HIV-positive nigerian adults harbor significantly higher serum lumefantrine levels than HIV-negative individuals seven days after treatment for Plasmodium falciparum infection.

Management of coinfection with malaria and HIV is a major challenge to public health in developing countries, and yet potential drug-drug interactions between antimalarial and antiviral regimens have not been adequately investigated in people with both infections. Each of the constituent components of artemether-lumefantrine, the first-line regimen for malaria treatment in Nigeria, and nevirapine, a major component of highly active antiretroviral therapy, are drugs metabolized by the cytochrome P450 3A4 isoenzyme system, which is also known to be induced by nevirapine. We examined potential interactions between lumefantrine and nevirapine in 68 HIV-positive adults, all of whom were diagnosed with asymptomatic Plasmodium falciparum infections by microscopy. Post hoc PCR analysis confirmed the presence of P. falciparum in only a minority of participants. Day 7 capillary blood levels of lumefantrine were significantly higher in HIV-positive participants than in 99 HIV-negative controls (P = 0.0011). Associations between day 7 levels of lumefantrine and risk of persistent parasitemia could not be evaluated due to inadequate power. Further investigations of the impact of nevirapine on in vivo malaria treatment outcomes in HIV-infected patients are thus needed

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

High sensitivity methods to quantify chloroquine and its metabolite in human blood samples using LC-MS/MS

AIM: Chloroquine is an antimalarial drug used in the treatment of Plasmodium vivax malaria. Three methods to quantify chloroquine and its metabolite in blood matrices were developed and validated. METHODOLOGY AND RESULTS: Different high-throughput extraction techniques were used to recover the drugs from whole blood (50 μl), plasma (100 μl) and dried blood spots (15 μl as punched discs) followed by quantification with LC-MS/MS. The intra- and inter-batch precisions were below 15%, and thus meet regulatory acceptance criteria. CONCLUSION: The developed methods demonstrated satisfactory validation performance with high sensitivity and selectivity. The assays used simple and easy to automate extraction techniques. All methods were reliable with robust performance and demonstrated to be suitable to implement into high-throughput routine analysis of clinical pharmacokinetic samples

Enantiospecific pharmacokinetics and drug-drug interactions of primaquine and blood-stage antimalarial drugs

Objectives Characterization of the pharmacokinetic properties of the enantiomers of primaquine and carboxyprimaquine following administration of racemic primaquine given alone and in combination with commonly used antimalarial drugs. Methods Enantiomeric pharmacokinetics were evaluated in 49 healthy adult volunteers enrolled in three randomized cross-over studies in which a single dose of primaquine was given alone and then, after a suitable washout period, in combination with chloroquine, dihydroartemisinin/piperaquine or pyronaridine/artesunate. Non-linear mixed-effects modelling was used to characterize pharmacokinetics and assess the impact of drug–drug interactions. Results The volume of distribution of racemic primaquine was decreased by a median (95% CI) of 22.0% (2.24%–39.9%), 24.0% (15.0%–31.5%) and 25.7% (20.3%–31.1%) when co-administered with chloroquine, dihydroartemisinin/piperaquine and pyronaridine/artesunate, respectively. The oral clearance of primaquine was decreased by a median of 19.1% (14.5%–22.8%) when co-administered with pyronaridine/artesunate. These interactions were enantiospecific with a relatively higher effect on (+)-S-primaquine than on (−)-R-primaquine. No drug–drug interaction effects were seen on the pharmacokinetics of either carboxyprimaquine enantiomer. Conclusions Population pharmacokinetic models characterizing the enantiospecific properties of primaquine were developed successfully. Exposure to primaquine, particularly to the (+)-S-primaquine but not the carboxy metabolites, increased by up to 30% when co-administered with commonly used antimalarial drugs. A better mechanistic understanding of primaquine metabolism is required for assessment of its efficacy and haematological toxicity in humans

Population pharmacokinetics and pharmacodynamics of lumefantrine in young Ugandan children treated with artemether-lumefantrine for uncomplicated malaria

The pharmacokinetics and pharmacodynamics of lumefantrine, a component of the most widely used treatment for malaria, artemether-lumefantrine, has not been adequately characterized in young children.Capillary whole-blood lumefantrine concentration and treatment outcomes were determined in 105 Ugandan children, ages 6 months to 2 years, who were treated for 249 episodes of Plasmodium falciparum malaria with artemether-lumefantrine.Population pharmacokinetics for lumefantrine used a 2-compartment open model with first-order absorption. Age had a significant positive correlation with bioavailability in a model that included allometric scaling. Children not receiving trimethoprim-sulfamethoxazole with capillary whole blood concentrations 200 ng/mL (P = .0007). However, for children receiving trimethoprim-sulfamethoxazole, the risk of recurrent parasitemia did not differ significantly on the basis of this threshold. Day 3 concentrations were a stronger predictor of 28-day recurrence than day 7 concentrations.We demonstrate that age, in addition to weight, is a determinant of lumefantrine exposure, and in the absence of trimethoprim-sulfamethoxazole, lumefantrine exposure is a determinant of recurrent parasitemia. Exposure levels in children aged 6 months to 2 years was generally lower than levels published for older children and adults. Further refinement of artemether-lumefantrine dosing to improve exposure in infants and very young children may be warranted

Self-reported data: a major tool to assess compliance with anti-malarial combination therapy among children in Senegal

Background: Although there are many methods available for measuring compliance, there is no formal gold standard. Different techniques used to measure compliance were compared among children treated by the anti-malarial amodiaquine/sulphadoxine-pyrimethamine (AQ/SP) combination therapy, in use in Senegal between 2004 and 2006. Methods: The study was carried out in 2004, in five health centres located in the Thies region (Senegal). Children who had AQ/SP prescribed for three and one day respectively at the health centre were recruited. The day following the theoretical last intake of AQ, venous blood, and urine samples were collected for anti-malarial drugs dosage. Caregivers and children above five years were interviewed concerning children's drug intake. Results: Among the children, 64.7% adhered to 80% of the prescribed dose and only 37.7% were strict full adherent to the prescription. There was 72.7% agreement between self-reported data and blood drug dosage for amodiaquine treatment. Concerning SP, results found that blood dosages were 91.4% concordant with urine tests and 90% with self-reported data based on questionnaires. Conclusion: Self-reported data could provide useful quantitative information on drug intake and administration. Under strict methodological conditions this method, easy to implement, can be used to describe patients' behaviors and their use of new anti-malarial treatment. Self-reported data is a major tool for assessing compliance in resource poor countries. Blood and urine drug dosages provide qualitative results that confirm any drug intake. Urine assays for SP could be useful to obtain public health data, for example on chemoprophylaxis among pregnant women

A high performance liquid chromatographic assay of Mefloquine in saliva after a single oral dose in healthy adult Africans

<p>Abstract</p> <p>Background</p> <p>Mefloquine-artesunate is a formulation of artemisinin based combination therapy (ACT) recommended by the World Health Organization and historically the first ACT used clinically. The use of ACT demands constant monitoring of therapeutic efficacies and drug levels, in order to ensure that optimum drug exposure is achieved and detect reduced susceptibility to these drugs. Quantification of anti-malarial drugs in biological fluids other than blood would provide a more readily applicable method of therapeutic drug monitoring in developing endemic countries. Efforts in this study were devoted to the development of a simple, field applicable, non-invasive method for assay of mefloquine in saliva.</p> <p>Methods</p> <p>A high performance liquid chromatographic method with UV detection at 220 nm for assaying mefloquine in saliva was developed and validated by comparing mefloquine concentrations in saliva and plasma samples from four healthy volunteers who received single oral dose of mefloquine. Verapamil was used as internal standard. Chromatographic separation was achieved using a Hypersil ODS column.</p> <p>Results</p> <p>Extraction recoveries of mefloquine in plasma or saliva were 76-86% or 83-93% respectively. Limit of quantification of mefloquine was 20 ng/ml. Agreement between salivary and plasma mefloquine concentrations was satisfactory (r = 0.88, <it>p </it>< 0.001). Saliva:plasma concentrations ratio was 0.42.</p> <p>Conclusion</p> <p>Disposition of mefloquine in saliva paralleled that in plasma, making salivary quantification of mefloquine potentially useful in therapeutic drug monitoring.</p

Development of Analytical Methods for the Determination of Antimalarials in Biological Fluids

The aim of this thesis was to develop analytical methods for measuring antimalarial drugs in biological fluids. Solid phase extraction (SPE) was used for the enrichment and purification of the drugs. Automatic extraction procedures using a SPE robot were developed to reduce the workload for the analyst and to minimize variations in the extraction procedure. Liquid chromatography (LC) with either UV or mass spectrometric (MS) detection was used to determine sample concentrations. Determination of Pyronaridine in whole blood utilised a weak cation exchanger to extract Pyronaridine from blood. To improve LC separation between Pyronaridine and the internal standard, ion-pairing was utilized. For the simultaneous quantification of the highly lipophilic Atovaquone and the strong basic drug Proguanil with metabolites, a novel mixed mode solid phase extraction column was used. It combines the properties of a carboxylic acid (CBA) column and a non-polar octyl-silica (C8) column to extract the compounds from plasma; it also required a gradient LC separation. Stability is an important factor when developing new methods. A new approach was used to evaluate the stability of Amodiaquine in blood and plasma. This included the use of a stability marker, a stable compound which was added together with Amodiaquine when preparing the stability samples. This eliminated between-run variations and variations associated with preparation of new stock solutions. Lumefantrine (LF) is one of the active components in a new drug combination recommended by the World Health Organization as a replacement for older drugs which have lost their effect. The first of the two methods described for this compound is the determination of LF and a possible metabolite in plasma with a calibration range suitable for pharmacokinetic studies. In the second method, a capillary sampling technique is used where the blood is dried on a sampling paper and sent to the laboratory where the extraction and determination of LF concentrations take place. This method facilitates sample collection and will enable drug efficacy studies conducted in rural settings. To monitor a current change in treatment policy and self medication, a screening assay was developed. Its purpose is to be a complement to interviewing patients about their previous medication (in the previous few weeks) and to detect some of the more common drugs which might have been used.</p