Dried whole plant Artemisia annua as a novel antimalarial therapy

Dried Whole plant Artemisia annua as a novel antimalarial therapy September 2014 Mostafa Ahmed Elfawal Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Stephen M. Rich Malaria is one of the worst vector-borne parasitic diseases in the developing world. The World Health Organization (WHO) estimated that 215 million cases of malaria occurred, with \u3e655,000 deaths; half the world’s population is at risk of contracting the disease. Drugs are primary weapons for reducing malaria in human populations. Successful drugs are highly efficacious and inexpensive to manufacture synthetically. However, emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently, the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model. We found that WP reduces parasitemia at least five fold more effectively than a comparable dose of purified drug, slows the evolution of malarial drug resistance in Plasmodium chabaudi infected mice, and is effective against already resistant Plasmodium yoelii (ART). This increased efficacy may result from the increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. When accompanied by plant material, more artemisinin enters the blood stream, demonstrating a beneficial effect of the plant matrix on the bioavailability of artemisinin. Increased efficacy and resilience against drug resistance may result from the synergistic benefits of other anti-malarial compounds in A. annua, such as flavonoids and terpenoids. Although effective against Plasmodium, neither WP nor artemisinin and artesunate are effective against Babesia microti. The differential response of B. microti and Plasmodium to artemisinins is likely the result of significant differences in their cell biology and metabolism of hemoglobin. Well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed whole plant A. annua therapy might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality

Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes

Soil-transmitted nematodes (STNs), namely hookworms, whipworms, and ascarids, are extremely common parasites, infecting 1-2 billion of the poorest people worldwide. Two benzimidazoles, albendazole and mebendazole, are currently used in STN mass drug administration, with many instances of low/reduced activity reported. New drugs against STNs are urgently needed. We tested various models for STN drug screening with the aim of identifying the most effective tactics for the discovery of potent, safe and broad-spectrum agents. We screened a 1280-compound library of approved drugs to completion against late larval/adult stages and egg/larval stages of both the human hookworm parasite Ancylostoma ceylanicum and the free-living nematode Caenorhabditis elegans, which is often used as a surrogate for STNs in screens. The quality of positives was further evaluated based on cheminformatics/data mining analyses and activity against evolutionarily distant Trichuris muris whipworm adults. From these data, two pairs of positives, sulconazole/econazole and pararosaniline/cetylpyridinium, predicted to target nematode CYP-450 and HSP-90 respectively, were prioritized for in vivo evaluation against A. ceylanicum infections in hamsters. One of these positives, pararosaniline, showed a significant impact on hookworm fecundity in vivo. Taken together, our results suggest that anthelmintic screening with A. ceylanicum larval stages is superior to C. elegans based on both reduced false negative rate and superior overall quality of actives. Our results also highlight two potentially important targets for the discovery of broad-spectrum human STN drugs

Anthelminthic Screening for Parasitic Nematodes

For many parasitic diseases, high-throughput phenotypic screening is an important tool in finding new drugs. Some of the most important parasitic diseases are caused by nematodes. However, these parasitic nematodes are not typically amenable to high throughput screening. Due to the ease of its maintenance and suitability for high throughput assay, the nematode Caenorhabditis elegans is instead used. To address whether C. elegans is a good model for nematode drug discovery, we compared the drug susceptibility of C. elegans relative to the human hookworm nematode parasite Ancylostoma ceylanicum at several developmental stages using a library of FDA approved drugs. I will present results of these studies that point to how well C. elegans efficacy correlates with hookworm efficacy and how early larval stages (easier to get) correlated with adult stages (more representative of what stage is targeted in human therapy). In addition, we are working on moderate-high throughput system for screening adult parasites. Murine Holigmosomoides polygyrus is a good model for human parasitic nematodes. Using Union Biometrica, Copas, worm sorter we were able to sort H. polygyrus into 384 well format. Here I will discuss the capabilities of this system as well as how we are building de novo, in collaboration with the Albrecht laboratory at WPI, an imaging and image analysis platform for screening adult stages of this parasite against large drug libraries

Use of whole plant Artemisia annua L. as an antimalarial therapy

Anti-malarial drugs are primary weapons for reducing Plasmodium transmission in human populations. Successful drugs have been highly efficacious and inexpensive to synthetically manufacture. Emergence of resistant parasites reduces the lifespan of each drug that is developed and deployed. Currently, the most effective anti-malarial is artemisinin (AN), which is extracted from the leaves of Artemisia annua. Because of its poor pharmacokinetic properties and prudent efforts to curtail emergence of resistance, AN is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant and the added cost of secondary anti-malarials in the ACT, make AN in the developing world a costly treatment. Here we show that dried leaves of A. annua administered orally are more effective at killing malaria parasites than a comparable dose of purified drug in a rodent malaria model (P. chabaudi). A single dose of whole plant (WP) A. annua containing 24 mg/kg AN clears 99% of parasites, where a comparable dose of pure drug has half that effect. This is consistent with findings that blood levels of AN are 40 times greater in mice receiving WP versus those given pure drug. We hypothesize that in addition to increasing bioavailability of AN, administration of WP alone may constitute a combination therapy because it contains other anti-malarial compounds that have been shown to synergize with AN. Inexpensive, efficacious, and resilient treatment for malaria based upon WP A. annua that can be grown and processed locally would be an effective addition to the global effort to reduce malaria morbidity and mortality

Cognitive and Microbiome Impacts of Experimental Ancylostoma ceylanicum Hookworm Infections in Hamsters

Hookworms are one of the most prevalent and important parasites, infecting ~500 million people worldwide. Hookworm disease is among the leading causes of iron-deficiency anemia in the developing world and is associated with significant growth stunting and malnutrition. In humans, hookworms appear to impair memory and other forms of cognition, although definitive data are hard to come by. Here we study the impact of a human hookworm parasite, Ancylostoma ceylanicum, on cognition in hamsters in a controlled laboratory setting. We developed tests that measure long-term memory in hamsters. We find that hookworm-infected hamsters were fully capable of detecting a novel object. However, hookworm-infected hamsters were impaired in detecting a displaced object. Defects could be discerned at even at low levels of infection, whereas at higher levels of infection, hamsters were statistically unable to distinguish between displaced and non-displaced objects. These spatial memory deficiencies could not be attributed to defects in infected hamster mobility or to lack of interest. We also found that hookworm infection resulted in reproducible reductions in diversity and changes in specific taxanomic groups in the hamster gut microbiome. These data demonstrate that human hookworm infection in a laboratory mammal results in a specific, rapid, acute, and measurable deficit in spatial memory, and we speculate that gut alterations could play some role in these cognitive deficits. Our findings highlight the importance of hookworm elimination and suggest that finer tuned spatial memory studies be carried out in humans

Developing a Whole Plant Artemisia annua Antimalarial Therapeutic: pACT

The GRAS plant Artemisia annua L. produces the sesquiterpene lactone, artemisinin. The current therapy for malaria is artemisinin + an older drug: artemisinin combination therapy (ACT). In Plasmodium chabaudi-infected mice, dried leaves of A. annua are more potent than equal amounts of pure artemisinin and may also prevent artemisinin drug resistance from emerging. This whole plant therapy is pACT: plant-based artemisinin combination therapy. Pharmacokinetics in healthy and infected mice given either pure artemisinin or pACT is different and showed that \u3e 40 fold more artemisinin enters the blood when plant material is present; plant matrix enhanced bioavailability into serum. Dried leaves as capsules or tablets given to African malaria patients were also efficacious. Flavonoids, phenolic acids, monoterpenes and other artemisinic metabolites found in the plant have mild antimalarial activity. Some may synergize with artemisinin to enhance its efficacy. In simulated digestion studies the effects of cellulose and gelatin capsules, sucrose, 4 oils, and 3 staple grains (rice, corn, and millet) were studied to determine their effect on AN and flavonoid release into the liquid phase of the intestinal stage of digestion. Compared to pACT alone: sucrose and oil enhanced release of flavonoids by 100%, but artemisinin was unaffected; both capsule types, and corn and millet meal significantly reduced artemisinin release, but had no effect on flavonoids. From field trials in MA, it was estimated that \u3e 500,000 patients could be treated from plants grown on 1 ac of land. Analysis of 10 crops of the high artemisinin-producing WPI clone of A. annua grown under different field and lab conditions showed there was consistent production of artemisinin at about 1.4% DW. Together these results show how a simple herbal remedy could be used as an efficacious, inexpensive, controlled and sustainable orally delivered therapeutic for treating malaria and other artemisinin-susceptible diseases

Small Molecule Inhibitors of Metabolic Enzymes Repurposed as a New Class of Anthelmintics

We thank Qi Wang for her technical assistance related to clustering compounds and identifying representatives for screening. This work was supported by National Institute of Allergy and Infectious Diseases (NIAID) grant AI081803 to M.M. The study was also partly supported by NIAID grant AI056189 to R.V.A.Peer reviewedPostprin

Dried Whole Plant Artemisia annua as an Antimalarial Therapy

Drugs are primary weapons for reducing malaria in human populations. However emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of the dried leaves of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model (Plasmodium chabaudi). We found that a single dose of WP (containing 24 mg/kg artemisinin) reduces parasitemia more effectively than a comparable dose of purified drug. This increased efficacy may result from a documented 40-fold increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. Synergistic benefits may derive from the presence of other anti-malarial compounds in A. annua. If shown to be clinically efficacious, well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed A. annua might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality

Genetic Diversity of Ixodid Ticks Parasitizing Eastern Mouse and Dwarf Lemurs in Madagascar, with Descriptions of the Larva, Nymph, and Male of Ixodes lemuris (Acari: Ixodidae)

The ixodid ticks parasitizing small-bodied nocturnal mouse and dwarf lemurs (Primates, Cheirogaleidae) in Madagascar are poorly documented. At Tsinjoarivo, a high-altitude eastern rain forest, mouse and dwarf lemurs were parasitized by ixodid ticks. At Ranomafana, a montane southeastern rain forest, dwarf lemurs hosted a species of Ixodes, whereas mouse lemurs were parasitized by Haemaphysalis lemuris. Ixodes specimens represent all active stages, and females are morphologically consistent with previous descriptions of Ixodes lemuris females, the only described stage in the literature. Morphological comparisons and genetic analysis using fragments of COI gene confirm that all Ixodes ticks from Tsinjoarivo and Ranomafana forests belong to the same species, i.e., Ixodes lemuris. Thus, we are able to provide descriptions of the previously unknown larva, nymph, and male. Mouse lemurs at both locations were parasitized only by immature stages of I. lemuris (at Tsinjoarivo) or H. lemuris (at Ranomafana), whereas dwarf lemurs were parasitized by all stages of I. lemuris. We suggest that ecological and biogeographical conditions may affect the pattern of tick infestation at Tsinjoarivo and Ranomafana. Additional studies are necessary to understand the tick-host associations of small-bodied nocturnal lemurs