A Quantitative Method for the Study of HIV-1 and Mycobacterium tuberculosis Coinfection.

Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) syndemic interactions are a major global health concern. Despite the clinical significance of coinfection, our understanding of the cellular pathophysiology and the therapeutic pharmacodynamic impact of coinfection is limited. Here, we use single-round infectious HIV-1 pseudotyped viral particles expressing green fluorescent protein alongside M. tuberculosis expressing mCherry to study pathogenesis and treatment. We report that HIV-1 infection inhibited intracellular replication of M. tuberculosis and demonstrate the therapeutic activity of antiviral treatment (efavirenz) and antimicrobial treatment (rifampicin). The described method could be applied for detailed mechanistic studies to inform the development of novel treatment strategies.

Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic and urgent treatment and prevention strategies are needed. Nitazoxanide, an anthelmintic drug, has been shown to exhibit in vitro activity against SARS-CoV-2. The present study used physiologically based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported SARS-CoV-2 EC90 . METHODS: A whole-body PBPK model was validated against available pharmacokinetic data for healthy individuals receiving single and multiple doses between 500 and 4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the EC90 in >90% of the simulated population. PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. RESULTS: The PBPK model was successfully validated against the reported human pharmacokinetics. The model predicted optimal doses of 1200 mg QID, 1600 mg TID and 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12 hours post dose was estimated. CONCLUSION: The PBPK model predicted tizoxanide concentrations within doses of nitazoxanide already given to humans previously. The reported dosing strategies provide a rational basis for design of clinical trials with nitazoxanide for the treatment or prevention of SARS-CoV-2 infection. A concordant higher dose of nitazoxanide is now planned for investigation in the seamless phase I/IIa AGILE trial.

Population Pharmacokinetic Properties of Sulfadoxine and Pyrimethamine: a Pooled Analysis To Inform Optimal Dosing in African Children with Uncomplicated Malaria.

Sulfadoxine-pyrimethamine with amodiaquine is recommended by the World Health Organization as seasonal malaria chemoprevention for children aged 3 to 59 months in the sub-Sahel regions of Africa. Suboptimal dosing in children may lead to treatment failure and increased resistance. Pooled individual patient data from four previously published trials on the pharmacokinetics of sulfadoxine and pyrimethamine in 415 pediatric and 386 adult patients were analyzed using nonlinear mixed-effects modeling to evaluate the current dosing regimen and, if needed, to propose an optimized dosing regimen for children under 5 years of age. The population pharmacokinetics of sulfadoxine and pyrimethamine were both best described by a one-compartment disposition model with first-order absorption and elimination. Body weight, age, and nutritional status (measured as the weight-for-age Z-score) were found to be significant covariates. Allometric scaling with total body weight and the maturation of clearance in children by postgestational age improved the model fit. Underweight-for-age children were found to have 15.3% and 26.7% lower bioavailabilities of sulfadoxine and pyrimethamine, respectively, for each Z-score unit below -2. Under current dosing recommendations, simulation predicted that the median day 7 concentration was below the 25th percentile for a typical adult patient (50 kg) for sulfadoxine for patients in the weight bands of 8 to 9, 19 to 24, 46 to 49, and 74 to 79 kg and for pyrimethamine for patients in the weight bands of 8 to 9, 14 to 24, and 42 to 49 kg. An evidence-based dosing regimen was constructed that would achieve sulfadoxine and pyrimethamine exposures in young children and underweight-for-age young children that were similar to those currently seen in a typical adult.

Genetic Determinants of the Pharmacokinetic Variability of Rifampin in Malawian Adults with Pulmonary Tuberculosis.

Variable exposure to antituberculosis (TB) drugs, partially driven by genetic factors, may be associated with poor clinical outcomes. Previous studies have suggested an influence of the SLCO1B1 locus on the plasma area under the concentration-time curve (AUC) of rifampin. We evaluated the contribution of single nucleotide polymorphisms (SNPs) in SLCO1B1 and other candidate genes (AADAC and CES-1) to interindividual pharmacokinetic variability in Malawi. A total of 174 adults with pulmonary TB underwent sampling of plasma rifampin concentrations at 2 and 6 h postdose. Data from a prior cohort of 47 intensively sampled, similar patients from the same setting were available to support population pharmacokinetic model development in NONMEM v7.2, using a two-stage strategy to improve information during the absorption phase. In contrast to recent studies in South Africa and Uganda, SNPs in SLCO1B1 did not explain variability in AUC0-∞ of rifampin. No pharmacokinetic associations were identified with AADAC or CES-1 SNPs, which were rare in the Malawian population. Pharmacogenetic determinants of rifampin exposure may vary between African populations. SLCO1B1 and other novel candidate genes, as well as nongenetic sources of interindividual variability, should be further explored in geographically diverse, adequately powered cohorts.

A simultaneous population pharmacokinetic analysis of rifampicin in Malawian adults and children.

AIMS: Low rifampicin plasma concentrations can lead to treatment failure and increased risk of developing drug resistant tuberculosis. The objectives of this study were to characterize the population pharmacokinetics (popPK) of rifampicin in Malawian children and adults with tuberculosis, simulate exposures under revised WHO dosing guidelines that aim to reduce the risk of low exposures of rifampicin and examine predicted exposures using weight- and age-based dosing bands under new dosing recommendations. METHODS: Patients were recruited at least two weeks after initiation of the intensive phase of treatment and received RIF in FDC of anti-TB drugs. A total of 5-6 rich and 1-2 sparse samples were collected. nonmem (v7.2) was used to build a population-PK model. RESULTS: A 165 TB patients, 115 adults and 50 children, aged 7 months to 65 years and weighing 4.8 to 87 kg, were included in the one compartment model with first order absorption best described the data. The mean population estimate for CL/F was 23.9 (l h(-1)  70 kg(-1) ) with inter-individual variability of 46.6%. Exposure was unaffected by HIV status. Relative bioavailability in children was estimated at 49% lower compared to adults (100% relative bioavailability). Simulations showed significantly lower rifampicin exposure in children vs. adults. In children average AUC was 13.5 mg l(-1) h, which was nearly half that was observed in adults (26.3 mg l(-1)  h). Using age as a surrogate for weight in dosing bands gave similar results compared with the weight bands. Increasing dose to approximately 15 mg kg(-1) , increased AUC in children to an average of 22 mgl(-1)  h. bringing expected exposures in children closer to those predicted for adults. CONCLUSION: The popPK model developed can be used to optimize rifampicin exposures through dosing simulations. WHO dosing recommendations may not be achieved using currently licensed fixed dose combination formulations of TB therapy.

CRIMALDDI: a prioritized research agenda to expedite the discovery of new anti-malarial drugs.

The CRIMALDDI Consortium has been a three-year project funded by the EU Framework Seven Programme. It aimed to develop a prioritized set of recommendations to speed up anti-malarial drug discovery research and contribute to the setting of the global research agenda. It has attempted to align thinking on the high priority issues and then to develop action plans and strategies to address these issues. Through a series of facilitated and interactive workshops, it has concluded that these priorities can be grouped under five key themes: attacking artemisinin resistance; creating and sharing community resources; delivering enabling technologies; exploiting high throughput screening hits quickly; and, identifying novel targets. Recommendations have been prioritized into one of four levels: quick wins; removing key roadblocks to future progress; speeding-up drug discovery; and, nice to have (but not essential). Use of this prioritization allows efforts and resources to be focused on the lines of work that will contribute most to expediting anti-malarial drug discovery. Estimates of the time and finances required to implement the recommendations have also been made, along with indications of when recommendations within each theme will make an impact. All of this has been collected into an indicative roadmap that, it is hoped, will guide decisions about the direction and focus of European anti-malarial drug discovery research and contribute to the setting of the global research agenda.

CRIMALDDI: platform technologies and novel anti-malarial drug targets.

The Coordination, Rationalization, and Integration of antiMALarial drug Discovery & Development Initiatives (CRIMALDDI) Consortium, funded by the EU Framework Seven Programme, has attempted, through a series of interactive and facilitated workshops, to develop priorities for research to expedite the discovery of new anti-malarials. This paper outlines the recommendations for the development of enabling technologies and the identification of novel targets.Screening systems must be robust, validated, reproducible, and represent human malaria. They also need to be cost-effective. While such systems exist to screen for activity against blood stage Plasmodium falciparum, they are lacking for other Plasmodium spp. and other stages of the parasite's life cycle. Priority needs to be given to developing high-throughput screens that can identify activity against the liver and sexual stages. This in turn requires other enabling technologies to be developed to allow the study of these stages and to allow for the culture of liver cells and the parasite at all stages of its life cycle.As these enabling technologies become available, they will allow novel drug targets to be studied. Currently anti-malarials are mostly targeting the asexual blood stage of the parasite's life cycle. There are many other attractive targets that need to be investigated. The liver stages and the sexual stages will become more important as malaria control moves towards malaria elimination. Sexual development is a process offering multiple targets, even though the mechanisms of differentiation are still not fully understood. However, designing a drug whose effect is not curative but would be used in asymptomatic patients is difficult given current safety thresholds. Compounds active against the liver schizont would have a prophylactic effect and Plasmodium vivax elimination requires effectors against the dormant liver hypnozoites. It may be that drugs to be used in elimination campaigns will also need to have utility in the control phase. Compounds with activity against blood stages need to be screened for activity against other stages.Natural products should also be a valuable source of new compounds. They often occupy non-Lipinski chemical space and so may reveal valuable new chemotypes.

A yeast expression system for functional and pharmacological studies of the malaria parasite Ca²âº/H⁺ antiporter.

BACKGROUND: Calcium (Ca²âº) signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca²âº is tightly regulated through the co-ordinated action of primary and secondary Ca²âº transporters. Identifying selective inhibitors of Ca²âº transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented. METHODS: The Plasmodium falciparum Ca²âº/H⁺ antiporter (PfCHA) was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese. RESULTS: Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca²âº, Mg²âº and Mn²âº in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca²âº transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca²âº-loading assays for further functional and pharmacological studies. DISCUSSION: The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent cation/H⁺ antiporter susceptible to the effects of cation/H⁺ inhibitors such as KB-R7943. This type of gene expression systems should advance the efforts for the screening of potential inhibitors of this type of divalent cation transporters as part of the malaria drug discovery initiatives and for functional studies in general. CONCLUSION: The expression and activity of the PfCHA detected in yeast by a bioluminescence assay that follows the levels of cytoplasmic Ca²âº as well as Mg²âº and Mn²âº lend itself to high-throughput and quantitative settings for pharmacological screening and functional studies.

CRIMALDDI: a co-ordinated, rational, and integrated effort to set logical priorities in anti-malarial drug discovery initiatives.

Despite increasing efforts and support for anti-malarial drug R&D, globally anti-malarial drug discovery and development remains largely uncoordinated and fragmented. The current window of opportunity for large scale funding of R&D into malaria is likely to narrow in the coming decade due to a contraction in available resources caused by the current economic difficulties and new priorities (e.g. climate change). It is, therefore, essential that stakeholders are given well-articulated action plans and priorities to guide judgments on where resources can be best targeted.The CRIMALDDI Consortium (a European Union funded initiative) has been set up to develop, through a process of stakeholder and expert consultations, such priorities and recommendations to address them. It is hoped that the recommendations will help to guide the priorities of the European anti-malarial research as well as the wider global discovery agenda in the coming decade.

Novel anti-Wolbachia drugs, a new approach in the treatment and prevention of veterinary filariasis?

Filarial nematodes are tissue-dwelling parasitic worms that can cause a range of disfiguring pathologies in humans and potentially lethal infections of companion animals. The bacterial endosymbiont, Wolbachia, is present within most human and veterinary filarial pathogens, including the causative agent of heartworm disease, Dirofilaria immitis. Doxycycline-mediated drug targeting of Wolbachia leads to sterility, clearance of microfilariae and gradual death of adult filariae. This mode of action is attractive in the treatment of filariasis because it avoids severe host inflammatory adverse reactions invoked by rapid-killing anthelmintic agents. However, doxycycline needs to be taken for four weeks to exert curative activity. In this review, we discuss the evidence that Wolbachia drug targeting is efficacious in blocking filarial larval development as well as in the treatment of chronic filarial disease. We present the current portfolio of next-generation anti-Wolbachia candidates discovered through phenotypic screening of chemical libraries and validated in a range of in vitro and in vivo filarial infection models. Several novel chemotypes have been identified with selected narrow-spectrum anti-Wolbachia specificity and superior time-to-kill kinetics compared with doxycycline. We discuss the opportunities of developing these novel anti-Wolbachia agents as either cures, adjunct therapies or new preventatives for the treatment of veterinary filariasis.

Measurement of adherence, drug concentrations and the effectiveness of artemether-lumefantrine, chlorproguanil-dapsone or sulphadoxine-pyrimethamine in the treatment of uncomplicated malaria in Malawi.

BACKGROUND: Sulphadoxine-pyrimethamine (SP) is the only single dose therapy for uncomplicated malaria, but there is widespread resistance. At the time of this study, artemether-lumefantrine (AL) and chlorproguanil-dapsone (CPD), both multi-dose regimes, were considered possible alternatives to SP in Malawi. The aim of this study was to investigate the impact of poor adherence on the effectiveness of AL and CPD. METHODS: Children > or =12 months and adults with uncomplicated malaria were randomized to receive AL, CPD or SP. Adherence was measured using a questionnaire and electronic monitoring devices, MEMS, pill bottles that recorded the date and time of opening. Day-7 plasma dapsone or lumefantrine concentrations were measured to examine their relationship with adherence and clinical response. RESULTS: 841 patients were recruited. The day-28 adequate clinical and parasitological response (ACPR) rates, using intention to treat analysis (missing data treated as failure), were AL 85.2%, CPD 63.7% and SP 50%. ACPR rates for AL were higher than CPD or SP on days 28 and 42 (p < or = 0.002 for all comparisons). CPD was more effective than SP on day-28 (p = 0.01), but not day-42.Very high adherence was reported using the questionnaire, 100% for AL treated patients and 99.2% for the CPD group. Only three CPD participants admitted missing any doses. 164/181 (90.6%) of CPD treated patients took all their doses out of the MEMS container and they were more likely to have a day-28 ACPR than those who did not take all their medication out of the container, p = 0.024. Only 7/87 (8%) AL treated patients did not take all of their doses out of their MEMS container and none had treatment failure.Median day-7 dapsone concentrations were higher in CPD treated patients with ACPR than in treatment failures, p = 0.012. There were no differences in day-7 dapsone or lumefantrine concentrations between those who took all their doses from the MEMS container and those who did not. A day-7 lumefantrine concentration reported to be predictive of AL treatment failure in Thailand was not useful in this population; only one of 16 participants with a concentration below this threshold (175 ng/ml) had treatment failure. CONCLUSION: This study provides reassurance of the effectiveness of AL, even with unsupervised dosing, as it is rolled out across sub-Saharan Africa. Self-reported adherence appears to be an unreliable measure of adherence in this population.

Pharmacokinetics and clinical efficacy of midazolam in children with severe malaria and convulsions.

AIM: To investigate the pharmacokinetics and clinical efficacy of intravenous (IV), intramuscular (IM) and buccal midazolam (MDZ) in children with severe falciparum malaria and convulsions. METHODS: Thirty-three children with severe malaria and convulsions lasting > or =5 min were given a single dose of MDZ (0.3 mg kg(-1)) IV (n = 13), IM (n = 12) or via the buccal route (n = 8). Blood samples were collected over 6 h post-dose for determination of plasma MDZ and 1'-hydroxymidazolam concentrations. Plasma concentration-time data were fitted using pharmacokinetic models. RESULTS: Median (range) MDZ C(max) of 481 (258-616), 253 (96-696) and 186 (64-394) ng ml(-1) were attained within a median (range) t(max) of 10 (5-15), 15 (5-60) and 10 (5-40) min, following IV, IM and buccal administration, respectively. Mean (95% confidence interval) of the pharmacokinetic parameters were: AUC(0,infinity) 596 (327, 865), 608 (353, 864) and 518 (294, 741) ng ml(-1) h; V(d) 0.85 l kg(-1); clearance 14.4 ml min(-1) kg(-1), elimination half-life 1.22 (0.65, 1.8) h, respectively. A single dose of MDZ terminated convulsions in all (100%), 9/12 (75%) and 5/8 (63%) children following IV, IM and buccal administration. Four children (one in the IV, one in the IM and two in the buccal groups) had respiratory depression. CONCLUSIONS: Administration of MDZ at the currently recommended dose resulted in rapid achievement of therapeutic MDZ concentrations. Although IM and buccal administration of MDZ may be more practical in peripheral healthcare facilities, the efficacy appears to be poorer at the dose used, and a different dosage regimen might improve the efficacy.

Safety and mosquitocidal efficacy of high-dose ivermectin when co-administered with dihydroartemisinin-piperaquine in Kenyan adults with uncomplicated malaria (IVERMAL): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Ivermectin is being considered for mass drug administration for malaria due to its ability to kill mosquitoes feeding on recently treated individuals. However, standard, single doses of 150-200 µg/kg used for onchocerciasis and lymphatic filariasis have a short-lived mosquitocidal effect (<7 days). Because ivermectin is well tolerated up to 2000 µg/kg, we aimed to establish the safety, tolerability, and mosquitocidal efficacy of 3 day courses of high-dose ivermectin, co-administered with a standard malaria treatment. METHODS: We did a randomised, double-blind, placebo-controlled, superiority trial at the Jaramogi Oginga Odinga Teaching and Referral Hospital (Kisumu, Kenya). Adults (aged 18-50 years) were eligible if they had confirmed symptomatic uncomplicated Plasmodium falciparum malaria and agreed to the follow-up schedule. Participants were randomly assigned (1:1:1) using sealed envelopes, stratified by sex and body-mass index (men: <21 vs ≥21 kg/m2; women: <23 vs ≥23 kg/m2), with permuted blocks of three, to receive 3 days of ivermectin 300 µg/kg per day, ivermectin 600 µg/kg per day, or placebo, all co-administered with 3 days of dihydroartemisinin-piperaquine. Blood of patients taken on post-treatment days 0, 2 + 4 h, 7, 10, 14, 21, and 28 was fed to laboratory-reared Anopheles gambiae sensu stricto mosquitoes, and mosquito survival was assessed daily for 28 days after feeding. The primary outcome was 14-day cumulative mortality of mosquitoes fed 7 days after ivermectin treatment (from participants who received at least one dose of study medication). The study is registered with ClinicalTrials.gov, number NCT02511353. FINDINGS: Between July 20, 2015, and May 7, 2016, 741 adults with malaria were assessed for eligibility, of whom 141 were randomly assigned to receive ivermectin 600 µg/kg per day (n=47), ivermectin 300 µg/kg per day (n=48), or placebo (n=46). 128 patients (91%) attended the primary outcome visit 7 days post treatment. Compared with placebo, ivermectin was associated with higher 14 day post-feeding mosquito mortality when fed on blood taken 7 days post treatment (ivermectin 600 µg/kg per day risk ratio [RR] 2·26, 95% CI 1·93-2·65, p<0·0001; hazard ratio [HR] 6·32, 4·61-8·67, p<0·0001; ivermectin 300 µg/kg per day RR 2·18, 1·86-2·57, p<0·0001; HR 4·21, 3·06-5·79, p<0·0001). Mosquito mortality remained significantly increased 28 days post treatment (ivermectin 600 µg/kg per day RR 1·23, 1·01-1·50, p=0·0374; and ivermectin 300 µg/kg per day 1·21, 1·01-1·44, p=0·0337). Five (11%) of 45 patients receiving ivermectin 600 µg/kg per day, two (4%) of 48 patients receiving ivermectin 300 µg/kg per day, and none of 46 patients receiving placebo had one or more treatment-related adverse events. INTERPRETATION: Ivermectin at both doses assessed was well tolerated and reduced mosquito survival for at least 28 days after treatment. Ivermectin 300 µg/kg per day for 3 days provided a good balance between efficacy and tolerability, and this drug shows promise as a potential new tool for malaria elimination. FUNDING: Malaria Eradication Scientific Alliance (MESA) and US Centers for Disease Control and Prevention (CDC).

Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy.

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.

Comparative folate metabolism in humans and malaria parasites (part II): activities as yet untargeted or specific to Plasmodium.

The folate pathway represents a powerful target for combating rapidly dividing systems such as cancer cells, bacteria and malaria parasites. Whereas folate metabolism in mammalian cells and bacteria has been studied extensively, it is understood less well in malaria parasites. In two articles, we attempt to reconstitute the malaria folate pathway based on available information from mammalian and microbial systems, in addition to Plasmodium-genome-sequencing projects. In part I, we focused on folate enzymes that are already used clinically as anticancer drug targets or that are under development in drug-discovery programs. In this article, we discuss mammalian folate enzymes that have not yet been exploited as potential drug targets, and enzymes that function in the de novo folate-synthesis pathway of the parasite--a particularly attractive area of attack because of its absence from the mammalian host.

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children.

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).

Reflections on the Nobel Prize for Medicine 2015--The Public Health Legacy and Impact of Avermectin and Artemisinin.

The award of the Nobel Prize to Dr Bill Campbell and Professor Satoshi Omura for their role in the discovery of avermectin and Professor Youyou Tu for her work on the development of artemisinin has been universally welcomed by the International Health community for what the Nobel Committee described as 'The discoveries of Avermectin and Artemisinin have revolutionized therapy for patients suffering from devastating parasitic diseases. Campbell, Omura and Tu have transformed the treatment of parasitic diseases. The global impact of their discoveries and the resulting benefit to mankind are immeasurable'.

Therapeutic potential of folate uptake inhibition in Plasmodium falciparum.

Plasmodium falciparum parasites resistant to the combination sulfadoxine-pyrimethamine are spreading in Africa, particularly in East Africa. This is a matter of concern because there are no other affordable drugs available. This article provides the evidence indicating that sulfadoxine-pyrimethamine resistance can be reversed in vitro and discusses how this information might be exploited to extend the therapeutic lifetime of sulfadoxine-pyrimethamine in vivo.

The folate metabolic network of Falciparum malaria.

The targeting of key enzymes in the folate pathway continues to be an effective chemotherapeutic approach that has earned antifolate drugs a valuable position in the medical pharmacopoeia. The successful therapeutic use of antifolates as antimalarials has been a catalyst for ongoing research into the biochemistry of folate and pterin biosynthesis in malaria parasites. However, our understanding of the parasites folate metabolism remains partial and patchy, especially in relation to the shikimate pathway, the folate cycle, and folate salvage. A sizeable number of potential folate targets remain to be characterised. Recent reports on the parasite specific transport of folate precursors that would normally be present in the human host awaken previous hypotheses on the salvage of folate precursors or by-products. As the parasite progresses through its life-cycle it encounters very contrasting host cell environments that present radically different metabolic milieus and biochemical challenges. It would seem probable that as the parasite encounters differing environments it would need to modify its biochemistry. This would be reflected in the folate homeostasis in Plasmodium. Recent drug screening efforts and insights into folate membrane transport substantiate the argument that folate metabolism may still offer unexplored opportunities for therapeutic attack.

Sulfadoxine-pyrimethamine-based combinations for malaria: a randomised blinded trial to compare efficacy, safety and selection of resistance in Malawi.

BACKGROUND: In Malawi, there has been a return of Plasmodium falciparum sensitivity to chloroquine (CQ) since sulfadoxine-pyrimethamine (SP) replaced CQ as first line treatment for uncomplicated malaria. When used for prophylaxis, Amodiaquine (AQ) was associated with agranulocytosis but is considered safe for treatment and is increasingly being used in Africa. Here we compare the efficacy, safety and selection of resistance using SP or CQ+SP or artesunate (ART)+SP or AQ+SP for the treatment of uncomplicated falciparum malaria. METHODOLOGY AND FINDINGS: 455 children aged 1-5 years were recruited into a double-blinded randomised trial comparing SP to the three combination therapies. Using intention to treat analysis with missing outcomes treated as successes, and without adjustment to distinguish recrudescence from new infections, the day 28 adequate clinical and parasitological response (ACPR) rate for SP was 25%, inferior to each of the three combination therapies (p<0.001). AQ+SP had an ACPR rate of 97%, higher than CQ+SP (81%) and ART+SP (70%), p<0.001. Nineteen children developed a neutropenia of