In vitro and in vivo leishmanicidal activity of Dysoxylum binectariferum and its fractions against Leishmania donovani

The leishmanicidal effect of crude ethanolic extract of stem bark of Dysoxylum binectariferum and its fractions has been investigated against Leishmania donovani, the causative agent of visceral leishmaniasis. Ethanolic extract was lethal to promastigotes as well as amastigote forms in macrophage system at the concentration of 100 microg/ml. Chloroform fraction significantly inhibited promastigote multiplication and was also active against amastigotes in infected J774A.1 macrophages at 100 microg/ml. Hexane fraction was moderately active and the other fractions were inactive against both the forms. When tested in vivo in hamsters, ethanolic extract was toxic at 500 mg/kg whereas exhibited marginal activity (67.7+/-5.3%) at 250 mg/kg x 5, p.o. on day 7 post treatment (p.t.) which increases slightly (69+/-4.7) by day 30 p.t. Chloroform and n-hexane fractions exhibited 64.3+/-4% and 47.8+/-4.6% parasite inhibition at the dose of 100 mg/kg x 5 p.o., respectively. The pure compound, rohitukine, obtained from chloroform fraction showed weaker in vitro activity and was ineffective in infected hamsters. The lead potential of this plant need further investigations.     

Design and synthesis of novel substituted quinazoline derivatives as antileishmanial agents

4-(Substituted-benzylidine)-2-substituted-5,6-dihydrobenzo[h]quinazoline (5a–p) and 4-(substituted-benzylidine)-2-substituted-3, 4, 5, 6-tetrahydrobenzo[h]quinazoline (6a–p) have been synthesized from 2-(substituted-benzylidine)tetralone-1(3a–d) and several substituted guanidine sulfates(4a–d).These compounds were tested for their in vitro antileishmanial activity. The compounds 6i, 6f, 6g show promising antileishmanial activity against Leishmania donovani.     

Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents

Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.     

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world''s most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly¹. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited ²;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance ³. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.In vitro promastigotes ⁴ and axenic amastigotes assays⁵ are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).     

Synthesis and antileishmanial activity of fluorinated rhodacyanine analogues: The ‘fluorine-walk’ analysis

In a search for potent antileishmanial drug candidates, eighteen rhodacyanine analogues bearing fluorine or perfluoroalkyl substituents at various positions were synthesized. These compounds were tested for their inhibitory activities against Leishmania martiniquensis and L. orientalis. This ‘fluorine-walk’ analysis revealed that the introduction of fluorine atom at C-5, 6, 5′, or 6′ on the benzothiazole units led to significant enhancement of the activity, correlating with the less negative reduction potentials of the fluorinated analogues confirmed by the electrochemical study. On the other hand, CF₃ and OCF₃ groups were found to have detrimental effects, which agreed with the poor aqueous solubility predicted by the in silico ADMET analysis. In addition, some of the analogues including the difluorinated species showed exceptional potency against the promastigote and axenic amastigote stages (IC₅₀ = 40–85 nM), with the activities surpassing both amphotericin B and miltefosine.