Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study.

BACKGROUND:HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS:In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION:The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues.

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Replacement of (R)-methadone by a double dose of (R,S)-methadone in addicts: interindividual variability of the (R)/(S) ratios and evidence of adaptive changes in methadone pharmacokinetics.

METHODS: Twenty-two patients receiving (R)-methadone maintenance treatment were switched to a double dose of (R,S)-methadone: blood samples were collected before and after the change, and the concentrations of the enantiomers were measured. In the second period, during racemic methadone treatment, important interindividual variability in the stereoselective disposition of the enantiomers of methadone was measured, with (R)/(S) ratios ranging from 0.63 to 2.40. This point should be taken into account particularly with respect to therapeutic drug monitoring of racemic methadone. RESULTS: A significant decrease P < 0.005 in the mean serum concentration/dose ratios of the active (R)-enantiomer before and after the change was measured (mean 3.97 and 3.33). CONCLUSION: Although of small amplitude (16%), this decrease confirms previously described adaptive changes in methadone pharmacokinetics during racemic methadone maintenance treatment and may necessitate, in some patients, a dose adjustment.

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m². The mean actual applied dose in Estancia Salazar was 11.2 ± 3.1 mg a.i./m² in walls and 11.9 ± 5.6 mg a.i./m² in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 ± 6.9 mg a.i./m² and 34.7 ± 10.4 mg a.i./m² in walls and 28.8 ± 19.2 mg a.i./m² and 24.9 ± 21.8 mg a.i./m² in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying,

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N′-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N′-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Delta-9-tetrahydrocannabinol accumulation, metabolism and cell-type-specific adverse effects in aggregating brain cell cultures.

Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type difference was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 microM in single treatment and of 1 microM and 2 microM in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 microM of THC or JWH 015, whereas the expression of TNF-alpha remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation.

Benznidazole levels in blood vary with age in rats

Benznidazole (Bz) exhibits toxic side effects in animal studies and clinical use. Reductive metabolism of Bz in liver microsomes modulates the duration of its chemotherapeutic effect and its toxicity. The rate of this metabolism depends on age and is less intense in newborns and youngsters than in adults. In the present study, we determined Bz blood levels in rats of different ages that received Bz intragastrically (100 mg/kg). We developed and validated a high-pressure liquid chromatography with UV detector method for determination of Bz levels in whole blood. Bz levels were significantly higher and persisted for longer periods of time in the blood of young rats when compared to that of adult animals.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests: a study in 9 Swiss laboratories.

INTRODUCTION: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43μg/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgens in humans.

Carbon isotope ratio of androgens in urine specimens is routinely determined to exclude an abuse of testosterone or testosterone prohormones by athletes. Increasing application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in the last years for target and systematic investigations on samples has resulted in the demand for rapid sample throughput as well as high selectivity in the extraction process particularly in the case of conspicuous samples. For that purpose, we present herein the complimentary use of an SPE-based assay and an HPLC fractionation method as a two-stage strategy for the isolation of testosterone metabolites and endogenous reference compounds prior to GC/C/IRMS analyses. Assays validation demonstrated acceptable performance in terms of intermediate precision (range: 0.1-0.4 per thousand) and Bland-Altman analyses revealed no significant bias (0.2 per thousand). For further validation of this two-stage analyses strategy, all the specimens (n=124) collected during a major sport event were processed.

Identification and in vitro reactivity of celiac immunoactive peptides in an apparent gluten-free beer.

Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific anti-gluten peptide antibody. However, in beverages or food suffering complex hydrolytic processes as beers, the relative proportion of reactive peptides for celiac patients and the analytical techniques may differ, because of the diversity of the resulting peptide populations after fermentations. A beer below 20 parts per million of gluten but yet detectable levels of gluten peptides by anti-gliadin 33-mer antibodies (G12 and A1) was analyzed. We identified and characterized the relevant peptides for either antibody recognition or immunoactivity in celiac patients. The beer was fractionated by HPLC. The relative reactivity of the different HPLC fractions to the G12/A1 antibodies correlated to the reactivity of peripheral blood mononuclear cells isolated from 14 celiac individuals. Peptides from representative fractions classified according to the relative reactivity to G12/A1 antibodies were identified by mass spectrometry. The beer peptides containing sequences with similarity to those of previously described G12 and A1 epitopes were synthesized and confirmed significant reactivity for the antibodies. The most reactive peptides for G12/A1 also confirmed the highest immunogenicity by peripheral blood mononuclear cell activation and interferon γ production from celiac patients. We concluded that preparative HPLC combined with anti-gliadin 33-mer G12/A1 antibodies were very sensitive and specific methods to analyze the relevant immunogenic peptides in hydrolyzed gluten.

Rapid profiling of intact glucosinolates in Arabidopsis leaves by UHPLC-QTOFMS using a charged surface hybrid column.

INTRODUCTION: The analysis of glucosinolates (GS) is traditionally performed by reverse-phase liquid chromatography coupled to ultraviolet detection after a time-consuming desulphation step, which is required for increased retention. Simpler and more efficient alternative methods that can shorten both sample preparation and analysis are much needed. OBJECTIVE: To evaluate the feasibility of using ultrahigh-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) for the rapid profiling of intact GS. METHODOLOGY: A simple and short extraction of GS from Arabidopsis thaliana leaves was developed. Four sub-2 µm reverse-phase columns were tested for the rapid separation of these polar compounds using formic acid as the chromatographic additive. High-resolution QTOFMS was used to detect and identify GS. RESULTS: A novel charged surface hybrid (CSH) column was found to provide excellent retention and separation of GS within a total running time of 11 min. Twenty-one GS could be identified based on their accurate mass as well as isotopic and fragmentation patterns. The method was applied to determine the changes in GS content that occur after herbivory in Arabidopsis. In addition, we evaluated its applicability to the profiling of other Brassicaceae species. CONCLUSION: The method developed can profile the full range of GS, including the most polar ones, in a shorter time than previous methods, and is highly compatible with mass spectrometric detection.

Evaluation of two HbA1c point-of-care analyzers.

BACKGROUND Measurement of HbA1c is the most important parameter to assess glycemic control in diabetic patients. Different point-of-care devices for HbA1c are available. The aim of this study was to evaluate two point-of-care testing (POCT) analyzers (DCA Vantage from Siemens and Afinion from Axis-Shield). We studied the bias and precision as well as interference from carbamylated hemoglobin. METHODS Bias of the POCT analyzers was obtained by measuring 53 blood samples from diabetic patients with a wide range of HbA1c, 4%-14% (20-130 mmol/mol), and comparing the results with those obtained by the laboratory method: HPLC HA 8160 Menarini. Precision was performed by 20 successive determinations of two samples with low 4.2% (22 mmol/mol) and high 9.5% (80 mmol/mol) HbA1c values. The possible interference from carbamylated hemoglobin was studied using 25 samples from patients with chronic renal failure. RESULTS The means of the differences between measurements performed by each POCT analyzer and the laboratory method (95% confidence interval) were: 0.28% (p<0.005) (0.10-0.44) for DCA and 0.27% (p<0.001) (0.19-0.35) for Afinion. Correlation coefficients were: r=0.973 for DCA, and r=0.991 for Afinion. The mean bias observed by using samples from chronic renal failure patients were 0.2 (range -0.4, 0.4) for DCA and 0.2 (-0.2, 0.5) for Afinion. Imprecision results were: CV=3.1% (high HbA1c) and 2.97% (low HbA1c) for DCA, CV=1.95% (high HbA1c) and 2.66% (low HbA1c) for Afinion. CONCLUSIONS Both POCT analyzers for HbA1c show good correlation with the laboratory method and acceptable precision.

Poly-epsilon-caprolactone intravitreous devices: an in vivo study.

PURPOSE: The objective of this study was to evaluate the long-term safety and pharmacokinetic profile of a dexamethasone-loaded poly-epsilon-caprolactone (PCL) intravitreous implant. METHODS: The PCL devices were prepared by compression and were inserted into the vitreous of pigmented rabbits. At different time points, vitreous samples were retrieved, and dexamethasone concentration was analyzed by high-performance liquid chromatography. The biodegradation of the implants was evaluated by scanning electron microscopy, and the dexamethasone remaining was evaluated at the end of follow-up. Clinical and histologic examinations were performed to evaluate the implant's tolerance. RESULTS: The PCL implant allows for a controlled and prolonged delivery of dexamethasone in rabbits eyes since it released the drug within the therapeutic range for at least 55 weeks. At 55 weeks approximately 79% of the drug was still present in the implant. Biodegradation study showed that PCL implants degradation is very slow. Clinical and histologic observations showed that the devices were very well tolerated in the rabbit eye. CONCLUSIONS: This study demonstrates the feasibility and tolerance of intravitreous PCL drug delivery systems, which can offer a wide range of applications for intraocular drug delivery because of their controlled and prolonged release over months or even years.

Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity.

The peptidoglycan of Gram-positive bacteria is known to trigger cytokine release from peripheral blood mononuclear cells (PBMCs). However, it requires 100-1000 times more Gram-positive peptidoglycan than Gram-negative lipopolysaccharide to release the same amounts of cytokines from target cells. Thus, either peptidoglycan is poorly active or only part of it is required for PBMC activation. To test this hypothesis, purified Streptococcus pneumoniae walls were digested with their major autolysin N-acetylmuramoyl-L-alanine amidase, and/or muramidase. Solubilized walls were separated by reverse phase high pressure chromatography. Individual fractions were tested for their PBMC-stimulating activity, and their composition was determined. Soluble components had a Mr between 600 and 1500. These primarily comprised stem peptides cross-linked to various extents. Simple stem peptides (Mr &lt;750) were 10-fold less active than undigested peptidoglycan. In contrast, tripeptides (Mr &gt;1000) were &gt;/=100-fold more potent than the native material. One dipeptide (inactive) and two tripeptides (active) were confirmed by post-source decay analysis. Complex branched peptides represented &lt;/=2% of the total material, but their activity (w/w) was almost equal to that of LPS. This is the first observation suggesting that peptidoglycan stem peptides carry high tumor necrosis factor-stimulating activity. These types of structures are conserved among Gram-positive bacteria and will provide new material to help elucidate the mechanism of peptidoglycan-induced inflammation.