Pharmacokinetics of artemether and dihydroartemisinin in healthy Pakistani male volunteers treated with artemether-lumefantrine

BACKGROUND: Artemether-lumefantrine is one of the most widely used anti-malarial drug combinations in the world with excellent tolerability and cure rates in adult and paediatric patients with uncomplicated falciparum malaria. The aim of this study was to evaluate the pharmacokinetics of artemether and its active metabolite, dihydroartemisinin, in healthy Pakistani volunteers. METHODS: Twelve healthy male Pakistani subjects, aged 20 to 50, were recruited into the study. A fixed oral combination of artemether-lumefantrine (80-480 mg) was given as a single oral dose. Frequent blood samples were collected and artemether and dihydroartemisinin were quantified in human plasma using solid-phase extraction and liquid chromatography coupled with tandem mass spectrometry. Drug concentration-time data were evaluated with non-compartmental analysis. RESULTS: Observed maximum concentrations (mean ± SD) of artemether and dihydroartemisinin were 184 ± 100 ng/mL and 126 ± 46 ng/mL, respectively. These concentrations were reached at 1.56 ± 0.68 hr and 1.69 ± 0.59 hr, respectively, after drug intake. The terminal elimination half-life of artemether and dihydroartemisinin were 2.00 ± 0.71 hr and 1.80 ± 0.31 hr, respectively. Apparent volume of distribution and oral clearance for artemether were estimated to 666 ± 220 L and 257 ± 140 L/hr. The same parameters were estimated to 702 ± 220 L and 269 ± 57 L/hr for dihydroartemisinin. CONCLUSIONS: The overall pharmacokinetic properties of artemether and dihydroartemisinin in healthy Pakistani subjects are comparable to healthy subjects and patients from other populations

Plasmodium falciparum drug resistance phenotype as assessed by patient antimalarial drug levels and Its association With pfmdr1 polymorphisms

Background. Multidrug-resistant Plasmodium falciparum is a major threat to global malaria control. Parasites develop resistance by gradually acquiring genetic polymorphisms that decrease drug susceptibility. The aim of this study was to investigate the extent to which parasites with different genetic characteristics are able to withstand individual drug blood concentrations. Methods. We analyzed 2 clinical trials that assessed the efficacy and effectiveness of artemether-lumefantrine. As a proof of concept, we used measured day 7 lumefantrine concentrations to estimate the concentrations at which reinfections multiplied. P. falciparum multidrug resistance gene 1 (pfmdr1) genotypes of these parasites were then correlated to drug susceptibility. Results. Reinfecting parasites with the pfmdr1 N86/184F/D1246 haplotype were able to withstand lumefantrine blood concentrations 15-fold higher than those with the 86Y/Y184/1246Y haplotype. Conclusions. By estimating drug concentrations, we were able to quantify the contribution of pfmdr1 single-nucleotide polymorphisms to reduced lumefantrine susceptibility. The method can be applied to all long-half-life antimalarial drugs, enables early detection of P. falciparum with reduced drug susceptibility in vivo, and represents a novel way for unveiling molecular markers of antimalarial drug resistance.Swedish Development Cooperation Agency-Department for Research Cooperation (SIDA-SAREC) [SWE 2004-3850, Bil-Tz 16/9875007059, SWE-2009-165]; World Health Organization MIM-TDR [[A60100] MAL IRM 06 03]; Goljes Foundation; Swedish medical research council [K2010-56X-21457-01-3]; Wellcome Trust of Great Britai

Artemisinin resistance in Plasmodium falciparum malaria.

BACKGROUND: Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance. METHODS: In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance. RESULTS: We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups. CONCLUSIONS: P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.

A high-throughput LC-MS/MS assay for piperaquine from dried blood spots: improving malaria treatment in resource-limited settings

Background: Malaria is a parasitic disease that affects many of the poorest economies, resulting in approximately 241 million clinical episodes and 627,000 deaths annually. Piperaquine, when administered with dihydroartemisinin, is an effective drug against the disease. Drug concentration measurements taken on day 7 after treatment initiation have been shown to be a good predictor of therapeutic success with piperaquine. A simple capillary blood collection technique, where blood is dried onto filter paper, is especially suitable for drug studies in remote areas or resource-limited settings or when taking samples from children, toddlers, and infants. Methods: Three 3.2 mm discs were punched out from a dried blood spot (DBS) and then extracted in a 96-well plate using solid phase extraction on a fully automated liquid handling system. The analysis was performed using LC-MS/MS with a calibration range of 3 – 1000 ng/mL. Results: The recovery rate was approximately 54–72 %, and the relative standard deviation was below 9 % for low, middle and high quality control levels. The LC-MS/MS quantification limit of 3 ng/mL is sensitive enough to detect piperaquine for up to 4–8 weeks after drug administration, which is crucial when evaluating recrudescence and drug resistance development. While different hematocrit levels can affect DBS drug measurements, the effect was minimal for piperaquine. Conclusion: A sensitive LC-MS/MS method, in combination with fully automated extraction in a 96-well plate format, was developed and validated for the quantification of piperaquine in DBS. The assay was implemented in a bioanalytical laboratory for processing large-scale clinical trial samples

A LC-MS/MS Assay for Quantification of Amodiaquine and Desethylamodiaquine in Dried Blood Spots on Filter Paper

Artesunate-amodiaquine (ARS-AQ) is a first-line antimalarial treatment recommended by the World Health Organization. AQ is the long acting partner drug in this combination, and therapeutic success is correlated with the terminal exposure to AQ. Dried blood spot (DBS) sampling for AQ is a convenient and minimally invasive technique, especially suitable for clinical studies in resource limited settings and pediatric studies. Our primary aim was to develop and validate a bioanalytical method for quantification of AQ and its active metabolite in capillary blood applied onto filter paper as a DBS sample. The separation was achieved using a reverse phase column (Zorbax SB-CN 50 × 4.6 mm, I.D. 3.5 μm) and a mobile phase consisting of acetonitrile:ammonium formate 20 mM with 0.5% formic acid (15:85, v/v). A 50 μL DBS was punctured with five 3.2 mm punches from the filter paper, and the punches collected correspond to approximately 15 μL of dried blood. The blood was then extracted using a mixture of 0.5% formic acid in water:acetonitrile (50:50, v/v), along with stable isotope-labeled internal standards (AQ-D10 and desethylamodiaquine [DAQ]-D5). Mass spectrometry was used for quantification over the range of 2.03-459 ng/mL for AQ and 3.13-1570 ng/mL for DAQ. The validation of the method was carried out in compliance with regulatory requirements. The intra- and interbatch precisions were below 15% and passed all validation acceptance criteria. No carryover and no matrix effects were detected. Normalized matrix factors (analyte/internal standard) ranged from 0.96 to 1.03 for all analytes, hence no matrix effects. AQ and DAQ were stable in all conditions evaluated. Long-term stability in DBS samples was demonstrated for up to 10 years when stored at -80°C and for 15 months when stored at room temperature. The developed method was demonstrated to be reliable and accurate. This assay may be particularly useful in the context of resource limited settings and in pediatric field studies

Dose‐Optimization of a Novel Co‐Formulated Triple Combination Antimalarial Therapy: Artemether‐Lumefantrine‐Amodiaquine

Artemisinin‐based combination therapy (ACT) is the first‐line therapy for uncomplicated falciparum malaria, but artemisinin resistance in Asia and now sub‐Saharan Africa is threatening our ability to control and eliminate malaria. Triple‐ACTs have emerged as a viable alternative treatment to combat declining ACT efficacy due to drug‐resistant malaria. In this study, we developed and evaluated an optimal fixed‐dose regimen of artemether‐lumefantrine‐amodiaquine through population pharmacokinetic modeling and simulation. Three published population‐based pharmacometric models and two large cohorts of observed adult subjects and pediatric malaria patients were used to simulate pharmacokinetic profiles of different dosing strategies. Based on simulated total exposure and peak concentrations, an optimal dose regimen was developed resulting in an extension of the current 4 weight bands to a total of 5 weight bands to generate equivalent exposures in all body weight groups and minimize the fluctuation in exposure between patients. The proposed drug‐to‐drug ratio of artemether‐lumefantrine‐amodiaquine (20:120:40 mg) was kept constant throughout the dosing bands in order to simplify manufacturing, implementation, and further development of a fixed‐dose co‐formulated product

Use of population pharmacokinetic‐pharmacodynamic modelling to inform antimalarial dose optimization in infants

Infants bear a significant malaria burden but are usually excluded from participating in early dose optimization studies that inform dosing regimens of antimalarial therapy. Unlike older children, infants' exclusion from early‐phase trials has resulted in limited evidence to guide accurate dosing of antimalarial treatment for uncomplicated malaria or malaria‐preventive treatment in this vulnerable population. Subsequently, doses used in infants are often extrapolated from older children or adults, with the potential for under‐ or overdosing. Population pharmacokinetic‐pharmacodynamic (PK‐PD) modelling, a quantitative methodology that applies mathematical and statistical techniques, can aid the design of clinical studies in infants that collect sparse pharmacokinetic data as well as support the analysis of such data to derive optimized antimalarial dosing in this complex and at‐risk yet understudied subpopulation. In this review, we reflect on what PK‐PD modelling can do in programmatic settings of most malaria‐endemic areas and how it can be used to inform antimalarial dose optimization for preventive and curative treatment of uncomplicated malaria in infants. We outline key developmental physiological changes that affect drug exposure in early life, the challenges of conducting dose optimization studies in infants, and examples of how PK‐PD modelling has previously informed antimalarial dose optimization in this subgroup. Additionally, we discuss the limitations and gaps of PK‐PD modelling when used for dose optimization in infants. To utilize modelling well, there is a need to generate useful, sparse, PK and PD data in this subpopulation to inform antimalarial optimal dosing in infancy

Evaluation of in vitro drug-drug interactions of ivermectin and antimalarial compounds

Background: Ivermectin is lethal to Anopheles mosquitoes and a novel approach to malaria transmission control. Ivermectin could be co-administered with antimalarial drugs in mass drug administration, seasonal malaria chemoprevention, or other chemoprevention approaches. Co-administration with antimalarial drugs may impact ivermectin metabolism and/or absorption, resulting in increased or decreased exposure to ivermectin. Methods: To evaluate potential CYP-mediated drug-drug interactions (DDIs), ivermectin (1 µM) was incubated with pooled human liver microsomes, with and without the most commonly used antimalarial drugs at concentrations approximating twofold to tenfold the peak concentrations achieved following standard treatment. The antimalarial drugs investigated were dihydroartemisinin, piperaquine, chloroquine, artesunate, pyronaridine, mefloquine, artemether, lumefantrine, primaquine, atovaquone, proguanil, tafenoquine, sulfadoxine, pyrimethamine, and amodiaquine. Samples (50 µL) were collected at 0, 15, 30, 45, 60, 90, 120, and 150 min of incubation and ivermectin concentrations were measured using liquid chromatography-mass spectrometry. The metabolism rate of ivermectin was evaluated based on the normalized peak area (%) of ivermectin over a total of 150 min of incubation, applying linear regression to derive the rate of metabolism. Antimalarial compounds resulting in notable impact on the rate of ivermectin metabolism with a relative difference ≥ 50% and ≥ 25% were considered to have a substantial and partial effect on the in vitro metabolism of ivermectin, respectively. Results: Compounds that had a substantial DDI effect on the in vitro metabolism of ivermectin included piperaquine (98%), mefloquine (91%), chloroquine (76%), proguanil (60%), and lumefantrine (51%). Compounds that a partial DDI effect on the in vitro metabolism of ivermectin included atovaquone (48%), artesunate (27%), and pyronaridine (25%). All other antimalarials evaluated showed an in vitro interaction of 8–23%. Conclusions: Several of the commonly used antimalarial drugs, are mostly or in part metabolized by CYP3A4 and showed a notable DDI effect on the in vitro metabolism of ivermectin. This could potentially lead to clinically important pharmacokinetic and pharmacodynamic DDIs if co-administered, and needs to be evaluated in prospective clinical trials

A physiologically-based pharmacokinetic framework for prediction of drug exposure in malnourished children

Malnutrition in children is a global health problem, particularly in developing countries. The effects of an insufficient supply of nutrients on body composition and physiological functions may have implications for drug disposition and ultimately affect the clinical outcome in this vulnerable population. Physiologically-based pharmacokinetic (PBPK) modeling can be used to predict the effect of malnutrition as it links physiological changes to pharmacokinetic (PK) consequences. However, the absence of detailed information on body composition and the limited availability of controlled clinical trials in malnourished children complicates the establishment and evaluation of a generic PBPK model in this population. In this manuscript we describe the creation of physiologically-based bridge to a malnourished pediatric population, by combining information on (a) the differences in body composition between healthy and malnourished adults and (b) the differences in physiology between healthy adults and children. Model performance was confirmed using clinical reference data. This study presents a physiologically-based translational framework for prediction of drug disposition in malnourished children. The model is readily applicable for dose recommendation strategies to address the urgent medicinal needs of this vulnerable population