Quantification of 4 antidepressants and a metabolite by LC-MS for therapeutic drug monitoring.

A liquid chromatography method coupled to mass spectrometry was developed for the quantification of bupropion, its metabolite hydroxy-bupropion, moclobemide, reboxetine and trazodone in human plasma. The validation of the analytical procedure was assessed according to Société Française des Sciences et Techniques Pharmaceutiques and the latest Food and Drug Administration guidelines. The sample preparation was performed with 0.5mL of plasma extracted on a cation-exchange solid phase 96-well plate. The separation was achieved in 14min on a C18 XBridge column (2.1mm×100mm, 3.5μm) using a 50mM ammonium acetate pH 9/acetonitrile mobile phase in gradient mode. The compounds of interest were analysed in the single ion monitoring mode on a single quadrupole mass spectrometer working in positive electrospray ionisation mode. Two ions were selected per molecule to increase the number of identification points and to avoid as much as possible any false positives. Since selectivity is always a critical point for routine therapeutic drug monitoring, more than sixty common comedications for the psychiatric population were tested. For each analyte, the analytical procedure was validated to cover the common range of concentrations measured in plasma samples: 1-400ng/mL for reboxetine and bupropion, 2-2000ng/mL for hydroxy-bupropion, moclobemide, and trazodone. For all investigated compounds, reliable performance in terms of accuracy, precision, trueness, recovery, selectivity and stability was obtained. One year after its implementation in a routine process, this method demonstrated a high robustness with accurate values over the wide concentration range commonly observed among a psychiatric population.

Single-dose oral amoxicillin or linezolid for prophylaxis of experimental endocarditis due to vancomycin-susceptible and vancomycin-resistant Enterococcus faecalis.

Endocarditis prophylaxis following genitourinary or gastrointestinal procedures targets Enterococcus faecalis. Prophylaxis recommendations advocate oral amoxicillin (2 g in the United States and 3 g in the United Kingdom) in moderate-risk patients and intravenous amoxicillin (2 g) or vancomycin (1 g) plus gentamicin in high-risk patients. While ampicillin-resistant (or amoxicillin-resistant) E. faecalis is still rare, there is a concern that these regimens might fail against vancomycin-resistant and/or aminoglycoside-resistant isolates. The present study tested oral linezolid as an alternative. Rats with catheter-induced aortic vegetations were given prophylaxis simulating human pharmacokinetics of oral amoxicillin (2- to 3-g single dose), oral linezolid (600 mg, single or multiple oral doses every 12 h), or intravenous vancomycin (1-g single dose). Rats were then inoculated with the minimum inoculum infecting 90% of the animals (90% infective dose [ID(90)]) or with 10 times the ID(90) of the vancomycin-susceptible E. faecalis strain JH2-2 or the vancomycin-resistant (VanA phenotype) E. faecalis strain UCN41. Amoxicillin was also tested with two additional vancomycin-susceptible E. faecalis strains, 309 and 1209. Animals were sacrificed 3 days later. All the tested bacteria were susceptible to amoxicillin and gentamicin. Single-dose amoxicillin provided 100% protection against all four isolates at both the ID(90) and 10 times the ID(90). In contrast, linezolid required up to four consecutive doses to provide full protection against the vancomycin-resistant isolate. Vancomycin protected only against the vancomycin-susceptible strain. The high efficacy of single-dose oral amoxicillin suggests that this regimen could be used for prophylaxis in both moderate-risk and high-risk patients without additional aminoglycosides. Linezolid appears to be less reliable, at least against the vancomycin-resistant strain.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Efficacy of daptomycin in the treatment of experimental endocarditis due to susceptible and multidrug-resistant enterococci.

OBJECTIVES: Daptomycin was tested in vitro and in rats with experimental endocarditis against the ampicillin-susceptible and vancomycin-susceptible Enterococcus faecalis JH2-2, the vancomycin-resistant (VanA type) mutant of strain JH2-2 (strain JH2-2/pIP819), and the ampicillin-resistant and vancomycin-resistant (VanB type) Enterococcus faecium D366. METHODS: Rats with catheter-induced aortic vegetations were treated with doses simulating intravenously kinetics in humans of daptomycin (6 mg/kg every 24 h), amoxicillin (2 g every 6 h), vancomycin (1 g every 12 h) or teicoplanin (12 mg/kg every 12 h). Treatment was started 16 h post-inoculation and continued for 2 days. RESULTS: MICs of daptomycin were 1, 1 and 2 mg/L, respectively, for strains JH2-2, JH2-2/pIP819 and D366. In time-kill studies, daptomycin showed rapid (within 2 h) bactericidal activity against all strains. Daptomycin was highly bound to rat serum proteins (89%). In the presence of 50% rat serum, simulating free concentrations, daptomycin killing was maintained but delayed (6-24 h). In vivo, daptomycin treatment resulted in 10 of 12 (83%), 9 of 11 (82%) and 11 of 12 (91%) culture-negative vegetations in rats infected with strains JH2-2, JH2-2/pIP819 and D366, respectively (P < 0.001 compared to controls). Daptomycin efficacy was comparable to that of amoxicillin and vancomycin for susceptible isolates. Daptomycin, however, was significantly (P < 0.05) more effective than teicoplanin against the glycopeptide-susceptible strain JH2-2 and superior to all comparators against resistant isolates. CONCLUSIONS: These results support the use of the newly proposed daptomycin dose of 6 mg/kg every 24 h for treatment of enterococcal infections in humans.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants' maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.

Transcriptional regulation of MDR1, a gene involved in antifungal drug resistance in Candida albicans

ABSTRACT Upregulation of the Major Facilitator transporter gene MDR1 (Multi_drug Resistance 1) is one of the mechanisms observed in Candida albicans clinical isolates developing resistance to azole antifungal agents. To better understand this phenomenon, the cis-acting regulatory elements present in a modulatable reporter system under the control of the MDR1 promoter were characterized. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H2O2, whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements, that are necessary and sufficient to convey the same transcriptional responses to a heterologous promoter (CDR2), were identified within the MDR1promoter. The first element, called BRE (for Benomyl Response Element, -296 to -260 with respect to the ATG start codon), is required for benomyl-dependent MDR1 upregulation and for constitutive high expression of MDR1 in FR2. The second element, termed HRE (for H2O2 Response Element, -561 to -520), is required for H2O2-dependent MDR1 upregulation, but is dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the blip transcription factor Cap1p lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H2O2 and diminished significantly the transient response to benomyl. Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a transacting and positive regulatory role in benomyl- and H2O2-dependent transcription of MDR1. However, it is not the only transcription factor involved in the response of MDR1 to benomyl. A minimal BRE element (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also delimited. Genome-wide transcript profiling analyses undertaken with a matched pair of clinical isolates, one of which being azole-resistant and upregulating MDR1, and with an azole-susceptible strain exposed to benomyl, revealed that genes specifically upregulated by benomyl harbour in their promoters Cap1p binding site(s). This strengthened the idea that Cap1p plays a role in benomyl-dependent upregulation of MDR1. BRE-like sequences were also identified in several genes co-regulated with MDR1 in both conditions, which was consistent with the involvement of the BRE in both processes. A set of 147 mutants lacking a single transcription factor gene was next screened for loss of MDR1response to benomyl. Unfortunately, none of the tested mutants showed a loss of benomyl-dependent MDR1 upregulation. Nevertheless, a significant diminution of the response was observed in the mutants in which the MADS-box transcription factor Mcm1p and the C2H2 zinc finger transcription factor orf19.13374p were inactivated, suggesting that Mcm1p and orf19.13374p are involved in MDR1response to benomyl. Interestingly, the BRE contains a perfect match to the binding consensus of Mcm1p, raising the possibility that MDR1may be a direct target of this transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools we have developed during characterization of the cis-acting elements of the MDR1promoter should now serve to elucidate the nature of the components that modulate its activity. RESUME La surexpression du gène MDR1 (pour Résistance Multidrogue 1), qui code pour un transporteur de la famille des Major Facilitators, est l'un des mécanismes observés dans les isolats cliniques de la levure Candida albicans développant une résistance aux agents antifongiques appelés azoles. Pour mieux comprendre ce phénomène, les éléments de régulation agissant en cis dans un système rapporteur modulable sous le contrôle du promoteur MDR1 ont été caractérisés. Dans une souche sensible aux azoles, la transcription de ce rapporteur est transitoirement surélevée en réponse soit au bénomyl soit à l'agent oxydant H2O2, alors que son expression est constitutivement élevée dans une souche résistante aux azoles (souche FR2). Deux éléments de régulation agissant en cis, nécessaires et suffisants pour transmettre les mêmes réponses transcriptionnelles à un promoteur hétérologue (CDR2), ont été identifiés dans le promoteur MDR1. Le premier élément, appelé BRE (pour Elément de Réponse au Bénomyl, de -296 à -260 par rapport au codon d'initiation ATG) est requis pour la surexpression de MDR1dépendante du bénomyl et pour l'expression constitutive de MDR1 dans FR2. Le deuxième élément, appelé HRE (pour Elément de Réponse à l'H2O2, de -561 à -520), est requis pour la surexpression de MDR1 dépendante de l'H2O2, mais n'est pas impliqué dans l'expression constitutive du gène MDR1. Deux sites de fixation potentiels (TTAG/CTAA) pour le facteur de transcription Cap1p ont été identifiés dans l'élément HRE. De plus, l'inactivation de CAP1 abolit la réponse transitoire à l'H2O2 et diminua significativement la réponse transitoire au bénomyl. Cap1p, qui est impliqué dans les réponses de la cellule au stress oxydatif, doit donc jouer un rôle positif en trans dans la surexpression de MDR1 dépendante du bénomyl et de l'H2O2. Cependant, ce n'est pas le seul facteur de transcription impliqué dans la réponse au bénomyl. Un élément BRE d'une longueur minimale (de -290 à -273) a également été défini et est suffisant pour détecter une interaction spécifique in vitro avec des protéines provenant d'extraits bruts de C. albicans. L'analyse du profil de transcription d'une paire d'isolats cliniques comprenant une souche résistante aux azoles surexprimant MDR1, et d'une souche sensible aux azoles exposée au bénomyl, a révélé que les gènes spécifiquement surexprimés par le bénomyl contiennent dans leurs promoteurs un ou plusieurs sites de fixation pour Cap1p. Ceci renforce l'idée que Cap1p joue un rôle dans la surexpression de MDR1dépendante du bénomyl. Une ou deux séquences ressemblant à l'élément BRE ont également été identifiées dans la plupart des gènes corégulés avec MDR1 dans ces deux conditions, ce qui était attendu compte-tenu du rôle joué par cet élément dans les deux processus. Une collection de 147 mutants dans lesquels un seul facteur de transcription est inactivé a été testée pour la perte de réponse au bénomyl de MDR1. Malheureusement, la surexpression de MDR1 dépendante du bénomyl n'a été perdue dans aucun des mutants testés. Néanmoins, une diminution significative de la réponse a été observée chez des mutants dans lesquels le facteur de transcription à MADS-box Mcm1p et le facteur de transcription à doigts de zinc de type C2H2 orf19.13374p ont été inactivés, suggérant que Mcm1p et orf19.13374p sont impliqués dans la réponse de MDR1au bénomyl. Il est intéressant de noter que la BRE contient une séquence qui s'aligne parfaitement avec la séquence consensus du site de fixation de Mcm1p, ce qui soulève la possibilité que MDR1 pourrait être une cible directe de cet activateur transcriptionnel. En conclusion, alors que l'identité des facteurs agissant en trans en se fixant à la BRE et à la HRE reste à être confirmée, les outils que nous avons développés au cours de la caractérisation des éléments agissant en cis sur le promoteur MDR1 peut maintenant servir à élucider la nature des composants modulant son activité.

Prescription des psychotropes au cabinet médical [Psychotropic drug prescription in general practice].

General practitioners treat patients with psychiatric disorders, for whom they have to evaluate the indication of a psychotropic medication. In addition to the patient's symptoms, the clinician has to take into account transferential and countertransferential elements linked to the prescription. Sociological factors also influence both the patient and the clinician, partly due to the western society's value of performance. Consistent with the bio-psycho-social model of disease, we recommend that the evaluation of the indication of a psychotropic medication includes the patient's symptoms, but also the psychological and sociological factors.

Profiles of drug users in Switzerland and effects of early-onset intensive use of alcohol, tobacco and cannabis on other illicit drug use.

QUESTIONS UNDER STUDY / PRINCIPLES: The main aim of this study was to investigate profiles of drug users, with a particular focus on illicit drugs other than cannabis, and to explore the effect of early-onset intensive use (drunkenness, daily smoking, high on cannabis) on profiles of illicit drug use. METHODS: Baseline data from a representative sample of 5,831 young Swiss men in the ongoing Cohort Study on Substance Use Risk Factors were used. Substance use (alcohol, tobacco, cannabis and 15 types of other illicit drug) and age of onset of intensive use were assessed. The Item Response Theory (IRT) and prevalence rates at different ages of onset were used to reveal different profiles of illicit drug use. RESULTS: In addition to cannabis, there were two profiles of other illicit drug use: (a) "softer" drug users (uppers, hallucinogens and inhaled drugs), among which ecstasy had the highest discriminatory potential (IRT slope = 4.68, standard error (SE) = 0.48; p <0.001); and (b) "harder" drug users (heroin, ketamine, gamma-hydroxybutyrate/gamma-hydroxylactone, research chemicals, crystal meth and spice), among which ketamine had the highest discriminatory potential (slope = 4.05; SE = 0.63; p <0.001). Onset of intensive use at the age of 12 years or younger also discriminated between these two profiles. CONCLUSION: Both the IRT model and the effect of onset of intensive use enabled two groups of illicit drugs to be identified. In particular, very early onset (at 12 years or younger) intensive use of any substance was a marker for later use of the second group of drugs.

Correlations between imatinib pharmacokinetics, pharmacodynamics, adherence, and clinical response in advanced metastatic gastrointestinal stromal tumor (GIST): An emerging role for drug blood level testing?

Imatinib is the standard of care for patients with advanced metastatic gastrointestinal stromal tumors (GIST), and is also approved for adjuvant treatment in patients at substantial risk of relapse. Studies have shown that maximizing benefit from imatinib depends on long-term administration at recommended doses. Pharmacokinetic (PK) and pharmacodynamic factors, adherence, and drug-drug interactions can affect exposure to imatinib and impact clinical outcomes. This article reviews the relevance of these factors to imatinib's clinical activity and response in the context of what has been demonstrated in chronic myelogenous leukemia (CML), and in light of new data correlating imatinib exposure to response in patients with GIST. Because of the wide inter-patient variability in drug exposure with imatinib in both CML and GIST, blood level testing (BLT) may play a role in investigating instances of suboptimal response, unusually severe toxicities, drug-drug interactions, and suspected non-adherence. Published clinical data in CML and in GIST were considered, including data from a PK substudy of the B2222 trial correlating imatinib blood levels with clinical responses in patients with GIST. Imatinib trough plasma levels <1100ng/mL were associated with lower rates of objective response and faster development of progressive disease in patients with GIST. These findings have been supported by other analyses correlating free imatinib (unbound) levels with response. These results suggest a future application for imatinib BLT in predicting and optimizing therapeutic response. Nevertheless, early estimates of threshold imatinib blood levels must be confirmed prospectively in future studies and elaborated for different patient subgroups.

Personalized modeling for drug concentration prediction using Support Vector Machine

Building a personalized model to describe the drug concentration inside the human body for each patient is highly important to the clinical practice and demanding to the modeling tools. Instead of using traditional explicit methods, in this paper we propose a machine learning approach to describe the relation between the drug concentration and patients' features. Machine learning has been largely applied to analyze data in various domains, but it is still new to personalized medicine, especially dose individualization. We focus mainly on the prediction of the drug concentrations as well as the analysis of different features' influence. Models are built based on Support Vector Machine and the prediction results are compared with the traditional analytical models.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

Current drug discovery strategies against arenavirus infections.

Arenaviruses are a large group of emerging viruses including several causative agents of severe hemorrhagic fevers with high mortality in man. Considering the number of people affected and the currently limited therapeutic options, novel efficacious therapeutics against arenaviruses are urgently needed. Over the past decade, significant advances in knowledge about the basic virology of arenaviruses have been accompanied by the development of novel therapeutics targeting different steps of the arenaviral life cycle. High-throughput, small-molecule screens identified potent and broadly active inhibitors of arenavirus entry that were instrumental for the dissection of unique features of arenavirus fusion. Novel inhibitors of arenavirus replication have been successfully tested in animal models and hold promise for application in humans. Late in the arenavirus life cycle, the proteolytic processing of the arenavirus envelope glycoprotein precursor and cellular factors critically involved virion assembly and budding provide further promising 'druggable' targets for novel therapeutics to combat human arenavirus infection.

Tigecycline in combination with other antimicrobials: a review of in vitro, animal and case report studies.

Tigecycline has been investigated in combination with other antibacterials against a wide range of susceptible and multiresistant Gram-positive and Gram-negative bacteria. Combinations have been analysed in vitro, in animal models and in human case reports. In vitro, tigecycline combined with other antimicrobials produces primarily an indifferent response (neither synergy nor antagonism). Nevertheless, synergy occurred when tigecycline was combined with rifampicin against 64-100% of Enterococcus spp., Streptococcus pneumoniae, Enterobacter spp. and Brucella melitensis isolates. Combinations of tigecycline with amikacin also showed synergy for 40-100% of Enterobacter spp., Klebsiella pneumoniae, Proteus spp. and Stenotrophomonas maltophilia isolates. Moreover, bactericidal synergisms occurred with tigecycline plus amikacin against problematic Acinetobacter baumannii and Proteus vulgaris, and with colistin against K. pneumoniae. Data from animal experiments and case reports, although limited, displayed consistent beneficial activity of tigecycline in combination with other antibacterials against multiresistant organisms, including vancomycin against penicillin-resistant S. pneumoniae in experimental meningitis, gentamicin against Pseudomonas aeruginosa in experimental pneumonia, daptomycin against Enterococcus faecium endocarditis, and colistin against K. pneumoniae bacteraemia and P. aeruginosa osteomyelitis. Antagonism was extremely rare in vitro and was not reported in vivo. Thus, tigecycline may be combined with a second antimicrobial as part of a combination regimen.