Ecotoxicological approaches to assess the long-term effects of four anticancer drugs and metabolites on Daphnia pulex

The aquatic environment is exposed continuously and increasingly to chemical substances such as pharmaceuticals. These medical compounds are released into the environment after having being consumed and body-excreted by patients. Pharmaceutical residues are synthetic molecules that are not always removed by traditional sewage treatment processes and thus escape degradation. Among pharmaceuticals that escape sewage treatment plants (STPs), the anticancer drugs were measured in STP effluents and natural waters. In the aquatic environment, their long-term effects at low concentrations are sparsely known on non-target species. Tamoxifen is an anticancer drug that is widely prescribed worldwide for the prevention and treatment of hormone receptor-positive breast cancers. Two of its metabolites, i.e., endoxifen and 4-hydroxy- tamoxifen (4OHTam), have high pharmacological potency in vivo and such as tamoxifen, they are excreted via faeces by patients. Tamoxifen was measured in STP effluents and natural waters but, to the best of our knowledge, its metabolites concentrations in waters have never been reported. Imatinib is another and recent anticancer compound that targets specific tumour cells. This pharmaceutical is also body excreted and because of its increasing use in cancer treatment, imatinib may reach the natural water. The effects of tamoxifen and imatinib are unknown upon more than one generation of aquatic species. And the effects of 4OHTam, endoxifen have never been studied in ecotoxicology so far. The aims of this thesis were threefold. First, the sensitivity of D. pulex exposed to tamoxifen, 4OHTam, endoxifen or imatinib was assessed using ecotoxicological experiments. Ecotoxicology is the science that considers the toxic effects of natural or synthetic substances, such as pharmaceuticals, on organisms, populations, community and ecosystem. Acute and multigenerational (2-4 generations) tests were performed on daphnids considering several studied endpoints, such as immobilisation, size, reproduction, viability and intrinsic rate of natural increase. Additional prospective assays were designed to evaluate whether 1) low concentrations of tamoxifen and 4OHTam were able to induce toxic effects when used in combination, and 2) daphnids were able to recover when offspring were withdrawn from solutions carrying the pharmaceutical. Second, the stability of tamoxifen, 4OHTam and endoxifen in incubation medium was evaluated in solution exempted from daphnids. Because the nominal concentrations of tamoxifen, 4OHTam and endoxifen did not correspond to the measured, we provide a predictive method to estimate the concentrations of these chemicals during long-term ecotoxicological tests. Finally, changes in protein expressions were analysed in D. pulex exposed 2 or 7 seven days to tamoxifen using ecotoxicoproteomic experiments with a shot-gun approach inducing a peptide fractionation step. Our results show that tamoxifen, 4OHTam and endoxifen induced adverse effects in D. pulex at environmentally relevant concentrations. At very low concentrations, these molecules displayed unusual and teratogenic effects because morphological abnormalities were observed in offspring, such as thick and short antennas, curved spines, premature neonates and aborted eggs. Tamoxifen was the most toxic compound among the test chemicals, followed by 4OHTam, endoxifen and imatinib. Tamoxifen no-observed effect concentrations (NOECs) that were calculated for size, reproduction and intrinsic rate were below or in the range of the concentrations measured in natural waters, i.e., between 0.12 µg/L and 0.67 µg/L. For instance, the tamoxifen NOECs that were calculated for reproduction were between 0.67 and 0.72 µg/L, whereas the NOEC was < 0.15 µg/L when based on morphological abnormalities. The NOECs of 4OHTam were higher but still in the same order of magnitude as tamoxifen environmental concentrations, with a value of 1.48 µg/L. Endoxifen NOEC for the intrinsic rate of natural increase (r) and the reproduction were 0.4 and 4.3 µg/L, respectively. Daphnids that were withdrawn from tamoxifen and 4OHTam were not able to recover. Also, the reproduction of D. pulex was reduced when the treated animals were exposed to the combination of tamoxifen and 4OHTam while no effects were observed when these chemicals were tested individually at the same concentration. Among the anticancer drugs that were tested during this thesis, imatinib was the less toxic molecule towards D. pulex. No effects on size and reproduction were observed within two generations, except for the first whose reproduction decreased at the highest test concentration, i.e., 626 µg/L. Our results also underline the need to use measured or predicted concentrations instead of the nominal during aquatic experiments, particularly when lipophilic molecules are tested. Indeed, notable differences between nominal (i.e., theoretical) and measured concentrations were found with tamoxifen, 4OHTam and endoxifen at all test concentrations. A cost and time sustainable method was proposed to predict the test exposure levels of these chemicals during long-term experiments. This predictive method was efficient particularly for low concentrations, which corresponded to the test concentrations in multigenerational tests. In the ecotoxicoproteomic experiments a total of 3940 proteins were identified and quantified in D. pulex exposed to tamoxifen. These results are currently the largest dataset from D. pulex that is published and the results of proteomic analyses are available for the scientific community. Among these 3940 proteins, 189 were significantly different from controls. After protein annotation, we assumed that treated daphnids with tamoxifen had shifted cost-energy functions, such as reproduction, to maintain their basic metabolism necessary to survive. This metabolic cost hypothesis was supported by the presence of proteins involved in oxidative stress. Biomarkers for early detection of tamoxifen harmful effects on D. pulex were not discovered but the proteins of the vitellogenin-2 family (E9H8K5) and the ryanodine receptor (E9FTU9) are promising potential biomarkers because their expression was already modified after 2 days of treatment. In this thesis, the effects of tamoxifen, 4OHTam and endoxifen on daphnids raise questions about the potential impact of tamoxifen and 4OHTam in other aquatic ecosystems, and therefore, about metabolites in ecotoxicology. Because the NOECs were environmentally relevant, these results suggest that tamoxifen and 4OHTam may be interesting pharmaceuticals to consider in risk assessment. Our findings also emphasize the importance of performing long-term experiments and of considering multi-endpoints instead of the standard reproductive endpoint. Finally, we open the discussion about the importance to measure test exposures or not, during ecotoxicological studies. -- Les milieux aquatiques sont exposés continuellement à un nombre croissant de substances chimiques, notamment les médicaments issus de la médecine vétérinaire et humaine. Chez les patients, les substances administrées sont utilisées par le corps avant d'être éliminées par l'intermédiaire des excrétas dans le système d'eaux usées de la ville. Ces eaux rejoignent ensuite une station de traitement afin d'y éliminer les déchets. Dans le cas des molécules chimiques, il arrive que les processus de traitement d'eaux usées ne soient pas suffisamment efficaces et que ces molécules ne soient pas dégradées. Elles sont alors libérées dans le milieu aquatique avec les effluents de la station d'épuration. Une fois dans l'environnement, ces résidus de médicaments sont susceptibles d'induire des effets sur la faune et la flore aquatique, dont les conséquences à long terme et à faibles concentrations sont peu connues. Les anticancéreux sont une famille de médicaments qui peuvent échapper aux traitements des stations d'épuration et qui sont retrouvées dans le milieu aquatique naturel. Parmi ces substances, le tamoxifen est une molécule utilisée dans le monde entier pour prévenir et traiter les cancers hormonaux dépendant du sein, notamment. Une fois ingéré, le tamoxifen est transformé par le foie en métabolites dont deux d'entre eux, le 4-hydroxy-tamoxifen (4OHTam) et l'endoxifen, possèdent un affinité pour les récepteurs aux estrogènes et une efficacité sur les cellules tumorales supérieure au tamoxifen lui- même. Tout comme la molécule mère, ces métabolites sont principalement éliminés par l'intermédiaire des fèces. Le tamoxifen a déjà été mesuré dans les effluents de stations d'épuration et dans les eaux naturelles, mais aucune valeur n'a été reportée pour ses métabolites jusqu'à présent. Un autre anticancéreux, également éliminé par voie biliaire et susceptible d'atteindre l'environnement, est l'imatinib. Cette récente molécule a révolutionné le traitement et la survie des patients souffrant de leucémie myéloïde chronique et de tumeur stromales gastrointestinales. Les effets du tamoxifen et de l'imatinib sur plusieurs générations d'organismes aquatiques, tels que les microcrustacés Daphnia, sont inconnus et le 4OHTam et l'endoxifen n'ont même jamais été testés en écotoxicologie. Cette thèse s'est articulée autour de trois objectifs principaux. Premièrement, la sensibilité des D. pulex exposés au tamoxifen, 4OHTam, endoxifen et imatinib a été évaluée par l'intermédiaire de tests aigus et de tests sur deux à quatre générations. La mobilité, la taille, la reproduction, la viabilité et la croissance potentielle de la population ont été relevées au cours de ces expériences. Des tests supplémentaires, à but prospectifs, ont également été réalisés afin d'évaluer 1) la capacité de récupération des daphnies, lorsque leurs descendants ont été placés dans un milieu exempté de tamoxifen ou de 4OHTam, 2) les effets chez les daphnies exposées à une solution contenant de faibles concentration de tamoxifen et de 4OHTam mélangés. Le deuxième objectif a été d'évaluer la stabilité du tamoxifen, 4OHTam et endoxifen dilué dans le milieu des daphnies. Après analyses, les concentrations mesurées ne correspondaient pas aux concentrations nominales (c.-à-d., théoriques) et il a été nécessaire de développer une méthode efficace de prédiction des niveaux d'exposition lors de tests de longue durée réalisés avec ces trois molécules. Finalement, des changements dans l'expression des protéines chez des daphnies exposées au tamoxifen ont été investigués par l'intermédiaire d'expériences écotoxicoprotéomiques avec une approche dite de shot-gun avec une étape de fractionnement des protéines. Les résultats obtenus dans cette thèse montrent que le tamoxifen, le 4OHTam et l'endoxifen induisent des effets indésirables chez les daphnies à des niveaux d'exposition proches ou identiques aux concentrations du tamoxifen mesurées dans l'environnement, c'est-à-dire 0.12 et 0.67 µg/L de tamoxifen. Ces molécules ont induit des effets inhabituels tels que la production de : nouveau-nés anormaux, avec des antennes et des queues déformées, des prématurés et des oeufs avortés. Le tamoxifen fut la molécule la plus toxique pour les D. pulex suivie du 4OHTam, de l'endoxifen et enfin de l'imatinib. Lors des expériences sur plusieurs générations, les concentrations n'ayant statistiquement pas d'effet (c.à.d. NOEC en anglais) sur la taille, la reproduction et la croissance intrinsèque de la population étaient du même ordre de grandeur que les concentrations environnementales du tamoxifen. Par exemple, les NOECs du tamoxifen calculées pour la reproduction étaient de 0.67 et 0.72 µg/L, tandis que celle calculée sur la base des anomalies chez les nouveau-nés était < 0.15 µg/L. Les NOECs du 4OHTam se situaient entre 0.16 et 1.48 µg/L et celles de l'endoxifen pour la croissance intrinsèque de la population, ainsi que pour la reproduction, étaient de 0.4 et 4.3 µg/L, respectivement. Dans l'expérience basée sur la récupération des daphnies, la taille et la reproduction ont diminué bien que la descendance fût placée dans un milieu sans substances chimiques. Les daphnies exposées au mélange de tamoxifen et de 4OHTam ont produit moins de nouveau-nés que les contrôles, alors que ces concentrations n'ont pas induit d'effets lorsque testées individuellement. Finalement, l'imatinib n'a pas montré d'effets sur les deux générations testées. Seule la première génération exposée à la plus haute concentration (626 µg/L) a montré une diminution de la reproduction. Les résultats obtenus lors de l'évaluation de la stabilité du tamoxifen, 4OHTam et endoxifen dans le milieu des daphnies ont souligné l'importance d'utiliser des concentrations mesurées ou prédites en écotoxicologie. En effet, des différences notables entre concentrations nominales et mesurées ont été observées à toutes les concentrations et l'hypothèse d'un phénomène d'adsorption sur le verre des récipients a été posée. De ce fait, il a été nécessaire d'élaborer une méthode prédictive efficace et acceptable, en terme de temps et de coûts. Une régression polynomiale basée sur des concentrations mesurées et nominales a permis de prédire avec efficacité les faibles niveaux d'exposition utilisés lors d'expériences écotoxicologiques à long terme, sur plusieurs générations. Suite aux expériences d'écotoxicoprotéomiques, un total de 3940 protéines ont été identifiées et quantifiées chez des daphnies exposées au tamoxifen. Ce nombre est actuellement la plus large série de données publiées et mises à disposition pour la communauté scientifique. Parmi ces protéines, 189 sont significatives et possiblement reliées à des processus de reproduction et de stress. Sur cette base, nous avons émis l'hypothèse que les individus subissant un stress, lié à l'exposition au tamoxifen, ont utilisé leur énergie de base pour favoriser leur survie plutôt que la reproduction. Enfin, la détermination de bio-marqueurs exprimant des dommages précoces des daphnies exposées au tamoxifen n'a pas abouti en tant que telle, mais des protéines prometteuses, telle que la famille de viellogenin-2 (E9H8K5) et le récepteur à la ryanodine (E9FTU9), ont été exprimées après deux jours d'exposition déjà. Ces protéines pourraient faire l'objet d'investigations écotoxicoprotéomiques futures. Les résultats de cette thèse posent certaines questions quant au risque du tamoxifen, du 4OHTam et de l'endoxifen sur la faune et la flore aquatique et plus particulièrement sur les anticancéreux présents dans l'environnement. Les effets toxiques de ces molécules ont été observés à des concentrations environnementales et sur plusieurs générations. La question de considérer les métabolites, et ainsi les pro-médicaments, en écotoxicologie est soulevée, notamment parce que ces molécules peuvent être plus actives et efficaces que la molécule mère. Les expériences chroniques, sur plusieurs générations sont également à favoriser car elles offrent un meilleur reflet de la réalité environnementale que des essais aigus ou d'une génération. L'utilisation de la protéomique permet d'agrandir les connaissances sur les effets des médicaments à un niveau inférieur de l'organisation biologique et ainsi, de mieux comprendre de potentiels mécanismes d'action ou de déterminer de potentiels biomarqueurs. Finalement, il semble important de discuter de l'opportunité de mesurer les concentrations qui sont testées en écotoxicologie afin de ne pas sous-estimer le risque pour la faune et la flore aquatique.

Concomitant administration of intravenous drugs in the ICU: evaluation of physico-chemical compatibilities

Background and objective: Patients in the ICU often get many intravenous (iv) drugs at the same time. Even with three-lumen central venous catheters, the administration of more than one drug in the same iv line (IVL) is frequently necessary. The objective of this study was to observe how nurses managed to administer these many medications and to evaluate the proportion of two-drugs associations (TDA) that are compatible or not, based on known compatibility data. Design: Observational prospective study over 4 consecutive months. All patients receiving simultaneously more than one drugs in the same IVL (Y-site injection or mixed in the same container) were included. For each patient, all iv drugs were recorded, as well as concentration, infusion solution, location on the IVL system, time, rate and duration of administration. For each association of two or more drugs, compatibility of each drug was checked with each other. Compatibilities between these pairs of drugs were assessed using published data (mainly Trissel LA. Handbook on Injectable Drugs and Trissel's Tables of Physical Compatibility) and visual tests performed in our quality control laboratory. Setting: 34 beds university hospital adult ICU. Main outcome measures: Percentage of compatibilities and incompatibilities between drugs administered in the same IVL. Results: We observed 1,913 associations of drugs administered together in the same IVL, 783 implying only two drugs. The average number of drugs per IVL was 3.1 ± 0.8 (range: 2-9). 83.2% of the drugs were given by continuous infusion, 14.3% by intermittent infusion and 2.5% in bolus. The associations observed allowed to form 8,421 pairs of drugs (71.7% drug-drug and 28.3% drug-solute). According to literature data, 80.2% of the association were considered as compatible and 4.4% incompatible. 15.4% were not interpretable because of different conditions between local practices and those described in the literature (drug concentration, solute, etc.) or because of a lack of data. After laboratory tests performed on the most used drugs (furosemide, KH2PO4, morphine HCl, etc.), the proportion of compatible TDA raised to 85.7%, the incompatible stayed at 4.6% and only 9.7% remain unknown or not interpretable. Conclusions: Nurses managed the administration of iv medications quite well, as only less than 5% of observed TDA were considered as incompatible. But the 10% of TDA with unavailable compatibility data should have been avoided too, since the consequences of their concomitant administration cannot be predictable. For practical reasons, drugs were analysed only by pairs, which constitutes the main limit of this work. The average number of drugs in the same association being three, laboratory tests are currently performed to evaluate some of the most observed three-drugs associations.

Synthesis of chemically functionalized superparamagnetic nanoparticles as delivery vectors for chemotherapeutic drugs.

Superparamagnetic iron oxide nanoparticles (SPIONs) are in clinical use for disease detection by MRI. A major advancement would be to link therapeutic drugs to SPIONs in order to achieve targeted drug delivery combined with detection. In the present work, we studied the possibility of developing a versatile synthesis protocol to hierarchically construct drug-functionalized-SPIONs as potential anti-cancer agents. Our model biocompatible SPIONs consisted of an iron oxide core (9-10 nm diameter) coated with polyvinylalcohols (PVA/aminoPVA), which can be internalized by cancer cells, depending on the positive charges at their surface. To develop drug-functionalized-aminoPVA-SPIONs as vectors for drug delivery, we first designed and synthesized bifunctional linkers of varied length and chemical composition to which the anti-cancer drugs 5-fluorouridine or doxorubicin were attached as biologically labile esters or peptides, respectively. These functionalized linkers were in turn coupled to aminoPVA by amide linkages before preparing the drug-functionalized-SPIONs that were characterized and evaluated as anti-cancer agents using human melanoma cells in culture. The 5-fluorouridine-SPIONs with an optimized ester linker were taken up by cells and proved to be efficient anti-tumor agents. While the doxorubicin-SPIONs linked with a Gly-Phe-Leu-Gly tetrapeptide were cleaved by lysosomal enzymes, they exhibited poor uptake by human melanoma cells in culture.

Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC-MS.

A simple and sensitive LC-MS method was developed and validated for the simultaneous quantification of aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL), clozapine (CLO), olanzapine (OLA), sertindole (STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) in human plasma. The above mentioned compounds and the internal standard (remoxipride) were extracted from 0.5 mL plasma by solid-phase extraction (mix mode support). The analytical separation was carried out on a reverse phase liquid chromatography at basic pH (pH 8.1) in gradient mode. All analytes were monitored by MS detection in the single ion monitoring mode and the method was validated covering the corresponding therapeutic range: 2-200 ng/mL for DUL, OLA, and STN, 4-200 ng/mL for DSTN, 5-1000 ng/mL for ARI, DARI and finally 2-1000 ng/mL for ATO, CLO, NCLO, VEN, OVEN. For all investigated compounds, good performance in terms of recoveries, selectivity, stability, repeatability, intermediate precision, trueness and accuracy, was obtained. Real patient plasma samples were then successfully analysed.

Novel anesthetics and other treatment strategies for refractory status epilepticus

Refractory status epilepticus (RSE)-that is, seizures resistant to at least two antiepileptic drugs (AEDs)-is generally managed with barbiturates, propofol, or midazolam, despite a low level of evidence (Rossetti, 2007). When this approach fails, the need for alternative pharmacologic and nonpharmacologic strategies emerges. These have been investigated even less systematically than the aforementioned compounds, and are often used, sometimes in succession, in cases of extreme refractoriness (Robakis & Hirsch, 2006). Several possibilities are reviewed here. In view of the marked heterogeneity of reported information, etiologies, ages, and comedications, it is extremely difficult to evaluate a given method, not to say to compare different strategies among them. Pharmacologic Approaches Isoflurane and desflurane may complete the armamentarium of anesthetics,' and should be employed in a ''close'' environment, in order to prevent intoxication of treating personnel. c-Aminobutyric acid (GABA)A receptor potentiation represents the putative mechanism of action. In an earlier report, isoflurane was used for up to 55 h in nine patients, controlling seizures in all; mortality was, however, 67% (Kofke et al., 1989). More recently, the use of these inhalational anesthetics was described in seven subjects with RSE, for up to 26 days, with an endtidal concentration of 1.2-5%. All patients required vasopressors, and paralytic ileus occurred in three; outcome was fatal in three patients (43%) (Mirsattari et al., 2004). Ketamine, known as an emergency anesthetic because of its favorable hemodynamic profile, is an N-methyl-daspartate (NMDA) antagonist; the interest for its use in RSE derives from animal works showing loss of GABAA efficacy and maintained NMDA sensitivity in prolonged status epilepticus (Mazarati & Wasterlain, 1999). However, to avoid possible neurotoxicity, it appears safer to combine ketamine with GABAergic compounds (Jevtovic-Todorovic et al., 2001; Ubogu et al., 2003), also because of a likely synergistic effect (Martin & Kapur, 2008). There are few reported cases in humans, describing progressive dosages up to 7.5 mg/kg/h for several days (Sheth & Gidal, 1998; Quigg et al., 2002; Pruss & Holtkamp, 2008), with moderate outcomes. Paraldehyde acts through a yet-unidentified mechanism, and appears to be relatively safe in terms of cardiovascular tolerability (Ramsay, 1989; Thulasimani & Ramaswamy, 2002), but because of the risk of crystal formation and its reactivity with plastic, it should be used only as fresh prepared solution in glass devices (Beyenburg et al., 2000). There are virtually no recent reports regarding its use in adults RSE, whereas rectal paraldehyde in children with status epilepticus resistant to benzodiazepines seems less efficacious than intravenous phenytoin (Chin et al., 2008). Etomidate is another anesthetic agent for which the exact mechanism of action is also unknown, which is also relatively favorable regarding cardiovascular side effects, and may be used for rapid sedation. Its use in RSE was reported in eight subjects (Yeoman et al., 1989). After a bolus of 0.3 mg/kg, a drip of up to 7.2 mg/kg/h for up to 12 days was administered, with hypotension occurring in five patients; two patients died. A reversible inhibition of cortisol synthesis represents an important concern, limiting its widespread use and implying a careful hormonal substitution during treatment (Beyenburg et al., 2000). Several nonsedating approaches have been reported. The use of lidocaine in RSE, a class Ib antiarrhythmic agent modulating sodium channels, was reviewed in 1997 (Walker & Slovis, 1997). Initial boluses up to 5 mg/kg and perfusions of up to 6 mg/kg/h have been mentioned; somewhat surprisingly, at times lidocaine seemed to be successful in controlling seizures in patients who were refractory to phenytoin. The aforementioned dosages should not be overshot, in order to keep lidocaine levels under 5 mg/L and avoid seizure induction (Hamano et al., 2006). A recent pediatric retrospective survey on 57 RSE episodes (37 patients) described a response in 36%, and no major adverse events; mortality was not given (Hamano et al., 2006 Verapamil, a calcium-channel blocker, also inhibits P-glycoprotein, a multidrug transporter that may diminish AED availability in the brain (Potschka et al., 2002). Few case reports on its use in humans are available; this medication nevertheless appears relatively safe (under cardiac monitoring) up to dosages of 360 mg/day (Iannetti et al., 2005). Magnesium, a widely used agent for seizures elicited by eclampsia, has also been anecdotally reported in RSE (Fisher et al., 1988; Robakis & Hirsch, 2006), but with scarce results even at serum levels of 14 mm. The rationale may be found in the physiologic blockage of NMDA channels by magnesium ions (Hope & Blumenfeld, 2005). Ketogenic diet has been prescribed for decades, mostly in children, to control refractory seizures. Its use in RSE as ''ultima ratio'' has been occasionally described: three of six children (Francois et al., 2003) and one adult (Bodenant et al., 2008) were responders. This approach displays its effect subacutely over several days to a few weeks. Because ''malignant RSE'' seems at times to be the consequence of immunologic processes (Holtkamp et al., 2005), a course of immunomodulatory treatment is often advocated in this setting, even in the absence of definite autoimmune etiologies (Robakis & Hirsch, 2006); steroids, adrenocorticotropic hormone (ACTH), plasma exchanges, or intravenous immunoglobulins may be used alone or in sequential combination. Nonpharmacologic Approaches These strategies are described somewhat less frequently than pharmacologic approaches. Acute implantation of vagus nerve stimulation (VNS) has been reported in RSE (Winston et al., 2001; Patwardhan et al., 2005; De Herdt et al., 2009). Stimulation was usually initiated in the operation room, and intensity progressively adapted over a few days up to 1.25 mA (with various regimens regarding the other parameters), allowing a subacute seizure control; one transitory episode of bradycardia/asystole has been described (De Herdt et al., 2009). Of course, pending identification of a definite seizure focus, resective surgery may also be considered in selected cases (Lhatoo & Alexopoulos, 2007). Low-frequency (0.5 Hz) transcranial magnetic stimulation (TMS) at 90% of the resting motor threshold has been reported to be successful for about 2 months in a patient with epilepsia partialis continua, but with a weaning effect afterward, implying the need for a repetitive use (Misawa et al., 2005). More recently, TMS was applied in a combination of a short ''priming'' high frequency (up to 100 Hz) and longer runs of low-frequency stimulations (1 Hz) at 90-100% of the motor threshold in seven other patients with simple-partial status, with mixed results (Rotenberg et al., 2009). Paradoxically at first glance, electroconvulsive treatment may be found in cases of extremely resistant RSE. A recent case report illustrates its use in an adult patient with convulsive status, with three sessions (three convulsions each) carried out over 3 days, resulting in a moderate recovery; the mechanism is believed to be related to modification of the synaptic release of neurotransmitters (Cline & Roos, 2007). Therapeutic hypothermia, which is increasingly used in postanoxic patients (Oddo et al., 2008), has been the object of a recent case series in RSE (Corry et al., 2008). Reduction of energy demand, excitatory neurotransmission, and neuroprotective effects may account for the putative mechanism of action. Four adult patients in RSE were cooled to 31_-34_C with an endovascular system for up to 90 h, and then passively rewarmed over 2-50 h. Seizures were controlled in two patients, one of whom died; also one of the other two patients in whom seizures continued subsequently deceased. Possible side effects are related to acid-base and electrolyte disturbances, and coagulation dysfunction including thrombosis, infectious risks, cardiac arrhythmia, and paralytic ileus (Corry et al., 2008; Cereda et al., 2009). Finally, anecdotic evidence suggests that cerebrospinal fluid (CSF)-air exchange may induce some transitory benefit in RSE (Kohrmann et al., 2006); although this approach was already in use in the middle of the twentieth century, the mechanism is unknown. Acknowledgment A wide spectrum of pharmacologic (sedating and nonsedating) and nonpharmacologic (surgical, or involving electrical stimulation) regimens might be applied to attempt RSE control. Their use should be considered only after refractoriness to AED or anesthetics displaying a higher level of evidence. Although it seems unlikely that these uncommon and scarcely studied strategies will influence the RSE outcome in a decisive way, some may be interesting in particular settings. However, because the main prognostic determinant in status epilepticus appears to be related to the underlying etiology rather than to the treatment approach (Rossetti et al., 2005, 2008), the safety issue should always represent a paramount concern for the prescribing physician. Conclusion The author confirms that he has read the Journal's position on issues involved in ethical publication and affirms that this paper is consistent with those guidelines.

Differential involvement of MEK kinase 1 (MEKK1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents.

MEK kinase 1 (MEKK1) is a 196-kDa enzyme that is involved in the regulation of the c-Jun N-terminal kinase (JNK) pathway and apoptosis. In cells exposed to genotoxic agents including etoposide and cytosine arabinoside, MEKK1 is cleaved at Asp874 by caspases. The cleaved kinase domain of MEKK1, itself, stimulates caspase activity leading to apoptosis. Kinase-inactive MEKK1 expressed in HEK293 cells effectively blocks genotoxin-induced apoptosis. Treatment of cells with taxol, a microtubule stabilizing agent, did not induce MEKK1 cleavage in cells, and kinase-inactive MEKK1 expression failed to block taxol-induced apoptosis. MEKK1 became activated in HEK293 cells exposed to taxol, but in contrast to etoposide-treatment, taxol failed to increase JNK activity. Taxol treatment of cells, therefore, dissociates MEKK1 activation from the regulation of the JNK pathway. Overexpression of anti-apoptotic Bcl2 blocked MEKK1 and taxol-induced apoptosis but did not block the caspase-dependent cleavage of MEKK1 in response to etoposide. This indicates Bcl2 inhibition of apoptosis is, therefore, downstream of caspase-dependent MEKK1 cleavage. The results define the involvement of MEKK1 in the induction of apoptosis by genotoxins but not microtubule altering drugs.

Inhibition of angiogenesis by non-steroidal anti-inflammatory drugs: from the bench to the bedside and back.

The formation of new blood vessels, a process globally referred to as angiogenesis, occurs in a number of pathological conditions, such as cancer and chronic inflammation. Recent findings indicate that cyclooxygenase-2 (COX-2), the inducible form of the cyclooxygenase (COX) isoenzymes, acts as a potent inducer of angiogenesis. Non-steroidal anti-inflammatory drugs (NSAIDs) are classical inhibitors of COX enzymes, which are widely prescribed for the treatment of inflammation, pain and fever. Selective COX-2 inhibitors (COXIBs) have been subsequently developed with the purpose to improve the safety profile of this class of therapeutics. More recently, substantial preclinical evidence demonstrated that NSAIDS and COXIBs have anti-angiogenic properties. This newly recognized activity opens the possibility of using these drugs for the treatment of angiogenesis-dependent diseases. In this article we review the most recent advances in understanding the mechanisms by which NSAIDs and COXIBs suppress angiogenesis, and we discuss their potential clinical use as anti-angiogenic drugs.

Citizens' preferences for brand name drugs for treating acute and chronic conditions: a pilot study.

Background: Generic drugs have been advocated to decrease the proportion of healthcare costs devoted to drugs, but are still underused. Objective: To assess citizens' preferences for brand name drugs (BNDs) compared with generic drugs for treating acute and chronic conditions. Methods: A questionnaire with eight hypothetical scenarios describing four acute and four chronic conditions was developed, with willingness to pay (WTP) determined using a payment card system randomized to ascending (AO) or descending order (DO) of prices. The questionnaire was distributed with an explanation sheet, an informed consent form and a pre-stamped envelope over a period of 3 weeks in 19 community pharmacies in Lausanne, Switzerland. The questionnaire was distributed to every third customer who also had health insurance, understood French and was aged =16 years (up to a maximum of ten customers per day and 100 per pharmacy). The main outcome measure was preferences assessed by WTP for BNDs as compared with generics, and impact of participants' characteristics on WTP. Results: Of the 1800 questionnaires, 991 were distributed and 393 returned (pharmacy participation rate?=?55%, subject participation rate?=?40%, overall response rate?=?22%); 51.7% were AO and 48.3% DO. Participants were predominantly women (62.6%) and of median age 62 years (range 16-90). The majority (70%) declared no WTP for BNDs as compared with generics. WTP was higher in people with an acute disease than in those with a chronic disease, did not depend on the type of chronic disease, and was higher in people from countries other than Switzerland. Conclusions: Most citizens visiting pharmacies attribute no added value to BNDs as compared with generics, although some citizen characteristics affected WTP. These results could be of interest to several categories of decision makers within the healthcare system.

Selectivity in the binding of psychotropic drugs to the variants of alpha-1 acid glycoprotein.

The S- and F-forms of alpha-1 acid glycoprotein (AAG) variants have been isolated by isoelectric focusing with immobilines from commercially available AAG. In equilibrium dialysis experiments using a multicompartmental system, a higher affinity for various basic drugs has been found with S- in comparison with F-AAG: Amitriptyline, nortriptyline, imipramine, desipramine, trimipramine, methadone, thioridazine, clomipramine, desmethylclomipramine, and maprotiline. The selectivity (binding to S- vs. F-AAG) is the most pronounced for methadone and the lowest for thioridazine, while it is absent for the acidic drug mephenytoin.

Effects on blood pressure and cardiovascular risk of variations in patients' adherence to prescribed antihypertensive drugs: role of duration of drug action.

P>Aim: To determine the effects of imperfect adherence (i.e. occasionally missing prescribed doses), and the influence of rate of loss of antihypertensive effect during treatment interruption, on the predicted clinical effectiveness of antihypertensive drugs in reducing mean systolic blood pressure (SBP) and cardiovascular disease (CVD) risk.Method:The effects of imperfect adherence to antihypertensive treatment regimens were estimated using published patterns of missed doses, and taking into account the rate of loss of antihypertensive effect when doses are missed (loss of BP reduction in mmHg/day; the off-rate), which varies between drugs. Outcome measures were the predicted mean SBP reduction and CVD risk, determined from the Framingham Risk Equation for CVD.Results:In patients taking 75% of prescribed doses (typical of clinical practice), only long-acting drugs with an off-rate of similar to 1 mmHg/day were predicted to maintain almost the full mean SBP-lowering effect throughout the modelled period. In such patients, using shorter-acting drugs (e.g. an off-rate of similar to 5-6 mmHg/day) was predicted to lead to a clinically relevant loss of mean SBP reduction of > 2 mmHg. This change also influenced the predicted CVD risk reduction; in patients with a baseline 10-year CVD risk of 27.0% and who were taking 75% of prescribed doses, a difference in off-rate from 1 to 5 mmHg/day led to a predicted 0.5% absolute increase in 10-year CVD risk.Conclusions:In patients who occasionally miss doses of antihypertensives, modest differences in the rate of loss of antihypertensive effect following treatment interruption may have a clinically relevant impact on SBP reduction and CVD risk. While clinicians must make every effort to counsel and encourage each of their patients to adhere to their prescribed medication, it may also be prudent to prescribe drugs with a low off-rate to mitigate the potential consequences of missing doses.

Fast quantification of ten psychotropic drugs and metabolites in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A sensitive and selective ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method was developed for the fast quantification of ten psychotropic drugs and metabolites in human plasma for the needs of our laboratory (amisulpride, asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, norquetiapine, olanzapine, paliperidone, quetiapine and risperidone). Stable isotope-labeled internal standards were used for all analytes, to compensate for the global method variability, including extraction and ionization variations. Sample preparation was performed by generic protein precipitation with acetonitrile. Chromatographic separation was achieved in less than 3.0min on an Acquity UPLC BEH Shield RP18 column (2.1mm×50mm; 1.7μm), using a gradient elution of 10mM ammonium formate buffer pH 3.0 and acetonitrile at a flow rate of 0.4ml/min. The compounds were quantified on a tandem quadrupole mass spectrometer operating in positive electrospray ionization mode, using multiple reaction monitoring. The method was fully validated according to the latest recommendations of international guidelines. Eight point calibration curves were used to cover a large concentration range 0.5-200ng/ml for asenapine, desmethyl-mirtazapine, iloperidone, mirtazapine, olanzapine, paliperidone and risperidone, and 1-1500ng/ml for amisulpride, norquetiapine and quetiapine. Good quantitative performances were achieved in terms of trueness (93.1-111.2%), repeatability (1.3-8.6%) and intermediate precision (1.8-11.5%). Internal standard-normalized matrix effects ranged between 95 and 105%, with a variability never exceeding 6%. The accuracy profiles (total error) were included in the acceptance limits of ±30% for biological samples. This method is therefore suitable for both therapeutic drug monitoring and pharmacokinetic studies.

Comparative cardiovascular effects of drugs used for hypertension.

Currently 4 classes of antihypertensive drugs - diuretics, beta-blockers, calcium channel blockers and angiotensin-converting enzyme (ACE) inhibitors - are most commonly used to treat hypertensive patients. Each class of drug has a distinctive cardiovascular pharmacodynamic profile and even within classes there exist agents with slightly different properties. The effects of the various drug classes on the heart and peripheral circulation, on the kidney and electrolyte metabolism, on the brain and on the renin-angiotensin system are now reasonably well described. Knowledge and understanding of these different cardiovascular effects are extremely important in order to adapt treatment to the needs of an individual patient. Furthermore, when combination therapy becomes necessary, the different cardiovascular aspects of the various drugs can be used to enhance antihypertensive efficacy and to attenuate adverse effects of separate compounds.