Pyrimethamine significantly lowers cerebrospinal fluid Cu/Zn superoxide dismutase in amyotrophic lateral sclerosis patients with SOD1 mutations.

OBJECTIVE: Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). METHODS: A multicenter (5 sites), open-label, 9-month-duration, dose-ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale-Revised, and McGill Quality of Life Single-Item Scale were measured at screening, visit 6, and visit 9. RESULTS: We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p < 0.001) with a mean reduction of 13.5% (95% confidence interval [CI] = 8.4-18.5) and at visit 9 (p < 0.001) with a mean reduction of 10.5% (95% CI = 5.2-15.8). INTERPRETATION: Pyrimethamine is safe and well tolerated in ALS. Pyrimethamine is capable of producing a significant reduction in total CSF SOD1 protein content in patients with ALS caused by different SOD1 mutations. Further long-term studies are warranted to assess clinical efficacy. Ann Neurol 2017;81:837-848.

Assessment of Clinical Pharmacokinetic Drug-Drug Interaction of Antimalarial Drugs α/ß-Arteether and Sulfadoxine-Pyrimethamine.

Antimalarial drug combination therapy is now being widely used for the treatment of uncomplicated malaria. The objective of the present study was to investigate the effects of coadministration of intramuscular α/ß-arteether (α/ß-AE) and oral sulfadoxine-pyrimethamine (SP) on the pharmacokinetic properties of each drug as a drug-drug interaction study to support the development of a fixed-dose combination therapy. A single-dose, open-label, crossover clinical trial was conducted in healthy adult Indian male volunteers (18 to 45 years, n = 13) who received a single dose of AE or SP or a combination dose of AE and SP. Blood samples were collected up to 21 days postadministration, and concentrations of α-AE, ß-AE, sulfadoxine, and pyrimethamine were determined by using a validated liquid chromatography-tandem mass spectrometry method. Pharmacokinetic parameters were calculated and statistically analyzed to calculate the geometric mean ratio and confidence interval. Following single-dose coadministration of intramuscular AE and oral SP, the pharmacokinetic properties of α/ß-AE were not significantly affected, and α/ß-AE had no significant effect on the pharmacokinetic properties of SP in these selected groups of healthy volunteers. However, more investigations are needed to explore this further. (This study has been registered in the clinical trial registry of India under approval no. CTRI/2011/11/002155.).

Development and validation of a generic liquid chromatographic method for the simultaneous determination of five commonly used antimalarial drugs: Application to pharmaceutical formulations and human plasma.

A simple, sensitive, and rapid liquid chromatographic method was developed and validated using diode array detection for the determination of five commonly used antimalarial drugs in pharmaceutical formulations and in human plasma. Chromatographic separation of antimalarial drugs and internal standard (ibuprofen) was achieved on a C18 column with a mobile phase composed of 10 mM dipotassium orthophosphate at pH 3.0, methanol, and acetonitrile in a ratio of 20:38:42 v/v, at a flow rate of 1 mL/min. The analytes were monitored at 220 nm and separated in ˂10 min. The method was validated for linearity, accuracy, precision, limit of quantification, and robustness. Both intra- and interday precisions (in terms of %RSD) were lower than 3% and accuracy ranged from 98.1 to 104.5%. Extraction recoveries were ≥96% in plasma. The limits of quantitation for artemether, lumefantrine, pyrimethamine, sulfadoxine, and mefloquine were 0.3, 0.03, 0.06, 0.15, and 0.15 µg/mL in human plasma. Stability under various conditions was also investigated. The method was successfully applied for quantification of antimalarial drugs in marketed formulations and in spiked human plasma. The method can be employed for routine QC purposes and in pharmacokinetic investigations.

A community-randomized evaluation of the effect of intermittent preventive treatment in infants on antimalarial drug resistance in southern Tanzania.

BACKGROUND: Intermittent preventive treatment in infants (IPTi) is the administration of sulfadoxine-pyrimethamine (SP) at 2, 3, and 9 months of age to prevent malaria. We investigated the influence of IPTi on drug resistance. METHODS: Twenty-four areas were randomly assigned to receive or not receive IPTi. Blood collected during representative household surveys at baseline and 15 and 27 months after implementation was tested for SP and resistance markers. RESULTS: The frequency of SP in blood was similar in the IPTi and comparison areas at baseline and at 15 months. dhfr and dhps mutations were also similar at baseline and then increased similarly in both arms after 15 months of SP-IPTi. First-line treatment was switched from SP to artemether-lumefantrine before the final survey, when SP positivity fell among infants in comparison areas but increased in IPTi areas. This was accompanied by an increase in dhfr but not dhps mutations in IPTi areas (P = .004 and P = .18, respectively). CONCLUSIONS: IPTi did not increase drug pressure or the selection on dhfr and dhps mutants, when SP was the first-line malaria treatment. Introduction of artemether-lumefantrine was followed by an increase in dhfr mutations, consistent with weak selection attributable to SP-IPTi, but not by an increase in dhps mutations, suggesting a fitness cost of this mutation.

Pharmacokinetic properties of conventional and double-dose sulfadoxine-pyrimethamine given as intermittent preventive treatment in infancy.

Intermittent preventive treatment in infancy (IPTi) entails routine administration of antimalarial treatment doses at specified times in at-risk infants. Sulfadoxine-pyrimethamine (SDX/PYR) is a combination that has been used as first-line IPTi. Because of limited pharmacokinetic data and suggestions that higher milligram/kilogram pediatric doses than recommended should be considered, we assessed SDX/PYR disposition, randomized to conventional (25/1.25 mg/kg of body weight) or double (50/2.5 mg/kg) dose, in 70 Papua New Guinean children aged 2 to 13 months. Blood samples were drawn at baseline, 28 days, and three time points randomly selected for each infant at 4 to 8 h or 2, 5, 7, 14, or 21 days. Plasma SDX, PYR, and N(4)-acetylsulfadoxine (NSX, the principal metabolite of SDX) were assayed by high-performance liquid chromatography (HPLC). Using population modeling incorporating hepatic maturation and cystatin C-based renal function, two-compartment models provided best fits for PYR and SDX/NSX plasma concentration profiles. The area under the plasma concentration-time curve from 0 h to infinity (AUC(0-∞)) was greater with the double dose versus the conventional dose of PYR (4,915 versus 2,844 µg/day/liter) and SDX (2,434 versus 1,460 mg/day/liter). There was a 32% reduction in SDX relative bioavailability with the double dose but no evidence of dose-dependent metabolism. Terminal elimination half-lives (15.6 days for PYR, 9.1 days for SDX) were longer than previously reported. Both doses were well tolerated without changes in hemoglobin or hepatorenal function. Five children in the conventional and three in the double-dose group developed malaria during follow-up. These data support the potential use of double-dose SDX/PYR in infancy, but further studies should examine the influence of hepatorenal maturation in very young infants.

An open-label Phase I/II clinical trial of pyrimethamine for the treatment of patients affected with chronic GM2 gangliosidosis (Tay-Sachs or Sandhoff variants).

Late-onset GM2 gangliosidosis is an autosomal recessive, neurodegenerative, lysosomal storage disease, caused by deficiency of ß-hexosaminidase A (Hex A), resulting from mutations in the HEXA (Tay-Sachs variant) or the HEXB (Sandhoff variant) genes. The enzyme deficiency in many patients with juvenile or adult onset forms of the disease results from the production of an unstable protein, which becomes targeted for premature degradation by the quality control system of the smooth endoplasmic reticulum and is not transported to lysosomes. In vitro studies have shown that many mutations in either the α or ß subunit of Hex A can be partially rescued, i.e. enhanced levels of both enzyme protein and activity in lysosomes, following the growth of patient cells in the presence of the drug, pyrimethamine. The objectives of the present clinical trial were to establish the tolerability and efficacy of the treatment of late-onset GM2 gangliosidosis patients with escalating doses of pyrimethamine, to a maximum of 100 mg per day, administered orally in a single daily dose, over a 16-week period . The primary objective, tolerability, was assessed by regular clinical examinations, along with a panel of hematologic and biochemical studies. Although clinical efficacy could not be assessed in this short trial, treatment efficacy was evaluated by repeated measurements of leukocyte Hex A activity, expressed relative to the activity of lysosomal ß-glucuronidase. A total of 11 patients were enrolled, 8 males and 3 females, aged 23 to 50 years. One subject failed the initial screen, another was omitted from analysis because of the large number of protocol violations, and a third was withdrawn very early as a result of adverse events which were not drug-related. For the remaining 8 subjects, up to a 4-fold enhancement of Hex A activity at doses of 50 mg per day or less was observed. Additionally marked individual variations in the pharmacokinetics of the drug among the patients were noted. However, the study also found that significant side effects were experienced by most patients at or above 75 mg pyrimethamine per day. We concluded that pyrimethamine treatment enhances leukocyte Hex A activity in patients with late-onset GM2 gangliosidosis at doses lower than those associated with unacceptable side effects. Further plans are underway to extend these trials and to develop methods to assess clinical efficacy.

Monitoring of plasma concentration of pyrimethamine (PYR) in infants with congenital Toxoplasma gondii infection--own observations.

The study objective was to determine plasma concentration of pyrimethamine in 24 infants aged 1-5 months, treated for congenital toxoplasmosis. Pyrimethamine was used in a single daily dose at an amount of 0.35-0.98 mg/kg daily, with sulfadiazine (50-100 mg/kg/day) in divided doses 2-3 times a day, and folinic acid given twice a week (7.5 mg). This regimen was continued for 2-6 months, then Fansidar was administered. Pyrimethamine concentration in plasma was measured using high-performance liquid chromatography method (HPLC). A total of 70 tests were performed. Concentration of pyrimethamine ranged from 0.01 to 1.2 microg/ml. In 14 children (58 tests) the concentration of pyrimethamine achieved therapeutic value. In 7 patients (8 tests) the concentration was below therapeutic level, and in 3 patients (4 tests) above therapeutic level. In 11/24 (46%) children transient moderate neutropenia was observed. Modification of therapy was necessary in 12 patients. Monitoring of pyrimethamine concentration in plasma improves safety and effectiveness of the therapy and is useful in obtaining correct individual dose of the drug. Neutropenia is the most common side-effect of pyrimethamine observed even when using the recommended dose.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K. In addition to the previously reported endogenous biomarker candidates (creatinine and N1 -methylnicotinamide (1-NMN)), N1 -methyladenosine (m1 A) was included as novel biomarkers. 1-NMN and m1 A presented as superior MATE1/2-K biomarkers since changes in their renal clearance (CLr ) along with pyrimethamine dose were well-correlated with metformin CLr changes. The CLr of creatinine was reduced by pyrimethamine, however, its changes poorly correlated with metformin CLr changes. Nonlinear regression analysis (CLr vs. mean total concentration of pyrimethamine in plasma) yielded an estimate of the inhibition constant (Ki ) of pyrimethamine and the fraction of the clearance pathway sensitive to pyrimethamine. The in vivo Ki value thus obtained was further converted to unbound Ki using plasma unbound fraction of pyrimethamine, which was comparable to the in vitro Ki for MATE1 (1-NMN) and MATE2-K (1-NMN and m1 A). It is concluded that 1-NMN and m1 A CLr can be leveraged as quantitative MATE1/2-K biomarkers for DDI risk assessment in healthy volunteers.

Sulfadiazine plasma concentrations in women with pregnancy-acquired compared to ocular toxoplasmosis under pyrimethamine and sulfadiazine therapy: a case-control study.

BACKGROUND: Dosing recommendations for the treatment of pregnancy-acquired toxoplasmosis are empirical and widely based on experimental data. There are no pharmacological data on pregnant women with acute Toxoplasma gondii infection under treatment with pyrimethamine (PY) and sulfadiazine (SA) and our study intends to tighten this gap. METHODS: In this retrospective case-control study, we included 89 pregnant women with primary Toxoplasma infection (PT) treated with PY (50 mg first dose, then 25 mg/day), SA (50 mg/kg of body weight/day), and folinic acid (10-15 mg per week). These were compared to a group of 17 women with acute ocular toxoplasmosis (OT) treated with an initial PY dose of 75 mg, thereafter 25 mg twice a day but on the same SA and folinic acid regimen. The exact interval between drug intake and blood sampling and co-medication had not been recorded. Plasma levels of PY and SA were determined 14 ± 4 days after treatment initiation using liquid chromatography-mass spectrometry and compared using the Mann-Whitney U test at a p < 0.05 level. RESULTS: In 23 PT patients (26%), SA levels were below 20 mg/l. Fifteen of these 23 patients (17% of all patients) in parallel presented with PY levels below 700 µg/l. Both drug concentrations differed remarkably between individuals and groups (PY: PT median 810 µg/l, 95% CI for the median [745; 917] vs. OT 1230 µg/l [780; 1890], p = 0.006; SA: PT 46.2 mg/l [39.9; 54.4] vs. OT 70.4 mg/l [52.4; 89], p = 0.015) despite an identical SA dosing scheme. CONCLUSIONS: SA plasma concentrations were found in the median 34% lower in pregnant women with PT compared to OT patients and fell below a lower reference value of 50 mg/l in a substantial portion of PT patients. The interindividual variability of plasma concentrations in combination with systematically lower drug levels and possibly a lower compliance in pregnant women may thus account for a still not yet supportable transmission risk. Systematic drug-level testing in PT under PY/SA treatment deserves to be considered.

High uptake of Intermittent Preventive Treatment of malaria in pregnancy is associated with improved birth weight among pregnant women in Ghana.

Despite the clinically proven advantages of intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP), utilisation has been low in many African countries. To increase uptake and achieve the desired effect, the World Health Organization revised the policy to a monthly administration. Assessing the coverage and impact of the revised policy on pregnancy and neonatal outcomes is, therefore, a necessity. A 2-parallel cross-sectional hospital-based study was carried out among pregnant women attending first antenatal care (ANC) and delivery. Maternal and cord blood samples were assayed for malaria parasites by quantitative PCR targeting both the 18S rDNA and the acidic terminal segment of Plasmodium falciparum var genes, and plasma SP levels were measured by liquid chromatography coupled to tandem mass spectrometry. Parasite prevalence was similar between the two study sites but decreased significantly between the first ANC (9% or 43%) and delivery (4% or 11%) based on the qPCR target. At delivery, 64.5% of women received ≥3 IPTp-SP dose, 15.5% received 2 doses and 6% had 1 dose. Taking ≥3 IPTp-SP doses was associated with an average birth weight increase of more than 0.165 kg. IPTp-SP uptake was associated with plasma SP level at delivery (OR = 32.3, p ≤ 0.005, 95% CI (13.3;78.4) for those that reported ≥3 IPTp-SP doses) while the same trend of improved birth weight was observed with high plasma SP levels. The new IPTp policy is well implemented and well utilised by women in the sites considered in this study and translates to the improved birth weight observed. This study confirms the interest and the clinical benefit expected from this policy change.

Adherence to intermittent preventive treatment for malaria in Papua New Guinean infants: A pharmacological study alongside the randomized controlled trial.

BACKGROUND: The intermittent preventive treatment in infants (IPTi) trial that took place in Papua New Guinea showed an overall reduction of 29% of the risk of malaria when delivering single-dose sulfadoxine-pyrimethamine (SP) associated to 3 days of amodiaquine (AQ) every three months to children during the first year of life. The aim of the present study was to assess if the last two doses of AQ were truly administered as prescribed by the parents at home based on drug level measurement and PK modelling, which is a good proxy of medication adherence. It provides also important information to discuss the efficacy of the intervention and on feasibility of self-administered preventive malaria treatment. METHODS AND FINDINGS: During the three-arm randomized double-blinded IPTi trial, each child was prescribed one dose of SP (day 0) and 3 doses of either AQ or artesunate (AS) at day 0, 1 & 2 adjusted to weight or placebo. Treatments were given at 3, 6, 9 and 12 months of age. The first day of treatment was delivered by nursing staff (initiation under directly observed treatment (DOT)) and the two last doses of AQ or AS by parents at home without supervision. For this cross-sectional study, 206 consecutive children already involved in the IPTi trial were enrolled over a 2-month period. At the time of the survey, allocation of the children to one of the three arms was not known. Blood samples for drug level measurement were collected from finger pricks one day after the planned last third dose intake. Only children allocated to the SP-AQ arm were included in the present analysis. Indeed, the half-life of AS is too short to assess if drugs were given on not. Because of the short half-life of AQ, desethyl-AQ (metabolite of AQ (DAQ)) measurements were used to investigate AQ medication adherence. Two PK (PK) models from previously published studies in paediatric populations were applied to the dataset using non-linear mixed effect modelling (NONMEM) to estimate the number of doses really given by the parents. The study nurse reported the administration time for the first AQ dose while it was estimated by the parents for the remaining two doses. Out of 206 children, 64 were in the SP-AQ arm. The adjusted dosing history for each individual was identified as the one with the lowest difference between observed and individual predicted concentrations estimated by the two PK models for all the possible adherence schemes. The median (range) blood concentration AQ in AQ arm was 9.3 ng/mL (0-1427.8 ng/mL), (Quartiles 1-3: 2.4 ng/mL -22.2 ng/mL). The median (range) for DAQ was 162.0 ng/mL (0-712 ng/mL), (Quartiles 1-3: 80.4 ng/mL-267.7 ng/mL). Under the assumption of full adherence for all participants, a marked underprediction of concentrations was observed using both PK models. Our results suggest that only 39-50% of children received the three scheduled doses of AQ as prescribed, 33-37% two doses and 17-24% received only the first dose administered by the study nurse. Both models were highly congruent to classify adherence patterns. CONCLUSIONS: Considering the IPTi intervention, our results seem to indicate that medication adherence is low in the ideal trial research setting and is likely to be even lower if given in day-to-day practice, questioning the real impact that this intervention might have. More generally, the estimation of the number of doses truly administered, a proxy measure of adherence and an assessment of the feasibility of the mode of administration, should be more thoroughly studied when discussing the efficacy of the interventions in trials investigating self-administered malaria preventive treatments.

dhfr and dhps genotype and sulfadoxine-pyrimethamine treatment failure in children with falciparum malaria in the Democratic Republic of Congo.

OBJECTIVE: To determine the relationship between mutations in dhfr and dhps and SP treatment failure in Plasmodium falciparum malaria in the Democratic Republic of the Congo (DRC). METHODS: Therapeutic efficacy trial was conducted in Rutshuru, Eastern DRC, between June and September 2002, comparing sulfadoxine-pyrimethamine (SP), SP plus amodiaquine (AQSP) and artesunate plus SP (ASSP) regimens for treating malaria in children under 5 years old. We genotyped 212 samples for mutations associated with SP resistance and investigated their association with treatment failure. RESULTS: In the SP arm, 61% of the subjects experienced treatment failure after 14 days. The failure rate was lower in the combination arms (AQSP: 32%, ASSP: 21%). The dhfr-108 and dhfr-51 mutations were nearly universal while 89% of the samples had at least one additional mutation at dhfr-59, dhps-437 or dhps-540. Dhps mutations had a bigger impact on treatment failure in children with high parasite density: for children with a parasite density <45 000 parasites/microl, the risk of treatment failure was 37% for mutations at dhps-437 and dhps-540 mutation and 21% for neither mutation [risk difference (RD) = 17%, 95% CI: -3%, 36%]. In children with a parasite density >45 000 parasites/microl, the treatment failure risk was 58% and 8% for children with both mutations or neither mutation, respectively (RD = 51%, 95% CI: 34%, 67%). CONCLUSIONS: Dhps-437 and dhps-540 are strongly associated with SP treatment failure and should be evaluated further as a method for surveillance of SP-based therapy in DRC.

Antimalarial resistance and DHFR/DHPS genotypes of Plasmodium falciparum three years after introduction of sulfadoxine-pyrimethamine and amodiaquine in rural Tanzania.

We assessed the efficacy of sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) and DHFR/DHPS genotypes of Plasmodium falciparum in rural Tanzania, 3 years after their introduction as first- and second-line treatments for uncomplicated malaria, respectively. Under five children with uncomplicated malaria were given standard treatments of either SP (n=66) or AQ (n=30) and treatment outcomes after 14 and 28 days were determined. Total treatment failure of 18 and 42.5% was observed for SP on days 14 and 28, respectively. For AQ, total treatment failure of 27 and 53% was found on day 14 and 28, respectively. On day 14, significantly lower SP total treatment failures were observed in 2004 compared with results from a study conducted in 1999 in the same location. No relationship was detected between clinical outcome and DHFR/DHPS genotypes, but the point mutation prevalence in parasites was higher than in 1999. Pre-treatment blood levels of SP were detected in a quarter of the study children: less than expected. We report unacceptably high levels of total treatment failures, both for first- and second-line treatments for uncomplicated malaria in Tanzania 3 years after their introduction, supporting the decision to replace them with artemisinin-based combination therapy.

Practical HPLC methods for the quantitative determination of common antimalarials in Africa.

This article describes high-performance liquid chromatographic assays for the quantification of sulfadoxine (SDX), pyrimethamine (PYM), chloroquine (CQ), amodiaquine (AQ) and desethylamodiaquine (AQM) from whole blood. All four assays were set up and validated in Malawi using a common high-performance liquid chromatography platform and column and involved the use of simple mobile phase and extraction reagents. Calibration curves were linear (r(2)>0.95) in the ranges 5-100microg/ml, 50-1000, 150-1500, 100-1000 and 100-1000ng/ml for SDX, PYM, CQ, AQ and AQM, respectively. Intra-assay and inter-assay coefficients of variation were <15% at 3 points spanning the concentration range and <20% at the lower limit of quantification. The assays were specific with no interference from the other antimalarials described in this report. All four assays use liquid-liquid extraction, reversed-phase chromatography and UV detection and require between 50 and 200microl of blood. Because the assays share common instruments and reagents, they are cost-efficient and could be used to optimise antimalarial drug therapies in other resource poor settings.

Pharmacokinetics of Sulfadoxine and Pyrimethamine for Intermittent Preventive Treatment of Malaria During Pregnancy and After Delivery.

Sulfadoxine/pyrimethamine is recommended for intermittent preventative treatment of malaria during pregnancy. Data from 98 women during pregnancy and 77 after delivery in four African countries were analyzed using nonlinear mixed-effects modeling to characterize the effects of pregnancy, postpartum duration, and other covariates such as body weight and hematocrit on sulfadoxine/pyrimethamine pharmacokinetic properties. During pregnancy, clearance increased 3-fold for sulfadoxine but decreased by 18% for pyrimethamine. Postpartum sulfadoxine clearance decreased gradually over 13 weeks. This finding, together with hematocrit-based scaling of plasma to whole-blood concentrations and allometric scaling of pharmacokinetics parameters with body weight, enabled site-specific differences in the pharmacokinetic profiles to be reduced significantly but not eliminated. Further research is necessary to explain residual site-specific differences and elucidate whether dose-optimization, to address the 3-fold increase in clearance of sulfadoxine in pregnant women, is necessary, viable, and safe with the current fixed dose combination of sulfadoxine/pyrimethamine.

Efficacy of sulfadoxine-pyrimethamine in the treatment of uncomplicated Plasmodium falciparum malaria in East Timor.

The efficacy of sulfadoxine-pyrimethamine (SP) in East Timor is unknown. We treated 38 individuals with uncomplicated Plasmodium falciparum malaria with SP and monitored the outcome for 28 days. Recrudescent parasitemia, confirmed by genotyping, were detected in three individuals resulting in a late treatment failure rate of 7.9% (95% confidence interval = 1.7-21.4%). The results suggest that SP is still efficacious in treating uncomplicated P. falciparum malaria in East Timor. However, the useful life of SP in East Timor may be limited because 80% of the parasites in our samples were found to already carry double mutations in P. falciparum dihydrofolate reductase (S108N/C59R). The data from this study also highlights that the presence of gametocytes may significantly influence the estimate of SP efficacy determined by genotyping.

Liquid chromatographic-mass spectrometric assay for simultaneous pyrimethamine and sulfadoxine determination in human plasma samples.

We present a liquid chromatographic-mass spectrometric assay for the simultaneous determination of sulfadoxine and pyrimethamine in human plasma samples. Sample clean-up was achieved by adding acetonitrile for protein precipitation. Gradient elution in only 10 min resulted in high throughput capability. Tandem mass spectrometric detection in multiple reaction monitoring was used for quantification. The developed analytical approach was successfully validated and was applied in the pharmacokinetic evaluation of the bioavailability between two sulfadoxine/pyrimethamine formulations available on the Eastern African market, using a cross-over design.

Simple and cost-effective liquid chromatographic method for determination of pyrimethamine in whole blood samples dried on filter paper.

A cost-effective HPLC method for determination of pyrimethamine (PYR) in human whole blood samples dried on filter paper (Whatman) is reported. Trimethoprim (TMP) was used as an internal standard. Whole blood spiked with PYR was transferred (100 microl) onto filter paper and dried at room temperature. Capillary blood samples (100 microl) after ingestion of three tablets of sulfadoxine-pyrimethamine (SP) by one subject were also tested. PYR and an internal standard (IS) TMP were extracted into di-isopropyl ether as bases and then re-extracted with 150 microl mobile phase. A C-18 column was used and the mobile phase consisted of phosphate buffer (0.05 M, pH 5):acetonitrile:concentrated perchloric acid (750:300:2.5, v/v/v). The absorbances of PYR and IS were monitored at 270 nm. The limit of quantification was 40 ng/ml. The within- and between-assay coefficient of variations were <10% at the limit of quantification.

Simultaneous determination of pyrimethamine, sulfadiazine and N-acetyl-sulfadiazine in plasma for monitoring infants in treatment of congenital toxoplasmosis.

A method for the simultaneous determination of pyrimethamine, sulfadiazine and its metabolite N-acetyl-sulfadiazine in small plasma samples from neonates in treatment for congenital toxoplasmosis has been developed. In this method only 25 microl of plasma is used and a simple sample preparation based on protein precipitation and centrifugation provides highly reliable data as the recovery is about 100% and the precision is good. The analysis is performed using high performance liquid chromatography with UV and mass spectrometric (MS) detection. Pyrimethamine was found to give a linear response using MS detection in the range 0.02-5 microg/ml. Sulfadiazine and its metabolite N-acetyl-sulfadiazine were preferably analysed by UV at 269 nm in the concentration ranges 0.2-200 microg/ml for sulfadiazine and 0.2-50 microg/ml for N-acetyl-sulfadiazine.

Pyrimethamine analysis by enzyme inhibition and HPLC assays.

Pyrimethamine is an antiparasitic agent currently used for therapy of central nervous system toxoplasmosis, a disease seen with increasing frequency in association with the AIDS epidemic. Monitoring of pyrimethamine levels may be particularly important because patients may be treated with high doses of the drug for extended periods of time. The authors have developed and validated both a new enzyme inhibition assay that can be run on an automated analyzer and an improved high performance liquid chromatography (HPLC) method. The calibration range of both methods is 100 to 3,000 micrograms/L. Both demonstrate good linearity, specificity, and precision, and correlate well with one another (r = 0.99). The CVs of the enzyme inhibition assay were < or = 8.6% and those of the HPLC method were < or = 5.4%. No interference was noted for a variety of drugs likely to be used concomitantly with or in lieu of pyrimethamine with the exception of a minor interference from trimethoprim in the enzyme inhibition assay. The major advantage of the enzyme inhibition assay is its ease of automation. The major advantages of the HPLC assay are its precision and relative simplicity. These methods should facilitate therapeutic monitoring of pyrimethamine.