Artifacts in the analysis of thioridazine and other neuroleptics.

Although the sensitivity to light of thioridazine and its metabolites has been described, the problem does not seem to be widely acknowledged. Indeed, a survey of the literature shows that assays of these compounds under light-protected conditions have been performed only in a few of the numerous analytical studies on this drug. In the present study, thioridazine, its metabolites, and 18 other neuroleptics were tested for their sensitivity to light under conditions used for their analysis. The results show that light significantly affects the analysis of thioridazine and its metabolites. It readily causes the racemization of the isomeric pairs of thioridazine 5-sulphoxide and greatly decreases the concentration of thioridazine. This sensitivity to light varied with the medium used (most sensitive in acidic media) and also with the molecule (in order of decreasing sensitivity: thioridazine > mesoridazine > sulforidazine). Degradation in neutral or basic media was slow, with the exception of mesoridazine in a neutral medium. Twelve other phenothiazines tested, as well as chlorprotixene, a thioxanthene drug, were found to be sensitive to light in acidic media, whereas flupenthixol and zuclopenthixol (two thioxanthenes), clozapine, fluperlapine, and haloperidol (a butyrophenone) did not seem to be affected. In addition to being sensitive to light, some compounds may be readily oxidized by peroxide-containing solvents.

Drug monographs on drugs which are frequently analysed in the context of Therapeutic Drug Monitoring = Arzneimittel-Monographien für Medikamente, die regelmässig im Rahmen des Therapeutic Drug Monitorings analysiert werden

In addition to the monographs which were published last year by the working group "Drug Monitoring" of the Swiss Society of Clinical Chemistry (SSCC) [1], new monographs have been written. The aim of these monographs is to give an overview of the most important information necessary for ordering a drug analysis or interpreting the results. Therefore, the targeted readers comprise laboratory health professionals and all receivers of laboratory reports. There is information provided on the indication for therapeutic drug monitoring, protein binding, metabolic pathways and enzymes involved, elimination half-life and elimination routes, and on therapeutic or toxic concentrations. Preanalytical considerations are of particular importance for therapeutic drug monitoring. Therefore, information is provided regarding a reasonable timing for the determination of drug concentrations as well as steady-state concentrations after changing the dose. Furthermore, the stability of the drug and its metabolite(s) after blood sampling is described. For readers with a specific interest in drug analysis, references to important publications are given. The number of monographs will be continuously enlarged. The updated files are presented on the homepage of the SSCC (www.sscc.ch).

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype-phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Minority quasispecies of drug-resistant HIV-1 that lead to early therapy failure in treatment-naive and -adherent patients.

BACKGROUND: Early virological failure of antiretroviral therapy associated with the selection of drug-resistant human immunodeficiency virus type 1 in treatment-naive patients is very critical, because virological failure significantly increases the risk of subsequent failures. Therefore, we evaluated the possible role of minority quasispecies of drug-resistant human immunodeficiency virus type 1, which are undetectable at baseline by population sequencing, with regard to early virological failure. METHODS: We studied 4 patients who experienced early virological failure of a first-line regimen of lamivudine, tenofovir, and either efavirenz or nevirapine and 18 control patients undergoing similar treatment without virological failure. The key mutations K65R, K103N, Y181C, M184V, and M184I in the reverse transcriptase were quantified by allele-specific real-time polymerase chain reaction performed on plasma samples before and during early virological treatment failure. RESULTS: Before treatment, none of the viruses showed any evidence of drug resistance in the standard genotype analysis. Minority quasispecies with either the M184V mutation or the M184I mutation were detected in 3 of 18 control patients. In contrast, all 4 patients whose treatment was failing had harbored drug-resistant viruses at low frequencies before treatment, with a frequency range of 0.07%-2.0%. A range of 1-4 mutations was detected in viruses from each patient. Most of the minority quasispecies were rapidly selected and represented the major virus population within weeks after the patients started antiretroviral therapy. All 4 patients showed good adherence to treatment. Nonnucleoside reverse-transcriptase inhibitor plasma concentrations were in normal ranges for all 4 patients at 2 separate assessment times. CONCLUSIONS: Minority quasispecies of drug-resistant viruses, detected at baseline, can rapidly outgrow and become the major virus population and subsequently lead to early therapy failure in treatment-naive patients who receive antiretroviral therapy regimens with a low genetic resistance barrier.

Toxic and drug-induced peripheral neuropathies: updates on causes, mechanisms and management.

PURPOSE OF REVIEW: This review discusses publications highlighting current research on toxic, chemotherapy-induced peripheral neuropathies (CIPNs), and drug-induced peripheral neuropathies (DIPNs). RECENT FINDINGS: The emphasis in clinical studies is on the early detection and grading of peripheral neuropathies, whereas recent studies in animal models have given insights into molecular mechanisms, with the discovery of novel neuronal, axonal, and Schwann cell targets. Some substances trigger inflammatory changes in the peripheral nerves. Pharmacogenetic techniques are underway to identify genes that may help to predict individuals at higher risk of developing DIPNs. Several papers have been published on chemoprotectants; however, to date, this approach has not been shown effective in clinical trials. SUMMARY: Both length and nonlength-dependent neuropathies are encountered, including small-fiber involvement. The introduction of new diagnostic techniques, such as excitability studies, skin laser Doppler flowmetry, and pharmacogenetics, holds promise for early detection and to elucidate underlying mechanisms. New approaches to improve functions and quality of life in CIPN patients are discussed. Apart from developing less neurotoxic anticancer therapies, there is still hope to identify chemoprotective agents (erythropoietin and substances involved in the endocannabinoid system are promising) able to prevent or correct painful CIPNs.

Reduced quality of life associated adverse events in intermediate hepatocellular carcinoma patients treated with PRECISION TACE with drug eluting beads: Results from the PRECISION V randomized trial

Purpose: To evaluate the extent of quality of life (QoL) associated adverse events (AEs) following PRECISION TACE with DC Bead compared with conventional transarterial chemoembolisation (cTACE). Methods and Materials: 201 intermediate HCC patients were treated with DC Bead (PRECISION TACE) or conventional TACE (cTACE) with doxorubicin in the PRECISION V clinical study. 93 patients were treated with DC Bead and 108 Patients with cTACE every 2 months and followed up for 6 months. AEs were classified according to the South West Oncology Group criteria. QoL associated AEs were defined as alopecia, constipation, nausea, vomiting, pyrexia, chills, asthenia, fatigue, and headache. Results: The biggest difference in QoL associated AEs was for alopecia: 2 patients (2.2%) for DC-Bead versus 21 patients (19.4%) for cTACE. For other clinical symptoms, constipation (n=10; 10.8% vs. n=13; 12%), vomiting (n=10; 10.8% vs. n=14; 13.0%), pyrexia (n=16; 17.2% vs. n=26; 24.1%), chills (n=1; 1.1% vs. n=5; 4.6%), and headache (n=2; 2.2% vs. n=8; 7.4%) showed lower incidence in the DC Bead group versus cTACE. Nausea, n= 15; 13.9% (n=15; 16.1%) and fatigue, n=6; 5.6% (n=13; 14.0%) were lower for cTACE. Total dose of doxorubicin was on average 35% higher in the DC Bead group. Conclusion: Although patients in the DC Bead group received a higher doxorubicin dose, less QoL associated AEs were reported for this group. Alopecia, the most obvious outward sign of toxicity, was only reported in a tenth of DC Bead patients. Thus, PRECISION TACE with DC Bead improves quality of life associated adverse events.

Ubiquitin-mediated protein degradation and methylation-induced gene silencing cooperate in the inactivation of the INK4/ARF locus in Burkitt lymphoma cell lines.

Burkitt lymphoma is one of the most aggressive tumors affecting humans. Together with the characteristic chromosomal translocation that constitutively activates the c-Myc oncogene, alterations in cellular tumor suppressor pathways are additionally required in order to allow the cells to overcome anti-oncogenic barriers and proliferate in an uncontrolled manner. The INK4a/ARF locus on chromosome 9p21 is considered a safeguard locus since it encodes the two important tumor suppressor proteins, p14 (ARF) and p16 (INK4a) . By regulating the p53 and Rb pathways p14 (ARF) and p16 (INK4a) respectively act as pro-apoptotic and cell cycle inhibitor proteins. The importance of the INK4a/ARF locus has been well documented in several human tumors as well as in Burkitt lymphoma. Although the mechanisms responsible for the transcriptional regulation of the INK4a/ARF locus have been thoroughly characterized, less is known about its posttranscriptional control. In this study we found that p16 (INK4a) and p14 (Arf) are concurrently inactivated in a panel of BL cell lines. We demonstrate that along with the epigenetic silencing of the p16INK4a gene, the complete inactivation of the locus is achieved by the improper turnover of INK4/ARF proteins by the ubiquitin-proteasome system (UPS), as the proteasome inhibitor MG-132 blocks p14 (ARF) degradation and induces a dramatic stabilization of the p16 (INK4a ) protein. We establish that the simultaneous deregulation of both DNA methylation patterns and the ubiquitin-dependent proteolysis system is required to completely inactive the INK4/ARF locus, opening new prospects for the understanding and treatment of Burkitt lymphoma.

Mapping HIV-related behavioural surveillance among injecting drug users in Europe, 2008.

The systematic collection of behavioural information is an important component of second-generation HIV surveillance. The extent of behavioural surveillance among injecting drug users (IDUs) in Europe was examined using data collected through a questionnaire sent to all 31 countries of the European Union and European Free Trade Association as part of a European-wide behavioural surveillance mapping study on HIV and other sexually transmitted infections. The questionnaire was returned by 28 countries during August to September 2008: 16 reported behavioural surveillance studies (two provided no further details). A total of 12 countries used repeated surveys for behavioural surveillance and five used their Treatment Demand Indicator system (three used both approaches). The data collected focused on drug use, injecting practices, testing for HIV and hepatitis C virus and access to healthcare. Eight countries had set national indicators: three indicators were each reported by five countries: the sharing any injecting equipment, uptake of HIV testing and uptake of hepatitis C virus testing. The recall periods used varied. Seven countries reported conducting one-off behavioural surveys (in one country without a repeated survey, these resulted an informal surveillance structure). All countries used convenience sampling, with service-based recruitment being the most common approach. Four countries had used respondent-driven sampling. Three fifths of the countries responding (18/28) reported behavioural surveillance activities among IDUs; however, harmonisation of behavioural surveillance indicators is needed.

Prevalence of social phobia in a clinical sample of drug dependent patients.

INTRODUCTION: Social phobia is among the most frequent psychiatric disorders and can be classified into two subtypes, nongeneralized and generalized. Whereas it significantly worsens the morbidity of comorbid substance abuse disorders, and it often is associated with reduced treatment responses, there is still lacking data on its prevalence in clinical populations of drug abusing patients. METHODS: The study sample consisted of 75 inpatients and 75 outpatients meeting DSM-IV criteria for drug dependence. Symptoms of social phobia were assessed with the French-language version of the Liebowitz Social Anxiety Scale (LSAS). RESULTS: Prevalence rate were 20% for the generalized subtype and 42.6% for the nongeneralized subtype. Gender difference emerged in the severity of fear, women reporting significantly greater fear relating to performance situations than men. CONCLUSIONS: An important proportion of patients with substance dependence present a comorbid generalized or nongeneralized social phobia. Early recognition of social phobia and adequate interventions is warranted for these patients in order to improve their treatment response with regard to quality of life and relapse prevention.

Genetic analysis of phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH.

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for alpha-ketoglutarate-dependent dioxygenases, sdpA(MH) and rdpA(MH), were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA(I/II)), two for dienelactone hydrolases (dccD(I/II)), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA(MH) was 99% identical to rdpA(MC1), an (R)-dichlorprop/alpha-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA(I) and DccA(II) did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.