphot B was 0.16 ± 0.32 µg/mL against stationary-phase promastigotes. In vivo evaluation in the L. donovani BALB/c mice model indicated that treatments with the combined drug therapy at doses of 12.5 mg/kg for 28 consecutive days significantly (p < 0.001) reduced parasite burden in the spleen as compared to the single drug treatments given at the same dosages. Although parasite burden was slightly lower (p < 0.05) in the Amphot B group than in the Dim-Art treatment group, the present study demonstrates the positive advantage and the potential use of the combined therapy of Dim-Art over the constituent drugs, Dim or Art when used alone. Further evaluation is recommended to determine the most efficacious combination ratio of the two compounds.

A atividade in vitro e in vivo de Diminazene (Dim), Artezunate (Art) e a combinação Dim e Art (Dim-Art) contra Leishmania donovani foi comparada com a droga de referência Anfotericina B. IC50 da Dim-Art foi 2,28 ± 0,24 µg/mL enquanto aquelas de Dim e Art foram 9,16 ± 0,3 µg/mL e 4,64 ± 0,48 µg/mL respectivamente. O IC50 da Anfotericina B foi 0,16 ± 0,32 µg/mL contra a fase estacionária de promastigotas. A avaliação in vivo do modelo de L. donovani em camundongos Balb/c indicou que os tratamentos com a terapêutica de drogas combinadas em doses de 12,5 mg/kg por 28 dias consecutivos significantemente (p < 0,001) reduziu a carga parasitária no baço quando comparada a tratamentos com uma única droga dada nas mesmas dosagens. Embora a carga parasitária tenha sido levemente mais baixa (p < 0.05) no grupo Anfotericina B quando comparada com o grupo tratado Dim-Art, o estudo presente demonstra a vantagem positiva do uso potencial da terapêutica combinada Dim-Art sobre drogas como Dim ou Art quando usadas isoladamente. Posterior avaliação é recomendada para determinar a média de combinação mais eficaz dos dois compostos.

Visceral leishmaniasis: Experimental models for drug discovery.

Visceral leishmaniasis (VL) or kala-azar is a chronic protozoan infection in humans associated with significant global morbidity and mortality. The causative agent is a haemoflagellate protozoan Leishmania donovani, an obligate intracellular parasite that resides and multiplies within macrophages of the reticulo-endothelial system. Most of the existing anti-leishmanial drugs have serious side effects that limit their clinical application. As an alternate strategy, vaccination is also under experimental and clinical trials. The in vitro evaluation designed to facilitate rapid testing of a large number of drugs has been focussed on the promastigotes milt little attention on the clinically relevant parasite stage, amastigotes. Screening designed to closely reflect the situation in vivo is currently time consuming, laborious, and expensive, since it requires intracellular amastigotes and animal model. The ability to select transgenic Leishmania expressing reporter proteins, such as the green fluorescent proteins (GFP) or the luciferase opened up new possibilities for the development of drug screening models. Many experimental animal models like rodents, dogs and monkeys have been developed, each with specific features, but none accurately reproduces what happens in humans. Available in vitro and in vivo methodologies for antileishmanial drug screening and their respective advantages and disadvantages are reviewed.

Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

Comparison of treatment regimens of kala-azar based on culture & sensitivity of amastigotes to sodium antimony gluconate.

BACKGROUND & OBJECTIVE: Present treatment strategies for kala-azar (visceral leishmaniasis, VL) include use of first line drug sodium antimony gluconate (SAG) to all patients but a large number of patients do not get relief with this drug. If a patient does not respond to a full course of SAG, a second or third line drug is given. We undertook this study to test whether an improved outcome can be achieved by employing a strategy of treatment based on culture and sensitivity of amastigotes to SAG compared with conventional empirical treatment. METHODS: In a double-blind, randomized, controlled trial done in Balaji Utthan Sansthan, Patna, of the 181 patients screened,140 were finally randomly allocated to two groups A and B; group A patients were treated with SAG if their amastigotes were sensitive to SAG, and all patients in group B were treated with SAG to start with. Primary outcome measured was as no relapse within 6 months of follow up after cure and other outcomes measured were period of stay of patients in hospital, expenditure involved in the treatment, and infectivity periods of two groups, two-third of treatment period and whole of untreated period were taken as infectivity period. SAG was used at a dosage of 20 mg/kg given deep intramuscular injections in buttock for 28 days, amphotericin B (AMB) given at a dose of 1 mg/kg body wt daily for 20 days as a slow intravenous infusion in 5 per cent dextrose. RESULTS: Of the 70 patients in group A, 29 patients whose amastigotes were sensitive to SAG were treated with SAG, 2 patients were withdrawn due to drug toxicity; and 2 relapsed within 6 months of follow up and ultimate cure occurred in 25 (86.2%) patients only. Of the 70 patients in group B treated with SAG, 5 (7.1%) patients withdrew due to drug toxicity, 35 patients (50%) did not respond to treatment, 5 (7.1%) relapsed during 6 months of follow up and thus only 25 patients (35.7%) were ultimately cured. The difference between the two groups was significant (P<0.001). No patient died during treatment due to any toxicity because of early withdrawal of patients from treatment apprehending toxicity. Patients whose amastigotes were resistant to SAG, withdrawn from the study due to SAG toxicity, relapsed after cure with SAG, and who did not respond to SAG in both the groups were treated with AMB and all were cured. Groups B and A patients spent 3065 and 2340 days respectively in hospital, group B 1.3 times more than group A. The likely period of spread of parasites in society was 1965 days in group B and 1644 days in group A, group B 1.4 times more than group A. The total expenditure on treatment in groups B and A was dollars 65,575 and dollars 50,590 respectively; group B patient had to spend 1.3 times more than group A. INTERPRETATION & CONCLUSION: A new strategy for treatment of kala-azar based on culture and sensitivity of amastigotes improved the cure rate, saved expenditure on the patient's treatment, patients had to stay for shorter periods in hospital and reduced the chance of spread of SAG resistant disease in society. Till the government opts for better drugs, the treatment based on culture and sensitivity of the parasites to SAG may be a better method.

Antileishmanial and trypanocidal activity of Brazilian Cerrado plants

The side effects and the emerging resistance to the available drugs against leishmaniasis and trypanosomiasis led to the urgent need for new therapeutic agents against these diseases. Thirty one extracts of thirteen medicinal plants from the Brazilian Cerrado were therefore evaluated in vitro for their antiprotozoal activity against promastigotes of Leishmania donovani, and amastigotes of Trypanosoma cruzi. Among the selected plants, Casearia sylvestris var. lingua was the most active against both L. donovani and T. cruzi. Fifteen extracts were active against promastigotes of L. donovani with concentrations inhibiting 50 percent of parasite growth (IC50) between 0.1-10 æg/ml, particularly those of Annona crassiflora (Annonaceae), Himatanthus obovatus (Apocynaceae), Guarea kunthiana (Meliaceae), Cupania vernalis (Sapindaceae), and Serjania lethalis (Sapindaceae). With regard to amastigotes of T. cruzi, extracts of A. crassiflora, Duguetia furfuracea (Annonaceae), and C. sylvestris var. lingua were active with IC50 values between 0.3-10 æg/ml. Bioassay fractionations of the more active extracts are under progress to identify the active antiparasite compounds.

Magnitude of unresponsiveness to sodium stibogluconate in the treatment of visceral leishmaniasis in Bihar.

BACKGROUND: The Indian government proposes to eliminate kala-azar, which has been a serious public health problem in Bihar. This study aimed to assess the magnitude of unresponsiveness to sodium stibogluconate in the treatment of new cases of visceral leishmaniasis and to identify the associated factors. METHODS: Patients with clinically and parasitologically confirmed visceral leishmaniasis (n = 182) who had received no prior treatment, were enrolled for the study. The patients were treated with sodium stibogluconate (20 mg/kg body weight; upper limit 850 mg), intramuscularly for 30 days. The vital parameters and side-effects, if any, were monitored. Patients who developed toxicity during treatment were excluded from the study but were given rescue treatment with liposomal amphotericin B. All patients who completed the treatment were followed up for 6 months. RESULTS: Unresponsiveness to sodium stibogluconate at the end of treatment was 43%. It was higher in women (48%) compared to men (40%). A significant association was observed between unresponsiveness and level of endemicity (p = 0.0002), large spleen size (p = 0.04) and immune response (migration inhibition factor) (p = 0.00002). At the end of 6 months' follow up, 27% of patients relapsed, giving a total unresponsiveness rate of 58%. CONCLUSION: Unresponsiveness to sodium stibogluconate is a serious problem in the management of patients with visceral leishmaniasis. In patients with factors associated with nonresponse to sodium stibogluconate, alternative drugs such as miltefosine or amphotericin B should be considered as first-line drugs.

Studies on stibanate unresponsive isolates of Leishmania donovani.

Visceral leishmaniasis, also known as kala-azar (KA) is generally caused by Leishmania donovani. Organic pentavalent antimonials (SbV) is the first line of treatment for KA. However, the number of KA patients unresponsive to treatment with Sb(V) is steadily increasing in India and elsewhere. The primary objective of this work is to determine the factor(s) associated with the rise of unresponsiveness. Analysis of the clonal population of parasites clearly indicated that wild type parasites isolated from KA patients who were clinically cured after treatment with Sb(V), were a mixture of resistant and sensitive cells. The resistant promastigotes were also resistant as amastigotes in vivo. It was further observed that Stibanate sensitive parasites can be made resistant to the drug by repeated passages in experimental animals followed by incomplete treatment with suboptimal doses of the drug. These results suggest that the steady rise in Sb(V) unresponsiveness of KA patients in India is due to infection with resistant parasites, generated as a result of irregular and often incomplete treatment of the patients

Studies on stibanate resistant Leishmania donovani isolates of Indian origin.

Studies with 26 clones of L. donovani promastigotes derived from three different Indian isolates indicated that wild type parasites are mixture of stibanate sensitive and resistant cells. Both forms of the parasite were resistant to the drug. Infection with resistant parasites appears to be the primary reason of high rate of pentavalent antimony unresponsiveness among Indian kala-azar patients. It was observed that the resistant parasites originated as a result of irregular and often incomplete treatment of kala-azar patients with pentavalent antimonials.

The effects of clofazimine, niclosamide & amphotericin B, on electron transport of Leishmania donovani promastigotes.

BACKGROUND & OBJECTIVES: The study was undertaken to explore the locus of interaction of clofazimine and niclosamide which showed substantial growth inhibition property in Leishmania donovani promastigotes. METHODS: The uptake of final electron acceptor oxygen and 2,6-dichlorophenolindophenol (DCPIP) reduction in the electron transport chain were measured by constant volume Warburg respirometer and monitoring absorbance at 600 nm, respectively. Irreversibility of O2 uptake inhibition by clofazimine and niclosamide was determined by dilution of cell suspension followed by centrifugation. RESULTS: Clofazimine and niclosamide showed their minimum inhibitory concentration (MIC) at 33 and 150 micrograms/ml, respectively. Oxygen uptake inhibition by clofazimine and niclosamide was not reversed by removal of the drug by centrifugation. Rotenone, a potent inhibitor of mammalian electron transport chain showed no inhibition on the electron transport chain of L. donovani promastigotes. Cyanide at 1 mM concentration showed partial inhibition in L. donovani promastigotes. Oxygen uptake and DCPIP reduction by L. donovani promastigotes were highly sensitive to sulphhydryl group inhibitors. Strong inhibition of oxygen uptake (80-100%) by L. donovani promastigotes was achieved by clofazimine, niclosamide and amphotericin B. Amphotericin B failed to inhibit DCPIP reduction by L. donovani promastigotes, whereas DCPIP reduction was inhibited by clofazimine and niclosamide, respectively. INTERPRETATION & CONCLUSION: DCPIP reduction was mediated by transplasma membrane electron transport as evidenced by its inhibition with membrane impermeable quinone 1,2-naphthoquinone-4-sulphonic acid (NQSA). Transplasma membrane electron transport requires b-cytochromes and sulphhydryl groups for its function and was inhibited by clofazimine and niclosamide.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58 percent) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Suppressor activity in Leishmania donovani-infected hamster serum: reversion by delipidated bovine serum albumin and role in cell cycle events

Visceral leishmaniasis caused by Leishmania donovani, is a chroníc disease with a high mortality rate. This protozoan induces a serious dysfunction of the immune system characterized by suppression of the cellular response to parasite antigens. We provide evidence for the involvement of lipids in the immunological alterations of experimental leishmaniasis. Sera obtained from 60-day-infected hamsters present increased triglyceride levels. Inhibition of cell proliferation was observed when splenocytes from normal hamsters were stimulated with concanavalin A in the presence of 3 per cent infected hamster serum (IHS) (Control 50 + 3 (x 10(3)) Cpm; IHS 5 ñ 1 (X 10(3)) cpm). This inhibition was reversed by the addition of 5 mg/ml of delipidated bovine serum albumin (BSA) to the cultures (Control 65 ñ 1 (X 10(3)) cpm; IHS 75 ñ 3 (x 10(3)) cpm). The inhibitory effect of IHS was demonstrable only when added to the culture simultaneously with the mitogen. This effect was not as intense on fresh, pre-activated cells or on the CTLL-2 cells. This cell line stimulated by IL-2 in the presence of IHS is only marginally inhibited (about 20 per cent inhibition). The suppressor effect on CTLL-2 was not reversed by the addition of increasing doses of IL-2 (up to 100 U/ml) to cultures. The inhibition of the proliferative response of the CTLL-2 cells caused by IHS was also reversed by the addition of delipidated BSA. Our data suggest a role for fatty acids in the infected hamster serum-induced suppression of normal or L. donovani-infected cell proliferation.

The FML vaccine (fucose-mannose ligand) protects hamsters from experimental kala-azar

Visceral leishmaniais or kala-azar, is a chronic and frequently lethal disease, caused by Leishmania donovani. Clinical signs include malaise, hepatosplenomegaly, hypergammaglobulinemia, fever, cachexia and progressive suppresion of the cellular immune response. Only few studies on prophylactic immunization against this disease have been understaken, mostly with crude antigens, and no vaccine against kala-azar is yet available. In previous studies, we have isolated the Fucose-Mannose Ligand (FML) of L. donovani that strongly and specifically inhibits the in vitro infection of macrophages by promastigotes and amastigotes. The FML behaves as a pontent immunogen for rabbits and mice, and is specifically recognized by kala-azar patient sera. The protective pontential of FML on kala-azar was now analyzed in the CB-hamster model. We studied the efect of three intraperitoneal weekly doses of FML (100 mg) in saponin (100 mg), folowed by an intracardiac injection of 107 amastigotes. Saponin- and saline-treated controls were also included. Protection was highly significant regarding the enhancement of anti-FML antibodies titers, the splenocyte proliferative response, and the intradermal delayed hypersensitivity reaction to antigen, as well as the decrease of the parasite burden in spleen and of splenomegaly. Protection to kala-azar was due to specific FML antigenic properties, since the results obtained by saponin alone were significantly different. We conclude that the use of FML and saponin as a vaccine reduced the disease impact and retarded its onset.

Visceral leishmaniasis in Gizan, Saudi Arabia

Visceral leishmaniasis is endemic in southern Saudi Arabia. We prospectively evaluated 121 patients with visceral leishmaniasis at King Fahad Hospital in Gizan. All patients were infants and children. Seasonal variation was observed with more cases presenting in late spring and summer and few in winter. The predominant clinical features in these patients were chronic fever, abdominal distention, weight loss and hepatosplenomegaly. Lymphadenopathy was rare in contrast to African kala-azar. Common laboratory abnormalities included anemia, leukopenia, thrombocytopenia, hypoalbuminemia and hypergammaglobulinemia. Liver function tests were deranged in one-third of patients. Leishmania hemagglutination test was positive in all patients and all of them had positive bone marrow smear or culture for Leishmania donovani. Patients responded well to stibogluconate [Pentostam] therapy with a cure rate of 96.7%. Four patients died in the first few days of therapy. Jaundice and grossly deranged liver function tests were found to be bad prognostic signs