N-Substituted Phenylhydrazones Kill the Ring Stage of Plasmodium falciparum.

Antimalarial resistance has hampered the effective treatment of malaria, a parasitic disease caused by Plasmodium species. As part of our campaign on phenotypic screening of phenylhydrazones, a library of six phenylhydrazones was reconstructed and evaluated for their in vitro antimalarial and in silico receptor binding and pharmacokinetic properties. The structures of the phenylhydrazone hybrids were largely confirmed using nuclear magnetic resonance techniques. We identified two compounds which exhibited significant antimalarial potential against the ring stage (trophozoite) of 3D7 chloroquine-sensitive (CS) strain and DD2 chloroquine-resistant (CR) strains of Plasmodium falciparum with monosubstituted analogs bearing meta or para electron-donating groups showing significant activity in the single-digit micromolar range. Structure activity relationship is presented showing that electron-donating groups on the substituent hydrophobic pharmacophore are required for antimalarial activity. Compounds PHN6 and PHN3 were found to be the most potent with pIC50s (calculated form in vitro IC50s) of 5.37 and 5.18 against 3D7 CS and DD2 CR strains, respectively. Our selected ligands (PHN3 and PHN6) performed better when compared to chloroquine regarding binding affinity and molecular stability with the regulatory proteins of Plasmodium falciparum, hence predicted to be largely responsible for their in vitro activity. Pharmacokinetic prediction demonstrated that the phenylhydrazones may not cross the blood-brain barrier and are not P-glycoprotein (P-gp) substrates, a good absorption of 62% to 69%, and classified as a category IV compound based on toxicity grading.

Alkaloidal Extracts from Avicennia africana P. Beauv. (Avicenniaceae) Leaf: An Antiplasmodial, Antioxidant, and Erythrocyte Viable.

Background: The emergence of drug-resistant parasites impedes disease management and eradication efforts. Hence, a reinvigorated attempt to search for potent lead compounds in the mangroves is imperative. Aim: This study evaluates in vitro antiplasmodial activity, antioxidant properties, and cytotoxicity of A. africana leaf alkaloidal extracts. Methods: The A. africana leaves were macerated with 70% ethanol to obtain a total crude extract. Dichloromethane and chloroform-isopropanol (3 : 1, v/v) were used to extract the crude alkaloids and quaternary alkaloids from the total crude. The antiplasmodial activities of the alkaloidal extracts were performed against 3D7 P. falciparum chloroquine-sensitive clone via the SYBR Green I fluorescence assay with artesunate serving as the reference drug. The alkaloidal extracts were further evaluated for antioxidant properties via the total antioxidant capacity (TAC), the total glutathione concentration (GSH), the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, and the ferric-reducing antioxidant power (FRAP) methods. The cytotoxic activity of the alkaloidal extracts was tested on erythrocytes using a 3-(4,5-dimethylthiazol-2-yl)-5-diphenyltetrazolium bromide-MTT assay with little modification. The phytocompounds in the alkaloidal extracts were identified via gas chromatography-mass spectrometry (GC-MS) techniques. Results: The total crude extract showed good antiplasmodial activity (IC50 = 11.890 µg/mL). The crude and quaternary alkaloidal extracts demonstrated promising antiplasmodial effects with IC50 values of 6.217 and 6.285 µg/mL, respectively. The total crude and alkaloidal extracts showed good antioxidant properties with negligible cytotoxicity on erythrocytes with good selectivity indices. The GC-MS spectral analysis of crude alkaloidal extracts gave indole and isoquinoline alkaloids and several other compounds. Dexrazoxane was found to be the main compound predicted, with an 86% peak area in the quaternary alkaloidal extract. Conclusion: The crude and quaternary alkaloidal extracts exhibited antiplasmodial activities and ability to inhibit oxidative stress with negligible toxicity on erythrocytes. This may be good characteristics to avoid oxidative stress related to Plasmodium infection in the treatment of malaria.

Anti-malarial activity of the alkaloid, heptaphylline, and the furanocoumarin, imperatorin, from Clausena anisata against human Plasmodium falciparum malaria parasites: ex vivo trophozoitocidal, schizonticidal and gametocytocidal approach.

BACKGROUND: The erythrocytic stage of the life cycle of the malaria parasite, Plasmodium falciparum, consists of trophozoite, schizont and gametocyte stages in humans. Various anti-malarial agents target different stages of the parasite to produce treatment outcomes. This study reports on the stage-specific anti-malarial activity of heptaphylline and imperatorin against human P. falciparum in addition to their cytotoxicity and selectivity indices (SI). METHODS: The compounds were isolated from Clausena anisata using column chromatography and their structures elucidated using NMR spectroscopy. The anti-malarial activity was determined by measuring the trophozoitocidal, schizonticidal and gametocytocidal activities of the compounds using the SYBR green assay. Cytotoxicity was evaluated using the tetrazolium-based colorimetric assay. RESULTS: Heptaphylline and imperatorin produced trophozoitocidal, schizonticidal and gametocytocidal activities with IC50s of 1.57 (0.2317)-26.92 (0.3144) µM with those of artesunate (the standard drug) being 0.00024 (0.0036)-0.0070 (0.0013) µM. In the cytotoxicity assay, the compounds produced CC50S greater than 350 µM and SI of 13.76-235.90. Also, the trophozoitocidal and schizonticidal activities of the compounds were more pronounced than their gametocytocidal activity. Imperatorin was 42.04% more trophozoitocidal than hepthaphyline. However, hepthaphyline has more schizonticidal and gametocytocidal properties than imperatorin. CONCLUSION: Heptaphylline and imperatorin are promising anti-malarial agents, since they possess potent anti-malarial activity with weak cytotoxicity on RBCs. However, imperatorin is a better anti-malarial prophylactic agent whereas heptaphylline is a better malaria treatment agent.

Assessment of artemisinin tolerance in Plasmodium falciparum clinical isolates in children with uncomplicated malaria in Ghana.

BACKGROUND: Artemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. The present study sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug sensitivity, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana. METHODS: Six months to fourteen years old children presenting with acute uncomplicated malaria (n = 115) were enrolled in two hospitals and a Health Centre in Ghana's Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitaemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 h SYBR Green I assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing. RESULTS: Of the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and 2/85 (2.4%) had parasitaemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum (Pf) kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates. CONCLUSIONS: The observed low proportion of participants with day-3 post-treatment parasitaemia is consistent with rapid ART clearance. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harboured by the two RSA positive isolates that had high ring survival in the present study, remains to be elucidated.

Estimation of parasite age and synchrony status in Plasmodium falciparum infections.

Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management.