Wolbachia depletion blocks transmission of lymphatic filariasis by preventing chitinase-dependent parasite exsheathment.

Lymphatic filariasis is a vector-borne neglected tropical disease prioritized for global elimination. The filarial nematodes that cause the disease host a symbiotic bacterium, Wolbachia, which has been targeted using antibiotics, leading to cessation of parasite embryogenesis, waning of circulating larvae (microfilariae [mf]), and gradual cure of adult infection. One of the benefits of the anti-Wolbachia mode of action is that it avoids the rapid killing of mf, which can drive inflammatory adverse events. However, mf depleted of Wolbachia persist for several months in circulation, and thus patients treated with antibiotics are assumed to remain at risk for transmitting infections. Here, we show that Wolbachia-depleted mf rapidly lose the capacity to develop in the mosquito vector through a defect in exsheathment and inability to migrate through the gut wall. Transcriptomic and Western blotting analyses demonstrate that chitinase, an enzyme essential for mf exsheathment, is down-regulated in Wolbachia-depleted mf and correlates with their inability to exsheath and escape the mosquito midgut. Supplementation of in vitro cultures of Wolbachia-depleted mf with chitinase enzymes restores their ability to exsheath to a similar level to that observed in untreated mf. Our findings elucidate a mechanism of rapid transmission-blocking activity of filariasis after depletion of Wolbachia and adds to the broad range of biological processes of filarial nematodes that are dependent on Wolbachia symbiosis.

AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

M1 macrophage features in severe Plasmodium falciparum malaria patients with pulmonary oedema.

BACKGROUND: Pulmonary oedema (PE) is a serious complication of Plasmodium falciparum malaria which can lead to acute lung injury in severe cases. Lung macrophages are activated during malaria infection due to a complex host-immune response. The molecular basis for macrophage polarization is still unclear but understanding the predominant subtypes could lead to new therapeutic strategies where the diseases present with lung involvement. The present study was designed to study the polarization of lung macrophages, as M1 or M2 macrophages, in the lungs of severe P. falciparum malaria patients, with and without evidence of PE. METHODS: Lung tissue samples, taken from patients who died from severe P. falciparum malaria, were categorized into severe malaria with PE and without PE (non-PE). Expression of surface markers (CD68+, all macrophages; CD40+, M1 macrophage; and CD163+, M2 macrophage) on activated lung macrophages was used to quantify M1/M2 macrophage subtypes. RESULTS: Lung injury was demonstrated in malaria patients with PE. The expression of CD40 (M1 macrophage) was prominent in the group of severe P. falciparum malaria patients with PE (63.44 ± 1.98%), compared to non-PE group (53.22 ± 3.85%, p < 0.05), whereas there was no difference observed for CD163 (M2 macrophage) between PE and non-PE groups. CONCLUSIONS: The study demonstrates M1 polarization in lung tissues from severe P. falciparum malaria infections with PE. Understanding the nature of macrophage characterization in malaria infection may provide new insights into therapeutic approaches that could be deployed to reduce lung damage in severe P. falciparum malaria.

Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis.

Elimination of filariasis requires a macrofilaricide treatment that can be delivered within a 7-day period. Here we have identified a synergy between the anthelmintic albendazole (ABZ) and drugs depleting the filarial endosymbiont Wolbachia, a proven macrofilaricide target, which reduces treatment from several weeks to 7 days in preclinical models. ABZ had negligible effects on Wolbachia but synergized with minocycline or rifampicin (RIF) to deplete symbionts, block embryogenesis, and stop microfilariae production. Greater than 99% Wolbachia depletion following 7-day combination of RIF+ABZ also led to accelerated macrofilaricidal activity. Thus, we provide preclinical proof-of-concept of treatment shortening using antibiotic+ABZ combinations to deliver anti-Wolbachia sterilizing and macrofilaricidal effects. Our data are of immediate public health importance as RIF+ABZ are registered drugs and thus immediately implementable to deliver a 1-wk macrofilaricide. They also suggest that novel, more potent anti-Wolbachia drugs under development may be capable of delivering further treatment shortening, to days rather than weeks, if combined with benzimidazoles.

Human Direct Skin Feeding Versus Membrane Feeding to Assess the Mosquitocidal Efficacy of High-Dose Ivermectin (IVERMAL Trial).

BACKGROUND: Ivermectin is being considered for mass drug administration for malaria, due to its ability to kill mosquitoes feeding on recently treated individuals. In a recent trial, 3-day courses of 300 and 600 mcg/kg/day were shown to kill Anopheles mosquitoes for at least 28 days post-treatment when fed patients' venous blood using membrane feeding assays. Direct skin feeding on humans may lead to higher mosquito mortality, as ivermectin capillary concentrations are higher. We compared mosquito mortality following direct skin and membrane feeding. METHODS: We conducted a mosquito feeding study, nested within a randomized, double-blind, placebo-controlled trial of 141 adults with uncomplicated malaria in Kenya, comparing 3 days of ivermectin 300 mcg/kg/day, ivermectin 600 mcg/kg/day, or placebo, all co-administered with 3 days of dihydroartemisinin-piperaquine. On post-treatment day 7, direct skin and membrane feeding assays were conducted using laboratory-reared Anopheles gambiae sensu stricto. Mosquito survival was assessed daily for 28 days post-feeding. RESULTS: Between July 20, 2015, and May 7, 2016, 69 of 141 patients participated in both direct skin and membrane feeding (placebo, n = 23; 300 mcg/kg/day, n = 24; 600 mcg/kg/day, n = 22). The 14-day post-feeding mortality for mosquitoes fed 7 days post-treatment on blood from pooled patients in both ivermectin arms was similar with direct skin feeding (mosquitoes observed, n = 2941) versus membrane feeding (mosquitoes observed, n = 7380): cumulative mortality (risk ratio 0.99, 95% confidence interval [CI] 0.95-1.03, P = .69) and survival time (hazard ratio 0.96, 95% CI 0.91-1.02, P = .19). Results were consistent by sex, by body mass index, and across the range of ivermectin capillary concentrations studied (0.72-73.9 ng/mL). CONCLUSIONS: Direct skin feeding and membrane feeding on day 7 resulted in similar mosquitocidal effects of ivermectin across a wide range of drug concentrations, suggesting that the mosquitocidal effects seen with membrane feeding accurately reflect those of natural biting. Membrane feeding, which is more patient friendly and ethically acceptable, can likely reliably be used to assess ivermectin's mosquitocidal efficacy. CLINICAL TRIALS REGISTRATION: NCT02511353.

Intracellular Pharmacodynamic Modeling Is Predictive of the Clinical Activity of Fluoroquinolones against Tuberculosis.

Clinical studies of new antitubercular drugs are costly and time-consuming. Owing to the extensive tuberculosis (TB) treatment periods, the ability to identify drug candidates based on their predicted clinical efficacy is vital to accelerate the pipeline of new therapies. Recent failures of preclinical models in predicting the activity of fluoroquinolones underline the importance of developing new and more robust predictive tools that will optimize the design of future trials. Here, we used high-content imaging screening and pharmacodynamic intracellular (PDi) modeling to identify and prioritize fluoroquinolones for TB treatment. In a set of studies designed to validate this approach, we show moxifloxacin to be the most effective fluoroquinolone, and PDi modeling-based Monte Carlo simulations accurately predict negative culture conversion (sputum sterilization) rates compared to eight independent clinical trials. In addition, PDi-based simulations were used to predict the risk of relapse. Our analyses show that the duration of treatment following culture conversion can be used to predict the relapse rate. These data further support that PDi-based modeling offers a much-needed decision-making tool for the TB drug development pipeline.

Intra-individual effects of food upon the pharmacokinetics of rifampicin and isoniazid.

Background: Poor response to TB therapy might be attributable to subtherapeutic levels in drug-compliant patients. Pharmacokinetic parameters can be affected by comorbidities or the interaction of drugs with food. Objectives: This study aimed to determine the effect of food intake upon pharmacokinetics of rifampicin and isoniazid in a Peruvian population with TB. Methods: Rifampicin and isoniazid levels were analysed at 2, 4 and 6 h after drug intake in both fasting and non-fasting states using LC-MS methods. Results: Sixty patients participated in the study. The median rifampicin Cmax and AUC0-6 were higher during fasting than non-fasting: 7.02 versus 6.59 mg/L (P = 0.054) and 28.64 versus 24.31 mg·h/L (P = 0.002). There was a statistically significant delay overall of non-fasting Tmax compared with the fasting state Tmax (P = 0.005). In the multivariate analysis, besides the effect of fasting, Cmax for females was 20% higher than for males (P = 0.03). Concerning isoniazid, there were significant differences in the Cmax during non-fasting (median = 3.51 mg/L) compared with fasting (4.54 mg/L). The isoniazid dose received had an effect upon the isoniazid levels (1.26, P = 0.038). In the multivariate analysis, isoniazid exposure during fasting was found to be 14% higher than during non-fasting (CI = 1.02-1.28, P < 0.001). Neither radiological extent of the disease nor consumption of food with drug intake nor pharmacokinetics of rifampicin or isoniazid was associated with a poorer treatment outcome. Conclusions: Rifampicin in particular and isoniazid pharmacokinetics were significantly affected by the intake of the drug with food between and within individuals.

Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7.

The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs.

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria.

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

Antimalarial 4(1H)-pyridones bind to the Qi site of cytochrome bc1.

Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Q(o) site (one of two potential binding sites within cytochrome bc1 using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Q(o) site but bind at the Q(i )site. The discovery that these compounds bind at the Q(i) site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Q(i) also explains the ability of this class to overcome parasite Q(o)-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles.

OptiMal-PK: an internet-based, user-friendly interface for the mathematical-based design of optimized anti-malarial treatment regimens.

BACKGROUND: The search for highly effective anti-malarial therapies has gathered pace and recent years have seen a number of promising single and combined therapies reach the late stages of development. A key drug development challenge is the need for early assessment of the clinical utility of new drug leads as it is often unclear for developers whether efforts should be focused on efficacy or metabolic stability/exposure or indeed whether the continuation of iterative QSAR (quantitative structure-activity and relationships) cycles of medicinal chemistry and biological testing will translate to improved clinical efficacy. Pharmacokinetic and pharmacodynamic (PK/PD)-based measurements available from in vitro studies can be used for such clinical predictions. However, these predictions often require bespoke mathematical PK/PD modelling expertise and are normally performed after candidate development and, therefore, not during the pre-clinical development phase when such decisions need to be made. METHODS: An internet-based tool has been developed using STELLA(®) software. The tool simulates multiple differential equations that describe anti-malarial PK/PD relationships where the user can easily input PK/PD parameters. The tool utilizes a simple stop-light system to indicate the efficacy of each combination of parameters. This tool, called OptiMal-PK, additionally allows for the investigation of the effect of drug combinations with known or custom compounds. RESULTS: The results of simulations obtained from OptiMal-PK were compared to a previously published and validated mathematical model on which this tool is based. The tool has also been used to simulate the PK/PD relationship for a number of existing anti-malarial drugs in single or combined treatment. Simulations were predictive of the published clinical parasitological clearance activities for these existing therapies. CONCLUSIONS: OptiMal-PK is designed to be implemented by medicinal chemists and pharmacologists during the pre-clinical anti-malarial drug development phase to explore the impact of different PK/PD parameters upon the predicted clinical activity of any new compound. It can help investigators to identify which pharmacological features of a compound are most important to the clinical performance of a new chemical entity and how partner drugs could potentially improve the activity of existing therapies.

PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC50. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development.

A Click Chemistry-Based Proteomic Approach Reveals that 1,2,4-Trioxolane and Artemisinin Antimalarials Share a Common Protein Alkylation Profile.

In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross-resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4-trioxolane activity based protein-profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi-quantitatively, suggesting a common mechanism of action that raises concerns about potential cross-resistance liabilities.

Baseline data of parasite clearance in patients with falciparum malaria treated with an artemisinin derivative: an individual patient data meta-analysis.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum manifests as slow parasite clearance but this measure is also influenced by host immunity, initial parasite biomass and partner drug efficacy. This study collated data from clinical trials of artemisinin derivatives in falciparum malaria with frequent parasite counts to provide reference parasite clearance estimates stratified by location, treatment and time, to examine host factors affecting parasite clearance, and to assess the relationships between parasite clearance and risk of recrudescence during follow-up. METHODS: Data from 24 studies, conducted from 1996 to 2013, with frequent parasite counts were pooled. Parasite clearance half-life (PC1/2) was estimated using the WWARN Parasite Clearance Estimator. Random effects regression models accounting for study and site heterogeneity were used to explore factors affecting PC1/2 and risk of recrudescence within areas with reported delayed parasite clearance (western Cambodia, western Thailand after 2000, southern Vietnam, southern Myanmar) and in all other areas where parasite populations are artemisinin sensitive. RESULTS: PC1/2 was estimated in 6975 patients, 3288 of whom also had treatment outcomes evaluate d during 28-63 days follow-up, with 93 (2.8 %) PCR-confirmed recrudescences. In areas with artemisinin-sensitive parasites, the median PC1/2 following three-day artesunate treatment (4 mg/kg/day) ranged from 1.8 to 3.0 h and the proportion of patients with PC1/2 >5 h from 0 to 10 %. Artesunate doses of 4 mg/kg/day decreased PC1/2 by 8.1 % (95 % CI 3.2-12.6) compared to 2 mg/kg/day, except in populations with delayed parasite clearance. PC1/2 was longer in children and in patients with fever or anaemia at enrolment. Long PC1/2 (HR = 2.91, 95 % CI 1.95-4.34 for twofold increase, p < 0.001) and high initial parasitaemia (HR = 2.23, 95 % CI 1.44-3.45 for tenfold increase, p < 0.001) were associated independently with an increased risk of recrudescence. In western Cambodia, the region with the highest prevalence of artemisinin resistance, there was no evidence for increasing PC1/2 since 2007. CONCLUSIONS: Several factors affect PC1/2. As substantial heterogeneity in parasite clearance exists between locations, early detection of artemisinin resistance requires reference PC1/2 data. Studies with frequent parasite count measurements to characterize PC1/2 should be encouraged. In western Cambodia, where PC1/2 values are longest, there is no evidence for recent emergence of higher levels of artemisinin resistance.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria.

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc(1). Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Effects of dosage, comorbidities, and food on isoniazid pharmacokinetics in Peruvian tuberculosis patients.

Poor response to tuberculosis (TB) therapy might be attributable to subtherapeutic levels in drug-compliant patients. Pharmacokinetic (PK) parameters can be affected by several factors, such as comorbidities or the interaction of TB drugs with food. This study aimed to determine the PK of isoniazid (INH) in a Peruvian TB population under observed daily and twice-weekly (i.e., biweekly) therapy. Isoniazid levels were analyzed at 2 and 6 h after drug intake using liquid chromatography mass spectrometric methods. A total of 107 recruited patients had available PK data; of these 107 patients, 42.1% received biweekly isoniazid. The mean biweekly dose (12.8 mg/kg of body weight/day) was significantly lower than the nominal dose of 15 mg/kg/day (P < 0.001), and this effect was particularly marked in patients with concurrent diabetes and in males. The median maximum plasma concentration (Cmax) and area under the concentration-time curve from 0 to 6 h (AUC0-6) were 2.77 mg/liter and 9.71 mg · h/liter, respectively, for daily administration and 8.74 mg/liter and 37.8 mg · h/liter, respectively, for biweekly administration. There were no differences in the Cmax with respect to gender, diabetes mellitus (DM) status, or HIV status. Food was weakly associated with lower levels of isoniazid during the continuation phase. Overall, 34% of patients during the intensive phase and 33.3% during the continuation phase did not reach the Cmax reference value. However, low levels of INH were not associated with poorer clinical outcomes. In our population, INH exposure was affected by weight-adjusted dose and by food, but comorbidities did not indicate any effect on PK. We were unable to demonstrate a clear relationship between the Cmax and treatment outcome in this data set. Twice-weekly weight-adjusted dosing of INH appears to be quite robust with respect to important potentially influential patient factors under program conditions.

Rapid kill of malaria parasites by artemisinin and semi-synthetic endoperoxides involves ROS-dependent depolarization of the membrane potential.

OBJECTIVES: Artemisinin and artemisinin semi-synthetic derivatives (collectively known as endoperoxides) are first-line antimalarials for the treatment of uncomplicated and severe malaria. Endoperoxides display very fast killing rates and are generally recalcitrant to parasite resistance development. These key pharmacodynamic features are a result of a complex mechanism of action, the details of which lack consensus. Here, we report on the primary physiological events leading to parasite death. METHODS: Parasite mitochondrial (ΔΨm) and plasma membrane (ΔΨp) electrochemical potentials were measured using real-time single-cell imaging following exposure to pharmacologically relevant concentrations of endoperoxides (artemisinin, dihydroartemisinin, artesunate and the synthetic tetraoxane RKA182). In addition, mitochondrial electron transport chain components NADH:quinone oxidoreductase (alternative complex I), bc1 (complex III) and cytochrome oxidase (complex IV) were investigated to determine their functional sensitivity to the various endoperoxides. RESULTS: Parasite exposure to endoperoxides resulted in rapid depolarization of parasite ΔΨm and ΔΨp. The rate of depolarization was decreased in the presence of a reactive oxygen species (ROS) scavenger and Fe(3+) chelators. Depolarization of ΔΨm by endoperoxides is not believed to be through the inhibition of mitochondrial electron transport chain components, owing to the lack of significant inhibition when assayed directly. CONCLUSIONS: The depolarization of ΔΨm and ΔΨp is shown to be mediated via the generation of ROS that are initiated by iron bioactivation of endoperoxides and/or catalysed by iron-dependent oxidative stress. These data are discussed in the context of current hypotheses concerning the mode of action of endoperoxides.

Pharmacokinetics of co-formulated mefloquine and artesunate in pregnant and non-pregnant women with uncomplicated Plasmodium falciparum infection in Burkina Faso.

OBJECTIVES: Mefloquine/artesunate has recently been developed as a fixed-dose combination, providing a promising rescue/alternative treatment for malaria during pregnancy. However, limited data are available on the effect of pregnancy on its pharmacokinetic properties. This study was conducted to assess the pharmacokinetic properties of mefloquine/carboxymefloquine and artesunate/dihydroartemisinin in pregnant and non-pregnant women with uncomplicated malaria. METHODS: Twenty-four women in their second and third trimesters of pregnancy and 24 paired non-pregnant women were enrolled. All patients were treated for uncomplicated Plasmodium falciparum malaria with a standard fixed-dose combination of oral mefloquine and artesunate one daily over 3 days. Frequent blood samples were collected before treatment and at scheduled times post-dose for the drug measurements and pharmacokinetic analyses. The study was registered at www.clinicaltrials.gov (identifier: NCT00701961). RESULTS: The total median exposure to mefloquine and dihydroartemisinin was not significantly different between the pregnant and non-pregnant women (P>0.05). There was a trend of higher exposure to mefloquine in the pregnant women, but this difference did not reach statistical significance (656700 versus 542400 h × ng/mL; P=0.059). However, the total exposure to carboxymefloquine was 49% lower during pregnancy (735600 versus 1499000 h × ng/mL; P<0.001) and the total drug exposure to artesunate was 42% higher during pregnancy (89.0 versus 62.9 h × ng/mL; P=0.039) compared with non-pregnant controls. CONCLUSIONS: The plasma levels of mefloquine and dihydroartemisinin appeared to be similar in both pregnant and non-pregnant women, but there were significant differences in carboxymefloquine and artesunate exposure. The data presented here do not warrant a dose adjustment in pregnant patients, but an extensive analysis of the data could provide a better understanding of these findings.

Anti-Wolbachia drug discovery and development: safe macrofilaricides for onchocerciasis and lymphatic filariasis.

Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4-6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA 'endgame scenarios', where test and treat strategies become more cost effective and deliverable.

Metabolite footprinting of Plasmodium falciparum following exposure to Garcinia mangostana Linn. crude extract.

Multidrug resistant Plasmodium falciparum is the major health problem in the tropics. Discovery and development of new antimalarial drugs with novel modes of action is urgently required. The aim of the present study was to investigate antimalarial activities of Garcinia mangostana Linn. crude ethanolic extract including its bioactive compounds as well as the metabolic footprinting of P. falciparum following exposure to G. mangostana Linn. extract. The median (range) IC50 (concentration that inhibits parasite growth by 50%) values of ethanolic extract of G. mangostana Linn., α-mangostin, ß-mangostin, gartanin, 9-hydroxycarbaxathone, artesunate, and mefloquine for 3D7 vs K1 P. falciparum clones were 12.6 (10.5-13.2) vs 4.5 (3.5-6.3) µg/ml, 7.3 (7.1-8.5) vs 5.0 (3.7-5.9) µg/ml, 47.3 (46.8-54.0) vs 35.0 (30.0-43.7) µg/ml, 9.2 (8.1-11.9) vs 6.8 (6.2-9.1) µg/ml, 0.6 (0.4-0.8) vs 0.5 (0.4-0.7) µg/ml, 0.4 (0.2-1.2) vs 0.7 (0.4-1.0)ng/ml, and 5.0 (4.2-5.0) vs 2.7 (2.5-4.6) ng/ml, respectively. The action of G. mangostana Linn. started at 12 h of exposure, suggesting that the stage of its action is trophozoite. The 12-h exposure time was used as a suitable exposure time for further analysis of P. falciparum footprinting. G. mangostana Linn. extract was found to target several metabolic pathways particularly glucose and TCA metabolisms. The malate was not detected in culture medium of the exposed parasite, which may indirectly imply that the action of G. mangostana Linn. is through interruption of TCA metabolism.