Sarah D’Alessandro

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.

Reinvestigating Old Pharmacophores: Are 4-Aminoquinolines and Tetraoxanes Potential Two-Stage Antimalarials?

The syntheses and antiplasmodial activities of various substituted aminoquinolines coupled to an adamantane carrier are described. The compounds exhibited pronounced in vitro and in vivo activity against Plasmodium berghei in the Thompson test. Tethering a fluorine atom to the aminoquinoline C(3) position afforded fluoroaminoquinolines that act as intrahepatocytic parasite inhibitors, with compound 25 having an IC50 = 0.31 μM and reducing the liver load in mice by up to 92% at 80 mg/kg dose. Screening our peroxides as inhibitors of liver stage infection revealed that the tetraoxane pharmacophore itself is also an excellent liver stage P. berghei inhibitor (78: IC50 = 0.33 μM). Up to 91% reduction of the parasite liver load in mice was achieved at 100 mg/kg. Examination of tetraoxane 78 against the transgenic 3D7 strain expressing luciferase under a gametocyte-specific promoter revealed its activity against stage IV-V Plasmodium falciparum gametocytes (IC50 = 1.16 ± 0.37 μM). To the best of our knowledge, compounds 25 and 78 are the first examples of either an 4-aminoquinoline or a tetraoxane liver stage inhibitors.

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.

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.

Hemozoin impairs cell cycle progression and promotes chemokine release in human microvascular endothelial cells

Background Cerebral malaria (CM) is a fatal complication of P. falciparum infection caused by the cytoadherence of infected erythrocytes to brain endothelial cells followed by microcirculatory obstruction, blood-brain barrier (BBB) damage, ring hemorrhages, inflammatory response and neurological sequelae. The combination of both parasite and host factors are involved in the pathogenesis of CM. In particular, malarial pigment, hemozoin (HZ) was shown to interfere with monocytes and endothelial cell functions. Recently our group demonstrated that HZ enhanced total gelatinolytic activity in endothelial cells by inducing ex novo matrix metalloproteinases-9 (MMP9) and promoting proMMP-9 protein expression (Prato et al., 2011).

Malaria Diagnosis, Therapy, Vaccines, and Vector Control

Malaria remains a massive public health problem in the world affecting more than 200 million people and causing 600,000 deaths every year, mostly in children under 5 years of age. Malaria control strategies include treatment of diagnosis-confirmed patients with artemisinin-based combination therapy, protection of individuals with insecticide-treated bed nets, and indoor residual spraying to fight against the vector. However, the emergence of artemisinin resistance in P. falciparum in Southeast Asia and the development of insecticide resistance in mosquitoes are putting these control tools at risk. An effective vaccine could be the tool to reach the eradication goal. However, only the RTS,S/AS01 vaccine is likely to be launched in 2015, despite its effectiveness is modest.

Novel Aminoquinoline Derivatives Significantly Reduce Parasite Load in Leishmania infantum Infected Mice

In this Letter, a detailed analysis of 30 4-aminoquinoline-based compounds with regard to their potential as antileishmanial drugs has been carried out. Ten compounds demonstrated IC50 < 1 μM against promastigote stages of L. infantum and L. tropica, and five compounds showed IC50 < 1 μM against intramacrophage L. infantum amastigotes. Two compounds showed dose-dependent enhancement of NO and ROS production by bone marrow-derived macrophages and remarkable reduction of parasite load in vivo, with advantage of being short-term and orally active. To the best of our knowledge, this is the first example of 4-amino-7-chloroquinoline derivatives active in Leishmania infantum infected mice.

Etiopathogenesis and Pathophysiology of Malaria

Malaria is a parasitic disease caused by Plasmodium protozoan parasites and transmitted by Anopheles mosquitoes. The disease is diffused in tropical areas, where it is associated with high morbidity and mortality. P. falciparum is the most dangerous species, mainly affecting young children. The parasite cycle occurs both in humans (asexual stages) and in mosquitoes (sexual stages). In humans, Plasmodium grows and multiplies within red blood cells using hemoglobin as essential source of nutrients and energy. However, this process generates toxic heme that the parasite aggregates into an insoluble inert biocrystal called hemozoin. This molecule sequesters in various organs (liver, spleen, and brain), potentially contributing to the development of malaria immunopathogenesis. Uncomplicated falciparum malaria clinical frame ranges from asymptomatic infection to classic symptoms such as fever, chills, sweating, headache, and muscle aches. However, malaria can also evolve into severe life-threatening complications, including cerebral malaria, severe anemia, respiratory distress, and acute renal failure.