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Background. We compared sulfadoxine-pyrimethamine (SP) with unsupervised artemether-lumefantrine (AL) and unsupervised amodiaquine-artesunate (ASAQ) fixed-dose formulation for the treatment of uncomplicated malaria in children in Benin. Methods. This open-label, noninferiority comparative trial included children aged 6―60 months. The follow-up period was 6 weeks, and the primary objective was a comparison of polymerase chain reaction (PCR)―adjusted effectiveness rates at day 28. Results. The study included 240 children (48 received SP, and 96 each received AL and ASAQ). The intention-to-treat analysis showed effectiveness rates on day 28 of 20.8%, 78.1 %, and 70.5% for SP, AL, and ASAQ, respectively. After adjustment for PCR results, these rates were 27.1%, 83.3%, and 87.4%, respectively. The per-protocol analysis (217 patients) showed effectiveness rates on day 28 of 21.7%, 88.0%, and 76.1% for SP, AL, and ASAQ, respectively. After adjustment for PCR results, these rates were 28.3%, 94.0%, and 93.2%, respectively. SP was less effective than the other drugs in the PCR-adjusted analysis, whereas AL and ASAQ were equally effective. The rate of new infection was higher among children treated with ASAQ than among those treated with AL. Conclusions. This was the first trial, to our knowledge, to compare unsupervised AL with unsupervised ASAQ fixed-dose formulation; both treatments provided high PCR-adjusted day 28 effectiveness rates. Efficacy rates for SP were surprisingly low. Clinical trials registration. NCT00460369. more...

Ifosfamide (IFOS) is a bifunctional alkylator with a wide spectrum of activity in solid tumors and has an autoinductive liver metabolism through P450 cytochromes. Autoinduction might permit a better therapeutic index for combination therapy. A phase I trial was investigated with interpatient dose escalation of a single dose of IFOS given every 2 weeks in advanced solid tumor patients. IFOS, its dechloroethylated and active 4-hydroxy metabolites, were measured at cycles 1 and 2 at the end of infusion, 2 and 5 h later, using gas chromatography. IFOS elimination was considered as following monocompartimental model kinetics. The results of 20 patients from January 2004 to June 2006 were included. The median of previous chemotherapies was 2 (0-5). The primary tumor was most often ovarian (5), peritoneal (3), sarcoma (2), melanoma (2) or miscellaneous (8). Ten patients received 2.5 g/m 2 and the other 10 patients received 3 g/m 2 . A total of 79 cycles were evaluable for toxicity. The median number of cycles was 4 (1-8). No grade 3-4 toxicity, no alopecia at first dose level and no toxicity-related fatal events were noted. One objective response was noted in a pancreatic cancer patient and one sustained CA125 decline in a heavily pretreated ovarian cancer patient. A slight (7-10%) but reproducible decrease of areas under the curve was detectable at cycle 2, at both dose levels, related to autoinductive metabolism. Intraindividual variations (large SD) were noticed for each pharmacokinetic parameter. A patient-dependent autoinduction of IFOS metabolism was detected rather than a slight nondose-dependent autoinduction. The toxicity profile allows the development of bi-weekly IFOS-based combination therapies. more...

A method for simultaneous analysis of chloroquine, proguanil and their metabolites from a whole blood sample (80 microL) dried on a filter paper was developed. Sample preparation included a liquid extraction from the filter paper, followed by a solid-phase extraction (C18 Bond Elut cartridge). Separation was obtained by reverse-phase liquid chromatography (HPLC) using a gradient elution on an X-Terra column; UV detection was made at 254 nm. This assay was linear between 150 and 2500 ng mL(-1) for chloroquine (and metabolite) and 300 and 2500 ng mL(-1) for proguanil and cycloguanil. The lower limit of quantification was close to 50 ng mL(-1) for chloroquine (and its metabolite) and 100 ng mL(-1) for proguanil (and its metabolite). No chromatographic interference from endogenous compounds or other tested anti-malarial drugs was evidenced. Chromatographic separation takes about 40 min with a coefficient of variation below 10.3% for within- and between-batch precision. The paper sampling method was validated in 10 healthy subjects treated by Savarine. The stability of compounds and metabolites on the filter paper was evaluated at four temperatures (-20, +4, 20 and 50 degrees C) and for 1, 5 and 20 days. Cycloguanil concentrations were not influenced by storage conditions, whereas, high temperatures and prolonged storage decreased chloroquine and proguanil levels. The proposed HPLC assay is accurate, precise and cost-effective; it can be used for pharmacokinetic and epidemiological studies on anti-malarial treatments. more...

La ketamine est un anesthesique dissociatif derive de la cyclohexanone, utilisee en medecine humaine et veterinaire pour induire l'endormissement et le maintenir lors de breves interventions chirurgicales. Inhibiteur non competitifdu glutamate au niveau des recepteurs NMDA centraux, la ketamine est rapidement metabolisee en norketamine, presentant une action pharmacologique, puis en dehydronorketamine eliminee par voie urinaire apres conjugaison. Les effets secondaires (delires, hallucinations) sont recherches au cours des raves parties. L'accroissement de son utilisation dans un but d'addiction est a l'origine d'un nombre eleve d'intoxications dont certaines mortelles. De nombreuses methodes chromatographiques en phase gazeuse ont ete rapportees utilisant toutes une etape de derivation et un volume d'echantillon important. Dans ce travail, nous decrivons une methode de dosage simultane de la ketamine et de la norketamine par chromatographie phase gazeuse sans derivation apres extraction liquide-liquide. L'ifosfamide est utilise comme etalon interne. La methode est lineaire entre 500 et 4000 μ g/L La limite de detection est de 160 et 180 μ g/L pour la ketamine et la norketamine, respectivement. La precision intra et inter series est satisfaisante ( more...

Objectives: Rasburicase (Fasturtec®) is used to prevent or treat hyperuricemia associated with chemotherapy. We developed a capillary zone electrophoresis method to measure urinary allantoin, the degradation product of uric acid by rasburicase. Design and methods: Electrophoresis was performed using a P/ACE 5500 system (Beckman) with a fused silica capillary tube and a UV–visible detector set at 214 nm. Urine samples from 10 patients with non-Hodgkin’s lymphoma were analyzed to validate the technique. Results: Using a sodium tetraborate running buffer, urinary allantoin was separated from related compounds and internal standard in less than 30 min. The method was linear up to 1.25 g/L (quantification limit: 30 mg/L); precision was below 10%. The total amount of allantoin excreted in patients treated by rasburicase ranged from 1.5 g to 7.9 g/4 days. Conclusion: This CZE assay is a simple, rapid and reproducible method to measure allantoin in urine. Different elimination profiles have been found in patients treated with rasburicase. more...

Pralidoxime methylsulfate (Contrathion) is widely used to treat organophosphate poisoning. Despite animal and human studies, the usefulness of Contrathion therapy remains a matter of debate. Therapeutic dosage regimens need to be clarified and availability of a reliable method for plasma pralidoxime quantification would be helpful in this process. We here describe a high-performance liquid chromatography technique with electrochemical detection to measure pralidoxime concentrations in human serum using guanosine as an internal standard. The assay was linear between 0.25 and 50 microg mL(-1) with a quantification limit of 0.2 microg mL(-1). The analytical precision was satisfactory, with variation coefficients lower 10%. This assay was applied to the analysis of a serum from an organophosphorate poisoned patient and treated by Contrathion infusions (100 and 200 mg h(-1)) after a loading dose (400 mg). more...

Background: We evaluated a new, automated multicapillary zone electrophoresis (CE) instrument (Capillarys®, 4.51 software version; Sebia) for human serum protein analysis.Methods: With the Capillarys β1-β2+® reagent set, proteins were separated at 7 kV for 4 min in 15.5 cm × 25 μm fused-silica capillaries (n = 8) at 35.5 °C in a pH 10 buffer with online detection at 200 nm. Serum samples with different electrophoretic patterns (n = 265) or potential interference (n = 69) were analyzed and compared with agarose gel electrophoresis (AGE; Hydrasys®-Hyrys®, Hydragel protein(e) 15/30® reagent set; Sebia).Results: CVs were Conclusions:The Capillarys instrument is a reliable CE system for serum protein analysis, combining advantages of full automation (ease of use, bar-code identification, computer-assisted correction of α1-globulins) with high analytical performances and throughput. more...

To prevent acquisition of HIV through oral sex, drugs used for preexposure prophylaxis (Prep) need to diffuse in saliva. We measured tenofovir (TFV) and emtricitabine (FTC) concentrations simultaneously in the plasma and saliva of 41 HIV-infected patients under stable antiretroviral treatment. Mean ratios of saliva/plasma concentration were 3% (±4%) and 86.9% (±124%) for TFV and FTC, respectively. Tenofovir disoproxil fumarate (TDF) should be used in combination with FTC to prevent oral acquisition of HIV. more...

We here describe an ion‐exchange high‐performance liquid chromatography technique with electrochemical detection for rapid quantification of glutathione, homocysteine, cysteinylglycine, and methionine. The analytical validation of the technique showed within‐assay and between‐assay coefficients of variation between 3.1 and 4.3%, and 3.7 and 8.6%, respectively. Percentages of recovery for overload and dilution tests were between 87 and 120%. Detection limits were 1 μmol/L for methionine and 0.5 μmol/L for other compounds. There was no interference with any physiological and pharmacological substances possessing a thiol function. Aminothiol concentrations determined in 100 control subjects (50 women and 50 men) showed no age‐ or sex‐rated differences for except for homocysteine which was increased (+ 28%) in oldest subjects of both sexes. In 60 patients at risk (30 with chronic renal failure, 30 with diabetes), homocysteine concentration was significantly increased. No variation in other aminothiols was observed in diabetic subjects. Methionine was decreased and cysteinylglycine was increased in patients with chronic renal failure. The present technique—rapid, easy to use, and reliable—appears suitable for routine application in the exploration of aminothiol metabolic pathways including mechanisms of hyperhomocysteinemia. J. Clin. Lab. Anal. 15:144–153, 2001. © 2001 Wiley‐Liss, Inc. more...

Stored plasma specimens from 164 participants in the ANRS 138 trial were analyzed to determine interleukin 6 (IL-6), high-sensitivity C-reactive protein (hsCRP), and D-dimer levels at baseline and weeks 24 and 48. These virologically suppressed, treatment-experienced patients were randomly assigned to undergo an immediate switch (IS) or a deferred switch (DS; at week 24) from an enfuvirtide-based antiretroviral therapy (ART) regimen to a raltegravir-based regimen. At week 24, a significant decrease from baseline was observed in the IS arm, compared with the DS arm, for IL-6 level (�30% vs +10%; P < .002), hsCRP level (�46% vs +15%; P < .0001), and D-dimer level (�40% vs +6%; P < .0001). At week 48, there was a reproducible decrease in levels of all biomarkers in the DS arm. more...

Vitamin A status in a sample of pregnant and lactating women living in several representative regions of Congo was assessed and compared between August and September 2004. This survey was conducted using a randomized two-stage cluster-sampling method with stratification on 90 clusters, each consisting of at least 15 women. Vitamin A status was determined in a total of 1,054 individuals, using the impression cytology with transfer (ICT) test, the modified relative dose response test (MRDR test) on dried blood spots (DBS), and clinical examination to detect signs of xerophthalmia. The clinical criterion defining vitamin A deficiency was the presence of active xerophthalmia (Bitot's spots [X1B]), active corneal disease), and/or night blindness (XN stage). The prevalence of clinical signs of stage XN and X1B xerophthalmia in the Republic of Congo was found to be 16% and 19% respectively. The prevalence of clinical signs (X1B) was greater in the rural north than in urban areas, with a gradient running from urban (5%) to rural area (33%); 27% of all the ICT tests showed that the subjects were suffering from vitamin A deficiency. The deficiency rates were significantly higher (p0.001) in urban surroundings (Brazzaville) than in the rural northern regions. The biochemical MRDR test showed the presence of vitamin A deficiency (or = 0.06) in 26% of the mothers in Brazzaville compared to 6% in the town of Kouilou; 44% of the women had retinol levels of10 microg/dL in the rural north whereas these percentages were significantly lower in the urban areas surveyed (chi-square = 62.30, p0.001). A significant correlation was found to exist (p0.001) between the ICT test and the MRDR test on DBS. In the population as a whole, 30% of the mothers suffering from malarial attack had abnormally low MRDR levels (or = 0.06) compared to no malaria. The results of the present study confirm that vitamin A deficiency is a serious public-health issue in pregnant and lactating mothers in the Republic of Congo. more...

Blood disorders caused by abnormal β-globin — β-thalassaemia and sickle cell disease — are the most prevalent inherited disorders worldwide, with patients often remaining dependent on blood transfusions throughout their lives. So a report of the successful use of gene therapy in a case of severe β-thalassaemia — using a lentiviral vector expressing the β-globin gene — is an eagerly awaited event. More than two years after gene transfer, the adult male patient has been transfusion-independent for 21 months. The therapeutic benefit seems to result from a dominant, myeloid-biased cell clone that may remain benign, although it could yet develop into leukaemia — a reminder that gene therapy is still at an early stage. Disorders caused by abnormal β-globin, such as β-thalassaemia, are the most prevalent inherited disorders worldwide. For treatment, many patients are dependent on blood transfusions; thus far the only cure has involved matched transplantation of haematopoietic stem cells. Here it is shown that lentiviral β-globin gene transfer can be an effective substitute for regular transfusions in a patient with severe β-thalassaemia. The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas1,2. The βE-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated βE-globin with partial instability1,2. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent1,2. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe βE/β0-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl−1, of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells. more...

International audience; Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid-induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells. more...

Differentiation therapy for acute myeloid leukemia uses transcriptional modulators to reprogram cancer cells. The most relevant clinical example is acute promyelocytic leukemia (APL), which responds dramatically to either retinoic acid (RA) or arsenic trioxide (As(2)O(3)). In many myeloid leukemia cell lines, cyclic adenosine monophosphate (cAMP) triggers growth arrest, cell death, or differentiation, often in synergy with RA. Nevertheless, the toxicity of cAMP derivatives and lack of suitable models has hampered trials designed to assess the in vivo relevance of theses observations. We show that, in an APL cell line, cAMP analogs blocked cell growth and unraveled As(2)O(3)-triggered differentiation. Similarly, in RA-sensitive or RA-resistant mouse models of APL, continuous infusions of 8-chloro-cyclic adenosine monophosphate (8-Cl-cAMP) triggered major growth arrest, greatly enhanced both spontaneous and RA- or As(2)O(3)-induced differentiation and accelerated the restoration of normal hematopoiesis. Theophylline, a well-tolerated phosphodiesterase inhibitor which stabilizes endogenous cAMP, also impaired APL growth and enhanced spontaneous or As(2)O(3)-triggered cell differentiation in vivo. Accordingly, in an APL patient resistant to combined RA-As(2)O(3) therapy, theophylline induced blast clearance and restored normal hematopoiesis. Taken together, these results demonstrate that in vivo activation of cAMP signaling contributes to APL clearance, independently of its RA-sensitivity, thus raising hopes that other myeloid leukemias may benefit from this therapeutic approach. more...

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P more...

Cytosine arabinoside (Ara-C) is widely used to induce remission in adult granulocytic leukemia. High doses can be infused in refractory leukemia or in relapse. After injection, Ara-C is quickly metabolized to uracil arabinoside (Ara-U), the main inactive metabolite. We here described a micellar electrokinetic capillary chromatography (MECC) method to simultaneously determine Ara-C/Ara-U in human serum using 6- O -methylguanine as an internal standard. The assay was linear from 6.25 to 200 μg/ml with a quantification limit between 3 and 6 μg/ml. The analytical precision was satisfactory between 2 and 4.3% (within-run) and 3.7 and 7.3% (between-runs). This assay was applied to the analysis of serum from acute granulocytic leukemia patient treated by high doses cytarabine (3 g/m 2 body surface). more...

A sensitive gas chromatographic (GC)/nitrogen phosphorus detection (NPD) system was developed for the determination of the antitumor drug ifosfamide (Ifos) and its 2-dechloroethylifosfamide (2-Difos), 3-dechloroethylifosfamide (3-Difos) and 4-hydroxyifosfamide (4-OHIfos) metabolites in human blood. 4-OHIfos was analyzed after coupling with a trapping agent and was used as an indicator of isophosphoramide mustard (IPM). Ifos and its metabolites 2-DIfos, 3-DIfos, 4-OHIfos and the internal standard (trofosfamide) were extracted into chloroform and then resolved by gas chromatography using a Hewlett Packard HP5 capillary column cross-linked with 5% phenyl methyl silicone (30 m; 530 microm I.D.; 2.65 microm film thickness). Precision and accuracy of the assay were determined over a three-day period and a concentration range of 3.25-50 microg/ml for Ifos, 0.8-14 microg/ml for 2D-Ifos, 0.6-10 microg/ml for 3D-Ifos and 0.08-1.40 microg/ml for 4-OHIfos. The limit of quantitation was set at 3.25, 0.80, 0.62 and 0.08 microg/ml, respectively, for Ifos, 2-DIfos, 3-DIfos and 4-OHIfos. The intra- and inter-day coefficients of variation and accuracies were less than 20%, except for a low concentration 4-OHIfos. This assay was then used to provide pharmacokinetic data on antitumor and toxicologic effects following intravenous infusion of Ifos. more...

Dimethyl sulfoxide (DMSO) is a chemical compound that is used to preserve haematopoietic stem cells during freezing at -180 degrees C. As DMSO is largely removed by washing before reinjection of cells into a patient, accidents (notably cardiovascular) are infrequent. The lack of a method for evaluating the residual quantities of this product led us to develop a technique for assaying DMSO by capillary zone electrophoresis without extraction. This simple, rapid and precise technique was applied to the supernatant of cell pellets of thirteen patients before and after washing. more...

Corrigendum: Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation

Simultaneous determination of progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone (DHEA) and 17-hydroxyprogesterone has been developed for human cerebral tissue. Before immunoassay, steroids were separated on a Celite column with propylene glycol as stationary phase with hexane containing increasing proportions of dichloromethane as mobile phase. This system allowed separation of steroids of similar polarity, especially of pregnenolone and progesterone. The brain regions studied cortex (prefrontal, parietal and temporal), cerebellum and corpus callosum, were obtained after autopsy from 9 women and 1 man between 76 and 93 years of age. Steroids were found in all regions. The overall concentrations expressed in nmol/kg of tissue were: 10.1, 7.6, 120.7, 19.6 and 10.4 respectively, for progesterone, androst-4-enedione, pregnenolone, dehydroepiandrosterone and 17-hydroxyprogesterone, corresponding to 7.3, 4.9, 74, 6.5 and 9.2 times the plasma levels. These very high concentrations, not previously described in human brain tissue, pose the question of the existence of local biosynthetic pathways independent of the peripheral endocrine gland system as well as that of progressive accumulation of steroids over a lifetime. Concentrations of each steroid in each subject varied little among the various brain regions studied, but there was much variation among the subjects with respect to the concentrations of a given steroid. more...

beta-Blockers are used as if they were equivalent. With ECG recordings in 42 patients we investigated the effect on sinus heart rate of four beta-blockers given at three successive daily doses. Heart rate was dose-dependently decreased by all drugs except acebutolol, the effect of which decreased at a higher dosage. The maximal effects of metoprolol, nadolol, and propranolol were similar but the drugs differed in potency (dosage producing 50% of maximal effect, calculated from the dose-effect relationships; nadolol, 0.3 mg/day; metoprolol, 120 mg/day; propranolol, 47 mg/day). Similar relationships were found with drug plasma concentrations (concentration producing 50% of maximal effect: nadolol, 3.5 ng/ml; metoprolol, 21 ng/ml; propranolol, 36 ng/ml) and with supine or upright heart rates and blood pressures. However, the drugs were not equivalent: In addition to its greater potency, nadolol differed from propranolol and metoprolol in the slope of its dose-response curve. We conclude that beta-blockers can be compared by ECG recordings and that nadolol is different from the other beta-blockers without intrinsic sympathomimetic activity. more...