Search

Simple Summary After the recent irruption of asciminib into the therapeutic arsenal for chronic myeloid leukemia, real-life data remain scarce to determine which patients may benefit most from this drug. Data on the efficacy of the drug in real-world setting have been reported, but a detailed analysis of the toxicity profile and the influence of prior intolerance to classical tyrosine kinase inhibitors (TKIs) has not been performed. The aim of the present analysis is to study in detail the toxicity profile of asciminib as well as to describe the risk of cross-toxicity with classical TKIs. These results may help to select the patient profile with the best chance of therapeutic success with asciminib monotherapy. (1) Background: Despite the prognostic improvements achieved with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML), a minority of patients still fail TKIs. The recent introduction of asciminib may be a promising option in intolerant patients, as it is a first-in-class inhibitor with a more selective mechanism of action different from the ATP-competitive inhibition that occurs with TKIs. Therefore, our goal was to analyze toxicities shown with asciminib as well as to study cross-toxicity with previous TKIs. (2) Methods: An observational, multicenter, retrospective study was performed with data from 77 patients with CML with therapeutic failure to second-generation TKIs who received asciminib through a managed-access program (MAP) (3) Results: With a median follow-up of 13.7 months, 22 patients (28.5%) discontinued treatment: 32% (7/22) due to intolerance and 45% (10/22) due to resistance. Fifty-five percent of the patients reported adverse effects (AEs) with asciminib and eighteen percent grade 3-4. Most frequent AEs were: fatigue (18%), thrombocytopenia (17%), anemia (12%), and arthralgias (12%). None of the patients experienced cardiovascular events or occlusive arterial disease. Further, 26%, 25%, and 9% of patients required dose adjustment, temporary suspension, or definitive discontinuation of treatment, respectively. Toxicities under asciminib seemed lower than with prior TKIs for anemia, cardiovascular events, pleural/pericardial effusion, diarrhea, and edema. Cross-toxicity risk was statistically significant for thrombocytopenia, anemia, neutropenia, fatigue, vomiting, and pancreatitis. (4) Conclusion: Asciminib is a molecule with a good safety profile and with a low rate of AEs. However, despite its new mechanism of action, asciminib presents a risk of cross-toxicity with classical TKIs for some AEs.

El Arrecife Verde, uno de los 45 arrecifes que actualmente se reconocen y que se encuentran en el área de Veracruz, Boca del Río y Antón Lizardo, es una pequeña superficie de alrededor de 726,000 m2, apenas visible sobre el horizonte desde la costa de la zona conurbada de la ciudad y puerto de Veracruz. Gracias a su cercanía con la costa, de apenas 6 km, y sobre todo a su pequeña isla de casi 35,000 m2, es particularmente importante por presentar un cayo con vegetación permanentemente verde, una posa con aguas salobres y cuatro especies de mangle. Se trata de un arrecife de coral de plataforma y eso le otorga un valor biótico extraordinario, además de tratarse de un ecosistema perturbado por diversas actividades humanas desde hace más de 500 años, como lo muestran los hallazgos arqueológicos prehispánicos dejados al descubierto por el impacto del huracán Karl. Además, tiene un interés excepcional por la posibilidad de ser zona experimental natural y accesible a la casi invisible vulnerabilidad a que son sujetos por los diversos efectos de las más variadas actividades antropogénicas. Con el propósito de dar a conocer la gran cantidad de información recabada, se invitó a diversos especialistas en oceanografía física, biológica y de contaminación, que tuvieran información o lotes biológicos del Arrecife Verde. Así, con la participación de 43 investigadores de 11 centros de investigación del país, en esta contribución se abordan diversos aspectos que permitirán encauzar diversos estudios en los otros arrecifes de Parque Nacional Sistema Arrecifal Veracruzano, como son los innumerables efectos antrópicos, el aspecto ambiental general, el patrón de corrientes en la región, las variaciones hidrográficas en cada arrecife, la riqueza de la flora en los arrecifes con cayo o isla, la flora marina y la diversidad de los grupos más conspicuos, considerando el inventario de 658 especies, como es el caso de las esponjas, ascidias, corales, moluscos, equinodermos, poliquetos, crustáceos y peces, entre otros.

Background – Albendazole is a benzoimidazole carbamate drug with anthelmintic and antiprotozoal activity against intestinal and tissue parasites. It has been described that the administration with meals increases albendazole absorption. Objective – Our aim was to compare the systemic exposure in healthy volunteers of two albendazole formulations after a single oral dose under fed conditions and to evaluate the effect of breakfast composition on its bioavailability. Methods – 12 healthy volunteers were included in a crossover, open, randomized comparative bioavailability trial including two stages. Single oral doses of 400 mg albendazole were administered under fed conditions (a light breakfast in first stage and a high-fat breakfast in the second) separated by 7-day washout periods. Plasma albendazole and albendazole sulfoxide concentrations were measured by HPLC-MS/MS. Results – Albendazole absorption was clearly influenced by the meal composition. A high-fat breakfast increased albendazole and albendazole sulfoxide area under the concentration-time curve (AUC) and maximum concentration (Cmax) by double compared to a light breakfast. The bioavailability of the two formulations was very similar, although the sample size was not sufficient to demonstrate bioequivalence because the intra-individual variability of albendazole was approximately 60%. Conclusions – The higher albendazole and albendazole sulfoxide levels when administered with a high-fat meal could be of importance in clinical practice. Since albendazole labelling recommends its administration with meals, it is necessary to insist the patient to take it with a fatty meal so that the effectiveness of albendazole is not compromised.

Clinical signs of syringomyelia and hydrocephalus occur secondary to cerebrospinal fluid (CSF) accumulation within the central nervous system. Omeprazole is recommended to treat these conditions despite little evidence of its capacity to decrease CSF production in the dog. Studies into new treatments are hampered by difficulties in measuring CSF production. The albumin quotient (QAlb), the ratio between CSF and serum albumin concentrations, may reflect CSF production and any decrease in CSF production should be associated with an increase in QAlb. The primary objective of this study was to determine CSF omeprazole concentration after administration of a high intravenous dose of omeprazole and to evaluate its impact on QAlb in the dog. The second aim was to validate QAlb as a surrogate marker of CSF production. Eighteen dogs were included in this prospective crossover placebo-controlled study. Each dog received omeprazole (10 mg/kg), acetazolamide (50 mg/kg) combined with furosemide (1 mg/kg) and saline. Blood and CSF samples were obtained on day 0 and then every 7 days, one hour after drug administration. Omeprazole concentrations (2.0 ± 0.4 μmol/L) reached in CSF after high dose omeprazole were lower than the concentrations previously described as decreasing CSF production in dogs. There was no significant increase in QAlb following administration of acetazolamide/furosemide, prohibiting validation of QAlb as a surrogate marker for CSF production. Several dogs presented transient mild side effects after injection of acetazolamide/furosemide. High dose omeprazole was well tolerated in all dogs.

Introduction: asciminib is a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor that potently inhibits aberrant kinase activity of the BCR-ABL1 oncoprotein via allosteric binding. asciminib has shown high efficacy profile in heavily pretreated Chronic Myeloid Leukemia (CML) patients with an adequate safety profile in phase I and III clinical trials. However, data from the use of asciminib in real life setting are still scarce. Methods: We gathered real-life retrospective data from 49 patients with BCR-ABL1 positive CML treated with asciminib (mean dose: 40 mg twice daily) between October 2018 and July 2021 at 33 institutions. The indication of asciminib was made according to the criterion of the attending physician and the drug was granted by Novartis under a controlled access program. Molecular biology tests were performed according to ELN guidelines and BCR-ABL/ABL ratios were expressed as % IS in all centers. Treatment responses were calculated with the patients at risk at each specific time points. For the event free survival (EFS), the events were treatment discontinuation due to any reason, progression or death. Data collection followed the local regulations for observational studies. Results: Median time on asciminib was 11,69 months for the entire cohort. Patients' characteristics are displayed on Table 1. Most patients were heavily pretreated with at least 3 prior TKI lines in 45 patients (91,83%), 18 of them receiving prior Ponatinib. Switch to asciminib occurred due to intolerance in 32 patients and due to resistance in the remaining 17. Fifteen patients (30,61%) harbored mutations in BCR-ABL1 (3 with a T315 mutation). Regarding efficacy (Table 2), probability of reaching or maintaining previous responses were 94%, 45% and 21% for complete hematological response (CHR), complete cytogenetic response (CCyR) and major molecular response (MMR), respectively. Considering probabilities of improving previous response, rates were 40%, 42% and 33% for the same parameters. Probabilities to obtain CCyR and MMR in resistant and intolerant patients were 29% (4/14) vs 55% (6/11) and 27% (4/15) vs 52% (11/21), respectively. Amid the patients previously treated with Ponatinib, probabilities of reaching or maintaining previous response were 53% (9/17) and 35% (6/17) for CCyR and MMR respectively, and 30% (3/10), 23% (3/13) displayed improvement of response. Regarding responses in patients with mutations, 39% (5/13) achieved or maintained CCyR and 31% (4/13) MMR; whereas 20% (2/10) and 18% (2/11) improved such responses. Of the three patients with T315I mutation, one discontinued due to progression to advanced stages, and the rest maintained the previous response. With a median follow-up of 11,69 months, the estimated EFS was 80% (figure 1). In terms of safety (Table 3), the most frequent extra-hematological adverse events (AE) were: fatigue (16,2%), joint pain (13,5%) and nausea (8,1%), most of them grade 1-2. Grade 3-4 AE were observed in 10% of patient (fatigue (2), cholestasis enzyme elevation (1), hypertension (1), pancreatitis (1) and pericardial effusion (1)). Thrombocytopenia was shown as the most frequent AE (16,3%), with 6% of patients suffering from grade 3-4. Dose reduction was required in 15 patients (30,6%). After a median follow up of 51 weeks, 73,5% of the patients remained on treatment. Only fourteen patients discontinued treatment due to progression or loss of efficacy, whereas 6% of patients discontinuing treatment due to intolerance. Conclusions: The results presented are in line with the data obtained in clinical trials, positioning asciminib as a potential safe and efficacious treatment for CML patients with failure to several TKI lines. Figure 1 Figure 1. Disclosures Sanchez-Guijo: Novartis: Consultancy, Honoraria, Research Funding; Celgene/Bristol-Myers-Squibb,: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Garcia Gutierrez: BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding.

ITH12674 is a multitarget drug, designed to exert a dual "drug-prodrug" mechanism of action, able to induce the phase II antioxidant and anti-inflammatory response for the treatment of brain ischemia. However, its physicochemical properties limit its potential preclinical development due to its low water solubility and instability towards heat and pH variations. In order to improve its properties, we prepared the inclusion complex of ITH12674 with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) by the freeze-drying method. The formation of the inclusion complex was confirmed by FT-IR spectroscopy, PXRD, DSC, 1H NMR and SEM techniques. Experimental results showed that the inclusion complex enhanced its water solubility and stability against heat, acidic and basic conditions. Furthermore, the inclusion complex, prepared in water solution, exerted the same potency to induce the phase II antioxidant response as the pure ITH12674. Thus the formation of the inclusion complex with HP-β-CD is a very effective method to stabilize and solubilize the active compound for its future preclinical development.

Therapeutic options for amyotrophic lateral sclerosis (ALS) are scarce and controversial. Although the aetiology of neuronal vulnerability is unknown, growing evidence supports a complex network in which multiple toxicity pathways, rather than a single mechanism, are involved in the pathogenesis of ALS. However, most cellular models only explain single pathogenic mechanisms. The present study proposes the two main cytotoxic mechanisms: (1) veratridine (VTD), which induced Na+ and Ca2+ overload; and (2) the TARD DNA-binding protein 43 (TDP-43) in NSC-34 cell line as an in vitro model of ALS. The study was carried out by MTT as an indirect measurement of cell viability and by flow cytometry to determine cell death stages. The impact of Ca2+ overload combined with TDP-43 overexpression increased early apoptosis of NSC-34 cells. Furthermore, we found that ITH33/IQM9.21 (ITH33) exerted a neuroprotective effect in this model by reducing activation of the apoptotic pathway. Therefore, treatment with VTD in TDP-43 overexpressing NSC-34 cells is a good in vitro ALS model that makes it possible to test new neuroprotective compounds such as ITH33.

The primary functions of adrenal medullary chromaffin cells are the synthesis and storage in their chromaffin vesicles of the catecholamines noradrenaline (NA) and adrenaline (AD), and their subsequent release into the bloodstream by Ca2+ -dependent exocytosis under conditions of fear or stress (fight or flight response). Several monoamines, nucleotides and opiates, such as leucine-enkephalin (LENK) and methionine-enkephalin (MENK), are also co-stored and co-released with the catecholamines. However, other neurotransmitters have not been studied in depth. Here, we present a novel high-resolution liquid chromatography-tandem mass spectrometry approach for the simultaneous monitoring of 14 compounds stored and released in bovine chromaffin cells (BCCs). We validated the analytical method according to the recommendations of the EMA and FDA by testing matrix effect, selectivity, sensitivity, precision, accuracy, stability and carry-over. After testing on six batches of BCCs from different cultures, the method enabled simultaneous quantitative determination of monoamines (AD, NA, dopamine, serotonin, 5-hydroxyindoleacetic acid, histamine and metanephrine), amino acids (L-glutamic acid, γ-aminobutyric acid), nucleotides (adenosine 5'-diphosphate, adenosine 5'-monophosphate, cyclic adenosine 5'-monophosphate) and neuropeptides (LENK and MENK) in the intracellular content, basal secretion and acetylcholine induced secretion of BBCs. The high-resolution approach used here enabled us to determine the levels of 14 compounds in the same BCC batch in only 16 min. This novel approach will make it possible to study the regulatory mechanisms of Ca2+ signaling, exocytosis and endocytosis using different neurotrophic factors and/or secretagogues as stimuli in primary BCC cultures. Our method is actually being applied to human plasma samples of different therapeutic areas where sympathoadrenal axis is involved in stress situations such as Alzheimer's disease, migraine or cirrhosis, to improve diagnosis and clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.

Agmatine, an endogenous neuromodulator, is a potential candidate to constitute an adjuvant/monotherapy for the management of depression. A recent study by our group demonstrated that agmatine induces Nrf2 and protects against corticosterone effects in a hippocampal neuronal cell line. The present study is an extension of this previous study by assessing the antidepressant-like effect of agmatine in an animal model of depression induced by corticosterone in mice. Swiss mice were treated simultaneously with agmatine or imipramine at a dose of 0.1 mg/kg/day (p.o.) and corticosterone for 21 days and the daily administrations of experimental drugs were given immediately prior to corticosterone (20 mg/kg/day, p.o.) administrations. Wild-type C57BL/6 mice (Nrf2 (+/+)) and Nrf2 KO (Nrf2 (−/−)) were treated during 21 days with agmatine (0.1 mg/kg/day, p.o.) or vehicle. Twenty-four hours after the last treatments, the behavioral tests and biochemical assays were performed. Agmatine treatment for 21 days was able to abolish the corticosterone-induced depressive-like behavior and the alterations in the immunocontent of mature BDNF and synaptotagmin I, and in the serotonin and glutamate levels. Agmatine also abolished the corticosterone-induced changes in the morphology of astrocytes and microglia in CA1 region of hippocampus. In addition, agmatine treatment in control mice increased noradrenaline, serotonin, and dopamine levels, CREB phosphorylation, mature BDNF and synaptotagmin I immunocontents, and reduced pro-BDNF immunocontent in the hippocampus. Agmatine’s ability to produce an antidepressant-like effect was abolished in Nrf2 (−/−) mice. The present results reinforce the participation of Nrf2 in the antidepressant-like effect produced by agmatine and expand literature data concerning its mechanisms of action., This work was supported by CNPq, CAPES/PDSE, CAPES/PROCAD, Núcleo de Excelência em Neurociências Aplicadas de Santa Catarina (NENASC) Project/ PRONEX Program CNPq/FAPESC (Brazil) to ALSR. The 8th Convocatoria de proyectos de Cooperación Interuniversitaria UAM-Santander con America Latina, Spanish Ministry of Economy and Competence Ref. SAF2012-32223 to MGL and ALSR. This work was also supported by FIS CA12/00122 to ARN and FPU12/02220 to AW.

It is well established that non-synaptic mechanisms can generate electrographic seizures after blockade of synaptic function. We investigated the interaction of intact synaptic activity with non-synaptic mechanisms in the isolated CA1 region of rat hippocampal slices using the “elevated-K+” model of epilepsy. Elevated K+ ictal bursts share waveform features with other models of electrographic seizures, including non-synaptic models where chemical synaptic transmission is suppressed, such as the low-Ca2+model. These features include a prolonged (several seconds) negative field shift associated with neuronal depolarization and superimposed population spikes. When population spikes are disrupted for up to several seconds, intracellular recording demonstrated that the prolonged suppression of population spikes during ictal activity was due to depolarization block of neurons. Elevated-K+ ictal bursts were often preceded by a build-up of “pre-ictal” epileptiform discharges that were characterized as either “slow-transition” (localized and with a gradual increase in population spike amplitude, reminiscent non-synaptic neuronal aggregate formation, presumed mediated by extracellular K+ concentrations ([K+])o accumulation), or “fast-transition” (with a sudden increase in population spike amplitude, presumed mediated by field effects). When ictal activity had a fast-transition it was preceded by fast-transition pre-ictal activity; otherwise population spikes developed gradually at ictal event onset. Addition of bicuculline, a GABAA receptor antagonist, suppressed population spike generation during ictal activity, reduced pre-ictal activity, and increased the frequency of ictal discharges. Nipecotic acid and NNC-711, both of which block GABA re-uptake, increased population spike amplitude during ictal bursts and promoted the generation of preictal activity. By contrast, addition of ionotropic glutamate-receptor antagonists (NBQX, D-APV) had no consistent effect on ictal burst waveform or frequency and did not fully suppress pre-ictal activity. Similarly, CGP 55848, a GABAB receptor antagonist, has no significant effect on pre-ictal activity or burst frequency (although it did increase burst duration slightly). Our results are consistent with the hypothesis that non-synaptic mechanisms underpin the generation of ictal bursts in CA1 and that GABAA synaptic mechanisms can shape event development by delaying event initiation and counteracting depolarization block.

Omeprazole (OME) is a proton pump inhibitor (PPI) with 58% bioavailability after single oral dose, presenting large inter-individual variations and significant drug-drug interactions. A simple and rapid liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with solid phase extraction (SPE) and isotope-labelled internal standard (IS) method was developed to monitor the plasma levels of OME for application in pharmacokinetics and drug-drug interactions studies. OME and its IS (OME-D3), were eluted with Zorbax extend C-18 rapid resolution (4.6 mm x 50 mm, 3.5 μm) at 25ºC, under isocratic conditions through a mobile phase consisting of 1 mM ammonium acetate, pH 8.5 (55%), and acetonitrile (ACN, 45%). The flow rate was 0.8 mL/min and the run time of chromatogram was 1.2 min. OME was detected and quantified by LC-MS/MS with positive electrospray ionization (ESI) that operates in multiple-reaction monitoring (MRM) mode. The method was linear in the range of 1.5- 2000 ng/mL for OME. The validation assays of accuracy and precision, matrix effect, extraction recovery and stability of the samples for OME did not deviate more than 20% for the lower limit of quantification (LLOQ) and no more than 15% for other quality controls (QCs), according to regulatory agencies., This work was also supported by FIS No. PI052124 and CA12/00122 to ARN and FPU12/02220 to AW.

Background Imatinib, dasatinib, and nilotinib are tyrosine kinase inhibitors (TKIs) used as first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring is important to achieve treatment efficacy in the case of imatinib and nilotinib, and to control toxicity in the case of dasatinib. New high-sensitivity methods to monitor those drugs are needed, especially for dasatinib. Thus, a simple method to determine plasma levels of imatinib, dasatinib, and nilotinib for application in clinical practice was developed. Methods TKIs were eluted with a Poroshell 120 EC-C18 column (2.1 × 75 mm, 2.7 μm) at 0.5 mL/min and 60°C, under gradient conditions through a mobile phase consisting of 4 mmol/L ammonium formate, pH 3.2 (65%), and acetonitrile (35%). TKIs were detected and quantified by liquid chromatography in tandem with mass spectrometry (LC/MS-MS) with positive electrospray ionization and analytes were extracted using solid phase extraction (Versaplate-SCX). Internal standards were isotope-labeled for each analyte. Results The method was linear in the range of 2.5-5000 ng/mL for imatinib, 0.75-400 ng/mL for dasatinib, and 2-4000 ng/mL for nilotinib. The validation assays for accuracy and precision, matrix effect, extraction recovery, carryover, and stability of the samples for all the TKIs were appropriate according to regulatory agencies. Furthermore, imatinib plasma samples, stored for 4 years at -80°C were quite stable in approximately half of the samples. Conclusions The method enables rapid quantification of TKI concentrations and is being applied to therapeutic drug monitoring to adjust dose and to manage adverse reactions in clinical practice.

Members of the Spanish Group of Chronic Myeloid Leukemia (GELMC) are: Valentín García-Gutiérrez (Hematology Department, Hospital Universitario Ramón y Cajal, Madrid, Spain). María T. Gómez-Casares (Hematology Department, Hospital Universitario Doctor Negrín, Las Palmas de Gran Canaria, Spain). José M. Puerta (Unidad de Gestión Clínica Hematología y Hemoterapia, Hospital Universitario Virgen de las Nieves, Granada, Spain). Juan M. Alonso-Domínguez (Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain). Santiago Osorio (Hematology Department, Hospital Universitario Gregorio Marañón, Madrid, Spain). Juan C. Hernández-Boluda (Hematology Department, Hospital Clínico, Valencia, Spain). Rosa Collado (Genetic Laboratory, Hematology Department, Consorcio Hospital General Universitario, Valencia, Spain). María J. Ramírez (Hematology Department, Hospital de Jerez de la Frontera, Jerez de la Frontera, Cádiz, Spain). Fátima Ibáñez (Hematology Department, Hospital San Pedro de Alcántara, Cáceres, Spain). Alicia Rodríguez (Hematology Department, Hospital Universitario Virgen Macarena, Sevilla, Spain). Concepción Ruiz-Nuño and Antonio Jiménez-Velasco (Hematology Department, Hospital Regional Universitario de Málaga, IBIMA, Spain). Juan L. Steegmann (Hematology Department & IIS-IP. Hospital Universitario de la Princesa, Madrid, Spain. In chronic myeloid leukemia (CML) patients, 3-month BCR-ABL1 levels have consistently been correlated with further outcomes. Monitoring molecular responses in CML using the GeneXpert (Cepheid) platform has shown an optimal correlation with standardized RQ-PCR (IS) when measuring BCR-ABL1 levels lower than 10%, as it is not accurate for values over 10%. The aim of the present study was to determine the predictive molecular value at three months on different outcome variables using the Xpert BCR-ABL1 MonitorTM assay (Xpert BCR-ABL1). We monitored 125 newly diagnosed consecutive CML patients in the chronic phase (CML-CP) using an automated method: Xpert BCR-ABL1. Only 5% of patients did not achieve an optimal response at 3 months, and the 10% BCR-ABL1 cutoff defined by RQ-PCR (IS) methods was unable to identify significant differences in the probabilities of achieving a complete cytogenetic response (CCyR) (50% vs. 87%, p = 0.1) or a major molecular response (MMR) (60% vs. 80%, p = 0.29) by 12 months. In contrast, a cutoff of 1.5% more accurately identified differences in the probabilities of achieving CCyR (98% vs. 54%, p

A simple, reproducible and fast (4 min chromatogram) method of liquid chromatography in tandem with mass spectrometry (LC/MS–MS) was developed to determine simultaneously the plasma levels of albendazole (ABZ) and its metabolite albendazole sulfoxide (ABZOX) for pharmacokinetic and clinical analysis. Each plasma sample was extracted by solid phase extraction (SPE) using phenacetin as internal standard (IS). The extracted sample was eluted with a Zorbax XDB-CN column using an isocratic method. The mobile phase consisting of water with 1% acetic acid (40%, A) and MeOH (60%, B), was used at a flow rate of 1 mL/min. ABZ and ABZOX were detected and identified by mass spectrometry with electrospray ionization (ESI) in the positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 5–1000 ng/mL for ABZ and 10–1500 ng/mL (full validation) or 10–5000 ng/mL (partial validation) for ABZOX, with 5 and 10 ng/mL lower limit of quantification (LLOQ) for ABZ and ABZOX, respectively. The tests of accuracy and precision, matrix effect, extraction recovery and stability of the samples for both ABZ and ABZOX did not deviate more than 20% for the LLOQ and no more than 15% for other quality controls (QCs), according to regulatory agencies.

The primary functions of adrenal medullary chromaffin cells are the synthesis and storage in their chromaffin vesicles of the catecholamines noradrenaline (NA) and adrenaline (AD), and their subsequent release into the bloodstream by Ca

Analysis of neurotransmitters and their metabolites is useful for the diagnosis of central nervous system diseases. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with protein precipitation was developed to monitor levels of adrenaline (AD), noradrenaline (NA), glutamic acid (Glu), gamma-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in rat brain tissue. Isoprenaline was used as an internal standard (IS). Neurotransmitters and metabolites were eluted with a reverse phase column under gradient conditions through a mobile phase consisting of 0.2% formic acid water solution/acetonitrile. The compounds were detected and quantified by LC-MS/MS with positive or negative electrospray ionization, which operates in multiple-reaction monitoring mode. The method was linear or polynomial (R-2 > 0.99) for AD, NA, Glu, GABA, DA, 5-HT, 5-HIAA, and MHPG in the range of 0.25-200, 0.5-200, 250-20,000, 250-20,000, 0.25-200, 10-3000, 1-50, and 1-50 ng/mL, respectively. The validation assays for accuracy and precision, matrix effect, extraction recovery, stability and carry-over of the samples for neurotransmitters and metabolites were consistent with the requirements of regulatory agencies. The method enables rapid quantification of neurotransmitters and their metabolites and has been applied in the nuclear factor (erythroid 2-derived)-like 2 (Nrf2) knockout mouse model of depression.

Mellström, Britt et al., [Background] Transcriptional repressor DREAM (downstream regulatory element antagonist modulator) is a Ca2+-binding protein that regulates Ca2+ homeostasis through gene regulation and protein-protein interactions. It has been shown that a dominant active form (daDREAM) is implicated in learning-related synaptic plasticity such as LTP and LTD in the hippocampus. Neuronal spines are reported to play important roles in plasticity and memory. However, the possible role of DREAM in spine plasticity has not been reported., [Results] Here we show that potentiating DREAM activity, by overexpressing daDREAM, reduced dendritic basal arborization and spine density in CA1 pyramidal neurons and increased spine density in dendrites in dentate gyrus granule cells. These microanatomical changes are accompanied by significant modifications in the expression of specific genes encoding the cytoskeletal proteins Arc, Formin 1 and Gelsolin in daDREAM hippocampus., [Conclusions] Our results strongly suggest that DREAM plays an important role in structural plasticity in the hippocampus., This work was funded by Instituto de Salud Carlos III/CIBERNED (to JRN, BM and JdF), Madrid Community/Neurodegmodels (to JRN), by Ministerio de Economia y Competitividad (grants SAF2010-21784 and SAF2014-53412-R, to JRN) and by Areces Foundation, the EU 6th Framework Program: Network of Excellence NeuroNE (JRN) and the ERA-NET Programs Neuron (JRN), grants from the Medical Research Council (TVPB) and by grants from the EJLB-CIHR Michael Smith Chair in Neurosciences and Mental Health, Canada Research Chair, Canadian Institute for Health Research operating grant (MOP-124807), NSERC Discovery Grant (RGPIN 402555) (to MZ). SK had a postdoctoral contract from the Ramón y Cajal Program of the Ministry of Science and Innovation., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

We have developed a method of liquid chromatography in tandem with mass spectrometry to monitor therapeu-tic levels of imatinib in plasma, a selective inhibitor of protein tyrosine kinase. After solid-phase extraction of plasmasamples, imatinib and its internal standard, imatinib-D8, were eluted with Zorbax SB-C 18 at 60 C, under isocratic conditionsthrough a mobile phase consisting of 4m M ammonium formate, pH: 3.2 (solution A) and acetonitrile solution B. The flow ratewas 0.8mL/min with 55% solution A+45% solution B. Imatinib was detected and quantified by mass spectrometry withelectrospray ionization operating in selected-reaction monitoring mode. The calibration curve was linear in the range10–5000ng/mL, the lower limit of quantitation being 10ng/mL. The method was validated according to the recommendationsof the Food and Drug Administration, including tests of matrix effect (bias 80 and

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.