Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring.

Dried blood spot (DBS) analysis has been introduced more and more into clinical practice to facilitate Therapeutic Drug Monitoring (TDM). To assure the quality of bioanalytical methods, the design, development and validation needs to fit the intended use. Current validation requirements, described in guidelines for traditional matrices (blood, plasma, serum), do not cover all necessary aspects of method development, analytical- and clinical validation of DBS assays for TDM. Therefore, this guideline provides parameters required for the validation of quantitative determination of small molecule drugs in DBS using chromatographic methods, and to provide advice on how these can be assessed. In addition, guidance is given on the application of validated methods in a routine context. First, considerations for the method development stage are described covering sample collection procedure, type of filter paper and punch size, sample volume, drying and storage, internal standard incorporation, type of blood used, sample preparation and prevalidation. Second, common parameters regarding analytical validation are described in context of DBS analysis with the addition of DBS-specific parameters, such as volume-, volcano- and hematocrit effects. Third, clinical validation studies are described, including number of clinical samples and patients, comparison of DBS with venous blood, statistical methods and interpretation, spot quality, sampling procedure, duplicates, outliers, automated analysis methods and quality control programs. Lastly, cross-validation is discussed, covering changes made to existing sampling- and analysis methods. This guideline of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology on the development, validation and evaluation of DBS-based methods for the purpose of TDM aims to contribute to high-quality micro sampling methods used in clinical practice.

Long-term adherence and effects on grip strength and upper leg performance of prescribed supplemental vitamin D in pregnant and recently pregnant women of Somali and Swedish birth with 25-hydroxyvitamin D deficiency: a before-and-after treatment study.

BACKGROUND: Muscular weakness and severe vitamin D deficiency is prevalent in Somali (veiled) pregnant women, Sweden. The study aims here were to explore adherence to prescribed supplemental vitamin D in new mothers with vitamin D deficiency and its effects on grip strength and upper leg performance in Somali (target group TG) and Swedish women (reference group RG) from spring through winter. METHODS: A before- and after study was designed. A cross-sectional sample of women in antenatal care with serum 25-OHD ≤50 nmol/L were prescribed one or two tablets daily (800 or 1600 IU vitamin D3 with calcium) for 10 months. Reminders were made by Somali nurses (TG) or Swedish doctors (RG). Baseline and 10 month measurements of plasma nmol/L 25-OHD, maximal grip strength held for 10 s (Newton, N) and ability to squat (yes;no) were done. Total tablet intake (n) was calculated. Outcome variables were changes from baseline in grip strength and ability to squat. Predicting variables for change in grip strength and ability to squat were calculated using linear and binary regression in final models. Undetectable 25-OHD values (<10 nmol/L) were replaced with '9' in statistic calculations. RESULTS: Seventy-one women (46 TG, 1/3 with undetectable baseline 25-OHD; 25 RG) participated. At the 10-month follow up, 17% TG and 8% RG women reported having refrained from supplement. Mean 25-OHD increased 16 to 49 nmol/L (TG) and 39 nmol/L to 67 nmol/L (RG), (both p < 0.001). Grip strength had improved from 153 to 188 N (TG) (p < 0.001) and from 257 to 297 N (RG) (p = 0.003) and inability to squat had decreased in TG (35 to 9, p < 0.001). Intake of number of tablets predicted increased grip strength (B 0.067, 95%CI 0.008-0.127, p = 0.027). One tablet daily (>300 in total) predicted improved ability to squat (OR 16; 95% CI 1.8-144.6). CONCLUSIONS: Adherence to supplemental vitamin D and calcium should be encouraged as an even moderate intake was associated to improved grip strength and upper leg performance, which was particularly useful for the women with severe 25-OHD deficiency and poor physical performance at baseline. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02922803 . Date of registration: September 28, 2016.

Population Pharmacokinetics and Pharmacodynamics of Lumefantrine in Young Ugandan Children Treated With Artemether-Lumefantrine for Uncomplicated Malaria.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 had a 3-fold higher hazard of 28-day recurrent parasitemia, compared with those with 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. CONCLUSIONS: 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.

High-Dose Chloroquine for Treatment of Chloroquine-Resistant Plasmodium falciparum Malaria.

BACKGROUND: Due to development of multidrug-resistant Plasmodium falciparum new antimalarial therapies are needed. In Guinea-Bissau, routinely used triple standard-dose chloroquine remained effective for decades despite the existence of "chloroquine-resistant" P. falciparum. This study aimed to determine the in vivo efficacy of higher chloroquine concentrations against P. falciparum with resistance-conferring genotypes. METHODS: Standard or double-dose chloroquine was given to 892 children aged <15 years with uncomplicated malaria during 3 clinical trials (2001-2008) with ≥ 35 days follow-up. The P. falciparum resistance-conferring genotype (pfcrt 76T) and day 7 chloroquine concentrations were determined. Data were divided into age groups (<5, 5-9, and 10-14 years) because concentrations increase with age when chloroquine is prescribed according to body weight. RESULTS: Adequate clinical and parasitological responses were 14%, 38%, and 39% after standard-dose and 66%, 84%, and 91% after double-dose chloroquine in children aged <5, 5-9, and 10-14 years, respectively, and infected with P. falciparum genotypes conferring chloroquine resistance (n = 195, P < .001). In parallel, median chloroquine concentrations were 471, 688, and 809 nmol/L for standard-dose and 1040, 1494, and 1585 nmol/L for double-dose chloroquine. CONCLUSIONS: Chloroquine resistance is dose dependent and can be overcome by higher, still well-tolerated doses.

Chloroquine is grossly under dosed in young children with malaria: implications for drug resistance.

BACKGROUND: Plasmodium falciparum malaria is treated with 25 mg/kg of chloroquine (CQ) irrespective of age. Theoretically, CQ should be dosed according to body surface area (BSA). The effect of dosing CQ according to BSA has not been determined but doubling the dose per kg doubled the efficacy of CQ in children aged <15 years infected with P. falciparum carrying CQ resistance causing genes typical for Africa. The study aim was to determine the effect of age on CQ concentrations. METHODS AND FINDINGS: Day 7 whole blood CQ concentrations were determined in 150 and 302 children treated with 25 and 50 mg/kg, respectively, in previously conducted clinical trials. CQ concentrations normalised for the dose taken in mg/kg of CQ decreased with decreasing age (p<0.001). CQ concentrations normalised for dose taken in mg/m(2) were unaffected by age. The median CQ concentration in children aged <2 years taking 50 mg/kg and in children aged 10-14 years taking 25 mg/kg were 825 (95% confidence interval [CI] 662-988) and 758 (95% CI 640-876) nmol/l, respectively (p = 0.67). The median CQ concentration in children aged 10-14 taking 50 mg/kg and children aged 0-2 taking 25 mg/kg were 1521 and 549 nmol/l. Adverse events were not age/concentration dependent. CONCLUSIONS: CQ is under-dosed in children and should ideally be dosed according to BSA. Children aged <2 years need approximately double the dose per kg to attain CQ concentrations found in children aged 10-14 years. Clinical trials assessing the efficacy of CQ in Africa are typically performed in children aged <5 years. Thus the efficacy of CQ is typically assessed in children in whom CQ is under dosed. Approximately 3 fold higher drug concentrations can probably be safely given to the youngest children. As CQ resistance is concentration dependent an alternative dosing of CQ may overcome resistance in Africa.

Physical performance and 25-hydroxyvitamin D: a cross-sectional study of pregnant Swedish and Somali immigrant women and new mothers.

BACKGROUND: Severe vitamin D deficiency can impair muscle strength. The study aims were to examine physical performance in the hands and upper legs, and analyze plasma 25-hydroxyvitamin D (25(OH)D) concentrations in women with presumably low (veiled, Somali-born) and high levels (unveiled, Swedish-born). METHODS: Women (n=123, 58% Swedish) enrolled at a Swedish antenatal clinic, latitude 60° N, were recruited. Plasma 25(OH) D was analyzed, measured as nmol/L, then categorized as <10 = undetectable, 10-24, 25-49, 50-74 or >75. Muscle strength was tested: maximal hand grip strength (in Newtons, N), and upper leg performance (categorized as able/unable to perform squatting, standing on one leg, standing from a chair, and lifting their hips). Social and anthropometric data were collected. Non-parametric statistics tested the data for differences in their ability to perform the tests across 25(OH)D categories. Undetectable values (<10 nmol/L) were replaced with '9' in the linear correlation statistics. A final main effect model for grip strength (in N) was calculated using stepwise linear regression for independent variables: country of birth, 25(OH)D levels, age, height, weight, physical activity, lactation status, parity, and gestational age. RESULTS: Somali participants (35%) had 25(OH)D levels of <10 nmol/L, and 90% had <25 nmol/L; 10% of Swedish participants had <25 nmol/L of 25(OH)D, and 54% had <50 nmol/L. Somali women had a relatively weak grip strength compared with Swedish women: median 202 N (inter-quartile range 167-246) vs. median 316 N (inter-quartile range 278-359), respectively. Somali women were also weak in upper leg performance: 73% were unable to squat, 29% unable to stand on one leg, and 21% could not lift their hips (not significant across 25(OH)D categories); most Swedish women could perform these tests. In the final model, grip strength (N) was significantly associated with 25(OH)D levels (B 0.94, p=0.013) together with Somali birth (B -63.9, p<0.001), age (B 2.5, p=0.02) and height (B 2.6, p=0.01). CONCLUSIONS: Many Somali women had undetectable/severely low 25(OH)D concentrations and pronounced hand and upper leg weakness; grip strength was strongly associated with 25(OH)D. Maternity health care personnel should be aware of this increased frequency and manage care accordingly.

Efficacy of artemether-lumefantrine in area of high malaria endemicity in India and its correlation with blood concentration of lumefantrine.

This study was conducted to correlate blood concentrations of lumefantrine with treatment outcome for patients with Plasmodium falciparum malaria when the drug was given without specific instructions for administration with food. Patients with P. falciparum malaria in the highly endemic state of Orissa, India, were enrolled during 2008 and followed-up for 28 days after admistration of artemether-lumefantrine for three days according to a World Health Organization protocol. Drug concentration in whole blood was determined by using blood spots placed on filter paper on day 7. The technology is suitable for field studies. One hundred percent of the patients had an adequate clinical and parasitological response. These results confirm the efficacy of artemether-lumefantrine in persons from poor tribal communities when given without specific instructions regarding co-administration with food, despite high inter-individual variability in blood concentrations of lumefantrine.

Determination of tafenoquine in dried blood spots and plasma using LC and fluorescence detection.

BACKGROUND: The growing problem of parasites developing resistance to the traditional antimalarial drugs makes the development of new effective and safe drugs crucial. Tafenoquine is a new promising antimalarial drug for prophylaxis and treatment. RESULTS: A bioanalytical method for the determination of tafenoquine in 100 µl of capillary blood applied onto sampling paper and in 100 µl of plasma has been developed and validated. The Whatman 31 ET Chr paper was treated with 0.6 mol/l tartaric acid to improve the extraction recovery and solid-phase extraction was used for cleanup procedure of the blood samples. Plasma samples were precipitated with methanol. Tafenoquine and internal standard were separated on a Zorbax SB-CN column by reversed-phase LC and detected with fluorescence detection at 262 and 470 nm. The within- and between-day variations were below 10 and 14%, respectively, over the range 50-200 nmol/l for capillary blood on sampling paper and below 6 and 10% for plasma samples. The LLOQ of the method was 50 nmol/l. CONCLUSION: The developed method has adequate sensitivity and is highly suitable for clinical studies in dried blood spots and plasma.

Effectiveness of artemether-lumefantrine provided by community health workers in under-five children with uncomplicated malaria in rural Tanzania: an open label prospective study.

BACKGROUND: Home-management of malaria (HMM) strategy improves early access of anti-malarial medicines to high-risk groups in remote areas of sub-Saharan Africa. However, limited data are available on the effectiveness of using artemisinin-based combination therapy (ACT) within the HMM strategy. The aim of this study was to assess the effectiveness of artemether-lumefantrine (AL), presently the most favoured ACT in Africa, in under-five children with uncomplicated Plasmodium falciparum malaria in Tanzania, when provided by community health workers (CHWs) and administered unsupervised by parents or guardians at home. METHODS: An open label, single arm prospective study was conducted in two rural villages with high malaria transmission in Kibaha District, Tanzania. Children presenting to CHWs with uncomplicated fever and a positive rapid malaria diagnostic test (RDT) were provisionally enrolled and provided AL for unsupervised treatment at home. Patients with microscopy confirmed P. falciparum parasitaemia were definitely enrolled and reviewed weekly by the CHWs during 42 days. Primary outcome measure was PCR corrected parasitological cure rate by day 42, as estimated by Kaplan-Meier survival analysis. This trial is registered with ClinicalTrials.gov, number NCT00454961. RESULTS: A total of 244 febrile children were enrolled between March-August 2007. Two patients were lost to follow up on day 14, and one patient withdrew consent on day 21. Some 141/241 (58.5%) patients had recurrent infection during follow-up, of whom 14 had recrudescence. The PCR corrected cure rate by day 42 was 93.0% (95% CI 88.3%-95.9%). The median lumefantrine concentration was statistically significantly lower in patients with recrudescence (97 ng/mL [IQR 0-234]; n = 10) compared with reinfections (205 ng/mL [114-390]; n = 92), or no parasite reappearance (217 [121-374] ng/mL; n = 70; p ≤ 0.046). CONCLUSIONS: Provision of AL by CHWs for unsupervised malaria treatment at home was highly effective, which provides evidence base for scaling-up implementation of HMM with AL in Tanzania.

Efficacy and effectiveness of artemether-lumefantrine after initial and repeated treatment in children <5 years of age with acute uncomplicated Plasmodium falciparum malaria in rural Tanzania: a randomized trial.

BACKGROUND: We assessed the efficacy, effectiveness and safety of artemether-lumefantrine, which is the most widely used artemisinin-based combination therapy in Africa, against Plasmodium falciparum malaria during an extended follow-up period after initial and repeated treatment. METHODS: We performed an open-label randomized trial of artemether-lumefantrine with supervised (n=180) and unsupervised intake (n=179) in children <5 years of age with uncomplicated falciparum malaria in rural Tanzania. Recurrent infections between day 14 and day 56 were retreated within the same study arm. Main end points were polymerase chain reaction (PCR)-corrected cure rates by day 56 and day 42 after initial and repeated treatment, respectively, as estimated by survival analysis. RESULTS: The PCR-corrected cure rate after initial treatment was 98.1% (95% confidence interval [CI], 94.2%-99.4%) after supervised and 95.1% (95% CI, 90.7%-98.1%) after unsupervised intake (P=.29). After retreatment of recurrent infections, the cure rates were 92.9% (95% CI, 81.8%-97.3%) and 97.6% (95% CI, 89.3%-98.8%), respectively (P=.58). Reinfections occurred in 46.9% (82 of 175) versus 50.9 % of the patients (relative risk [RR], 0.92 [95% CI, 0.74-1.14]; P=.46) after initial therapy and 32.4% (24 of 74) versus 39.0% (32 of 82) (RR, 0.83 [95% CI, 0.54-1.27]; P=.39) after retreatment. Median blood lumefantrine concentrations in supervised and unsupervised patients on day 7 were 304 versus 194 ng/mL (P<.001) after initial treatment and 253 versus 164 ng/mL (P=.001) after retreatment. Vomiting was the most commonly reported drug-related adverse event (in 1% of patients) after both initial and repeated treatment. CONCLUSIONS: Artemether-lumefantrine was highly efficacious even after unsupervised administration, despite significantly lower lumefantrine concentrations, compared with concentration achieved with supervised intake, and was well-tolerated and safe after initial and repeated treatment. CLINICAL TRIAL REGISTRATION: ISRCTN69189899.

Confirmed vivax resistance to chloroquine and effectiveness of artemether-lumefantrine for the treatment of vivax malaria in Ethiopia.

Chloroquine (CQ) is still the drug of choice for the treatment of vivax malaria in Ethiopia, whereas artemether-lumefantrine (AL) is for falciparum malaria. In this setting, clinical malaria cases are treated with AL. This necessitated the need to assess the effectiveness of AL for the treatment of Plasmodium vivax with CQ as a comparator. A total of 57 (80.3%) and 75 (85.2%) cases treated with CQ or AL, respectively, completed the study in an outpatient setting. At the end of the follow-up period of 28 days, a cumulative incidence of treatment failure of 7.5% (95% confidence interval [CI] = 2.9-18.9%) for CQ and 19% (95% CI = 11-31.6%) for AL was detected. CQ resistance was confirmed in three of five CQ treatment failures cases. The effectiveness of AL seems lower than CQ; however, the findings were not conclusive, because the AL evening doses were not supervised.

Assay for screening for six antimalarial drugs and one metabolite using dried blood spot sampling, sequential extraction and ion-trap detection.

BACKGROUND: More parasites are becoming resistant to antimalarial drugs, and in many areas a change in first-line drug treatment is necessary. The aim of the developed assay is to help determine drug use in these areas and also to be a complement to interviewing patients, which will increase reliability of surveys. RESULTS: This assay detects quinine, mefloquine, sulfadoxine, pyrimethamine, lumefantrine, chloroquine and its metabolite desethylchloroquine in a 100-µl dried blood spot. Most of the drugs also have long half-lives that make them detectable at least 7 days after administration. The drugs are extracted from the dried blood spot with sequential extraction (due to the big differences in physicochemical properties), solid-phase extraction is used as sample clean-up and separation is performed with gradient-LC with MS ion-trap detection. CONCLUSION: Detection limits (S/N > 5:1) at 50 ng/ml or better were achieved for all drugs except lumefantrine (200 ng/ml), and thus can be used to determine patient compliance. A major advantage of using the ion-trap MS it that it will be possible to go back into the data and look for other drugs as needed.

Chloroquine is grossly overdosed and overused but well tolerated in Guinea-bissau.

High chloroquine doses are commonly prescribed in Guinea-Bissau. Double-dose chloroquine has been shown to be more efficacious (92% efficacy) than the standard dose (80% efficacy). However, chloroquine is toxic when overdosed, and it was not known if the high doses prescribed in Guinea-Bissau were taken or whether they caused adverse effects. We aimed to determine the dosage of chloroquine commonly prescribed, the doses commonly taken, and whether concentration-dependent adverse events occurred in routine practice. Chloroquine prescriptions by eight physicians and chloroquine intake by 102 children were recorded. Chloroquine intake and adverse events were assessed by questioning. Chloroquine concentrations in whole blood were measured. The median total chloroquine dose prescribed and that reportedly taken were 81 and 77 mg kg(-1), respectively. The total dose was usually split into two to three daily doses of 6.6 mg kg(-1) each. These were taken unsupervised for a median of 5 days. Forty percent of the study children had chloroquine concentrations in the same range as those found in a previous study in which double the normal dose (50 mg kg(-1)) of chloroquine was taken. Only 3/102 children had Plasmodium falciparum in the blood at the time of diagnosis and treatment. No severe adverse events were reported. No adverse events were associated with higher chloroquine concentrations. High doses of chloroquine are commonly taken and well tolerated in Guinea-Bissau. Malaria diagnostics are poor, and chloroquine is commonly prescribed to children without parasitemia. Use of high-dose chloroquine is concurrent with an exceptionally low prevalence of chloroquine-resistant P. falciparum.

Low validity of caretakers' reports on use of selected antimalarials and antibiotics in children with severe pneumonia at an urban hospital in Uganda.

Febrile children in low-income countries receive care from multiple sources, and caretakers' ability to report drug intake is crucial for appropriate prescription of drugs when reaching health facilities. This study describes and validates caretakers' reported use of sulfamethoxazole, chloroquine and sulfadoxine in their children. We performed a cross-sectional study in 139 children diagnosed with severe pneumonia at hospital in Kampala, Uganda. Caretakers were interviewed regarding treatments given prior to arrival at the hospital. Reported drug intake was compared to drug levels in blood sampled on filter paper, analyzed by HPLC methods. Caretakers under-reported intake of the studied drugs. Positive and negative predictive values were 67 and 64% for sulfamethoxazole, 69 and 52% for chloroquine and 85 and 62% for sulfadoxine. Many caretakers were unaware of what drug had been given to the child, and more so if treated outside the home (risk ratio 2.6, 95% CI 1.2-5.6). We conclude that caretakers' reports of drug intake have limited validity. Health workers need to improve counseling of caretakers during drug dispensing, especially for antibiotics. The roles and names of different drugs should be emphasized during counseling, and existing information systems such as immunization cards should be considered for record-keeping of treatment given.

Determination of pyronaridine in whole blood by automated solid phase extraction and high-performance liquid chromatography.

A new extraction procedure for the analysis of pyronaridine in whole blood is presented. A weak cation exchanger with a carboxylic acid (CBA) sorbent was found to be a suitable solid phase sorbent for the extraction of pyronaridine. High-performance liquid chromatography with UV detection at 278 nm and an electrochemical detector at +0.75 V is used. The electrochemical detector gives higher selectivity than the UV detector. The separation was performed using a C18 reversed phase column with mobile phase of acetonitrile-phosphate buffer (0.01 mol/L, pH 2.5)- sodium perchlorate (1.0 mol/L; 22:77:1, v/v/v). The within-day RSDs were below 5% at all concentration levels between 75 nmol/L and 1500 nmol/L, and the between-day RSDs were below 14% at all concentration levels. The limit of quantification was about 50 nmol/L in 1000 microL whole blood with an RSD of 20% or less on a day-to-day basis. The stability of pyronaridine is increased if the pH is less than 3 in water solutions. In whole blood, the concentration decreases by about 10% for each freeze-thaw cycle performed. At room temperature (about 22 degrees C), pyronaridine concentration in whole blood decreases by about 10% within 12 to 24 hours.

Population pharmacokinetic and pharmacodynamic modelling of artemisinin and mefloquine enantiomers in patients with falciparum malaria.

OBJECTIVES: The aims of this study were to investigate whether artemisinin influences the pharmacokinetics of mefloquine enantiomers or vice versa and to model the antiparasitic effect of these drugs alone and in combination in Plasmodium falciparum malaria patients. METHODS: Forty-two male and female patients were randomised to treatment with either oral artemisinin 500 mg daily for 3 days followed by oral mefloquine 750 mg on day 4, oral artemisinin 500 mg daily for 3 days plus oral mefloquine 750 mg on day 1 or a single 750-mg oral dose of mefloquine. The data was modelled using NONMEM. RESULTS: All patients were successfully treated regardless of treatment. The fastest parasite clearance rates were observed in patients receiving artemisinin together with mefloquine on the first day of treatment. A pharmacodynamic model based on the life cycle of P. falciparum successfully described the efficacy of artemisinin, mefloquine and the combination. The time artemisinin concentration stays above a minimum inhibitory concentration was estimated to 2.97 h (relative standard error 4.7 h). The two mefloquine enantiomers exhibited different pharmacokinetics, with an oral clearance of 3.51 (7.9) l/h and 0.602 (6.9) l/h for RS-mefloquine and SR-mefloquine, respectively. In patients receiving only artemisinin the first 3 days, artemisinin oral clearance was 6.9-fold higher the last day of treatment compared with the first day. There was no difference in the pharmacokinetics of mefloquine enantiomers when mefloquine was given alone, in combination with artemisinin or after a 3-day regimen of artemisinin. There was a tendency towards, although non-significant, higher artemisinin concentrations when artemisinin was given together with mefloquine compared with when given alone. CONCLUSIONS: No significant pharmacokinetic interactions were observed after co-administration of artemisinin and mefloquine. The P. falciparum malaria pharmacodynamic model successfully described the antimalarial effect of artemisinin, mefloquine and a combination of the two drugs.

Time-dependent pharmacokinetics and drug metabolism of atovaquone plus proguanil (Malarone) when taken as chemoprophylaxis.

OBJECTIVE: To determine the pharmacokinetic profiles of atovaquone (ATO), proguanil (PROG) and its active metabolite cycloguanil (CYCLO) with respect to possible accumulation and kinetic interaction upon repeated dosing with Malarone. METHODS: Thirteen healthy volunteers first received a single dose and then after 1 week, repetitive daily doses of Malarone (one tablet) for 13 days. For analysis of plasma drug concentrations, blood samples were collected at regular intervals over 8 days after a single dose and over 12 days after the last day of multiple dosing. Single-dose and steady-state pharmacokinetic parameters were determined for each individual. Genotyping of the gene coding for CYP2C19, a major enzyme catalyzing PROG metabolism, was performed using polymerase chain reaction, and in vitro enzyme kinetic experiments were carried out to study the possible effect of ATO on the catalytic activities of CYP2C19 and 3A4 using fluorometric assays. RESULTS: For ATO, the ratio of the area under the concentration-time curve (AUC) during the last dose interval to the AUC after the single dose (AUC(0- tau)/AUC(0- infinity)) was found to be 0.90 [95% confidence interval (CI) 0.56, 1.24] indicating absence of undue accumulation. AUC(0- tau), and peak plasma concentration at steady state (C(max,ss)) values were, however, threefold lower than those reported in human immunodeficiency virus-infected subjects after 12 multiple daily doses of 250 mg ATO alone. Four volunteers, with mean CYCLO/PROG AUC(0-tau) of 0.03 (-0.23, 0.09) were classified as poor metaboliser (PM) phenotypes. There was a significant increase in the AUC of PROG at steady state with a PROG AUC(0-tau)/AUC(0-infinity) ratio of 1.38 (1.07, 1.69) in extensive metaboliser (EM) phenotypes. CYCLO/PROG AUC ratios were significantly lower 0.67 (0.54, 0.81) at steady state than that after the first single dose in EM phenotypes. The in vitro kinetic experiments on recombinant enzymes (CYP2C19 and CYP3A4) suggested a possible inhibition of catalytic activity of CYP3A4 by ATO. CONCLUSIONS: There was no unexpected accumulation of ATO following repeated administrations of the combination. In EM phenotypes, PROG elimination was reduced at steady state. Also, at steady state, either the elimination of CYCLO was increased or its formation clearance decreased the latter possibly by inhibition of CYP3A4 by ATO.