Towards a physio-cognitive model of slow-breathing

How may controlled breathing be beneficial, or detrimental to behavior? Computational process models are useful to specify the potential mechanisms that lead to behavioral adaptation during different breathing exercises. We present a physio-cognitive model of slow breathing implemented within a hybrid cognitive architecture, ACT-R/Φ. Comparisons to data from an experiment indicate that the physiological mechanisms are operating in a manner that is consistent with actual human function. The presented computational model provides predictions of ways that controlled breathing interacts with mechanisms of arousal to mediate cognitive behavior. The increasing use of breathing techniques to counteract effects of stressors makes it more important to have a detailed mechanistic account of how these techniques may affect behavior, both in ways that are beneficial and detrimental. This multi-level understanding is useful for adapting to changes in our physical and social environment, not only for performance, but for physical and mental health

Design, Synthesis, and Neurobehavioral Assessment of Novel Piracetam Derivatives for Alleviating Peripheral Neuropathy

Peripheral neuropathy is a common neurological disorder characterized by damage to the peripheral nerves, leading to symptoms such as pain, tingling, and numbness. Current treatment options are limited and often provide only symptomatic relief. This research paper explores the design, synthesis, and neurobehavioral assessment of novel piracetam derivatives as potential therapeutic agents for alleviating peripheral neuropathy. As peripheral neuropathy poses a considerable burden on affected individuals and lacks adequate treatment options, the study focuses on the design and synthesis of compounds with enhanced neuroprotective properties. The approach involves rational drug design to modify the piracetam structure, with subsequent in vitro and in vivo assessments of neuroprotective effects and neurobehavioral outcomes. The designed compounds are synthesized using established organic chemistry techniques, and their purity and identity are confirmed through rigorous spectroscopic and chromatographic methods. In vivo assessments on animal models of peripheral neuropathy include behavioral assays to measure pain thresholds, motor function, and cognitive performance. The anticipated results aim to provide insights into the potential therapeutic efficacy of the novel piracetam derivatives. Chemical structures, physicochemical properties, and neuroprotective outcomes will be discussed, considering the implications for developing innovative treatments for peripheral neuropathy. The abstract emphasizes the significance of addressing the unmet clinical needs of individuals suffering from peripheral neuropathy through the development of safe and effective therapeutic interventions. The conclusion underscores the importance of future research to delve into mechanistic aspects, conduct additional preclinical assessments, and ascertain the long-term safety and efficacy of the synthesized compounds before advancing to clinical trials

Toxicological profile for toluene

A Toxicological Profile for Toluene, Draft for Public Comment was released in September 2015. This edition supersedes any previously released draft or final profile.Chemical manager(s)/author(s): Jessilynn Taylor, Mike Fay, Robert Williams, Sharon Wilbur, ATSDR, Division of Toxicology and Human Health Sciences, Atlanta, GA; Peter McClure, Kimberly Zaccaria, H. Danielle Johnson, Mario Citra, SRC, Inc., North Syracuse, NY.tp56.pd

Study and evaluation of key factors in food and environment for human health concerns

La presente tesi, sviluppata in collaborazione con il Key Laboratory of Natural Resources of the Changbai Mountain and Functional Molecules - Yanbian University Ministry of Education (Yanji City, Jilin) e il centro di analisi BonassisaLab srl (Foggia, Italia), è incentrata sullo sviluppo e l’ottimizzazione di metodi di analisi adatti alla identificazione e quantificazione di contaminanti emergenti in matrici alimentari e ambientali. Un composto può essere definito contaminante emergente se: è apparso solo recentemente, anche grazie allo sviluppo di nuove metodologie analitiche e di indagine; i meccanismi di tossicità non sono ancora completamente compresi; è oggetto di revisioni legislative che includono nuove informazioni, non prese in considerazione precedentemente. Il loro studio è quindi di elevato interesse per la ricerca, e la determinazione analitica per la maggior parte di tali contaminanti rappresenta una sfida per i laboratori e gli enti governativi. Inoltre, i potenziali rischi per la salute umana a seguito di una loro assunzione e/o esposizione hanno portato ad un maggiore interesse nei loro confronti anche da parte delle organizzazioni dei consumatori e dei produttori e, conseguentemente, alla approvazione di leggi sempre più restrittive per la loro determinazione e quantificazione. Gli studi in questo settore sono attualmente incentrati su: a) classificazione di nuovi contaminanti emergenti, composti o molecole non noti per i quali è stata comprovata la loro tossicità; b) ricerche su contaminanti già noti per i quali gli effetti sugli ecosistemi non sono stati pienamente studiati e compresi; c) studio di problemi emergenti relativi a contaminanti noti, per una migliore comprensione dei rischi ad essi correlati dal punto di vista ambientale e per la salute umana. Il presente progetto di dottorato si innesta in questa tematica di ricerca ed è consistito nello sviluppo e l’ottimizzazione di metodi analitici per la determinazione di alcuni contaminanti emergenti in matrici alimentari ed ambientali mediante tecniche cromatografiche (GC, LC) abbinate a spettrometria di massa (MS). Nello specifico, cinque metodi analitici innovativi sono stati proposti per la determinazione dei piretroidi (PYR) in prodotti di origine animale (1), policlorobifenili (PCB) in campioni di uova (2) e latte (3), matrina e ossimatrina (MT, OMT ) in prodotti di origine vegetale (4) e derivati del benzene (BD) in campioni di acqua marina (5). Il primo lavoro menzionato ha riguardato la determinazione di sei PYRs (fenotrina, permetrina, ciflutrin, cipermetrina, deltametrina, fenvalerato) in campioni di uova e carne utilizzando un metodo analitico di screening sensibile e riproducibile mediante gascromatografia e rivelazione a cattura di elettroni (GC-ECD). Per la fase di preparazione del campione, è stata ottimizzata una procedura di purificazione basata sull'estrazione in fase solida (SPE); è stata ottenuta una separazione efficiente con un tempo di analisi totale inferiore a 60 minuti, comprendendo tutte le fasi analitiche dalla estrazione-purificazione fino alla determinazione cromatografica. Il metodo è stato ampiamente validato seguendo le direttive europee, dimostrandone la conformità in termini di selettività, sensibilità, recupero, precisione e incertezza di misura. Sono stati ottenuti ottimi risultati in termini di linearità in un intervallo di concentrazione compreso tra 50 e 500 μg L-1, con coefficienti lineari superiori a 0.9992. I limiti strumentali di rivelabilità sono risultati compresi tra 0.22 e 0.63 μg L-1, corrispondenti a 0.04 e 0.13 μg kg-1 in matrice. I limiti di rivelabilità nelle uova di gallina e in vari campioni di carne, calcolati su campioni additivati a concentrazioni note di piretroidi, sono rispettivamente compresi nell'intervallo di 0.05 e 0.25 μg kg-1 e 0.07 e 0.23 μg kg-1. I risultati della validazione analitica hanno confermato che il metodo proposto può essere utilizzato in modo affidabile per la determinazione dei piretroidi nelle analisi di controllo ufficiali. Il secondo lavoro ha riguardato un metodo analitico di screening, sensibile e riproducibile, per la determinazione e la quantificazione di sei policlorobifenili non diossino-simili (NDL-PCB, congeneri 28, 52, 101, 138, 153, 180) in uova di gallina basato sull'estrazione accelerata con solvente (ASE) per la determinazione della quantità di grasso da sottoporre ad un processo di purificazione tramite estrazione in fase solida (SPE) con analisi gascromatografica accoppiata ad una rivelatore a cattura di elettroni (GC-ECD). La separazione cromatografica ottimizzata, della durata inferiore ai 25 min, garantisce buone risposte strumentali per i sei NDL-PCB nell'intervallo tra 2.5 e 60.0 μg L-1, con coefficienti di correlazione sempre superiori a 0.9995. I limiti strumentali di rivelabilità sono risultati compresi tra 0.08 e 0.35 μg L-1, corrispondenti a 0.05 e 0.23 ng g-1 di grasso nella matrice, mentre i limiti di rivelabilità del metodo, calcolati su campioni di uova fortificate con concentrazioni note di analita, variano da 1.6 a 3.5 ng g-1 di grasso. Infine, il metodo è stato ampiamente validato in termini di selettività, sensibilità, recupero, precisione, robustezza e incertezza di misura, seguendo le linee guida europee. Nel terzo lavoro, lo studio ha riguardato la determinazione dei sei NDL-PCB analizzati precedentemente in campioni di latte mediante GC-ECD e spettrometria di massa, previa ottimizzazione multivariata della procedura di estrazione del campione attraverso il disegno sperimentale di Box-Behnken (BBD) e la funzione di desiderabilità. Nella definizione delle condizioni di estrazione sono stati considerati tre fattori: la percentuale di acetone nella miscela di estrazione, il rapporto campione/solvente e il tempo di estrazione. Il design a tre fattori ha richiesto 26 esperimenti che sono stati eseguiti in duplicato e in ordine randomizzato per ridurre al minimo gli effetti di distorsione delle variabili non controllate. I fattori ottimizzati (percentuale di acetone: 30%; rapporto campione/solvente: 0.11 g mL-1; tempo di estrazione: 45 min) hanno assicurato un basso consumo di solvente e un tempo di estrazione molto ridotto, consentendo una preparazione rapida e simultanea dei campioni. Il metodo è stato validato secondo le direttive europee attraverso la valutazione della linearità, selettività, LOD, LOQ, recupero, precisione e robustezza. Il quarto lavoro ha riguardato la determinazione quantitativa di biopesticidi a base di matrina in campioni di frutta e verdura tramite cromatografia liquida accoppiata a spettrometira di massa tandem. È stato sviluppato un processo di estrazione molto rapido mediante metanolo acidificato che garantisce tempi e consumi di solvente altamente ridotti e consente la preparazione simultanea di più campioni. Ottimi risultati cromatografici sono stati ottenuti in meno di 10 min. La validazione del metodo è stata eseguita secondo le direttive europee attraverso la valutazione della selettività, della linearità, dei limiti di rivelabilità e quantificazione, di recupero e precisione. Nelle condizioni ottimizzate di estrazione e separazione cromatografica sono state osservate buone risposte nell'intervallo di calibrazione 5-200 μg L-1 ottenendo coefficienti di correlazione superiori a 0.9984. I limiti strumentali di rivelabilità sono rispettivamente pari a 0.032 e 0.033 μg L-1 per la matrina e l'ossimatrina. I limiti di rivelabilità in arance, mele, finocchi e pomodori, calcolati su campioni fortificati con soluzioni standard di matrina e ossimatrina sono compresi rispettivamente nell'intervallo 0.13-1.5 μg kg-1 e 0.17-0.30 μg kg-1. Il metodo, dopo la validazione, è stato applicato con successo alla determinazione di matrina e ossimatrina in 102 campioni di frutta e verdura disponibili in commercio. Infine, l'ultimo capitolo ha riguardato i derivati del benzene (BD), una classe di contaminanti ambientali la cui esposizione rappresenta un grave rischio per la salute umana. Questi composti si diffondono molto rapidamente dall'atmosfera all'ecosistema marino e, per questo motivo, il loro monitoraggio in acqua di mare è molto importante. Per le analisi in traccia di questi composti nei campioni di acqua marina, è stata messa a punto una metodica di nanoestrazione in fase liquida nanoconfinata (NLPNE) abbinata alla determinazione gascromatografica e spettrometria di massa (GC/MS). Le procedure NLPNE ex-situ e in-situ sono state sviluppate e ottimizzate in termini di capacità di estrazione, tempo di analisi, precisione e accuratezza. Rispetto alle tradizionali procedure di estrazione, basate sulla microestrazione in fase solida (SPME) e sull'estrazione liquido-liquido (LLE), i metodi NLPNE qui proposti hanno consentito una rapida analisi in loco dei composti benzenici con basso consumo di solvente, fattori di arricchimento più elevati e con ottimi livelli di automazione. Sono stati ottenuti coefficienti di determinazione compresi tra 0.9929 e 0.9997 per tutti i BD nell'intervallo tra 0.10 e 500 ng mL-1 e 5.00 e 500 ng mL-1, rispettivamente utilizzando la tecnica NLPNE ex situ e in situ. I limiti di rivelabilità ex-situ e in-situ variano da 0.2 a 7.6 ng mL-1 e 0.04-1.00 ng mL-1. I risultati ottenuti suggeriscono che la metodica NLPNE accoppiata a GC-MS può essere considerato una tecnica performante per l’analisi ad alto rendimento di composti in tracce in campioni ambientali, alimentari e biologici.This thesis, developed in collaboration with the Key Laboratory of Natural Resources of the Changbai Mountain and Functional Molecules - Yanbian University Ministry of Education (Yanji City, Jilin) and the BonassisaLab srl analysis centre (Foggia, Italy), is focused on the development and optimization of analytical methods suitable for the identification and quantification in food and environmental matrices of emerging contaminants. A compound can be defined as an emerging contaminant if: it has appeared only recently thanks to the development of new analytical and investigation methodologies; the toxicity mechanisms are not fully understood; it is under legislative revisions to include new information not previously taken into account. Emerging contaminants are considered a fashionable research topic to the present day and most of them give a challenge for laboratories and regulatory agencies. Furthermore, the potential risks for human health related to their intake and/or exposure have led to deeper interest by consumer organizations and producers and are leading to the approval of more restrictive laws about their determination and quantification. Then, the studies are currently focused on: a) classification of new emerging contaminants, unknown compounds or molecules for which their toxicity has been proven; b) research on contaminants already known for which the effects on ecosystems have not been fully studied and understood; c) study of emerging issues related to known contaminants, for a better understanding of the risks related to them considering the environment and human health. In this research project, the development and optimization of analytical methods for the determination of some emerging contaminants in food and environmental matrices coupling liquid and gas chromatography with mass spectrometry were described. Five novel analytical methods for the determination and quantification of pyrethroids (PYRs) in animal-origin products (1), polychlorinated biphenyls (PCBs) in egg (2) and milk samples (3), matrine and oxymatrine (MT, OMT) in vegetables (4) and benzene derivatives (BDs) in sea-water (5) were developed. The first work mentioned deals with the determination of six PYRs (phenothrin, permethrin, cyfluthrin, cypermethrin, deltamethrin, fenvalerate) in egg and meat samples by a sensitive and reproducible screening analytical method based on gas chromatography and electron capture detection (GC-ECD). A fast cleanup procedure based on solid-phase extraction was used. An efficient separation was obtained with a total analysis time of less than 60 min, including the extraction-purification steps. Good responses for the six pyrethroids were obtained in a range of 50−500 μg L−1, with linear coefficients higher than 0.9992. Instrumental limits of detection were between 0.22 and 0.63 μg L−1, corresponding to 0.04 and 0.13 μg kg−1 in the matrix. Detection limits in chicken eggs and various meat samples, calculated on spiked samples, were in the range of 0.05−0.25 μg kg−1 and 0.07−0.23 μg kg−1, respectively. The validation results confirmed that the proposed GC-ECD method can be used as a reliable screening tool for the determination of pyrethroids in official check analyses. The method was extensively validated following the European directives, demonstrating its conformity in terms of selectivity, sensitivity, recovery, precision, and measurement uncertainty. The second work was about a sensitive and reproducible screening analytical method for the determination of six non-dioxin-like polychlorinated biphenyls (NDL-PCBs, congener 28, 52, 101, 138, 153, 180) in chicken eggs based on accelerated solvent extraction (ASE) procedure for the fat extraction and determination, a solid-phase extraction (SPE) sample clean-up process, and a gas chromatography – electron capture detection (GC-ECD) analysis. The optimized chromatographic separation, in less than 25 min, returned good responses for the six NDL-PCBs in the range of 2.5–60.0 μg L−1, with correlation coefficients always higher than 0.9995. Instrumental limits of detection were between 0.08–0.35 μg L−1, corresponding to 0.05 and 0.23 ng g−1 fat in the matrix, while method detection limits, calculated on spiked egg samples, ranged from 1.6 to 3.5 ng g−1 fat. The method has been extensively validated in terms of selectivity, sensitivity, recovery, precision, ruggedness, and measurement uncertainty, following the European Directives. In the third work, the study regarded the determination of the six NDL-PCBs in milk samples by GC-ECD and mass spectrometry, following a multivariate optimization process of the sample extraction procedure by Box-Behnken design through the global desirability function. Three factors were involved in refining the extraction conditions: the acetone percentage in the extraction mixture, the sample/solvent ratio, and the extraction time. The three-factor design required 26 experiments that were carried out in duplicate and in a randomized order to minimize the bias effects of uncontrolled variables. The optimized factors (acetone percentage: 30%; sample-to-solvent ratio: 0.11 g mL-1; extraction time: 45 min) ensured a low solvent consumption and a reduced extraction time, allowing a rapid and simultaneous preparation of multiple sample extracts. The method was validated according to the European directives through the evaluation of linearity, selectivity, LOD, LOQ, recovery, precision, and ruggedness. The fourth work consisted of the development of a rapid LC/tandem MS method for the quantitative determination of matrine-based biopesticides for the analysis of fruit and vegetable samples. A simple extraction process by acidified methanol was selected, ensuring reduced solvent consumption and time, and allowing a simultaneous preparation of multiple sample extracts. Excellent chromatographic results were obtained in terms of peak shape and efficiency, in less than 10 min. Method validation was performed according to the European directives through the evaluation of selectivity, linearity, detection and quantification limits, recovery, and precision. Under the optimal extraction and chromatographic conditions, good responses were observed in the range of 5–200 μg L-1, with correlation coefficients higher than 0.9984. Instrumental limits of detection were 0.032 and 0.033 μg L-1 for matrine and oxymatrine, respectively. Detection limits in oranges, apples, fennels, and tomatoes, calculated for matrine and oxymatrine on spiked samples, were in the range 0.13–1.5 μg kg-1 and 0.17-0.30 μg kg-1, respectively. The validated method was then successfully applied to the determination of matrine and oxymatrine in 102 commercially available samples of fruit and vegetables. Finally, the last chapter concerned the Benzene Derivatives (BDs), a class of environmental pollutants whose exposure poses a grave risk to human health. These compounds rapidly diffuse from the atmosphere to the marine ecosystem: for this reason, their monitoring in seawater is every day more compelling. For their determination in seawater at trace levels, nanoconfined liquid phase nano extraction (NLPNE) has been applied to the gas chromatography coupled with mass spectrometry analyses (GC/MS). Ex-situ and in-situ NLPNE procedures have been developed and optimized in terms of extraction capabilities, analysis time, precision, and accuracy. Compared to the traditional extraction procedures, based on solid-phase microextraction (SPME) and liquid-liquid extraction (LLE), the proposed NLPNE methods allowed rapid on-site analysis of benzene compounds with low solvent consumption, higher enrichment factors, and improved automation grade. Determination coefficients ranging from 0.9929 to 0.9997 were obtained for all BDs in the range 0.10–500 ng mL-1 and 5.00–500 ng mL-1, for ex-situ and in-situ NLPNE, respectively. Ex-situ and in-situ limits of detection ranged from 0.2 to 7.6 ng mL-1 and 0.04–1.00 ng mL-1. Our results suggest that NLPNE coupled to GC-MS can be considered a powerful technique for high-throughput analyses of trace compounds in environmental, food, and biological samples

Development of new antiepileptic drug candidates: a set of lamotrigine-related compounds

Epilepsy is one of the most common, chronic and serious neurological disorder, affecting million people worldwide. This brain disorder is characterised by recurrent spontaneous seizures, which have a considerable impact in the patients’ quality of life. The pharmacological therapy has been, and is likely to remain, the mainstay of treatment for this disorder. Although a large number of new antiepileptic drugs (AEDs) has been introduced into the market in the last years, about 30-40% of epileptic patients are still inadequately controlled by standard drug therapy. For this reason, it continues to be important to develop new and improved chemical entities through which epilepsy could be effectively controlled. In this context, the main objective of the present work was to discover new lead compounds with anticonvulsant properties for further development as AEDs. To achieve this goal, fifty dihydropyrimidin(thi)ones [DHPM(t)s] were synthesized through the Biginelli reaction, which consists in a one-pot cyclocondensation reaction among an aldehyde, a β-ketoester/acetylacetone and urea or thiourea. The products were purified and characterised by infrared and 1H- and 13C-nuclear magnetic resonance spectroscopy. High resolution mass spectrum was also obtained for the novel compounds. Afterwards, the anticonvulsant activity of the compounds was evaluated against electrically [maximal electroshock seizure (MES) test] and chemically [subcutaneous pentylenetetrazole (scPTZ) test] induced seizures in rodent models. The initial anticonvulsant screening was performed in CD-1 mice (n = 4/group) at 30 min and 4 h after the intraperitoneal administration of 30, 100 and 300 mg/kg of each compound. The investigated compounds were also evaluated in mice for neuromotor impairment (as a surrogate of minimal neurological deficit) on the rotarod performance test. Then, selected compounds previously identified as anticonvulsants in mice at the minimum dose tested were further assessed in Wistar rats (n = 4/group) at 30 min, 2 h and 4 h after the oral administration of 30 mg/kg. Additionally, the fifty DHPM(t)s were evaluated for their in vitro cytotoxicity in rat mesencephalic dopaminergic (N27), human hepatocellular carcinoma (HepaRG), human colorectal adenocarcinoma (Caco-2) and normal human dermal fibroblasts (NHDF) cell lines, through the well-established 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at the concentration of 30 μM. Moreover, as the efficacy of a molecule is strongly dependent on its pharmacokinetics, several kinetic properties were also investigated in in vitro and in silico models. Thus, all compounds were subjected to a set of in vitro screening assays performed on a cell line overexpressing the drug efflux transporter P-glycoprotein (MDCK-MDR1 cells) and on two models of parallel artificial membrane permeability assay (PAMPA) preditive of the apparent permeability (Papp) through intestinal membrane (intestinal PAMPA model) and blood-brain barrier (PAMPA-BBB model). Lastly, several physicochemical properties of the compounds were also calculated in silico and a set of pharmacokinetic and toxicity properties were estimated employing the new computational tool, pkCSM. The target molecules that were synthesized were mainly selected based on the structure of clinically relevant AEDs, in particular the structure of lamotrigine, aiming to discover new candidates for the development of improved AEDs. The majority of the chemical reactions occurred fastly and the products were obtained in good yields. The synthetic procedure used was also extended using additional specific reagents, being the respective products, which are new to the best of our knowledge, successfully synthesized. Due to practical considerations, only forty-two compounds (twenty-eight urea derivatives and fourteen thiourea derivatives) proceeded to in vivo experiments. The results of the initial pharmacological screening in mice revealed anticonvulsant protection in the MES model for twenty-four compounds showed anticonvulsant protection in the MES model, being nine of them active at the lowest dose tested (30 mg/kg). Structurally, the most promising compounds present smaller chains at the C5 of the dihydropyrimidine ring and an unsubstituted phenyl or a para-tolyl ring at the C4. In addition, the thiourea analogues also presented slightly increased anticonvulsant activity comparing with the corresponding urea analogues. The results of the minimal neuromotor impairment obtained through the rotarod assay showed that approximately 52% of the compounds are less toxic than lamotrigine, carbamazepine and phenytoin. Compounds MM 17, MM 19 and MM 83 also protected against MES-induced seizures in 50-75% of rats after the oral administration of 30 mg/kg. Furthermore, the most active compounds did not show notable cytotoxicity in in vitro experiments conducted in the several cell lines (relative cell proliferation higher than 50% at 30 μM), which can be relevant due to the fact that the toxicity is a common problem of the available AEDs. The data obtained showed that 82% of the investigated compounds are expected to have good intestinal permeability (Papp > 1.1×10-6 cm/s), and 66% of which good brain penetration (Papp > 2.0×10-6 cm/s), which can suggest a high passive transcellular permeability. In both cases, thiourea derivatives presented higher permeability values than the respective urea analogues, which can be associated with their higher lipophilicity. This finding can explain, at least in part, the higher activity of the thiourea derivatives in the anticonvulsant screening after both intraperitoneal and oral administrations. In addition, 44% of the compounds did not significantly modulate (inhibit or induce) P-glycoprotein at 10 and 50 μM. This is an interesting finding since P-glycoprotein is physiologically expressed in several tissues and organs relevant from a pharmacokinetics perspetive. Finally, in silico studies indicated that all compounds respect the Lipinski’s rule-of-five, suggesting that they possess favourable properties that fulfil the druglikeness criteria. The pkCSM in silico tool also estimated that the DHPM(t)s have good human intestinal absorption (67.73-93.91%) and an apparent volume of distribution at the steady-state in the same range of values of the AEDs. The in silico predictions also suggested a low plasma protein binding percentage for the target compounds, which is considered to be therapeutically favourable, minimizing the risk of drug interactions. These results corroborate those obtained with the intestinal PAMPA assay that showed that probably none of the tested compounds have a binding to plasma proteins higher than 90% (Papp ≤ 1.0×10−5 cm/s). The thiourea derivatives were also predicted as compounds that permeate better through biological barriers (e.g., Caco-2 cell monolayers and blood-brain barrier), similarly to the observed in the experimental PAMPA assays. However, the prediction model suggested that 14% of the urea derivatives have tendency for cytochrome P450 inhibition versus 36% of the thiourea derivatives. On the other hand, concerns on the disruption of normal liver function were predicted for half of the compounds. Overall, the set of studies carried out provide new information about the anticonvulsant activity of this class of heterocycles, along with pharmacokinetic and toxicity data. More than half of the investigated molecules showed anticonvulsant protection against electrically-induced seizures (MES model), confirming the interest of the pharmacophoric model for the design of new anticonvulsant agents. The data gathered here allowed to identify important structural features of this attractive scaffold that can be responsible for the anticonvulsant activity, which should be maintained or better explored in order to produce more active analogues in further hit-to-lead optimization. However, the results presented in this thesis are just the “tip of the iceberg” in the discovery and development of the DHPM(t)s as potential AEDs.A epilepsia é uma perturbação neurológica crónica que afeta milhões de pessoas em todo o mundo. Esta perturbação é caracterizada por crises epiléticas espontâneas e recorrentes, muito variadas na sua origem e apresentação clínica, as quais têm um impacto significativo na qualidade de vida dos doentes. A farmacoterapia tem sido, e provavelmente continuará a ser, o pilar da terapia da epilepsia. Todavia, embora um número considerável de novos fármacos antiepiléticos tenha sido introduzido no mercado nos últimos anos, cerca de 30-40% dos doentes epiléticos não alcançam um controlo apropriado das suas crises, mesmo quando são adequadamente tratados com os fármacos antiepiléticos disponíveis atualmente. Por esta razão, a descoberta e desenvolvimento de novas possibilidades farmacoterapêuticas que sejam mais seguras e, principalmente, mais eficazes, constituem um desafio e são de extrema importância. É neste contexto que surge o principal objetivo do presente trabalho; descobrir novos compostos com propriedades anticonvulsivantes para posterior desenvolvimento de novos fármacos antiepiléticos. Para atingir este objetivo, o design dos compostos selecionados baseou-se essencialmente na estrutura química da lamotrigina e levou à síntese de cinquenta dihidropirimidinonas/dihidropirimidinationas [DHPM(t)s] através da reação de Biginelli, a qual consiste numa reação de ciclocondensação entre um aldeído, um β-cetoester/acetilacetona e ureia ou tioureia. Depois de sintetizados, todos os compostos foram purificados e caracterizados através de espetros de infravermelhos e de ressonância magnética nuclear (protão e carbono-13); espetros de massa de alta resolução também foram obtidos para os compostos novos, ou seja, aqueles que não se encontravam descritos. Posteriormente, a atividade anticonvulsivante dos compostos foi avaliada em modelos animais de crises agudas induzidas eletricamente [teste do eletrochoque máximo (MES)] e quimicamente [teste do pentilenotetrazole subcutâneo (scPTZ)]. O screening inicial da atividade anticonvulsivante foi realizado em murganhos CD-1 (n = 4/grupo) e os compostos foram avaliados aos 30 min e às 4 h após a sua administração intraperitoneal nas doses de 30, 100 e 300 mg/kg. Paralelamente, os compostos em investigação também foram avaliados em murganhos, quanto à sua toxicidade neuromotora (traduzida pelo deficit neurológico mínimo), através do teste do aparelho rotativo. Posteriormente, alguns dos compostos identificados previamente como anticonvulsivantes nos murganhos na mínima dose testada foram ainda selecionados e testados em ratos Wistar (n = 4/grupo) aos 30 min, 2 h e 4 h após administração oral de uma dose de 30 mg/kg. Adicionalmente, as cinquenta DHPM(t)s foram avaliadas relativamente à sua citotoxicidade em sistemas in vitro de linhas celulares, concretamente em células dopaminérgicas mesencefálicas de rato (N27), células de carcinoma hepatocelular humano (HepaRG), células de adenocarcinoma coloretal humano (Caco-2) e fibroblastos normais da derme humana (NHDF), através do ensaio bem estabelecido do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT) e a uma concentração de 30 μM. Além disso, como a eficácia de uma molécula está fortemente dependente da sua farmacocinética, várias propriedades cinéticas também foram investigadas em modelos in vitro e in silico. Assim, todos os compostos sintetizados foram sujeitos a um conjunto de ensaios de screening in vitro realizados numa linha celular que sob-expressa o transportador de efluxo glicoproteína-P (células MDCK-MDR1) e em dois modelos de ensaios de permeabilidade em membrana artificial paralela (PAMPA), preditivos da permeabilidade aparente dos compostos através da membrana intestinal (PAMPA intestinal) e da barreira hematoencefálica (PAMPA-BBB). Por último, diversas propriedades físico-químicas dos compostos também foram calculadas in silico e um conjunto de propriedades farmacocinéticas e de toxicidade foram estimados com a uma nova ferramenta computacional, pkCSM. As moléculas alvo sintetizadas foram selecionadas principalmente com base na estrutura de fármacos antiepiléticos clinicamente relevantes, em particular a estrutura da lamotrigina, com o objetivo de descobrir novos candidatos para o desenvolvimento de fármacos antiepiléticos melhorados. A maioria das reações químicas ocorreram rapidamente e obtiveram-se bons rendimentos (acima de 60-70%) para grande parte dos produtos sintetizados. O procedimento de síntese química utilizado foi estendido também a reagentes específicos adicionais, tendo sido sintetizados com sucesso os produtos respetivos, os quais são novos (não descritos na literatura). Devido a questões práticas (baixos rendimentos na sua obtenção e fraca solubilidade aquosa), apenas quarenta e dois compostos (vinte e oito derivados de ureia e catorze derivados de tioureia) prosseguiram para os estudos in vivo. Os modelos animais usados foram os modelos gold standard para a identificação de novos compostos com propriedades anticonvulsivantes, sendo, desta forma, os modelos melhor validados. Os resultados do screening farmacológico inicial em murganhos revelaram proteção anticonvulsivante no modelo MES para vinte e quatro compostos, sendo nove deles ativos na dose mais baixa testada (30 mg/kg). Em termos estruturais, os compostos mais promissores apresentam cadeias mais curtas (provenientes da acetilacetona ou do acetoacetato de metilo) e um anel aromático não substituído ou substituído na posição para com um grupo metilo ligados, respetivamente, ao C5 e ao C4 do anel dihidropirimidínico. Referir também que os derivados tioureia apresentaram uma atividade anticonvulsivante ligeiramente superior comparativamente aos análogos correspondentes da série ureia. Os resultados de toxicidade neuromotora obtidos através do teste do aparelho rotativo evidenciaram que aproximadamente 52% dos compostos são menos tóxicos que a lamotrigina, carbamazepina e fenitoína. Os compostos MM 17, MM 19 (derivados de ureia) e MM 83 (derivado de tioureia) também protegeram contra as crises induzidas pelo MES em 50-75% dos ratos após administração oral (gavage) na dose de 30 mg/kg. Para além da atividade anticonvulsivante, os compostos mais ativos não exibiram citotoxicidade marcada nos estudos in vitro realizados nas diversas linhas celulares (proliferação celular relativa superior a 50% a 30 μM), o que pode ser relevante devido ao facto da toxicidade ser um problema comum aos fármacos antiepiléticos disponíveis. Neste contexto, as células N27 foram utilizadas por serem células neuronais e os alvos de ação dos fármacos antiepiléticos estarem localizados no sistema nervoso central. Por outro lado, procedeu-se à avaliação da citotoxicidade em células hepáticas (HepaRG) e intestinais (Caco-2), respetivamente, porque alguns fármacos antiepiléticos têm sido associados a hepatotoxicidade severa e porque a via oral é a via de administração desejada. Como estas duas linhas celulares são cancerígenas, considerou-se incluir também uma linha celular humana não cancerígena (NHDF). Relativamente aos estudos farmacocinéticos, os dados obtidos mostraram que 82% dos compostos investigados devem apresentar boa permeabilidade intestinal (Papp > 1,1×10-6 cm/s), 66% dos quais poderão ter boa penetração cerebral (Papp > 2,0×10-6 cm/s), o que pode sugerir uma elevada permeabilidade passiva transcelular passiva. Em ambos os ensaios, os derivados tioureia apresentaram valores de permeabilidade superiores em relação aos respetivos análogos da série ureia, o que pode estar associado à sua natureza mais lipofílica. Estes resultados podem explicar, pelo menos em parte, a maior atividade observada para os derivados tioureia no screening anticonvulsivante após as administrações por via intraperitoneal e via oral. Notar também que 44% dos compostos não modularam significativamente a glicoproteína-P (por inibição ou indução) a 10 μM e 50 μM. Este foi um achado importante porque a glicoproteína-P está fisiologicamente expressa em vários tecidos e órgãos relevantes de um ponto de vista farmacocinético. Finalmente, os estudos in silico indicaram que todos os compostos respeitam a regra dos cinco de Lipinski, sugerindo que eles possuem propriedades intrínsecas favoráveis de forma a preencher os critérios de druglikeness. A ferramenta in silico pkCSM também estimou que as DHPM(t)s têm boa absorção intestinal no homem (67,73-93,91%) e um volume aparente de distribuição no estado estacionário na mesma gama de valores encontrados para os fármacos antiepiléticos. As predições in silico também sugeriram uma percentagem baixa de ligação às proteínas plasmáticas para os compostos em estudo, o que é considerado favorável terapeuticamente, minimizando-se assim o risco de interações. Estes resultados corroboram os resultados obtidos no ensaio de PAMPA intestinal que mostrou que provavelmente nenhum dos compostos testados têm uma ligação às proteínas plasmáticas superior a 90% (Papp ≤ 1,0×10−5 cm/s). Os derivados tioureia foram ainda considerados como os compostos que permeiam melhor através de barreiras biológicas (p.e., em monocamadas de células Caco-2 e barreira hematoencefálica), de modo similar ao obtido nos estudos experimentais de PAMPA. Contudo, o modelo preditivo utilizado sugeriu que 14% dos derivados ureia têm tendência para inibir o citocromo P450 versus 36% dos derivados tioureia. Por outro lado, preocupações com a disrupção da função hepática normal foram preditas para metade dos compostos. Globalmente, os estudos levados a cabo fornecem novas informações sobre a atividade anticonvulsivante desta classe de heterociclos, bem como dados farmacocinéticos e de toxicidade. Mais de metade das moléculas investigadas apresentaram proteção anticonvulsivante contra as crises induzidas eletricamente (no modelo do MES), confirmando o interesse do modelo farmacofórico para o design de novos agentes anticonvulsivantes. Os dados aqui reunidos permitiram a identificação de características estruturais importantes destas moléculas que podem ser responsáveis pela atividade anticonvulsivante, as quais devem ser mantidas ou melhor exploradas para produzir análogos mais ativos nos próximos passos do desenvolvimento destes candidatos a fármacos. No entanto, os resultados apresentados nesta tese constituem apenas a “ponta do iceberg” no que diz respeito à descoberta e desenvolvimento de DHPM(t)s como potenciais fármacos antiepiléticos.Centro de Investigação em Ciências da Saúde da Faculdade de Ciências da Saúde da Universidade da Beira Interior; Laboratório de Farmacologia da Faculdade de Farmácia da Universidade de Coimbra

Case Record

UNDIFFERENTIATED SCHIZOPHRENIA: Mr. R was reported to be normal 4 years back, he was found to be preoccupied and not communicating well with his family members and preferred to stay alone. He was talking and laughing to self. When asked he said that he heard voices speaking to him and he were replying to it. Gradually his sleep decreased and he would sleep only for 3 to 4 hours at night. He did not attend classes. He was started washing his room many times. He was taking bath frequently. He was started worshiping god Anjanaya many times a day and also avoiding to speak with female family members. This continued for 2 months after which he started to be abusive and assaultive others for no reason. So he was taken to a psychiatrist, was admitted for 10 days and treated with ECT. After discharge he would discontinue medications on and off during which his symptoms would get exacerbated. He continued his studies but performance was poor. He also exhibited suicidal gestures on three occasions in the form of cutting his harms and carrying kerosene and matchbox inside the bathroom, saved by family members. Again the mother took him to psychiatrist and continued medication the symptoms were under the control. He completed his course but did not complete his studies. Then he got employment into Ford Company, his performance was average, he continued his job for 5 months with medication. After discontinue of treatment he became self-withdrawn, slowly he was neglecting self-care, attempted suicide, coworkers informed to his parents. Hence he was brought to IMH and admitted. No H/O sad mood, crying spells. No H/O tall claims, spending spree. No H/O thoughts being known to others or withdrawn. No H/O substance use. No H/O head injury, LOC, seizures. No H/O fever or any prolonged drug intake. SUBSTANCE INDUCED MOOD DISORDER: The patient was introduced to using betelnut along with his friends around 18 years of age. Later be continued to use tobacco in the form of Panparag, Hans, Shanthi betelnut 4-6 packets per day. Later after 3 years of tobacco intake, he started consuming alcohol for the first time along with his friends on some occasion, he consumed beer around 200ml. As he enjoyed the high produced by the drink, he continued to take alcohol at regular interval. After 1 year of beginning alcohol intake, he got married, after 6months of marriage life he started consuming alcohol in the form of brandy almost every night. He would become intoxicated, come home, abuse and assault his wife frequently. Due to frequent marrital dishormony his wife left and living with her grand parants for past 8 months. Now according to his be continued to drink alcohol, but the past two months he was engaged in some temple work, where he is supposedto have been introduced to cannabis.After consuming cannabis, his behavior became unmanageble. He frequently keep standing in the middle of the road and appear to make gesturesas if regulating the traffic. He would keep talking excessively and laugh for unprovoked reasons. His sleep patterns also worsened. All through out the night he would keep wandering in the street. And also he was started talking irrelevantly and would not be able to brought back home. He would also talk high about himself. He would claim himself to be God and capable of doing lot of things and able to grant wishes to all people. His selfcare also deteriorated, he started picking up quarrles and assaulted others. Nighbours made complaint against him. So, the family members brought him in the confused state to IMH. He was treated with Ing. Lorazepam 4 mg IM stat and referred to GGH for favour ofadmission and rule out other causes of delirium. He was treated at GGH for one week with Inj. Haloperidal 5 mg, Inj. Lorazepam 4 mg im, Inj.Thiamine and has been referred to IMH for further management. No h/o head injury/LOC/seizures. No h/o low mood/crying spells/suicidal attempts. No h/o hearing voices No h/o repititive washing/cheeking etc., DELUSIONAL DISORDER-MIXED: The patient was reported to be normal till one year eight months back. She claimed hat her co-tenant Mr. V called her for sexual relationship and she refused after that she started telling that he is setting people against her to harm her and also setup prostitutes as a co-tenants, to move her away from the place She also says that he tried to kill her with ambulance 108 and milk van by using his political influence. Patient gave complaint in nearby police station about Mr. Enquiry done. But the police Also turned against her by his political influence. So, she used to go to SP office, collector office daily and shout to arrest Mr. V. Mean while she vacated that house and shifted to Mr. S house who is friend of her brother. He is a widower, living alone. After 2 months of shifting to new house she started believing that the house owner was deeply loved with her whom she understands by his Gestures. And he did not admit his love for her has he did not want others to know. She was fought with the co-tenant once for silly reason. They were assaulted her with an aluminum mug and broken house hold article. And she assumed that Mr. V. only arranged them to fight with her. And also without any reason the patient was fought with the female Co-tenants whoever talking to Mr. S. She uses to tell everybody that the Mr. S. loves with her. The house owner warned them to vacate the house. But she did not vacate the house. The house owner slowly cut power supply, water supply to her portion. After 4 months she vacated the house to Next Street. Even after vacating, patient goes to Mr. S. house and starts quarrel with the new tenants that they should vacate and only because of them he is avoiding her. Every day she was going to her old house and tells everybody that the house owner loves with her. The husband told her not to go there, but she poured kerosene on him and try to kill him. So, the house owner filed a case against her, she was arrested and kept in observation at IMH. During observation she was continuously blaming the old co-tenant that all because him only it happen. Still the house owner his loves with her, she also loves him deeply. DEMENTIA IN ALZHEIMER’S DISEASE: The patient was reported to be normal till one year back. Then, her daughter noticed that the patient repeatedly searched for certain things in the house. She would forget simple things in the house like the way for going to toilet. At times she also found it difficult to return to her house after going for a walk. In course of time, she was not able to identify her close relatives. She was not able to remember whether she had taken her food or not. Her personal hygiene decreased gradually. She did not take bath and did not dress properly. She would pass urine inside the house itself at times. She slept for very little time and would wake up in the middle of the night and keep pacing inside the house.Slowly she was not able to identify her own family members. MENTAL RETARDATION-MILD: Patient was born out of non consanguineous marriage, full term normal delivery. Mother was 22 yrs and fathers age was 298 yrs. No history of any drug intake, fever or exanthematous eruptions in the ante natal period. No ante natal checkup was done. No history of radiation, injury, malnutrition, or vaginal bleeding. Delivery was conducted by local dhai; h/o prolonged 2nd stage of labor, the baby cried soon after birth and was breast fed after a short while. No h/o neonatal seizures or difficulty in feeding. No h/o of jaundice, breast fed up to 10 months, and there were no weaning difficulties

The human early-life exposome (HELIX): project rationale and design

Background: Developmental periods in early life may be particularly vulnerable to impacts of environmental exposures. Human research on this topic has generally focused on single exposure–health effect relationships. The “exposome” concept encompasses the totality of exposures from conception onward, complementing the genome. Objectives: The Human Early-Life Exposome (HELIX) project is a new collaborative research project that aims to implement novel exposure assessment and biomarker methods to characterize early-life exposure to multiple environmental factors and associate these with omics biomarkers and child health outcomes, thus characterizing the “early-life exposome.” Here we describe the general design of the project. Methods: In six existing birth cohort studies in Europe, HELIX will estimate prenatal and postnatal exposure to a broad range of chemical and physical exposures. Exposure models will be developed for the full cohorts totaling 32,000 mother–child pairs, and biomarkers will be measured in a subset of 1,200 mother–child pairs. Nested repeat-sampling panel studies (n = 150) will collect data on biomarker variability, use smartphones to assess mobility and physical activity, and perform personal exposure monitoring. Omics techniques will determine molecular profiles (metabolome, proteome, transcriptome, epigenome) associated with exposures. Statistical methods for multiple exposures will provide exposure–response estimates for fetal and child growth, obesity, neurodevelopment, and respiratory outcomes. A health impact assessment exercise will evaluate risks and benefits of combined exposures. Conclusions: HELIX is one of the first attempts to describe the early-life exposome of European populations and unravel its relation to omics markers and health in childhood. As proof of concept, it will form an important first step toward the life-course exposome