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La inteligencia artificial tiene el potencial de transformar la forma en que se brinda la atención médica. Puede respaldar mejoras en los resultados y aumentar la productividad y la eficiencia de la prestación de los servicios. En servicios de las diferentes especialidades los avances realizados a nivel hardware deben desarrollarse en paralelo con los métodos de aprendizaje automático, aspectos que la inteligencia artificial contribuye para promover un cambio de paradigma significativo en las más diversas áreas de la medicina. Es importante en la educación médica como eje para el conocimiento y en la toma de decisiones que pueden mejorar el desempeño de los profesionales. Los estudiantes de medicina de nueva generación pueden adaptarse perfectamente a los nuevos métodos digitalizados en un contexto médico globalizado, incluida la inteligencia artificial. Por ello es importante tener como objetivos a implementar en los planes de estudio e introducir programas educativos representativos de esta tecnología. Es fundamental que todas las áreas del Sistema de Salud tengan confianza en los sistemas informáticos específicamente en el aprendizaje profundo, no solo por la información concreta y objetiva que de él se deriva sino también por la posibilidad de predecir eventos futuros, brindando alta certeza en cuanto al diagnóstico y tratamiento de enfermedades.

Cold-restraint stress reduces gastric blood flow and produces acute gastric ulcers. We studied the role of Angiotensin II (Ang II) on gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats, a stress-sensitive strain, were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 blockade increased gastric blood flow 40% to 50%; prevented gastric ulcer formation by 70% to 80%; reduced the increase in adrenomedullary epinephrine and TH mRNA without preventing the stress-induced increase in adrenal corticosterone; decreased the stress-induced expression of tumor necrosis factor (TNF-α) and adhesion protein ICAM-1 in arterial endothelium, and neutrophil infiltration in the gastric mucosa; and decreased PGE2 content. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, anti-inflammatory effects with reduction in TNF-α, and ICAM-1 expression, leading to reduced neutrophil infiltration while maintaining the protective glucocorticoid effects and PGE2 release. Ang II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit. [ABSTRACT FROM AUTHOR]

The sympathoadrenal response to stress includes a profound increase in adrenomedullary catecholamine synthesis driven by stimulation of tyrosine hydroxylase (TH) transcription. We studied the role of Angiotensin II type 1 and 2 (AT1 and AT2) receptors during isolation stress, and under basal conditions. Pretreatment of rats with the AT1 receptor antagonist candesartan for 14 days prior to isolation completely prevented the stress-induced stimulation of catecholamine synthesis, decreasing tyrosine hydroxylase transcription by preventing the expression of the transcriptional factor, Fos-related antigen 2 (Fra-2). In addition, AT1 receptor antagonism prevented the stress-induced increase in adrenomedullary AT2 receptor binding and protein. Treatment of non-stressed, grouped animals under basal conditions with the AT1 receptor or with PD 123319, an AT2 receptor antagonist, decreased the adrenomedullary norepinephrine (NE) content and TH transcription. While AT1 receptor antagonism decreased the levels of Fra- 2 and the phosphorylated forms of cAMP responsive element binding protein (pCREB) and EKR2 (p-ERK2, phosphor-p42 MAP kinase), the AT2 antagonist decreased Fra-2 with no change in the phosphorylation of CREB or EKR2. Our results demonstrate that both adrenomedullary AT1 and AT2 receptor types maintain and promote the adrenomedullary catecholamine synthesis and the transcriptional regulation of TH. Instead of opposing effects, however, our results indicate a complex synergistic regulation between the AT1 and AT2 receptor types. [ABSTRACT FROM AUTHOR]

Background: Angiotensin II, is critical in regulating the sympathetic and neuroendocrine systems through angiotensin II type 1 receptors (AT 1 -R). Angiotensin II intracerebral administration increases water and sodium intake, as well as renal sodium excretion. Previously, our group has shown that AT 1 -R is involved in behavioral and neurochemical sensitization induced by amphetamine. We aimed to assess the physiological output, behavioral, and neurochemical responses to intracerebral angiotensin II administration, via the AT 1 -R, twenty-one days after amphetamine administration., Material and Methods: Male Wistar rats received daily vehicle or AT 1 -R antagonist (oral) for 10 days, and amphetamine or saline intraperitoneal (i.p.) from day 6 to 10. On day 25 they were implanted with an intracerebral cannula. On day 32, the animals received intracerebral angiotensin II. First group: the animals were tested in a free choice paradigm for 2% NaCl and water intake, and sacrificed for neuronal activity assessment via c-Fos immunohistochemistry. Second group: urine samples were collected for electrolyte determination. Third group: the animals were tested in the plus maze or the hole board for anxiety and working memory evaluation, respectively, and sacrificed for c-Fos immunohistochemistry., Results: Amphetamine exposure blunted the increase in sodium intake ( p = 0.0022), and potentiated the natriuretic ( p = 0.0043) and kaliuretic effect ( p = 0.0002) induced by angiotensin II. Moreover, amphetamine exposure prevented the expression of the anxiogenic effect (drug effect p < 0.0001) and the memory deficit ( p = 0.1314) induced by cerebral angiotensin II administration. Amph decreased c-Fos immunoreactivity in nucleus tractus solitarii (NTS) p = 0.0037; paraventricular nucleus (PVN) p = 0.0047; Central amygdala (CeA) p = 0.0008; Basolateral amygdala (BLA) p = 0.0018; increased in hippocampus region CA1 p = 0.0043; CA3 p = 0.026; and dentate gyrus (DG) p = 0.0057. The blockade of AT 1 -R prevented these alterations (sodium intake p = 0.0421 natriuresis p = 0.019; kaliuresis p = 0.196; working memory ( p < 0.0001); but no the anxiogenic response to angiotensin II (drug effect p < 0.0001); as well as the c-Fos changes (NTS p = 0.0052; PVN p = 0.029; CeA p = 0.0002; BLA p = 0.0021; CA1 p = 0.0026; CA3 p = 0.022; and DG p = 0.0016)., Conclusion: Most of the long-lasting AT 1 -R altered responses to brain angiotensin II administration induced by repeated amphetamine exposure could be prevented by AT 1 -R blockade.

Background: Dalbavancin, an antimicrobial lipoglycopeptide, is authorized in Europe for treating acute bacterial infections of the skin and skin structures in adults and pediatric patients aged 3 months and older. However, off-label dosing regimens have been proposed for various indications beyond acute bacterial infections of the skin and skin structures. This study presents a novel bioanalytical method using liquid chromatography-tandem mass spectrometry to quantify dalbavancin in low-volume plasma samples (50 μL)., Methods: The method underwent validation in accordance with international guidelines for bioanalytical method validation and was applied to 9 clinical samples obtained from pediatric and young adult patients undergoing dalbavancin therapy. Liquid chromatography-tandem mass spectrometry analyses were conducted at the G. Gaslini Institute in Genoa, Italy, utilizing an Ultimate 3000 ultra high performance liquid chromatography system coupled to a TSQ Quantiva Triple Quadrupole system (Thermo Fisher Scientific, Milan, Italy). The analytical procedure involved the addition of deuterated dalbavancin as internal standard and a rapid extraction from 50 µL of human plasma, followed by chromatographic separation on a Thermo Scientific Accucore Polar Premium column. Accurate quantification of the analyte was achieved through multiple reaction monitoring detection., Results: The assay exhibited linearity within the concentration range of 0.66-400 mcg/mL in plasma, demonstrating accuracy and reproducibility in the absence of matrix effects. Stability testing was conducted on both quality controls and real samples to establish a robust protocol under real-life conditions., Conclusions: This fast and reliable dalbavancin quantitation method could improve current pediatric clinical practice by enabling data collection for future dose recommendations in special patient populations., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Apixaban, a direct oral anticoagulant drug (DOAC), typically does not require routine therapeutic drug monitoring (TDM), yet recent guidelines propose its use in specific clinical scenarios. While various antifactor Xa (anti-FXa) chromogenic assays serve as useful proxies for measuring plasma exposure to apixaban in emergencies, they lack specificity compared with chromatographic methods. This research project is intended to the development and validation of a standardized protocol of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conformity with the ICH guidelines M10 for the measurement of apixaban in both plasma and dried plasma spots (DPSs). Samples preparation included protein precipitation after the addition of a deuterated internal standard (IS), and the chromatographic separation was carried out on a Thermo Scientific™ Accucore™ Polar Premium column (50 mm × 2.1 mm, i.d. 2.6 m). The newly developed LC-MS/MS method for apixaban mesurement from both plasma and DPS resulted linear over a wide concentration range (31.25-500 ng/mL), accurate, and reproducible without matrix effects, allowing for specific and rapid quantification. Stability was assessed on quality controls and a real sample, allowing the setting up of a robust TDM protocol that was applied to five anonymized plasma samples obtained from adult patients undergoing apixaban treatment at steady-state. In conclusion our novel LC-MS/MS method is adequate for accurate apixaban quantitation from both plasma and DPS matrixes, and may thus facilitate the guidelines suggested implementation of apixaban TDM, even in peripheral hospitals through shipment of DPS at reference laboratories., (© 2024 The Author(s). Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

Background and Aims: Ceftobiprole is a recent 5 th generation parenteral cephalosporin with antibacterial activity against a large range Gram+ and Gram- bacteria. Therapeutic drug monitoring (TDM) is an essential tool for maintaining plasma concentrations of antibiotics above the MIC by the end of the dosing interval, thus preventing the resistant strain diffusion. TDM is already recommended for other cephalosporins, and it is a reasonable tool contributing to the safety and efficacy of these drugs. During the treatment of patients in real-life, a number of pharmacokinetic (PK) changes not normally seen in healthy volunteers can occur which can impair the pharmacokinetic/pharmacodynamic target attainment. We aimed to develop simple and rapid HPLC-UV method for determination of ceftobiprole in human serum to implement TDM in clinical practice and support PKs and pharmacokinetic/pharmacodynamic (PK/PD) studies., Materials and Methods: Samples preparation of calibration standards, QC, and anonymous patients serum samples was performed by protein precipitation by adding 0.01 ml of sulphosalicylic acid at 30 % to 0.1 ml of each sample. Then samples were vortexed and the centrifuged at 12,000 rpm for 10 min at 4 °C. Fifty microlitres of clear supernatant were diluted 1:1 with mobile phase and transferred into HPLC autosampler held at 8 °C. Chromatographic separation was carried out in a gradient mode at 35 °C on an ultra-Biphenyl column using a Thermo Scientific chromatographic system with a Diode array. Data management was performed with Chromeleon 7.4 software., Results: The HPLC-UV method proved to be linear over wide concentration ranges (0.5-50.0 mg/L) and was accurate and reproducible in the absence of matrix effects, allowing for robust, specific, and rapid quantification of ceftobiprole from a low amount of serum (0.1 mL). The mean steady state C trough and C end values measured in the anonymous patients' samples were 6.26 ± 3.81 mg/L and 22.56 ± 15.69 mg/L, respectively., Conclusions: We report a broadened simple and fast HPLC with UV detection method for quantification of ceftobiprole in human serum to implement ceftobiprole TDM as clinical routine, and support future (PK/PD) studies in special patients' population., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Amphetamine (AMPH) exposure induces behavioural and neurochemical sensitization observed in rodents as hyperlocomotion and increased dopamine release in response to a subsequent dose. Brain Angiotensin II modulates dopaminergic neurotransmission through its AT 1 receptors (AT 1 -R), positively regulating striatal dopamine synthesis and release. This work aims to evaluate the AT 1 -R role in the development and maintenance of AMPH-induced sensitization. Also, the AT 1 -R involvement in striatal dopamine reuptake was analysed. The sensitization protocol consisted of daily AMPH administration for 5 days and tested 21 days after withdrawal. An AT 1 -R antagonist, candesartan, was administered before or after AMPH exposure to evaluate the participation of AT 1 -R in the development and maintenance of sensitization, respectively. Sensitization was evaluated by locomotor activity and c-Fos immunostaining. Changes in dopamine reuptake kinetics were evaluated 1 day after AT 1 -R blockade withdrawal treatment, with or without the addition of AMPH in vitro. The social interaction test was performed as another behavioural output. Repeated AMPH exposure induced behavioural and neurochemical sensitization, which was prevented and reversed by candesartan. The AT 1 -R blockade increased the dopamine reuptake kinetics. Neither the AMPH administration nor the AT 1 -R blockade altered the performance of social interaction. Our results highlight the AT 1 -R's crucial role in AMPH sensitization. The enhancement of dopamine reuptake kinetics induced by the AT 1 -R blockade might attenuate the neuroadaptive changes that lead to AMPH sensitization and its self-perpetuation. Therefore, AT 1 -R is a prominent candidate as a target for pharmacological treatment of pathologies related to dopamine imbalance, including drug addiction and schizophrenia., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Vancomycin is a glycopeptide antibiotic that has been adopted in clinical practice to treat gram-positive infections for more than 70 years. Despite vancomycin's long history of therapeutic use, optimal dose adjustments and pharmacokinetic/pharmacodynamic (PK/PD) target attainment in children are still under debate. Therapeutic drug monitoring (TDM) has been widely integrated into pediatric clinical practice to maximize efficacy and safety of vancomycin treatment. Area under the curve (AUC)-guided TDM has been recently recommended instead of trough-only TDM to ensure PK/PD target attainment of AUC 0-24h /minimal inhibitory concentration (MIC) > 400 to 600 and minimize acute kidney injury risk. Bayesian forecasting in pediatric patients allows estimation of population PK to accurately predict individual vancomycin concentrations over time, and consequently total vancomycin exposure. AUC-guided TDM for vancomycin, preferably with Bayesian forecasting, is therefore suggested for all pediatric age groups and special pediatric populations. In this review we aim to analyze the current literature on the pediatric use of vancomycin and summarize the current knowledge on dosing optimization for target attainment in special patient populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Janus kinase inhibitors are antirheumatic immunosuppressive drugs that target intracellular Janus kinases (JAKs). Baricitinib is a selective and reversible orally administered JAK1/JAK2 inhibitor approved for treating rheumatoid arthritis, atopic dermatitis, and alopecia areata in adult patients. Expanded access to baricitinib has been approved for treating pediatric patients affected by rare Mendelian autoinflammatory diseases with type I interferon-mediated damage. Knowledge of the pharmacokinetic properties and target plasma levels of baricitinib in pediatric patients is limited. In this study, a novel LC-MS/MS method for measuring baricitinib in plasma, validated according to the ICH M10 guidelines, is presented., Methods: Sample preparation was performed by adding 10 µL of IS working solution (150 ng/mL) and 200 µL of MeOH to each plasma sample. Chromatographic separation was conducted using a Thermo Scientific Accucore Polar Premium column (50 mm × 2.1 mm, i.d. 2.6 m). This method was applied to 7 real anonymous plasma samples obtained from pediatric patients treated with baricitinib at IRCCS Istituto Giannina Gaslini (Genoa, Italy). Patients of both sexes had a median age of 14 years (range, 10-17 years)., Results: The LC-MS/MS method resulted linear over wide concentration ranges (1.024-100 ng/mL) and was accurate and reproducible in the absence of matrix effects, allowing for robust, specific, and rapid quantification of baricitinib from a low amount of plasma (50 µL). The plasma concentration of baricitinib in the samples of the patients, expressed as mean ± SD, was 11.25 ± 10.86 ng/mL., Conclusions: This novel LC-MS/MS method is suitable for the therapeutic drug monitoring of baricitinib and can help guide therapy optimization in pediatric patients., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.)

Oritavancin (ORI) is a semisynthetic lipoglycopeptide approved as a single 1200 mg dose intravenous infusion for the treatment of acute bacterial skin and skin structure infections (ABSSSIs) caused by Gram-positive organisms in adults. The pharmacokinetic/pharmacodynamic (PK/PD) linear kinetic profile and long terminal half-life (~393 h) of ORI make it therapeutically attractive for the treatment of other Gram-positive infections for which prolonged therapy is needed. Multidose regimens are adopted in real-world clinical practice with promising results, but aggregated efficacy data are still lacking. A comprehensive search on PubMed/Medline, Scopus, Cochrane and Google Scholar databases was performed to include papers published up to the end of January 2023. All articles on ORI multiple doses usage, including case reports, with quantitative data and relevant clinical information were included. Two reviewers independently assessed papers against the inclusion/exclusion criteria and for methodological quality. Differences in opinion were adjudicated by a third party. From 1751 potentially relevant papers identified by this search, a total of 16 studies met the inclusion criteria and were processed further in the final data analysis. We extracted data concerning clinical response, bacteriologic response, mortality and adverse events (AEs). From the 16 included papers, 301 cases of treatment with multidose ORIs were identified. Multidose regimens comprised an initial ORI dose of 1200 mg followed by 1200 mg or 800 mg subsequent doses with a varying total number and frequency of reinfusions. The most often treated infections and isolates were osteomyelitis (148; 54.4%), ABSSSI (35; 12.9%) and cellulitis (14; 5.1%); and MRSA (121), MSSA (66), CoNS (17), E. faecalis (13) and E. faecium (12), respectively. Clinical cure and improvement by multidose ORI regimens were observed in 85% (231/272) and 8% (22/272) patients, respectively. Multidose ORI was safe and well tolerated; the most frequent AEs were infusion-related reactions and hypoglycemia. A multidose ORI regimen may be beneficial in treating other Gram-positive infections besides ABSSSIs, with a good safety profile. Further studies are warranted to ascertain the superiority of one multidose ORI scheme or posology over the other., Competing Interests: The authors declare no conflicts of interest.

Altered direct oral anticoagulant (DOAC) plasma levels can lead either to spontaneous hemorrhagic or thrombotic complications. We describe a case of suspected altered apixaban disposition in a patient with an upper gastrointestinal cancer resection treated with apixaban for non-valvular atrial fibrillation. Diagnosis of ischemic stroke for left hemiparesis was confirmed due to recent emergence of a hypodense area in the posterior capsular nucleus of ischemic reference in a context of binuclear capsular lacunar lesions. Thus, apixaban underexposure was suspected from anamnestic data and oral anticoagulation was switched to parenteral at the next scheduled dose for stroke recurrence. Before switching apixaban pharmacokinetic analysis was performed and unexpectedly showed apixaban plasma overexposure. After 3 days from the switch, the patient experienced spontaneous bleeding complications, for which the risk-benefit profile of continuing anticoagulant treatment for stroke recurrences warranted treatment discontinuation. Unexpected DOAC plasma exposure may present in special patient populations with thrombotic and bleeding complications. Though universally recognized therapeutic ranges have yet to be established for DOACs, periodic drug monitoring may aid in guiding optimization of DOAC therapy and reduce the risk of adverse events in special patient populations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Fluoropyrimidines (FPs) form still nowadays the backbone of chemotherapic schemes in colorectal cancer (CRC). Inter-patient variability of the toxicity profile of FPs may be partially accounted for by variable expression of dihydropyrimidine dehydrogenase (DPD). DPD rate activity is genetically determined by its extremely polymorphic coding gene DPYD. In spite of pharmacogenetic guideline-directed-dosing of FPs based regimens treating carrier of multiple variants of DPYD gene remains still challenging., Case Presentation: We present a case of a 48-year-old Caucasian man, compound heterozygous variant carrier of the DPYD gene (HapB3 and c.2194G>A) who had a diagnosis of adenocarcinoma of the left colon and was safely treated with a pharmacogenetic-guided 25% dose reduction of the standard CAP adjuvant treatment. Compound heterozygosis may have been responsible for an earlier over exposure to CAP resulting into low-grade toxicity with an anticipated median time to toxicity of the c.2194G>A variant to the 4th vs. 6th cycles. Some haplotypes of DPYD variants may have an advantage in terms of survival compared to wild-type patients. Our patient may also have benefitted from compound heterozygosis, as shown by no evidence of disease (NED) at 6-month follow-up., Conclusion: Pharmacogenetic-guided dosing of DPYD intermediate metabolizer compound heterozygous HapB3 and c.2194G>A variant carries should be managed by a multidisciplinary team with a dose reduction ranging from 25 to 50% to maintain effectiveness and close clinical monitoring for early detection of ADRs., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The prescriptive appropriateness of Proton Pump Inhibitors (PPIs) in polypharmacy is controversial. PPIs are often overprescribed and the risk of prescribing errors and adverse drug reactions increases for each additional drug added to therapy. Hence, guided deprescription should be considered and easily implementable in ward practice. This observational prospective study evaluated the implementation of a validated PPIs deprescription flow chart to real-life internal ward activity through the presence of a clinical pharmacologist as an enhancing additional factor by assessment of inhospital prescriber's adherence to the proposed flow chart. Patients' demographics and prescribing trends of PPIs prescriptions were analyzed by descriptive statistics. The final analysis of data included ninety-eight patients (forty-nine male and forty-nine female), aging 75.6 ± 10.6 years; 55.1% of patients had home-PPIs prescriptions, while 44.9% received inhospital-PPIs prescriptions. Evaluation of prescriber's adherence to the flow chart revealed that the percentage of patients with a prescriptive/deprescriptive pathway conforming to that of the flow chart was 70.4%, with low symptomatologic recurrences. The clinical pharmacologists' presence and influence in ward activity may have contributed to this finding, since continuous training of the prescribing physicians is deemed a success-related factor in the deprescribing strategy. Multidisciplinary management of PPIs deprescription protocols shows high adherence by prescribers in real-life hospital settings and low recurrence events.

Proton pump inhibitors (PPIs) are among the most controversially prescribed drugs in polypharmacy. This observational prospective study assessed the PPI prescriptive trend during hospitalization before and after implementation of a prescribing/deprescribing algorithm in a real-life hospital setting and the related clinical-economic benefit at discharge. PPI prescriptive trends were compared between three quarters of 2019 (9 months) and the same period of 2018 by a chi-square test with a Yate's correction. The proportions of treated patients in the two years (1120 discharged patients in 2018 and 1107 in 2019) were compared by the Cochran-Armitage trend test. DDDs (defined daily doses) were compared between 2018 and 2019 by the non-parametric Mann-Whitney test and normalizing DDD/DOT (days of therapy) and DDD/100 bd (bed days) for each patient. Multivariate logistic regression was performed on PPI prescriptions at discharge. The distribution of patients with PPIs at discharge was significantly different in the two years ( p = 0.0121). There was a downward trend in the number of PPI prescriptions (29.9%) in the third trimester of 2019 compared to the others of the same year (first trimester: 34.1%, second trimester: 36.0%) and by contrast with the same periods of 2018 (29.4, 36.0, and 34.7%) ( p = 0.0124). DDDs/patient did not differ between 2018 and 2019 nor across the three trimesters. However, both DDD/DOT and DDD/100 bd showed a decrease in the third trimester of 2019, with a marked difference for DDD/DOT ( p = 0.0107). The reduction in consumption detected in the last phase of 2019 in terms of DDD/DOT was 0.09 with a consequent containment of pharmaceutical spending. The development and implementation of multidisciplinary prescribing/deprescribing protocols in both hospital and community settings could lead to a reduction in the misuse of PPIs, with significant savings in healthcare resources.

The new breakthrough cystic fibrosis (CF) drug combination of ivacaftor (IVA), tezacaftor (TEZ), and elexacaftor (ELX), namely "caftor" drugs, directly modulates the activity and trafficking of the defective CF transmembrane conductance regulator protein (CFTR) underlying the CF disease. The role of therapeutic drug monitoring (TDM) of caftor drugs in clinical settings has recently been established. The availability of reliable and robust analytical methods for the quantification of IVA, TEZ, and ELX is essential to support dose-concentration-effect studies. We have developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the rapid and simultaneous quantification of IVA, TEZ, and ELX from the plasma of CF patients. The method was based on a rapid extraction protocol from 50 μL human plasma and separation on a reversed-phase C-18 HPLC column after the addition of deuterated internal standards. Accurate analyte quantification using multiple reaction monitoring (MRM) detection was then obtained using a Thermofisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. The method has been validated following international (EMA) guidelines for bioanalytical method validation and has been tested on plasma samples from 62 CF patients treated with the three-drug combination IVA/TEZ/ELX, marketed as Kaftrio ® or Trikafta ® , in steady-state condition. The assay was linear over wide concentration ranges (0.008-12 mg/L) in plasma for IVA, TEZ, and ELX, suitable for a broad range of plasma concentrations, and accurate and reproducible in the absence of matrix effects. The stability of analytes for at least 30 days at room temperature could allow for cost-effective shipment and storage. On the same day of sample collection, a sweat test was evaluated for 26 associated patients' samples, FEV1 (%) for 58, and BMI was calculated for 62. However, Spearman correlation showed no correlation between C through plasma concentrations of analytes (IVA, TEZ, ELX) and sweat test, FEV1 (%), or BMI. Our method proved to be suitable for TDM and could be helpful in assessing dose-concentration-response correlations in larger studies.

Glioblastoma (GBM) is characterized by fast-growing cells, genetic and phenotypic heterogeneity, and radio-chemo-therapy resistance, contributing to its dismal prognosis. Various medical comorbidities are associated with the natural history of GBM. The most disabling and greatly affecting patients' quality of life are neurodegeneration, cognitive impairment, and GBM-related epilepsy (GRE). Hallmarks of GBM include molecular intrinsic mediators and pathways, but emerging evidence supports the key role of non-malignant cells within the tumor microenvironment in GBM aggressive behavior. In this context, hyper-excitability of neurons, mediated by glutamatergic and GABAergic imbalance, contributing to GBM growth strengthens the cancer-nervous system crosstalk. Pathogenic mechanisms, clinical features, and pharmacological management of GRE with antiepileptic drugs (AEDs) and their interactions are poorly explored, yet it is a potentially promising field of research in cancer neuroscience. The present review summarizes emerging cooperative mechanisms in oncogenesis and epileptogenesis, focusing on the neuron-to-glioma interface. The main effects and efficacy of selected AEDs used in the management of GRE are discussed in this paper, as well as their potential beneficial activity as antitumor treatment. Overall, although still many unclear processes overlapping in GBM growth and seizure onset need to be elucidated, this review focuses on the intriguing targeting of GBM-neuron mutual interactions to improve the outcome of the so challenging to treat GBM.

Personalization of busulfan (Bu) exposure via therapeutic drug monitoring (TDM) is recommended for patients treated with high-dose conditioning regimens. Several laboratories' developed methods are available in the literature with a lack of standardization. The aim of this study is to develop a new standardized LC-MS/MS method and validate it according to the international ICH M10 (EMA) guidelines. Our method is based on rapid protein precipitation from 50 µL plasma followed by separation on a reversed-phase C-18 UHPLC column after the addition of deuterated internal standard and has been tested on real samples from pediatric patients treated with myeloablative conditioning regimens, including Bu, before autologous or allogeneic hematopoietic stem cell transplantation (HSCT). The validated LC-MS/MS method is linear over wide concentration ranges (125-2000 ng/mL), accurate, and reproducible in the absence of matrix effects, allowing for the specific and rapid quantification of Bu and allowing next-dose recommendations to be made in a timely fashion to answer clinicians' needs. Given the lack of data on the stability of Bu in real clinical samples, stability was assessed both on quality controls and on real samples to set up a robust protocol in real-life conditions. This novel LC-MS/MS method is suitable for application to the TDM-guided personalization of conditioning treatments with high-dose busulfan in pediatric patients undergoing HSCT.

Objectives: to assess the clinical care impact resulting from the lack of a regional reference Centre for Paediatric Poisoning in Liguria Region (Northern Italy) and to describe the demographic and clinical characteristics of paediatric patients who accessed the Emergency Department of the 'Gaslini' Paediatric Hospital (Genoa, Liguria Region) for intoxication., Design: retrospective cohort study., Setting and Participants: patients' cases of both sexes, <18 years old, who accessed the Emergency Department of the 'Gaslini' Paediatric Hospital between January 2017 and December 2019 for intoxication., Main Outcome Measures: the Poisoning Severity Score (PSS), a simple and reliable scoring system to describe poisonings and define their severity, was used. The primary objective was pursued by investigating the percentage of cases of intoxication which followed, in the study period, a clinical care pathway inconsistent with the degree of severity ascertained through the retrospective application of the PSS. Clinical-demographic data, triage tag color-coding, and causes of intoxication of cases were also collected. Descriptive statistics were used to summarize results., Results: a total of 172 cases were identified over the study period; 28 did not meet the inclusion criteria. The final analysis involved 144 cases of intoxication, 70 were from females and 74 from males, with a median age of 3 years-old; 60% of study cases followed a clinical care pathway consistent with the intoxication severity ascertained trough the PSS, in 40% of study cases the clinical care pathway was inconsistent with PSS. The triage tag colour-code assigned was green in 16% of accesses, yellow in 82%, and red in only 2%. Out of the total of accesses, 40% of cases were attributed to drug intoxication in which the agents most involved were analgesics and sedative-hypnotic drugs, 30% to carbon monoxide and fumes poisoning, 23% to food/other substance intoxication, and 7% to alcohol intoxication., Conclusions: implementing a referral Centre for Paediatric Poisoning could potentially affect 40% of access to the Emergency Department. Further analysis should be carried out to clarify whether an integrated Telemedicine Service could guide the correct management of intoxicated paediatric patients by referring them, through the Poisoning Severity Score system, for home monitoring or immediate hospitalization, if necessary.

Computerized techniques for image analysis are critical for progress in cell biology. The complexity of the data in current methods eliminates the need for manual image analysis and usually requires the application of multiple algorithms sequentially to the images. Our aim was to develop a software for immunohistochemical analysis of brain dopaminergic neurons combining several computational approaches to automatically analyze and quantify their number in the substantia nigra after a neurotoxic injury. For this purpose, we used a Parkinson's disease animal model to test our application. The dopaminergic neurotoxin, 6-hydroxydopamine, was administered in adult male rats to damage dopaminergic neurons in substantia nigra and to induce hemiparkinsonism. The lesion was corroborated by behavioral evaluation in response to apomorphine and amphetamine. The animals were euthanized and their brains processed for tyrosine hydroxylase immunohistochemistry for dopamine neuron identification. Neurons positive for tyrosine hydroxylase were evaluated in substantia nigra by light microscopy. The images were used to show quantification applicability. To test our software counting accuracy and validity, automatic dopamine neuron number was correlated with the data obtained by three independent observers. Several parameters were used to depict neuronal function in dataset images from control and lesioned brains. In conclusion, we could perform an automated quantification of dopaminergic neurons and corroborate the validity and accuracy of a freely available software., (© 2022. International Federation for Medical and Biological Engineering.)

Neurodegeneration with brain iron accumulation (NBIA) comprises various rare clinical entities with brain iron overload as a common feature. Magnetic resonance imaging (MRI) allows diagnosis of this condition, and genetic molecular testing can confirm the diagnosis to better understand the intracellular damage mechanism involved. NBIA groups disorders include: pantothenate kinase-associated neurodegeneration (PKAN), mutations in the gene encoding pantothenate kinase 2 (PANK2); neuroferritinopathy, mutations in the calcium-independent phospholipase A2 gene (PLA2G6); aceruloplasminemia; and other subtypes with no specific clinical or MRI specific patterns identified. There is no causal therapy, and only symptom treatments are available for this condition. Promising strategies include the use of deferiprone (DFP), an orally administered bidentate iron chelator with the ability to pass through the blood-brain barrier. This is a prospective study analysis with a mean follow-up time of 5.5 ± 2.3 years (min-max: 2.4-9.6 years) to define DFP (15 mg/kg bid)'s efficacy and safety in the continuous treatment of 10 NBIA patients through clinical and neuroradiological evaluation. Our results show the progressive decrease in the cerebral accumulation of iron evaluated by MRI and a substantial stability of the overall clinical neurological picture without a significant correlation between clinical and radiological findings. Complete ferrochelation throughout the day appears to be of fundamental importance considering that oxidative damage is generated, above, all by non-transferrin-bound iron (NTBI); thus, we hypothesize that a (TID) administration regimen of DFP might better apply its chelating properties over 24 h with the aim to also obtain clinical improvement beyond the neuroradiological improvement.

Schizophrenia is a chronic disease affecting 1% worldwide population, of which 30% are refractory to the available treatments: thus, searching for new pharmacological targets is imperative. The acute and repeated ketamine administration are validated preclinical models that recreate the behavioral and neurochemical features of this pathology, including the parvalbumin-expressing interneurons dysfunction. Angiotensin II, through AT 1 receptors (AT 1 -R), modulates the dopaminergic and GABAergic neurotransmission. We evaluated the AT 1 -R role in the long-term neuronal activation and behavioral alterations induced by repeated ketamine administration. Adult male Wistar rats received AT 1 -R antagonist candesartan/vehicle (days 1-10) and ketamine/saline (days 6-10). After 14 days of drug-free, neuronal activation and behavioral analysis were performed. Locomotor activity, social interaction and novel object recognition tests were assessed at basal conditions or after ketamine challenge. Immunostaining for c-Fos, GAD67 and parvalbumin were assessed after ketamine challenge in cingulate, insular, piriform, perirhinal, and entorhinal cortices, striatum, and hippocampus. Additionally, to evaluate the AT 1 -R involvement in acute ketamine psychotomimetic effects, the same behavioral tests were performed after 6 days of daily-candesartan and a single-ketamine administration. We found that ketamine-induced long-lasting schizophrenia-like behavioral alterations, and regional-dependent neuronal activation changes, involving the GABAergic neurotransmission system and the parvalbumin-expressing interneurons, were AT 1 -R-dependent. The AT 1 -R were not involved in the acute ketamine psychotomimetic effects. These results add new evidence to the wide spectrum of action of ketamine and strengthen the AT 1 -R involvement in endurable alterations induced by psychostimulants administration, previously proposed by our group, as well as their preponderant role in the development of psychiatric pathologies., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Astrocytes play an essential role in the genesis, maturation and regulation of the neurovascular unit. Multiple evidence support that astrocyte reactivity has a close relationship to neurovascular unit dysfunction, oxidative stress and inflammation, providing a suitable scenario for the development of mental disorders. Ketamine has been proposed as a single-use antidepressant treatment in major depression, and its antidepressant effects have been associated with anti-inflammatory properties. However, Ketamine long-lasting effects over the neurovascular unit components remain unclear. Angiotensin II AT 1 receptor (AT 1 -R) blockers have anti-inflammatory, antioxidant and neuroprotective effects. The present work aims to distinguish the acute and long-term Ketamine effects over astrocytes response extended to other neurovascular unit components, and the involvement of AT 1 -R, in prefrontal cortex and ventral tegmental area. Male Wistar rats were administered with AT 1 -R antagonist Candesartan/Vehicle (days 1-10) and Ketamine/Saline (days 6-10). After 14 days drug-free, at basal conditions or after Ketamine Challenge, the brains were processed for oxidative stress analysis, cresyl violet staining and immunohistochemistry for glial, neuronal activation and vascular markers. Repeated Ketamine administration induced long-lasting region-dependent astrocyte reactivity and morphological alterations, and neuroadaptative changes observed as exacerbated oxidative stress and neuronal activation, prevented by the AT 1 -R blockade. Ketamine Challenge decreased microglial and astrocyte reactivity and augmented cellular apoptosis, independently of previous treatment. Overall, AT 1 -R is involved in the development of neuroadaptative changes induced by repeated Ketamine administration but does not interfere with the acute effects supporting the potential use of AT 1 -R blockers as a Ketamine complementary therapy in mental disorders., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Amphetamine (AMPH) alters neurons, glia and microvessels, which affects neurovascular unit coupling, leading to disruption in brain functions such as attention and working memory. Oxidative stress plays a crucial role in these alterations. The angiotensin type I receptors (AT 1 -R) mediate deleterious effects, such as oxidative/inflammatory responses, endothelial dysfunction, neuronal oxidative damage, alterations that overlap with those observed from AMPH exposure. Aims: The aim of this study was to evaluate the AT 1 -R role in AMPH-induced oxidative stress and glial and vascular alterations in the prefrontal cortex (PFC). Furthermore, we aimed to evaluate the involvement of AT 1 -R in the AMPH-induced short-term memory and working memory deficit. Methods: Male Wistar rats were repeatedly administered with the AT 1 -R blocker candesartan (CAND) and AMPH. Acute oxidative stress in the PFC was evaluated immediately after the last AMPH administration by determining lipid and protein peroxidation. After 21 off-drug days, long-lasting alterations in the glia, microvessel architecture and to cognitive tasks were evaluated by GFAP, CD11b and von Willebrand immunostaining and by short-term and working memory assessment. Results: AMPH induced acute oxidative stress, long-lasting glial reactivity in the PFC and a working memory deficit that were prevented by AT 1 -R blockade pretreatment. Moreover, AMPH induces transient angiogenesis in PFC via AT 1 -R. AMPH did not affect short-term memory. Conclusion: Our results support the protective role of AT 1 -R blockade in AMPH-induced oxidative stress, transient angiogenesis and long-lasting glial activation, preserving working memory performance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Basmadjian, Occhieppo, Marchese, Silvero C., Becerra, Baiardi and Bregonzio.)

Amphetamine-induced neuroadaptations involve vascular damage, neuroinflammation, a hypo-functioning prefrontal cortex (PFC), and cognitive alterations. Brain angiotensin II, through angiotensin type 1 receptor (AT 1 -R), mediates oxidative/inflammatory responses, promoting endothelial dysfunction, neuronal oxidative damage and glial reactivity. The present work aims to unmask the role of AT 1 -R in the development of amphetamine-induced changes over glial and vascular components within PFC and hippocampus. Attention deficit was evaluated as a behavioral neuroadaptation induced by amphetamine. Brain microvessels were isolated to further evaluate vascular alterations after amphetamine exposure. Male Wistar rats were administered with AT 1 -R antagonist, candesartan, followed by repeated amphetamine. After one week drug-off period, animals received a saline or amphetamine challenge and were evaluated in behavioral tests. Afterward, their brains were processed for cresyl violet staining, CD11b (microglia marker), GFAP (astrocyte marker) or von Willebrand factor (vascular marker) immunohistochemistry, and oxidative/cellular stress determinations in brain microvessels. Statistical analysis was performed by using factorial ANOVA followed by Bonferroni or Tukey tests. Repeated amphetamine administration increased astroglial and microglial markers immunoreactivity, increased apoptotic cells, and promoted vascular network rearrangement at the PFC concomitantly with an attention deficit. Although the amphetamine challenge improved the attentional performance, it triggers detrimental effects probably because of the exacerbated malondialdehyde levels and increased heat shock protein 70 expression in microvessels. All observed amphetamine-induced alterations were prevented by the AT 1 -R blockade. Our results support the AT 1 -R involvement in the development of oxidative/inflammatory conditions triggered by amphetamine exposure, affecting cortical areas and increasing vascular susceptibility to future challenges., (© 2019 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: The functioning of the central nervous system is complex and it implies tight and coordinated interactions among multiple components. Neurotransmitters systems imbalance is a hallmark in the central nervous system (CNS) disorders. These pathologies profoundly impact the social, cultural, and economic perspective worldwide. The etiopathology of CNS illnesses is still poorly understood, making their treatment difficult. Brain angiotensin II (Ang II), through its AT1 receptors, modulates dopaminergic, glutamatergic and GABAergic neurotransmission, which are responsible for movement control, cognition, emotions and stress responses. Alterations of these functions, concomitant with modified brain reninangiotensin system (RAS) components, have been described in CNS pathologies like depression, Parkinson, Alzheimer, and schizophrenia. In this sense, altered functionality of angiotensin I converting enzyme and AT1 receptors, is associated with augmented susceptibility to the occurrence of these pathologies. Moreover, some epidemiological data showed lower incidence of Alzheimer disease in hypertensive patients under treatment targeting RAS; meanwhile preclinical studies relate RAS with Parkinson and depression. Little is known about schizophrenia and RAS; however, Ang II is closely related to dopamine and glutamate pathways, which are mainly altered in this pathology., Conclusion: The available evidences, together with the results obtained by our group, open the possibility to postulate brain Ang II as a possible therapeutic target to treat the above-mentioned CNS disorders., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The use of psychostimulants, such as amphetamine (Amph), is associated with inflammatory processes, involving glia and vasculature alterations. Brain Angiotensin II (Ang II), through AT 1 -receptors (AT 1 -R), modulates neurotransmission and plays a crucial role in inflammatory responses in brain vasculature and glia. Our aim for the present work was to evaluate the role of AT 1 -R in long-term alterations induced by repeated exposure to Amph. Astrocyte reactivity, neuronal survival and brain microvascular network were analysed at the somatosensory cortex. Thermal nociception was evaluated as a physiological outcome of this brain area. Male Wistar rats (250-320 g) were administered with AT 1 -R antagonist Candesartan/vehicle (3 mg/kg p.o., days 1-5) and Amph/saline (2.5 mg/kg i.p., days 6-10). The four experimental groups were: Veh-Sal, CV-Sal, Veh-Amph, CV-Amph. On day 17, the animals were sacrificed and their brains were processed for Nissl staining and immunohistochemistry against glial fibrillary acidic protein (GFAP) and von Willebrand factor. In another group of animals, thermal nociception was evaluated using hot plate test, in the four experimental groups, on day 17. Data were analysed with two-way anova followed by Bonferroni test. Our results indicate that Amph exposure induces an increase in: neuronal apoptosis, astrocyte reactivity and microvascular network, evaluated as an augmented occupied area by vessels, branching points and their tortuosity. Moreover, Amph exposure decreased the thermal nociception threshold. Pretreatment with the AT 1 -R blocker prevented the described alterations induced by this psychostimulant. The decreased thermal nociception and the structural changes in somatosensory cortex could be considered as extended neuroadaptative responses to Amph, involving AT 1 -R activation., (© 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Rationale: Angiotensin II, by activation of its brain AT1-receptors, plays an active role as neuromodulator in dopaminergic transmission. These receptors participate in the development of amphetamine-induced behavioral and dopamine release sensitization. Dopamine is involved in cognitive processes and provides connectivity between brain areas related to these processes. Amphetamine by its mimetic activity over dopamine neurotransmission elicits differential responses after acute administration or after re-exposure following long-term withdrawal periods in different cognitive processes., Objective: The purpose of this study is to evaluate the AT1-receptor involvement in the acute and long-term amphetamine-induced alterations in long-term memory and in cellular-related events., Methods: Male Wistar rats (250-300 g) were used in this study. Acute effects: Amphetamine (0.5/2.5 mg/kg i.p.) was administered after post-training in the inhibitory avoidance (IA) response. The AT1-receptor blocker Losartan was administered i.c.v. before a single dose of amphetamine (0.5 mg/kg i.p.). Long-term effects: The AT1-receptors blocker Candesartan (3 mg/kg p.o.) was administered for 5 days followed by 5 consecutive days of amphetamine (2.5 mg/kg/day, i.p.). The neuroadaptive changes were evidenced after 1 week of withdrawal by an amphetamine challenge (0.5 mg/kg i.p.). The IA response, the neuronal activation pattern, and the hippocampal synaptic transmission were evaluated., Results: The impairing effect in the IA response of post-training acute amphetamine was partially prevented by Losartan. The long-term changes induced by repeated amphetamine (resistance to acute amphetamine interference in the IA response, neurochemical altered response, and increased hippocampal synaptic transmission) were prevented by AT1-receptors blockade., Conclusions: AT1-receptors are involved in the acute alterations and in the neuroadaptations induced by repeated amphetamine associated with neurocognitive processes.

A single or repeated exposure to psychostimulants induces long-lasting neuroadaptative changes. Different neurotransmitter systems are involved in these responses including the neuropeptide angiotensin II. Our study tested the hypothesis that the neuroadaptative changes induced by amphetamine produce alterations in brain RAS components that are involved in the expression of the locomotor sensitization to the psychostimulant drug. Wistar male rats, pretreated with amphetamine were used 7 or 21 days later to study AT1 receptors by immunohistochemistry and western blot and also angiotensinogen mRNA and protein in caudate putamen and nucleus accumbens. A second group of animals was used to explore the possible role of Ang II AT1 receptors in the expression of behavioral sensitization. In these animals treated in the same way, bearing intra-cerebral cannula, the locomotor activity was tested 21 days later, after an amphetamine challenge injection and the animals received an AT1 blocker, losartan, or saline 5min before the amphetamine challenge. An increase of AT1 receptor density induced by amphetamine was found in both studied areas and a decrease in angiotensinogen mRNA and protein only in CPu at 21 days after treatment; meanwhile, no changes were established in NAcc. Finally, the increased locomotor activity induced by amphetamine challenge was blunted by losartan administration in CPu. No differences were detected in the behavioral sensitization when the AT1 blocker was injected in NAcc. Our results support the hypothesis of a key role of brain RAS in the neuroadaptative changes induced by amphetamine., (Published by Elsevier B.V.)

Central nucleus of the amygdala (CeA) is one of the most important regulatory centres for the emotional processes. Among the different neurotransmitter systems present in this nucleus, AT1 receptors have been also found, but their role in the generation and modulation of emotions is not fully understood. The present work evaluated the effect of intra-amygdalar injection of losartan (AT1 receptor antagonist) and angiotensin II (Ang II) in the anxiety state induced by fear-potentiated plus maze in male Wistar rats. Fear in the elevated plus maze can be potentiated by prior inescapable footshock stress. The decrease in the time spent in the open arms induced by the inescapable footshock was totally prevented by losartan (4 pmol) administration in CeA. It was also found that Ang II (48 fmol) administration decreased the time spent in the open arms in animals with or without previous footshock exposure. The locomotor activity and grooming behaviour were also evaluated. The results obtained from the different parameters analyzed allowed us to conclude that the Ang II AT1 receptors in CeA are involved in the anxiety state induced by stress in the fear-potentiated plus-maze behaviour.

The purpose of this study was to examine the effect of orally administered Uncaria tomentosa aqueous extracts (UTE) (Willd. ex Roem. & Schult.) DC. (Rubiaceae) during 7, 15, 30 and 90 days of treatment on the expression of anxiety, as expressed in the elevated plus maze test in male Albino Swiss mice. UTE revealed an anxiogenic effect in relation to the control group at 15 and 30 days, but it was reversed after 90 days of administration, without affecting the locomotor activity or any deleterious effects on the overall performance of the animal, either for its ambulation, or clinical status, and body weight and organ weight/body weight from liver, lung and kidney were unaffected. These biphasic effects are usually indicative of heterogeneity in sites of action due to the presence of many alkaloids (speciophylline, uncarine F and uncarine E) and flavanols (catechin and epigallocatechin) identified and isolated from UTE.

It has been recognized that the stress-related peptides are involved in anxiety states. Angiotensin II receptor blockade by systemic administration of the AT(1) receptor antagonists has been proposed as a new treatment possibility for anxiety disorders. For better understanding of the related mechanisms, in this study we evaluated effects of bilateral intraamygdaloid injections of 2 (LOS 2) and 4 (LOS 4) μg of losartan (LOS), a selective AT(1) receptor antagonist, on the behavior of the not stressed and acutely stressed rats in an elevated "plus" maze. Under non-stress conditions, LOS 4 increased time spent in the open arms (p < 0.01), number of extreme open arm arrivals (p < 0.05), time per entry (p < 0.01), and the number of total arm entries (p < 0.05) showing thus considerable anxiolytic activity. The open arm extreme arrivals were increased by LOS 4 in both not stressed (p < 0.05) and stressed (p < 0.05) rats. When no stressed and stressed LOS 4 animals were compared, time per entry and the number of closed arm entries (p < 0.05, both) were decreased in the latter group. Moreover, the LOS 4 stressed rats had significantly increased open/closed arm quotient (p < 0.05) as compared to the both control and LOS 4 non-stress group (p < 0.05, both). These findings suggest that the AT(1) receptor blockade in amygdala is important for the anxiolytic action of LOS (and probably other AT(1) receptor blockers) under both non-stress and stress conditions.