Liposomes in leishmaniasis: Therapeutic effects of antimonial drugs, 8-aminoquinolines,and tetracycline

This study investigated the role of liposomes in changing the pharmacological efficacy of anti-leishmanial drugs in hamsters infected with visceral leishmaniasis. Enhanced anti-leishmanial activity could be accounted for only by liposome-encapsulated drugs. “Empty” liposomes (lacking anti-leishmanial drug) gave no therapeutic benefit by themselves, nor did they enhance the effectiveness of concurrently administered drugs. In the absence of additional drugs, empty liposomes actually resulted in a higher mortality due to endstage leishmaniasis. Mortality associated with chronic leishmaniasis, including that induced by empty liposomes, was reduced approximately 50% by orally administered unencapsulated tetracycline. Liposome-encapsulated tetracycline, given i.c., had no anti-leishmanial activity, thus indicating that tetracycline did not have inherent anti-leishmanial properties, and was beneficial because of its anti-bacterial effects. Liposomes containing an antimonial drug were effective when given i.c., i.p., or i.m., but not when given s.c. or p.o. Liposome-encapsulated antimonial drug had prophylactic activity and was effective when administered 8 days prior, but not 17 days prior, to infection. Unencapsulated antimonial drug had no prophylactic effect. In addition to antimonials, another class of compounds, 8-aminoquinolines, had marked anti-leishmanial activity in liposomes. One of these, WR 6026, was 700 to 1800 times more effective than an antimonial drug alone.     

Modification at the C9 position of the marine natural product isoaaptamine and the impact on HIV-1, mycobacterial, and tumor cell activity

As part of an investigation to generate optimized drug leads from marine natural pharmacophores for the treatment of neoplastic and infectious diseases, a series of novel isoaaptamine analogs were prepared by coupling acyl halides to the C9 position of isoaaptamine (2) isolated from the Aaptos sponge. This library of new semisynthetic products was evaluated for biological activity against HIV-1, Mtb, AIDS-OI, tropical parasitic diseases, and cancer. Compound 4 showed potent activity against HIV-1 (EC₅₀ 0.47 μg/mL), compound 19 proved to possess remarkable activity against Mycobacterium intracellulare with an IC₅₀ and MIC value of 0.15 and 0.31 μg/mL, while compounds 4 and 17 possessed anti-leishmanial activity with IC₅₀ values of 0.1 and 0.4 μg/mL, respectively. Compounds 16 and 17 showed antimalarial activity with EC₅₀ values of 230 and 240 ng/mL, respectively, and compound 14 exhibited an EC₅₀ of 0.05 μM against the Leukemia cell line K-562.     

A natural product inspired hybrid approach towards the synthesis of novel pentamidine based scaffolds as potential anti-parasitic agents

A natural product inspired molecular hybridization approach led us to a series of novel pentamidine based pyrimidine and chalcone scaffolds. All the hybrids were evaluated for their anti-leishmanial potential. Most of the screened compounds have showed significant in vitro anti-leishmanial activity with less cytotoxicity in comparison to the standard drugs (pentamidine, sodium stibogluconate, and miltefosine). Additionally, anti-malarial screening of these compounds was also done and four compounds have shown superior activity against chloroquine resistance strain (K1) of Plasmodium falciparum.     

Leishmania major and Leishmania tropica: I the in vitro effects of an immunomodulator, S2-Complex

This study was undertaken to try to determine the possible anti-leishmanial activity of S2-Complex, an organic complex of copper chloride, ascorbic acid, and nicotinamide. The promastigotes, axenic amastigotes, and intracellular amastigotes of both Leishmania major and Leishmania tropica were incubated with different concentrations of S2-Complex. The EC50 for each form was calculated. Results show that all forms of the parasites were dose dependently inhibited by S2-Complex. The promastigotes of both parasites were the most resistant with highest EC50 followed by axenic amastigotes. While intracellular amastigotes were the most sensitive with the lowest EC50.These results indicate that S2-Complex has a direct anti-leishmanial effect. When mice were treated with S2-Complex or BCG for four days before harvesting the macrophages, and the macrophages infected with both L. major and L. tropica, they showed increased phagocytosis and increased parasite killing. The results of S2-Complex were not statistically different from the immunomodulating agent BCG. These results indicate that S2-Complex has an immunomodulating effect in addition to the direct anti-leishmanial effect.     

Berberine chloride causes a caspase-independent,apoptotic-like death in Leishmania donovani promastigotes

Berberine chloride, a quarternary isoquinoline alkaloid, is a promising anti-leishmanial compound, IC₅₀ being 7.1 µM in L. donovani promastigotes. This leishmanicidal activity was initiated by its pro-oxidant effect, evidenced by enhanced generation of reactive oxygen intermediates that was accompanied by depletion of thiols; pre-incubation in N-acetyl cysteine, attenuated its cell viability, corroborating that generation of free radicals triggered its parasiticidal activity. Externalization of phosphatidylserine and elevation of intracellular calcium preceded depolarization of the mitochondrial membrane potential, which translated into an increase in the sub G₀/G₁ population and was accompanied by DNA laddering, hallmarks of apoptosis. Berberine chloride failed to induce caspase activity and anti-leishmanial activity in the presence of a pan caspase inhibitor, Z-Val-Ala-DL-Asp (methoxy)-fluoromethylketone remained unchanged, which indicated that the apoptosis was caspase independent. Collectively, the data indicates that Berberine chloride triggers an apoptosis-like death following enhanced generation of reactive oxygen species, thus meriting further pharmacological investigations.     

Mycobacterium indicus pranii (Mw) re-establishes host protective immune response in Leishmania donovani infected macrophages: critical role of IL-12

Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

4′,7-dihydroxyflavone conjugated carbon nanotube formulation demonstrates improved efficacy against Leishmania parasite

One of the global problems of rising concern is the spread of the neglected tropical disease, leishmaniasis. There are several drugs used for the treatment of the disease but the repertoire of drugs has drawbacks like toxicity and low therapeutic value. Considering the need for new drugs, we studied the synthesis of 4′,7-dihydroxyflavone conjugated multi-walled carbon nanotubes (47DHF-MWCNTs) and evaluated their anti-leishmanial activity against Leishmania donovani. The compound 47DHF was conjugated to the acid oxidized MWCTNs by Steglich esterification. The synthesized 47DHF-MWCNTs were characterized by UV spectroscopy, and, from the zeta value of 35 mV, they were found to be stable. 47DHF-MWCNTs possessed 84% drug loading efficiency and 63% cumulative drug release at intra-macrophage pH of 5.8. Moreover, the evaluation of 47DHF-MWCNTs for activity showed an IC₅₀ value of 0.051 ± 0.01 μg/ml and 0.072 ± 0.01 μg/ml against the promastigote and amastigote form, respectively. 47DHF-MWCNTs exhibited an infectivity index of 42 and selectivity index of 95, suggesting the activity of 47DHF-MWCNTs against intracellular amastigotes in the study. The 47DHF-MWCNTs also had low cytotoxicity towards macrophage cells. Fascinatingly, the 47DHF-MWCNTs treatment causes a high accumulation of ROS in the promastigotes suggesting the mechanism of anti-leishmanial activity to be ROS mediated. Summarizing from our results, we propose for the first time a novel 47DHF conjugated MWCNTs capable of anti-leishmanial activity with lower cytotoxicity that has a huge potential to be a formulation against leishmaniasis.     

Leishmania donovani: assessment of leishmanicidal effects of herbal extracts obtained from plants in the visceral leishmaniasis endemic area of Bihar, India

One obstacle faced in the effective control of visceral leishmaniasis (VL) is the limited number of available treatment options. Furthermore, control efforts have been hindered further by the emergence of Leishmania resistance to many of the available drugs. In this study, we investigated the anti-leishmanial properties of 30 medicinally important plants from the VL endemic area of Bihar, India and compared them to two available anti-leishmanial drugs (sodium antimony gluconate and amphotericin B) and two plant lectins (phytohemagglutinin and concanavalin A) on Leishmania donovani promastigotes in vitro at 24 and 48 h after initiation of culture. We identified eight plant extracts in addition to phytohemagglutinin and amphotericin B that significantly inhibited the growth of promastigotes (p < 0.03). We further studied the minimum effective concentrations as well as the effect on axenic amastigotes viability and the cell cytotoxicity on human peripheral blood of four (Agave americana, Azadirachta indica, Eclipta alba and Piper longum) of the eight plant extracts that induced significant promastigotes killing (p = 0.00098). Effect-based dose finding analysis revealed that the threshold concentration of A. americana required to eliminate L. donovani after 24 h was 0.05 mg/ml. A. indica and P. longum plant extracts eliminated L. donovani promastigotes after 48 h at concentrations of 0.1 and 0.5 mg/ml, respectively. E. alba eliminated the promastigotes at a concentration of 0.5 mg/ml within 24 h. The axenic amastigote killing response was 1.90-, 2.52- and 1.3-fold higher than the promastigote killing response with A. indica, A. americana and E. alba plant extracts, respectively. A. americana and A. indica, respectively, led to approximate 2.5- and 1.3-fold declines in mitochondrial dehydrogenase activity compared with control. E. alba stimulation resulted in an up-regulation of dehydrogenase activity (p = 0.00329). The CSA from P. longum was found to be least cytotoxic; the observed difference in mitochondrial activity was insignificant (p = 0.16314). Further studies may reveal the pharmacological significance of many of the plants with anti-leishmanial properties identified in the present study.     

The amino aldehydes produced by spermine and spermidine oxidation with maize polyamine oxidase have anti-leishmanial effect.

Purified maize polyamine oxidase bound to hydroxylapatite was utilized as an enzymatic reactor to produce oxidized spermidine and spermine. The oxidation products showed a consistent inhibitory activity on Leishmania infantum in mice. 1,3-diaminopropane showed a much lower activity. Moreover chemically prepared 4-aminobutyraldehyde had a similar ED50 to that of oxidized spermidine. It is concluded that among the compounds arising from polyamine oxidation those with the greatest anti-leishmanial effect are 4-aminobutyraldehyde and 1-(3-aminopropyl)-4-aminobutyraldehyde.     

2-(Bipiperidin-1-yl)-5-(nitroaryl)-1,3,4-thiadiazoles: Synthesis,evaluation of in vitro leishmanicidal activity,and mechanism of action

The development of novel leishmanicidal agents that are capable of being replaced by the available therapeutic options has become a priority. In the present study, the synthesis and leishmanicidal activity of a series of 5-(nitroheteroaryl-2-yl)-1,3,4-thiadiazole derivatives are described. All compounds appeared to be potent anti-leishmanial agents against both promastigote and amastigote forms of Leishmania major (L. major). Amongst the synthesized compounds, 2-([1,4′-bipiperidin]-1′-yl)-5-(5-nitrofuran-2-yl)-1,3,4-thiadiazole (IIa) and 1-(5-(1-methyl-5-nitro-1H-imidazole-2-yl)-1,3,4-thiadiazol-2-yl)-4-(piperidine-1-yl) piperidine (IIc) are the most effective. Infection index was statistically declined in the presence of all compounds. The analysis of redox-related factors revealed that exposure of L. major cells to IIa and IIc led to an increase in reactive oxygen species (ROS). Furthermore, two compounds were able to increase ROS and NO levels in infected macrophages in a dose-independent manner. In addition, we showed that these compounds induced cell death in promastigotes. Altogether, our results indicated the anti-leishmanial potential of IIa and IIc is mediated by apoptosis through an imbalance in the redox system resulting in the elevation of ROS. This new class of compound seems to hold great promise for the development of new and useful anti-leishmanial agents.     

Leishmania tropica and Leishmania donovani: solid phase radioimmunoassay using leishmanial excreted factor

A radioimmunoassay for the quantitative determination of anti-leishmanial excreted factor (EF) antibody in rabbit sera was developed. The assay, using Leishmania tropica and Leishmania donovani promastigotes EF, purified by either extraction with phenol followed by fractionation on a Sephadex G-100 column or by the dissociation of EF antibody complexes, was shown to be sensitive and reproducible. Using monospecific anti-EF antibodies, levels of as low as 0.06-0.12 micrograms/ml of anti-EF IgG could be detected. The specificity of the assay was assessed by inhibition with homologous and heterologous EF. Only minor cross-reactivity with heterologous EF was observed, and as little as 2.5 micrograms/ml of EF could be detected. Sera from kala-azar patients showed only 1.8-3.1 times more anti-EF activity, as compared with uninfected controls. No specificity was observed with sera from kala-azar patients with regard to the type of EF used. Almost the same activity was obtained with both EF from L. tropica and L. donovani. No anti-EF antibodies were detected in sera from patients with cutaneous leishmaniasis.     

Decrease of intestinal P-glycoprotein activity by 2n-propylquinoline,a new oral treatment for visceral leishmaniasis

Drugs currently available for visceral leishmaniasis treatment are potentially toxic, have to be administered by parenteral route and frequently give rise to drug resistance, due to the involvement of P-glycoproteins (P-gp) in Leishmania. The purpose of this study was to investigate a possible inhibitory effect of 2n-propylquinoline (2nPQ) on P-gp activity. 2nPQ is a new oral anti-leishmanial drug that has demonstrated its efficacy in BALB/c infected mice with Leishmania donovani [Antimicrob. Agents Chemother. 37 (1993) 859]. Rat everted gut sacs and human intestinal Caco-2 cell lines were used to study the effect of 2nPQ on P-gp activity. Our results demonstrate an inhibitory effect of 2nPQ on the P-gp activity with two P-gp substrates (rhodamine 123 and digoxin), two P-gp inhibitors (cyclosporin A and verapamil), and in two different species. Alone or associated with other active drugs, 2nPQ would be very useful to control Leishmania Multi-Drug-Resistance and intestinal P-gp in humans with kala-azar.     

Nuphar lutea: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

Several anti-leishmanial drugs of choice are of plant origin. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The development of new compounds is urgently required.Aims of the studyTo determine the leishmanicidal activity of the Nuphar lutea plant extract against Leishmania major in vitro.Materials and methodsThe leishmanicidal activity of methanolic plant extract against L. major free living promastigotes and intracellular amastigotes was evaluated, using microscopic examinations and the enzymatic XTT assay.ResultsMethanolic extract of N. lutea was highly effective against both Leishmania promastigotes and L. amastigotes (IC₅₀=2±0.12 μg/ml; ID₅₀=0.65±0.023 μg/ml; LD₅₀=2.1±0.096 μg/ml, STI=3.23). The extract at 1.25 μg/ml totally eliminated the intracellular parasites within 3 days of treatment. Also, a synergistic anti-leishmanial activity was demonstrated with N. lutea extract combined with the anti-leishmanial drug – paromomycin. The partially purified N. lutea active component was found to be a thermo-stable alkaloid(s) with no electrical charge and is resistant to boiling and to methanol, dichloromethane and xylene treatment.ConclusionsThe present study suggests that N. lutea might be a potential source of anti-leishmanial compounds.     

Synthesis and anti-leishmanial activity of 1-aryl-β-carboline derivatives against Leishmania donovani

β-carbolines from various natural and synthetic sources have been known to show diverse biological activities. As a part of our current ongoing project to search for potent natural product-derived anti-leishmanial compounds, we have synthesized a series of substituted 1-aryl-β-carboline derivatives. A total of 22 compounds were synthesized and tested in vitro against Leishmania donovani, out of which 6 compounds (4, 5, 10, 11, 19 and 22) showed notably more activity than the standard miltefosine (IC(50) 12.07±0.82 μM), with compound 4 being the most potent (IC(50) 2.16±0.26 μM).     

Leishmania major: Anti-leishmanial activity of Nuphar lutea extract mediated by the activation of transcription factor NF-κB

Here we report the effect of a partially purified alkaloid fraction (NUP) of Nuphar lutea on nuclear factor kappa B (NF-κB) expression and studied its mechanism of toxicity against Leishmania major in C3H mice peritoneal macrophages. NUP was found to be a mixture of thermo-stable dimeric sesquiterpene thioalkaloids containing mainly thionupharidines. The anti-leishmanial activity was shown to be mediated through the activation of NF-κB and increased iNOS production. Additionally, the nitric oxide inhibitor, N^G-monomethyl-l-arginine (0.5 mM) totally reverted the anti-leishmanial effect of NUP (0.25 and 0.5 μg/ml). NUP was also shown to act as an anti-oxidant, almost completely inhibiting the macrophage respiratory burst activity. However, no elevated lysozyme (EC3.2.1.17) or β-galactosidase (EC3.2.1.23) activities were demonstrated in macrophages treated with NUP. This study suggests, that the activity of NUP is mediated by NF-κB activation and the production of nitric oxide which is dependent on the l-arginine:NO pathway.     

Ivermectin, levamisole and thymic extract for chemotherapy and immunostimulation of visceral leishmaniasis in hamsters and mice

The anti-leishmanial activity of ivermectin, pentostam or combination of pentostam with either levamisole or thymic extract was tested against Leishmania donovani infection in hamsters and mice. In vitro peritoneal macrophage-parasite interaction, the effect of splenic cells on the interaction of macrophages and parasites, spleen weight, parasite burden in spleen tissue as well as the antibody titers using micro-ELISA were used as parameters for evaluating the efficacy of these chemotherapeutic regimens. Treatment with ivermectin and immunoenhancement with pentostam combined with levamisole gave best results in both animal models. Furthermore, regimens used in this work were all active in reducing phagocytosis of promastigotes by macrophages in vitro.     

Pharmacokinetic / pharmacodynamic relationships of liposomal amphotericin B and miltefosine in experimental visceral leishmaniasis

BackgroundThere is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL.Methodology / Principal findingsBALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates.Conclusion / SignificanceUsing a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.     

Characterization and stability studies on surfactant,detergent and oxidant stable α-amylase from marine haloalkaliphilic Saccharopolyspora sp. A9

A halopalkaliphilic marine Saccharopolyspora sp. stain A9 with an ability to produce surfactants, oxidant and detergent stable α-amylase was isolated from marine sediments collected from west coast of India. The α-amylase from strain A9 was purified to homogeneity with the aid of ammonium sulfate precipitation and gel filtration chromatography by using Sephadex G-75, insoluble corn starch and sephacryl S-100 column, with a 39.01-fold increase in specific activity. SDS-PAGE and zymogram activity staining showed a single band equal to molecular mass of 66 kDa. Enzyme was found to be stable in presence of wide range of NaCl concentration with maximum activity found at 11% (w/v) of NaCl. Enzyme showed remarkable stability towards laboratory surfactants, detergents and oxidants. Glucose, maltose and maltotriose were the main end product of starch hydrolysis, indicating it is α-amylase.