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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.

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 (p < 0.001) 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 of < 10 microg/dL in the rural north whereas these percentages were significantly lower in the urban areas surveyed (chi-square = 62.30, p < 0.001). A significant correlation was found to exist (p < 0.001) 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.

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.

A nationally-representative sample of 2,696 preschool children living in Congo was examined during Au gust-September 2003 to determine the rates of vitamin A deficiency. Ninety clusters of 30 children, aged six months to six years, were selected, using a randomized two-level cluster-sampling method. Vitamin A deficiency was determined by assessing the prevalence of active xerophthalmia (nightblindness and/or Bitot spots) in the cross-over sample of 2,696 individuals. A semi-quantitative seven-day dietary questionnaire was concurrently applied to the mothers of children enrolled to estimate the latter's consumption of vitamin A-rich food. Vitamin A status was assessed by performing the modified relative dose-response test (MRDR) on dried blood spots (DBS) from a subsample of 207 children aged less than six years and the impression cytology with transfer (ICT) test on a subsample of 1,162 children. Of the children enrolled, 5.2% suffered from nightblindness, 8.0% had Bitot spots, and 2.5% had other vitamin A deficiency sequellae. Fifty-three percent of the ICT tests showed the presence of vitamin A deficiency. The biochemical MRDR test showed that the vitamin A status of 30% of the study children was critical. Twenty-seven of them had retinol levels of < 10 microg/dL [mean +/- standard deviation (SD) 7.02 +/- 2.0 microg/dL], and 50% had retinol levels of 10-20 microg/dL (mean +/- SD 14.2 +/- 2.83 microg/dL). The poor health status and low rates of consumption of vitamin A-rich food are the main factors determining critical status. Vitamin A deficiency, reflecting poor nutrition and health, is a serious public-health issue among children aged less than six years in Congo.

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 β(E)/β(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The β(E)-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 β(E)-globin with partial instability. When this is compounded with a non-functional β(0) allele, a profound decrease in β-globin synthesis results, and approximately half of β(E)/β(0)-thalassaemia patients are transfusion-dependent. 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 β(E)/β(0)-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.

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.

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.

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/m2 and the other 10 patients received 3 g/m2. 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.

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.

Objectives: A representative sample of 5722 pre-school children living in rural and urban areas of the Congo was examined between July and September 1999 for assessing Vitamin A deficiency., Methods: Using a randomized two-level cluster sampling method, 190 clusters of 30 children aged from 6 months to 6 years were selected in order to assess the prevalence of active xerophthalmia (night blindness and/or Bitot spots). Concurrently, the children's height and weight were determined. A semi-quantitative seven-day dietary questionnaire was applied to the mothers of 5722 children to estimate the latter's consumption of Vitamin A rich foodstuffs. The prevalence of biochemical deficiency was assessed based on the serum retinol concentrations analyzed in dried blood spots from a sub-sample of 300 children living in the Pointe-Noire area., Results: Among the 5722 children studied, 0.7% were found to suffer from night blindness and 7.7% had Bitot spots. The weekly intake of Vitamin A rich foods was estimated in 5722 children. Our data suggest that Vitamin A rich food consumption was lower in rural zones than in urban area according to the food frequency method threshold values. The serum retinol levels were lower than 10 microg/dl in 18% (95% confidence interval [C.I.]: 13.7, 22.3) [8.04+/-2.87 microg/dl] and less than 20 microg/dl in 49% (95% C.I.: 43.4, 54.6) [15.05+/-2.76 microg/dl] of the 300 studied children. We have established a significant relation between mean serum retinol levels and high rate of Vitamin A food intake (chi-square=59.64, 2 d.d.l., p<0.05) in the sample studied. The mean serum retinol concentrations did not differ significantly between the various Z-scores of weight for age (W/A) and height for age (H/A) patterns. But children with a weight for height (W/H) ratio below -2 standard deviation (S.D.) had significantly lower serum retinol values [9.33+/-1.3 microg/dl] than those with a W/H ratio greater than or equal to -2S.D. [10.82+/-4.84 microg/dl]., Conclusion: These data suggest that Vitamin A deficiency is still a serious public health problem in rural areas of the Congo in which this study was carried out.

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.

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).

In tropical countries. vitamin A deficiency is one of the most important dietary deficiencies. Its monitoring usually involves analysis of retinol after venipuncture with some difficulties (disease transmission, religious belief). Sample collection on Dried Blood Spot (DBS) is less invasive and safer. Sample storage is easier. We developed a liquid chromatography method with electrochemical detection to measure DBS retinol. Retinol acetate was used as an internal standard. The method is linear up to 2.5 microM with a detection limit of 0.04 microM. Precision is below 10% and DBS retinol recovery overage is 90%. DBS retinol concentration decreased during 7 days after sampling, it is necessary to wait this delay before to determine vitamin A concentrations. In Congolese children DBS retinol measurement showed a severe vitamin A deficiency in 8% of them. This percentage is closely correlated with clinical parameters.

The measurement of iodine is a widely accepted method to explore iodine disorders. The most precise estimation is the determination of the urinary iodine in 24-hour collections. Urine collection is notoriously difficult to obtain, specially in children. In these conditions, serum measurement could be a method to overcome these limitations. We describe a colorimetric method adapted on a microtiter plate, with optimized serum mineralization conditions. The method is linear to 2400 nmol/L with a detection limit of 75 nmol/L. Precision is below 10%. The method was validated against one automatic technique. We conclude that this relatively simple method could be an additional tool to explore dysthyroidism.

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 beta1-beta2+ reagent set, proteins were separated at 7 kV for 4 min in 15.5 cm x 25 micro m fused-silica capillaries (n = 8) at 35.5 degrees 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 <3.5% for albumin, <11% for alpha(1)-globulin, <4.1% for alpha(2)-globulin, <7.4% for beta-globulin, and <5.8% for gamma-globulin (3 control levels); measured throughput was 60 samples/h. In patients without paraprotein (n = 116), the median differences between CE and AGE were -5.4 g/L for albumin, 4.0 g/L for alpha(1)-globulin, 0.7 g/L for alpha(2)-globulin, 0.6 g/L for beta-globulin (P <0.001 for all fractions), and -0.1 g/L for gamma-globulin (not significant). More samples had at least one gamma-migrating peak detected by CE (n = 135 vs 130; paraprotein detection limit, approximately 0.5-0.7 g/L), but fewer were quantified (n = 84 vs 91) because of gamma- to beta-migration shifts. There was a 1.2 g/L median difference between CE and AGE for gamma-migrating paraprotein quantification (n = 69; P <0.001). Several ultraviolet-absorbing substances (lipid emulsion, hemoglobin) or molecules (contrast agent, gelatin-based plasma substitute) induced CE artifacts., 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 alpha(1)-globulins) with high analytical performances and throughput.

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.

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<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE., (Copyright 2002 Elsevier Science BV.)

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 microg/ml with a quantification limit between 3 and 6 microg/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/m2 body surface).

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 micromol/L for methionine and 0.5 micromol/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.

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.

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.

Ifosfamide is one of the most efficient antimitotic in soft tissue sarcoma. To try to find a possible dose-effect, 10 patients with advanced pretraited relapsed soft tissue sarcoma received 15 g/m2/cycle ifosfamide in continuous infusion during 5 days. A pharmacokinetic study was done for 2 patients. All patients received growth factors, ondansetron and 8 clonazepam. Renal toxicity was evaluated after the first and the second cycle. Twenty two cycles were delivered to patients who have been already treated with ifosfamid (10 patients with 15 g/m2 to 54 g/m2, median 27 g/m2) or cis platinum (2 patients). No major renal or neurologic toxicity was observed; only subclinical modifications of urinary enzymes excretion were found. Two patients had visual hallucinations at the end of a cycle and just in the 2 following days; another presented a neuropathy of inferior limbs. Hematological toxicity was very limited. Pharmacokinetic study did not show induction mechanism at this dosage and with this type of administration. So ifosfamide 3 g/m2 during 5 days is feasible. The few level of complications observed is perhaps linked to the daily dose of 3 g/m2 instead of 4 g/m2 or more used in the other studies.

Purpose: This study investigated the in vitro pharmacologic behavior and disposition kinetics of all-trans retinoic acid (ATRA) in acute myeloid leukemic (AML) cells, their sensitivity to its differentiating effect, and the in vivo response of acute promyelocytic leukemia (APL) patients after therapy., Patients and Methods: Fresh leukemic cells from 14 AML patients (nine APL and five non-APL), were incubated in suspension culture in the absence or presence of 10(-6) mol/L ATRA. Intracellular ATRA concentration and ATRA metabolism was determined by high-performance liquid chromatography (HPLC)., Results: Immediate uptake is observed with maximal intracellular levels (Cmax) achieved after 24 hours of incubation. At this time, ATRA levels were variable, ranging from 20 to 230 pmol/10(6) cells (median, 100 pmol/10(6) cells). Comparison of ATRA intracellular levels with the in vitro response of patients' cell samples as measured by the percentage of nitro blue tetrazolium (NBT)-positive cells after a 3-day incubation period allowed us to discriminate a group of APL patients (n = 6) with high Cmax (group A; median, 200 pmol/10(6) cells) and maximal differentiation at day 3 (median, 80%), and a group of patients (n = 8, three APL and five non-APL) with low Cmax (group B; median, 35 pmol/10(6) cells) and poor in vitro response (median, 40%; APL cases only). Interestingly, all APL patients, except one included in group A (rapid in vitro ATRA uptakers), achieved a complete remission., Conclusion: These findings suggest that intracellular ATRA concentrations are determinant for ATRA response and should be taken into account when monitoring the efficacy of ATRA differentiation therapeutic trials in malignant disorders.

The diverse effects of all-trans retinoic acid (ATRA) on growth, differentiation and homeostasis of vertebrate organisms are mediated by three distinct isoforms of retinoic acid receptors (RARs). Although it is not known to what extent each RAR contributes to the different effects of ATRA, several studies have demonstrated that ATRA induced granulocytic differentiation in human myeloid leukemic cell lines is mediated by RAR alpha. In this study, we investigated ATRA binding affinity of the endogenous nuclear receptors of HL-60 and NB4 leukemic cells. Scatchard plot analysis yielded an apparent dissociation constant of 5 +/- 0.3 nM and 1400 +/- 80 receptor sites per cell in HL-60 cells, whereas the NB4 promyelocytic leukemic cell line showed a lower affinity (8.5 +/- 0.5 nM and 900 +/- 30 receptor sites per cell). Modulation of RAR alpha protein (5 fold excess) was found in NB4 cells after 24 hours ATRA exposure, whereas HL-60 cells required a 72-hour culture period to weakly increase the RAR alpha protein level. These data were closely related to the ATRA intracellular concentration and kinetics of terminal differentiation of the cells.

All-trans retinoic acid (all-trans RA), the active metabolite of vitamin A, has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL), the AML3 subtype of the FAB cytological classification. Complete remission is obtained by inducing terminal granulocytic differentiation of the leukemic cells. To study all-trans RA pharmacokinetics in patients with APL, a rapid, precise and selective high-performance liquid chromatographic (HPLC) assay was developed. This method is easy and shows good repeatability (C.V. = 8.41-12.44%), reproducibility (C.V. = 9.19-14.73%), accuracy (C.V. = 3.5-11%) and sensitivity with a detection limit of 5 pmol/ml. The analysis is performed using normal-phase HPLC in an isocratic mode with UV detection after solid-phase extraction on octadecyl (C18) columns. The mobile phase is hexane-dichloromethane-dioxane (78:18:4, v/v) containing 1% acetic acid.

All-trans retinoic acid (ATRA) has been demonstrated to be an efficient alternative to chemotherapy in the treatment of acute promyelocytic leukemia (APL or AML3). Complete remission is obtained by inducing granulocytic differentiation of the leukemic cells. To date, the exact mechanism through which ATRA exerts its differentiating effect is not known. The present investigation was initiated to characterize ATRA intracellular concentrations achieved in human myeloid leukemic cells in relation to their different sensitivity to ATRA differentiating effect. During the first 24 h of incubation, a significant decrease of ATRA in the culture medium and a marked increase in the intracellular concentrations were observed. Maximal uptake by the leukemic cells was reached within minutes, with levels between 20 and 260 pmol/10(6) cells (median = 100). Interestingly, a correlation between ATRA-induced differentiation and the intracellular ATRA concentration achieved was observed. In fact, patients with intracellular levels below 60 pmol/10(6) cells defined slow uptakers, never exceeded 40% differentiated cells at day 3. On the other hand, cells with 2-4-fold higher concentration (100-250 pmol/10(6) cells) achieved 100% differentiated cells at day 3. This report suggests that intracellular ATRA concentration is a key pharmacological parameter that should be taken into account to gain further insights into ATRA sensitivity in APL patients.

Acute promyelocytic leukemia (APL), is a homogeneous subgroup of acute myelogenous leukemias characterized by phenotypic and genetic markers. APL is associated with a reciprocal chromosomal translocation t(15,17) which has been shown to disrupt the retinoic acid receptor alpha (RAR alpha) gene. As a result, a portion of the RAR alpha gene becomes fused with a chromosome 15 locus termed PML (promyelocytic myeloid leukemia) from which chimeric PML/RAR alpha fusion mRNAs are expressed. The presence of these fusion transcripts in APL patients strongly support the hypothesis that both the t(15;17), and thus PML/RAR alpha, play a crucial role in the leukemogenesis of this disease. APL cells are specifically responsive to all-trans retinoic acid (ATRA) and this characteristic has allowed the first differentiation therapy with retinoic acid. However, failure or partial responses are observed and, though this has most frequently been reported in patients at second or third relapse. The molecular basis of the absence of ATRA response in these patients has not been determined.

It has been shown that patients with acute promyelocytic leukemia (AML3 subtype) treated with all-trans retinoic acid (all-trans RA), 45 mg/m2/day, achieve complete remission through differentiation of the leukemic clone to mature myeloid cells, which die spontaneously. The pharmacokinetics of all-trans RA given by mouth were studied in 15 AML3 patients. Blood samples were drawn for 24 h following a single oral dose of 45 mg/m2 and assayed for all-trans RA and 13-cis retinoic acid (13-cis RA) plasma concentrations by specific high-performance liquid chromatography. In one patient all-trans RA and 13-cis RA levels were below the detection limits at all times. In the other patients, the time to peak concentration of all-trans RA was between 60 and 210 min (median 90 min) after ingestion, with maximum concentrations between 0.03 and 2.5 micrograms/ml (median 0.4 micrograms/ml). These concentrations were within the in vitro differentiating concentration range of all-trans RA for these patients' cells. In nine patients, enterohepatic cycling was suggested by the presence on the concentration versus time curve of a secondary peak that occurred at meal times. The apparent plasma elimination half-life was between 16.8 and 77.4 min (median 30 min). Detectable plasma levels of 13-cis RA in 12 patients indicated in vivo isomerization of all-trans RA. Despite the high inter-individual variability of all-trans RA pharmacokinetics in these patients, high blast cell counts and failure to respond to differentiation treatment tended to be associated with low all-trans RA Cmax values and high clearance estimates.

We report six women with severe acne lesions associated with taking amineptine, a tricyclic antidepressant. The lesions appeared after self-administration of high doses of the drug over long periods of time. They mainly occurred on the face, back, and thorax, but were also found on the extremities and in the perineal region. In five of the six cases, severity of cutaneous lesions appeared to be correlated with degree of overdose. The sixth patient never admitted having taken amineptine. Most of the patients had been unsuccessfully treated with isotretinoin for 18 months. In all six cases, chromatography of urinary 17-ketosteroids showed abnormal peaks and retention times which were different from those usually found for known steroids. In addition, the areas under these peaks were found to be a function of the degree of intoxication and of the clinical severity of the lesions. Mass spectrometry was used to qualitatively study urinary amineptine metabolites, disclosing compounds normally found only in trace amounts, as well as certain others heretofore not described in man. In two of the three patients who stopped taking amineptine, cutaneous lesions subsequently diminished, totally disappearing in the least severe case.

Antisera against acebutolol were produced in rabbits immunized by means of this drug conjugated with bovine serum albumin. These antisera were used to develop a method of radioimmunoassay for acebutolol. The plasma radioimmunoassay, described here, requires no extraction and is very easy to perform besides being quick, specific and sensitive. As little as 2.97 X 10(-9) mol/l of acebutolol can be detected. This radioimmunoassay is suitable for assaying the large number of samples usually measured in pharmacological studies.

Sperm examinations and/or measurements of plasma FSH, LH and testosterone were performed on 54 patients with Hodgkin's disease before and/or after 3 or 6 courses of multiagent chemotherapy (MOPP). In 24 patients without constitutional symptoms the results of fresh and frozen sperm examination were similar to those of a fertile control population. In 23 patients, pretreatment hormone levels were identical with those of controls. During the first year following completion of chemotherapy all patients had azoospermia and high FSH levels. From the second year onwards, 2 out of 16 patients had normal spermatogenesis (the wife of one of these gave birth to a healthy child), while 4 had grossly deficient spermatogenesis and 10 azoospermia. All male patients suffering from Hodgkin's disease should be asked to give sperm for freezing before chemotherapy and should be informed that spermatogenesis may be resumed from the second posttreatment year.

The distribution of 5 alpha-dihydrotestosterone to the blood following application of a solution of this androgen to the skin in a hydro-alcoholic gel was studied in order to evaluate the adequacy of the percutaneous route in correcting androgen deficiencies. In 14 adult men, daily percutaneous administration of 5 alpha-dihydrotestosterone (125 mg in 5 g gel) increases, on the average, 4 to 5 times its initial concentration in plasma. On the 14th day of treatment, repeated evaluations of plasma 5 alpha-dihydrotestosterone, between 2 and 21 h after final administration of gel, demonstrated the stability of diurnal 5 alpha-dihydrotestosterone levels and showed the regular distribution of the steroid from a presumably cutaneous reservoir. Plasma 5 alpha-androstane-3 alpha,17 beta-diol levels evolve parallel to those of 5 alpha-dihydrotestosterone. Plasma testosterone and luteinizing hormone, on the contrary, decrease considerably. No variation of follicle-stimulating-hormone is observed during treatment. The percutaneous absorption represents an interesting method for administration of a natural androgen in men, particularly because one avoids the deleterious effects of supra-physiological levels in the liver achieved with oral administration.

A radioimmunological method for measuring saliva cortisol was worked out. It includes one extraction and one radiocompetitive reaction with an anticortisol 3-CMO:BSA immunoserum. Frequent values for saliva cortisol were determined in various physiological and pathological conditions and compared to plasma cortisol values. In adult men, the mean concentrations at 8 a.m. and midnight were respectively 14.50 +/- 7.10 and 1.40 +/- 1.21 nmol/l (mean +/- SD). In untreated women, plasma and saliva cortisol levels were not significantly different from those measured in women taking estroprogestative drugs or in pregnant women. Following stimulation by synthetic beta 1-24 ACTH (Synacthen), the saliva cortisol determined 90 min after the 8 a.m. sampling rose by over 500% whereas plasma cortisol only rose by 120%. Similarly the drop in 8 a.m. cortisol, which followed midnight administration of 1 mg of dexamethasone was larger for saliva than for plasma cortisol. In patients with adrenal hyperactivity the circadian rhythm disappeared from saliva cortisol and patients with deficient adrenal activity displayed drastically reduced levels for saliva cortisol. Saliva cortisol seems thus to be more helpful to diagnosis than plasma cortisol.

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.

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.

A simultaneous radioimmunoassay for delta 4-androstenedione (delta 4), dehydroepiandrosterone (DHA) and 11 beta-hydroxyandrostenedione (11 beta OH delta 4) in plasma is described. This involved preparing first an anti-11 beta-hydroxyandrostenedione-3-0-carboxymethyl oxime/BSA antiserum which binds both delta 4 and 11 beta OH delta 4, and an anti-dehydrosterone-7-0-carboxymethyl oxime/BSA antiserum. A chromatographic step using celite minicolumns separates these three steroids. The method was applied to the measurement of the plasma basal values of these three androgens in control subjects. Mean concentrations (ng/ml) of delta 4, DHA and 11 beta OH delta 4 were respecstively 1.35, 6.63 and 3.13 in males; 1.35, 6.65 and 2.59 in premenopausal females; 0.46, 1.53 and 1.38 in post-menopausal females, and 0.39, 0.73 and 1.78 in children 1--6 years of age. Dynamic tests were also carried out: ACTH stimulation was found to increase delta 4, DHA and 11 beta OH delta 4. Dexamethasone had a reverse effect causing a 50% diminution in delta 4 levels, a marked decrease in DHA levels, and a 90% decrease in 11 beta OH delta 4 levels. Metyrapone test was found to produce a 223% increase in delta 4 levels, a 196% increase in DHA levels, and a decrease of more than 90% in the 11 beta OH delta 4 levels. Estroprogestative drug treatment was accompanied by a decrease of not only delta 4, but also of DHA and 11 beta OH delta 4. Preliminary clinical results concerning these steroids show a parallel increase or decrease of delta 4 and 11 beta OH delta 4 in adrenal pathology. In ovarian hyperandrogeny, delta 4 is increased and 11 beta OH delta 4 is unchanged.

We report the effects of sublingual absorption of a single dose (0.5 mg) of oestradiol-17 beta (E2) in 8 post-menopausal women, on plasma E2 and oestrone (E1), urine elimination of total E2 + E1 and on plasma FSH and LH. The results show that sublingual absorption of E2 occurs and that plasma concentrations of E2 obtained (between 133.2 to 320 pmol/l) in this way were higher than those obtained after percutaneous absorption of a single dose (3 mg) of E2. The ratio E1/E2 in plasma is close to that of pre-menopausal women.