Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.

Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi, disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.

Rapid and quantitative antimalarial drug efficacy testing via the magneto-optical detection of hemozoin.

Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magneto-optical detection of hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying hemozoin production over the intraerythrocytic cycle, we reveal that hemozoin formation is already initiated by ~ 6-12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6-10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.

Open-source discovery of chemical leads for next-generation chemoprotective antimalarials.

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

Ethnomedicines and anti-parasitic activities of Pakistani medicinal plants against Plasmodia and Leishmania parasites.

BACKGROUND: Leishmaniasis and malaria are the two most common parasitic diseases and responsible for large number of deaths per year particularly in developing countries like Pakistan. Majority of Pakistan population rely on medicinal plants due to their low socio-economic status. The present review was designed to gather utmost fragmented published data on traditionally used medicinal plants against leishmaniasis and malaria in Pakistan and their scientific validation. METHODS: Pub Med, Google Scholar, Web of Science, ISI Web of knowledge and Flora of Pakistan were searched for the collection of data on ethnomedicinal plants. Total 89 articles were reviewed for present study which was mostly published in English. We selected only those articles in which complete information was given regarding traditional uses of medicinal plants in Pakistan. RESULTS: Total of 56 plants (malaria 33, leishmaniasis 23) was found to be used traditionally against reported parasites. Leaves were the most focused plant part both in traditional use and in in vitro screening against both parasites. Most extensively used plant families against Leishmaniasis and Malaria were Lamiaceae and Asteraceae respectively. Out of 56 documented plants only 15 plants (Plasmodia 4, Leishmania 11) were assessed in vitro against these parasites. Mostly crude and ethanolic plant extracts were checked against Leishmania and Plasmodia respectively and showed good inhibition zone. Four pure compounds like artemisinin, physalins and sitosterol extracted from different plants proved their efficacy against these parasites. CONCLUSIONS: Present review provides the efficacy and reliability of ethnomedicinal practices and also invites the attention of chemists, pharmacologist and pharmacist to scientifically validate unexplored plants that could lead toward the development of novel anti-malarial and anti-leishmanial drugs.

Mechanical clearance of red blood cells by the human spleen: Potential therapeutic applications of a biomimetic RBC filtration method.

During their lifespan, circulating RBC are frequently checked for their deformability. This mechanical quality control operates essentially in the human spleen. RBC unable to squeeze though narrow splenic slits are retained and cleared from the blood circulation. Under physiological conditions this prevents microvessels from being clogged by senescent, rigid RBC. Retention of poorly deformable RBC is an important determinant of pathogenesis in malaria and may also impact the clinical benefit of transfusion. Modulating the splenic retention of RBC has already been proposed to support therapeutic approaches in these research fields. To this aim, the development of microplates for high throughput filtration of RBC through microsphere layers (microplate-based microsphiltration) has been undertaken. This review focuses on potential therapeutic applications provided by this technology in malaria chemotherapy and transfusion.

Progress toward elimination of malaria in Nigeria: uptake of artemisinin-based combination therapies for the treatment of malaria in households in Benin City.

BACKGROUND: The Roll Back Malaria (RBM) Partnership converged in Abuja in 2000. In 2005, Nigeria adopted artemisinin-based combination therapies (ACTs) as first-line therapy for uncomplicated malaria. It was determined that by 2010, 80% of persons with malaria would be effectively treated. OBJECTIVES: To describe household practices for malaria treatment in Benin City; to explore demographic characteristics that may influence use of ACTs. MATERIALS AND METHODS: Multistage sampling technique was used to select households from each of the three local government areas in Benin City. Adult respondents were interviewed. Household reference persons (HRPs) were defined by International Labour Organization categories. Data were collected between December 2009 and February 2010 and were analyzed using Statistical Package for the Social Sciences Version 16.0, at a significance level of P < 0.05 (2-tailed). RESULTS: Of the 240 households selected, 217 were accessible, and respondents from 90% of these recalled the most recent episode (s) of malaria. One-third of malaria episodes had occurred in children younger than 5 years. ACTs were used in 4.9% of households; sulfadoxine-pyrimethamine was the chief non-ACT antimalarial, followed by artemisinin monotherapies. Patent medicine stores were the most common sources of antimalarial medicines (38.2%), followed by private hospitals (20.3%) and private pharmacies (10.6%). Only 8.3% of households got their medicines from government hospitals. Having a HRP in managerial or professional categories was associated with a 6 times higher odds of using ACTs, compared to other occupational categories [odds ratio (OR) 5.8; confidence interval (CI) 1.470-20.758, P = 0.016]. Fathers' tertiary or higher education was significantly associated with ACT use, but not mothers' (OR 0.054, CI 0.006-0.510; P = 0.011 and OR 0.905, CI 0.195-4.198; P = 0.898, respectively). CONCLUSION: Ten years after the historic Abuja meeting, only 5% of households in Benin City used ACTs for the treatment of malaria, sourcing medicines chiefly from patent medicine stores and private hospitals. Fathers' level of education was significantly associated with ACT use. Interventions to eliminate malaria from Nigeria should mainstream the men folk and health care providers outside government hospitals, in line with the Nigerian reality.

Dihydroquinazolinone inhibitors of proliferation of blood and liver stage malaria parasites.

Drugs that target both the liver and blood stages of malaria will be needed to reduce the disease's substantial worldwide morbidity and mortality. Evaluation of a 259-member library of compounds that block proliferation of the blood stage of malaria revealed several scaffolds--dihydroquinazolinones, phenyldiazenylpyridines, piperazinyl methyl quinolones, and bis-benzimidazoles--with promising activity against the liver stage. Focused structure-activity studies on the dihydroquinazolinone scaffold revealed several molecules with excellent potency against both blood and liver stages. One promising early lead with dual activity is 2-(p-bromophenyl)-3-(2-(diethylamino)ethyl)-2,3-dihydroquinazolin-4(1H)-one with 50% effective concentrations (EC50s) of 0.46 µM and 0.34 µM against liver stage Plasmodium berghei ANKA and blood stage Plasmodium falciparum 3D7 parasites, respectively. Structure-activity relationships revealed that liver stage activity for this compound class requires a 3-dialkyl amino ethyl group and is abolished by substitution at the ortho-position of the phenyl moiety. These compounds have minimal toxicity to mammalian cells and are thus attractive compounds for further development.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Screening medicinal plants for the detection of novel antimalarial products applying the inhibition of ß-hematin formation.

The identification of novel scaffolds for the development of effective and safe treatments to fight malaria is urgently needed. One of the main opportunities is the discovery of new molecules from natural origin. A simple, robust and cost-effective colorimetric assay based on the inhibition of ß-hematin has been adapted to routinely screen plant extracts with the ultimate goal to identify novel antimalarial ingredients. The development of this assay has included a careful optimization of all critical experimental parameters. The ß-hematin assay can be completed in less than one working day, requiring a 96-well UV-vis plate reader and low-cost commercially available reagents using a standard operating protocol. It can be used on its own or in combination with the well-known Plasmodium growth inhibition assay and has the obvious merit to be informative at the early stage of drug discovery regarding the mechanism of action of the actives. A total of 40 diverse natural products and 219 plants extracts were tested. Good correlations in respect with specificity (pure compounds 85%, extracts 93%) and positive predictive value (pure compounds 72%, extracts 50%) were obtained in comparison with Plasmodium growth inhibition assay that was used as the reference assay.

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

Leveraging cell cycle analysis in anticancer drug discovery to identify novel plasmodial drug targets.

Cancer and malaria are life threatening diseases killing millions of people each year. In spite of our best efforts, both continue to resist full control and eradication. If untreated, both malaria and cancer can lead to death. Only a few antimalarial drugs have been developed over the last decades and new drugs are urgently needed to combat drug-resistant parasites. Significant progress has been made in understanding the molecular mechanisms of cancer and designing new anticancer therapies. However, similar to malaria, majority of cancers quickly develop resistance to single target-based therapy. Novel cancer therapeutics are being developed with the aim of targeting multiple signalling pathways in tumour cells, an approach that may be applicable to antimalarial therapy. In this review we compare cell signalling pathways targeted by cancer drugs with similar pathways in the malaria parasite. We placed particular emphasis on cell cycle regulation and cell cycle checkpoints since the associated molecular machinery controlling these processes are conserved in Plasmodium. Furthermore, a large number of cancer drugs target cell cycle control mechanisms and, therefore, these compounds may possess antimalarial activity. We tried to demonstrate that promising areas of anticancer drug development can be incorporated in the existing antimalarial drug discovery program as well as deepen our understanding of parasite biology.

Iron metabolism and malaria.

Recent evidence from a large, randomized, controlled trial has suggested that the universal administration of iron to children in malaria-endemic areas is associated with an increase in adverse health outcomes. The purpose of this paper is to summarize the available ecologic and intervention trials related to iron and malaria in children, and to set these against current knowledge of the biology of host-pathogen interactions involving iron metabolism. We conclude that, although not fully consistent, the balance of evidence confirms that administration of iron (usually in combination with folic acid) increases the incidence of malaria when given without prophylaxis and in the absence of universal access to treatment. The mechanisms by which additional iron can benefit the parasite are far from clear. There is evidence to suggest that the apparent detrimental effect of iron supplementation may vary according to levels of antecedent iron status, the presence of hemoglobinopathies and glucose-6-phosphate dehydrogenase (G6PD) deficiency, and other host genetic variants, such as variants in haptoglobin. The effects of malaria on host iron metabolism are also reviewed and reveal that the key cause of malaria-induced anemia is a maldistribution of iron and suppression of erythropoiesis rather than an exacerbation of gross iron deficiency. We tentatively conclude that, if it is to be recommended, universal iron supplementation in malarious areas should only be considered in conjunction with some form of prophylaxis (e.g., intermittent preventive therapy [IPT]) or in the context of good health services with ready access to facilities for malaria diagnosis and treatment. An alternative approach would be to screen for anemia and target supplementation only to anemic children. With regard to treatment, there is good evidence that iron supplementation should be withheld until the treatment schedule is complete, both because iron may inhibit treatment and because the absorption of oral iron is blocked by the inflammatory response.

Comments on background papers related to iron, folic acid, malaria and other infections.

This review comments on and summarizes five expert presentations and reports made at a meeting hosted by the World Health Organization (WHO) in Lyon, France, 12-14 June 2006, related to iron and folate supplementation and their interactions with infection. The meeting was called because of the mortality implications of the Pemba iron study and the possible need to change WHO policy as soon as possible. Six tabled presentations were reviewed. A majority of these expert reviews regarded the Pemba study as indicating a specific adverse interaction between iron supplementation and malaria. A majority regarded such an effect as already reviewed, demonstrated, and predicted in existing literature published prior to the Pemba study. A majority concluded that there was a risk of malarial morbidity associated with oral iron supplementation. A majority made recommendations for change, indicating either that the 1998 WHO/UNICEF recommendation for iron supplementation in malarious areas should be suspended pending further research or that it should be stopped. A majority felt that folate supplementation was a less likely cause of the Pemba result; two mentioned the interference of oral folate with antifolate antimalarials; a majority suggested suspension of folic acid supplementation to children in malarious areas. Only one presentation argued for net population benefits of folate and none for iron.

Critical review of strategies to prevent and control iron deficiency in children.

Iron deficiency is prevalent in infants and young children in developing countries and is associated with adverse developmental outcomes. The routine provision of additional iron by food fortification or the use of iron supplements is generally recommended. The wisdom of this approach in regions where the transmission of Plasmodium falciparum malaria is perennial and intense is now being questioned, because a large trial in Pemba, Tanzania, demonstrated an increased risk of serious morbidity among children under the age of 3 years who were given routine daily iron and folic acid supplements. However, the results of a concurrent substudy suggest that the untoward effects occurred in children who were not iron deficient, and that iron deficiency itself is associated with an increased risk of severe morbidity that can be reduced by iron and folic acid supplementation. There is an urgent need for additional research to confirm these observations, to establish the role, if any, of the concurrent folic acid supplementation, to evaluate the risk of alternative methods for delivering iron that, on theoretical grounds, could be safer, and to establish the programmatic feasibility of targeting iron fortificants or supplements to iron-deficient children. It is evident that a single strategy for ensuring adequate iron nutrition in young children in different parts of the world is no longer likely to be satisfactory. Moreover, integration with other health-related strategies, particularly malaria control programs, will be essential.

[Combined antimalarial therapy using artemisinin]. / Terapia antimalarica combinata con derivati dell'artemisinina.

The existing armamentarium of drugs for the treatment and prevention of malaria is limited primarily by resistance (and cross-resistance between closely related drugs). However, most of these drugs still have a place and their life-span could be prolonged if better deployed and used, and also by rationally combining them based on pharmacodynamic and pharmacokinetic properties. Newer compounds are also being developed. The nature of malaria disease and its prevalence in the developing world call for innovative approaches to develop new affordable drugs and to safeguard the available ones. According to WHO, the concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also delay the development of resistance to the individual components of the combination. Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. In the context of this definition, multiple-drug therapies that include a nonantimalarial drug to enhance the antimalarial effect of a blood schizontocidal drug are not considered combination therapy. Similarly, certain antimalarial drugs that fit the criteria of synergistic fixed-dose combinations are operationally considered as single products in that neither of the individual components would be given alone for anti-malarial therapy. An example is sulfadoxine-pyrimethamine. Artemisinin-based combination therapies have been shown to improve treatment efficacy and also contain drug resistance in South-East Asia. However, major challenges exist in the deployment and use of antimalarial drug combination therapies, particularly in Africa. These include: 1) the choice of drug combinations best suited for the different epidemiological situations; 2) the cost of combination therapy; 3) the timing of the introduction of combination therapy; 4) the operational obstacles to implementation, especially compliance. As a response to increasing levels of antimalarial resistance, the World Health Organization (WHO) recommends that all countries experiencing resistance to conventional monotherapies, such as chloroquine, amodiaquine or sulfadoxine/pyrimethamine, should use combination therapies, preferably those containing artemisinin derivatives (ACTs--artemisinin-based combination therapies) for malaria caused by Plasmodium falciparum. There is a promising role of such compounds in replacing or complementing current options. Since 1979, several different formulations of artemisinin and its derivatives have been produced and studied in China in several thousand patients for either P. falciparum or P. vivax malaria. To date, there is no evidence of drug resistance to these compounds. The use of artemisinin, artemether, arteether and artesunate for either uncomplicated or severe malaria is now spreading through almost all malarious areas of the world, although some of they have no patent protection, their development (with few exceptions) has not followed yet full international standards. Both artesunate, artemether and arteether are rapidly and extensively converted to their common bioactive metabolite, dihydroarte-misinin. WHO currently recommends the following therapeutic options: 1) artemether/lumefantrine; 2) artesunate plus amodiaquine; 3) artesunate plus sulfadoxine/pyrimethamine (in areas where SP efficacy remains high); 4) artesunate plus mefloquine (in areas with low to moderate transmission); and 5) amodiaquine plus sulfadoxine/pyrimethamine, in areas where efficacy of both amodiaquine and sulfadoxine/pyrimethamine remains high (mainly limited to countries in West Africa). This non artemisinin-based combination therapy is reserved as an interim option for countries, which, for whatever reason, are unable immediately to move to ACTs.

Chronobiology and chronotherapy of malaria: investigations with murine malaria models.

The schizogonic rhythm of rodent malarias in the blood of mice varies from one species or subspecies to the other. Synchronous strains enable a precise study of the sensitive stage of the parasite to antimalarial drugs. Asynchronous strains are less sensitive than synchronous strains because of the delayed penetration of merozoites into red blood cells.

[Cultivation of exo-erythrocytic schizonts of rodent Plasmodium in hepatocytes: a new experimental model for chemotherapy of malaria]. / La culture des schizontes exo-érythocytaires des Plasmodium de rongeurs dans des hépatocytes: un nouveau modèle expérimental pour la chimiothérapie du paludisme.

An in vitro experimental model using primary cultures of laboratory bred Thamnomys gazellae's hepatocytes and Plasmodium yoelii yoelii' sporozoites was set up for chemotherapeutic studies. The surface of the culture was reduced (0.5 cm) and allowed the rapid performance and analysis of schizonticide activity tests, with a reduced biological material (Rodents, Anopheles, sporozoites). Fifteen compounds were tested. When activity of molecules is known in vivo, both results in vivo and in vitro are parallel.

Plasmodium gallinaceum: avian screen for drugs with radical curative properties.

Existing primary screens for radical curative antimalarial drugs fail to adequately detect many compounds which affect the latent, exoerythrocytic hypnozoite, the stage of the parasite responsible for relapse. At the same time, these screens falsely identify a wide range of compounds with no radical curative activity. The avian malaria, Plasmodium gallinaceum, and Aedes aegypti mosquitos were used in a screen which measures the effects of candidate compounds on gametocytes and their development within the mosquito. Sporontocidal and gametocytocidal effects could be differentiated by this screen. In a blind study, those compounds shown to be exclusively gametocytocidal were those same drugs which had previously been shown to have radical curative effects against true relapsing malarias. The chicken malaria gametocyte screen was more sensitive than the rodent screens in detecting useful compounds, with a minimum of false positive identifications.