Yolanda Corbett

Antiplasmodial activity of Punica granatum L. fruit rind.

AIM OF THE STUDY Sun-dried rind of the immature fruit of Punica granatum L. (Punicaceae) (Pg) is presently used as a herbal formulation (OMARIA) in Orissa, India, for the therapy and prophylaxis of malaria. The aims of this study were (i) to assess in vitro the antiplasmodial activity of the methanolic extract, of a tannin enriched fraction and of compounds/metabolites of the antimalarial plant, (ii) to estimate the curative efficacy of the Pg extracts and (iii) to explore the mechanism of action of the antiplasmodial compounds. Urolithins, the ellagitannin metabolites, were also investigated for antiplasmodial activity. MATERIALS AND METHODS Chloroquine-susceptible (D10) and -resistant (W2) strains of Pf were used for in vitro studies and the rodent malaria model Plasmodium berghei-BALB/c mice was used for in vivo assessments. Recombinant plasmepsins 2 and 4 were used to investigate the interference of Pg compounds with the metabolism of haemoglobin by malaria parasites. RESULTS The Pg methanolic extract (Pg-MeOH) inhibited parasite growth in vitro with a IC(50) of 4.5 and 2.8 microg/ml, for D10 and W2 strain, respectively. The activity was found to be associated to the fraction enriched with tannins (Pg-FET, IC(50) 2.9 and 1.5 microg/ml) in which punicalagins (29.1%), punicalins, ellagic acid (13.4%) and its glycoside could be identified. Plasmepsin 2 was inhibited by Pg-MeOH extract and by Pg-FET (IC(50) 7.3 and 3.0 microg/ml), which could partly explain the antiparasitic effect. On the contrary, urolithins were inactive. Both Pg-MeOH extract and Pg-FET did not show any in vivo efficacy in the murine model. CONCLUSIONS The in vitro studies support the use of Pg as antimalarial remedy. Possible explanations for the negative in vivo results are discussed.

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection

OBJECTIVES Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

Dihydroartemisinin inhibits the human erythroid cell differentiation by altering the cell cycle

Artemisinin derivatives such as dihydroartemisinin (DHA) induce significant depletion of early embryonic erythroblasts in animal models. We have reported previously that DHA specifically targets pro-erythroblasts and basophilic erythroblasts, when human CD34+ stem cells are differentiated toward the erythroid lineage, indicating that a window of susceptibility to artemisinins may exist also in human developmental erythropoiesis during pregnancy. To better investigate the toxicity of artemisinin derivatives, the structure-activity relationship was evaluated against the K562 leukaemia cell line, used as a model for differentiating early human erythroblasts. All artemisinins derivatives, except deoxyartemisinin, inhibited both spontaneous and induced erythroid differentiation, confirming that the peroxide bridge is responsible for the erythro-toxicity. On the contrary, cell growth was markedly reduced by DHA, artemisone and artesunate but not by artemisinin, 10-deoxoartemisinin or deoxy-artemisinin. The substituent at position C-10 is responsible only for the anti-proliferative effect, since 10-deoxoartemisinin did not reduce cell growth but arrested the differentiation of K562 cells. In particular, the results showed that DHA resulted the most potent and rapidly acting compound of the drug family, causing (i) the decreased expression of GpA surface receptors and the down regulation the γ-globin gene; (ii) the alteration of S phase of cell cycle and (iii) the induction of programmed cell death of early erythroblasts in a dose dependent manner within 24h. In conclusion, these findings confirm that the active metabolite DHA is responsible for the erythro-toxicity of most of artemisinins used in therapy. Thus, as long as no further clinical data are available, current WHO recommendations of avoiding malaria treatment with artemisinins during the first trimester of pregnancy remain valid.

cAMP-Signalling Regulates Gametocyte- Infected Erythrocyte Deformability Required for Malaria Parasite Transmission

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

A new class of antimalarial dioxanes obtained through a simple two-step synthetic approach: rational design and structure-activity relationship studies.

A new series of simple endoperoxides, characterized by a 3-methoxy-1,2-dioxane scaffold, was designed on the basis of a previously developed pharmacophore. Through a simplified and versatile scheme of synthesis, which utilizes cheap and commercially available starting materials, it was possible to obtain several structurally and stereochemically different compounds that were tested against P. falciparum. Most of compounds showed antimalarial activity in the low micromolar range and no cellular toxicity, all being significantly more active on chloroquine resistant (CQ-R) than on chloroquine sensitive (CQ-S) strains. Resulting structure-activity relationships were analyzed by means of experimental and computational techniques, validating our design rationale and tailoring it for the new scaffold. Our study demonstrated that according to the hypothesized mechanism of action, the antimalarial activity can be improved through rational structural modifications, paving the way for the development of new simplified antimalarial endoperoxides.

Salinomycin and Other Ionophores as a New Class of Antimalarial Drugs with Transmission-Blocking Activity

ABSTRACT The drug target profile proposed by the Medicines for Malaria Venture for a malaria elimination/eradication policy focuses on molecules active on both asexual and sexual stages of Plasmodium, thus with both curative and transmission-blocking activities. The aim of the present work was to investigate whether the class of monovalent ionophores, which includes drugs used in veterinary medicine and that were recently proposed as human anticancer agents, meets these requirements. The activity of salinomycin, monensin, and nigericin on Plasmodium falciparum asexual and sexual erythrocytic stages and on the development of the Plasmodium berghei and P. falciparum mosquito stages is reported here. Gametocytogenesis of the P. falciparum strain 3D7 was induced in vitro, and gametocytes at stage II and III or stage IV and V of development were treated for different lengths of time with the ionophores and their viability measured with the parasite lactate dehydrogenase (pLDH) assay. The monovalent ionophores efficiently killed both asexual parasites and gametocytes with a nanomolar 50% inhibitory concentration (IC50). Salinomycin showed a fast speed of kill compared to that of standard drugs, and the potency was higher on stage IV and V than on stage II and III gametocytes. The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito midgut, confirming their transmission-blocking activity. Potential toxicity due to hemolysis was excluded, since only infected and not normal erythrocytes were damaged by ionophores. Our data strongly support the downstream exploration of monovalent ionophores for repositioning as new antimalarial and transmission-blocking leads.

Antiplasmodial and anti-inflammatory activities of Canthium henriquesianum (K. Schum), a plant used in traditional medicine in Burkina Faso

ETHNOPHARMACOLOGICAL RELEVANCE Canthium henriquesianum (K. Schum) is traditionally used in Burkina Faso for the treatment of malaria, but has not been properly investigated, yet. The aim of this study was to characterize in vitro the antiplasmodial and the anti-inflammatory activity of extracts from Canthium henriquesianum (K. Schum). In parallel, extracts of Gardenia sokotensis (Hutch) and Vernonia colorata (Willd), also traditionally used together in Burkina Faso and already reported with antimalarial activity, were compared. MATERIALS AND METHODS Plant extracts were tested in vitro for antimalarial activity against chloroquine susceptible (D10) and resistant (W2) strains of Plasmodium falciparum using the lactate dehydrogenase assay. Cell cytotoxicity was assessed on human dermal fibroblast (HDF) by the MTT assay. The selectivity index (SI) was used as the ratio of the activity against the parasites compared to the toxicity of the plant extract against HDF. In vitro cytokine production was assessed by ELISA technique. RESULTS Canthium henriquesianum aqueous extract had a moderate antimalarial activity (IC50<50 µg/ml) with a good selectivity index (SI=HDF/D10>7). Canthium henriquesianum diisopropyl ether extract was the most potent inhibitor of parasite growth with an IC50 9.5 µg/ml on W2 and 8.8 µg/ml on D10 and limited toxicity (SI>2). Gardenia sokotensis and Vernonia colorata aqueous extracts were shown to be significantly less active (IC50≥50 µg/ml) with substantial toxicity. In addition, when the production of IL-1β and TNFα by lipopolysaccharide (LPS) or hemozoin (malaria pigment) stimulated human THP-1 monocytes was assayed, it was found that the extract of Canthium henriquesianum induced a dose-dependent inhibition of IL-1β, but not of TNFα production, thus confirming its traditional use as antipyretic. By NMR analysis, the chromone was identified as the mostly represented compound in the diisopropyl ether extract of Canthium henriquesianum. Chromone however, was less active as antimalarial than the crude extract and it did not inhibit cytokine production at not toxic doses, indicating that other molecules in the total extracts contribute to the antiplasmodial and anti-inflammatory activity. CONCLUSION Canthium henriquesianum seems to possess antimalarial activity in vitro and the ability to inhibit the production of the pyrogenic cytokine IL-1β.

A new P. falciparum gametocyte drug screening assay based on pLDH detection

Plasmodium gametocytes (GCT) have recently been proposed as a crucial target for the development of new antimalarials in order to achieve malaria elimination and eventually eradication. At present, however, a widely accepted and routinely used screening method for potential gametocytocidal drugs does not exist. The aim of our work was to adapt the parasite lactate dehydrogenase (pLDH), already standardised for drug screening on asexual stages, to measure gametocyte drug sensitivity. In clinics the GCT- pLDH, which is present during all the five stages of gametocyte development, can be measured with good sensitivity through OptiMAL, an immunochromatographic diagnostic test. The pLDH assay is fast, simple, not expensive and does not require complex equipment or special waste disposal. It can be applied to field isolates since transgenesis is not needed. Gametocytogenesis of two different strains of P. falciparum, 3D7 and NF54, was induced in vitro using a standardized protocol, asexual parasites were removed by N-acetylglucosamine treatment, and GCT were seeded in 96well plates. A linear correlation between the percentage of gametocytemia, microscopically counted by Giemsa staining, and the optical density, measured spectroscopically by pLDH assay, was demonstrated. A good signal to noise ratio was obtained with the pLDH assay, and the Z’factor was calculated as indicator of the robustness of the method. Our data also indicate that GCT have a pLDH activity higher than asexual parasites. GCT were treated for 48-72h with primaquine, the gold standard against mature gametocytes in vivo, which was used to validate most of the GCT screening methods in literature; dihydroartemisinin, active on young GCT; and methylene blue, an old antimalarial recently characterised also for its anti-GCT activity. Finally, epoxomicin was tested since its strong gametocytocidal effect has been recently reported. Dose-response curves were obtained with all the four drugs. However, some discrepancies were observed between the Giemsa staining and the pLDH detection at high concentrations of the drugs, suggesting that morphological abnormalities, detected microscopically, precede the decay of pLDH activity in drug-treated GCT. In order to better understand these observations, we prolonged the treatment for further 72h. This extra-incubation period allowed us to calculate, from the pLDH assay, the IC50 (as the 50% inhibition compared to control untreated GCT) of the tested compounds, which were comparable to those obtained by Giemsa staining. These results demonstrate the feasibility of pLDH assay to measure GCT content in culture. Although more specific probes for GCT viability need to be standardized for measuring stage-specific drug activity, pLDH can be used as the first, fast and cheap screening method to find potential gametocitocydal drugs.

D-Glucose-Derived 1,2,4-Trioxepanes: Synthesis, Conformational Study, and Antimalarial Activity.

New enantiomerically pure 1,2,4-trioxepanes 10a,b/11a,b were synthesized from D-glucose. Their conformational behavior was studied by low-temperature NMR and substantiated by DFT calculations. On evaluation of in vitro antimalarial activity, the adamantyl derivative 11b showed IC50 values in the low micromolar range, particularly against the W2 chloroquine-resistant Plasmodium falciparum strain (IC50 = 0.15 ± 0.12 μM).

Malaria pigment stimulates chemokine production by human microvascular endothelium

Severe falciparum malaria is characterized by the sequestration of infected erythrocytes and leukocyte recruitment in the microvasculature, resulting in impaired blood flow and metabolic disturbances. Which parasite products cause chemokine production, thus contributing to the strong host inflammatory response and cellular recruitment are not well characterized. Here, we studied haemozoin (Hz), the end-product of haem, a ferriprotoporphyrin-IX crystal bound to host and parasite lipids, DNA, and proteins. We found that natural Hz isolated from Plasmodium falciparum cultures induces CXCL8 and CCL5 production in human dermal microvascular endothelial cells (HMEC-1) in a time-dependent manner. This up-regulation is not caused by haem but rather by Hz-generated lipoperoxidation products (15-HETE) and fibrinogen associated to Hz, and is, at least in part, triggered by the activation of NF-κB, as it was significantly inhibited by artemisinin and other NF-κB pathway inhibitors.