Introdução: pacientes com epilepsia em tratamento com politerapia podem manifestar sinais de efeitos adversos e/ou ineficácia terapêutica decorrentes das possíveis interações entre as diferentes drogas antiepilépticas (DAEs) que compõem o esquema terapêutico. Para contornar esta situação, as DAEs desenvolvidas atualmente apresentam perfil farmacocinético com menor potencial para interações farmacológicas. O levetiracetam é uma nova DAE aprovada para utilização como terapia adjuntiva no tratamento de crises focais em adultos. Seu metabolismo, por não depender de forma significativa do sistema oxidativo microssomal hepático, proporciona associações positivas com outras DAEs. Entretanto, observações clínicas de que a associação entre levetiracetam e DAEs indutoras enzimáticas (carbamazepina, fenitoína, fenobarbital e primidona) implicaria em menor disposição plasmática do levetiracetam têm sido confirmadas por alguns estudos e consideradas irrelevantes por outros. Objetivo: caracterizar e comparar o perfil farmacocinético do levetiracetam entre pacientes adultos com epilepsia em tratamento regular com DAEs indutoras enzimáticas e pacientes que estejam ou em tratamento com DAEs que não alteram a atividade das enzimas de metabolismo ou sem tratamento farmacológico. Casuística e Métodos: trinta pacientes foram selecionados, tendo sido alocados quinze em cada grupo, de acordo com o perfil das DAEs em uso regular (grupo indutor enzimático e grupo controle). A todos foi administrada dose única oral de levetiracetam 1000 mg. Ao longo de 24 horas foram coletadas sete amostras de sangue para determinação da concentração plasmática do levetiracetam e três amostras de urina para quantificação do levetiracetam eliminado inalterado e de seu principal metabólito inativo, o ucb L057. As amostras foram encaminhadas à Universidade de Pavia, Itália, e analisadas por cromatografia líquida de alta eficiência (HPLC). Resultados: foram calculados os seguintes parâmetros farmacocinéticos: concentração plasmática máxima de levetiracetam e o tempo decorrido até seu alcance; meia-vida de eliminação; constante de velocidade de eliminação; área sob a curva de concentração plasmática versus tempo; clearance oral aparente e clearance renal do levetiracetam; volume aparente de distribuição e quantidades excretadas na urina como fármaco inalterado e como ucb L057. Comparações entre os grupos foram feitas a partir dos testes t de Student ou Mann-Whitney, conforme apropriado. O grupo em tratamento com DAEs indutoras enzimáticas apresentou clearance oral aparente do levetiracetam significativamente maior e meia-vida de eliminação significativamente menor do que o grupo controle (p < 0,05). As quantidades tanto de levetiracetam quanto de ucb L057 eliminadas na urina não divergiram significativamente entre os dois grupos (p > 0,05). Discussão e Conclusões: estudos têm evidenciado o potencial das DAEs indutoras enzimáticas tanto para estimular a atividade de enzimas hidrolíticas, como as responsáveis pela conversão do levetiracetam a ucb L057, quanto para inibir e/ou competir pelos sítios de ligação dos transportadores presentes nos túbulos renais responsáveis pela secreção ativa do ucb L057. Embora o presente estudo não tenha objetivado identificar e caracterizar as vias de metabolismo e eliminação do levetiracetam, os dados encontrados evidenciam a diferença na disposição plasmática deste fármaco quando associado às DAEs indutoras enzimáticas. Considerando que o levetiracetam é majoritariamente prescrito em associações, as quais geralmente envolvem ao menos uma DAE indutora enzimática, o sucesso da terapêutica dos pacientes em que o levetiracetam for adicionado ao esquema medicamentoso prévio ou em que as DAEs indutoras enzimáticas tenham suas posologias modificadas pode ser prejudicado caso não haja o reconhecimento da possibilidade de ocorrência da alteração de perfil farmacocinético evidenciada. / Introduction: patients with epilepsy treated with two or more antiepileptic drugs (AEDs) associated (politherapy) can show signs of adverse effects and/or therapeutic inefficacy due to possible interactions among the different combined AEDs. As an attempt to handle this problem, the AEDs developed nowadays are showing pharmacokinetic characteristics that decrease their potential to get involved in pharmacological interactions. Levetiracetam is a new AED approved as add-on therapy for the treatment of focal seizures in adults. Its metabolism does not rely significantly on the hepatic microssomal oxidative system, what has been considered a positive aspect in favor of its use in association with other AEDs. However, clinical observations of decreased levetiracetam plasma disposition when it is associated with enzyme inducing AEDs (carbamazepine, phenytoin, phenobarbital and/or primidone) has been confirmed by some studies and considered irrelevant by others. Purpose: to describe and compare the pharmacokinetic profile of levetiracetam among adult patients with epilepsy in treatment with enzyme-inducers AEDs and patients in treatment with AEDs without any impact on enzymes activity or with no pharmacological treatment. Patients and Methods: a single oral dose of levetiracetam 1000 mg was administered for the thirty selected patients (fifteen per group). Over 24 hours, seven blood samples were collected to have their levetiracetam concentrations quantified, and three urine samples were collected to have their levetiracetam and ucb L057 (the main levetiracetam inactive metabolite) amounts quantified. The samples were sent to University of Pavia, Italy, to be analyzed by high performance liquid chromatography (HPLC). Results: the following pharmacokinetics parameters were calculated: the maximum plasma levetiracetam concentration and the time it occurred, elimination half-life, elimination rate constant, area under the curve, levetiracetam apparent oral clearance and renal clearance, volume of distribution and amount excreted in urine as unchanged drug and as ucb L057. Comparisons between the two groups were performed by Student t-test or Mann-Whitney test, as appropriate. The group of patients treated with enzyme-inducers AEDs showed the levetiracetam apparent oral clearance significantly higher and elimination half-life significantly lower than those from control group (p < 0,05). The amount excreted in urine as unchanged drug and as ucb L057 were not significantly different between the two groups (p > 0,05). Discussion: some studies highlight the capacity of enzyme inducing AEDs both to increase the activity of hydrolysis enzymes, such as those responsible for converting levetiracetam to ucb L057, and to inhibit and/or to compete for the binding sites on the transporters responsible for active tubular secretion of ucb L057. Although the present study did not aim to identify and describe the metabolic and elimination pathways of levetiracetam, the data found clearly show the difference in levetiracetam disposition when it is associated with enzyme inducing AEDs. Considering that levetiracetam is mainly prescribed in association with other drugs, and in most of these associations at least one drug is an enzyme inducer, neglecting this evident change in levetiracetam pharmacokinetic in cases such as those which levetiracetam is added to a previous regimen, or those which enzyme-inducers have their prescription changed, can negatively affect the success of the treatment and consequently the patients quality of life.

Introdução: pacientes com epilepsia em tratamento com politerapia podem manifestar sinais de efeitos adversos e/ou ineficácia terapêutica decorrentes das possíveis interações entre as diferentes drogas antiepilépticas (DAEs) que compõem o esquema terapêutico. Para contornar esta situação, as DAEs desenvolvidas atualmente apresentam perfil farmacocinético com menor potencial para interações farmacológicas. O levetiracetam é uma nova DAE aprovada para utilização como terapia adjuntiva no tratamento de crises focais em adultos. Seu metabolismo, por não depender de forma significativa do sistema oxidativo microssomal hepático, proporciona associações positivas com outras DAEs. Entretanto, observações clínicas de que a associação entre levetiracetam e DAEs indutoras enzimáticas (carbamazepina, fenitoína, fenobarbital e primidona) implicaria em menor disposição plasmática do levetiracetam têm sido confirmadas por alguns estudos e consideradas irrelevantes por outros. Objetivo: caracterizar e comparar o perfil farmacocinético do levetiracetam entre pacientes adultos com epilepsia em tratamento regular com DAEs indutoras enzimáticas e pacientes que estejam ou em tratamento com DAEs que não alteram a atividade das enzimas de metabolismo ou sem tratamento farmacológico. Casuística e Métodos: trinta pacientes foram selecionados, tendo sido alocados quinze em cada grupo, de acordo com o perfil das DAEs em uso regular (grupo indutor enzimático e grupo controle). A todos foi administrada dose única oral de levetiracetam 1000 mg. Ao longo de 24 horas foram coletadas sete amostras de sangue para determinação da concentração plasmática do levetiracetam e três amostras de urina para quantificação do levetiracetam eliminado inalterado e de seu principal metabólito inativo, o ucb L057. As amostras foram encaminhadas à Universidade de Pavia, Itália, e analisadas por cromatografia líquida de alta eficiência (HPLC). Resultados: foram calculados os seguintes parâmetros farmacocinéticos: concentração plasmática máxima de levetiracetam e o tempo decorrido até seu alcance; meia-vida de eliminação; constante de velocidade de eliminação; área sob a curva de concentração plasmática versus tempo; clearance oral aparente e clearance renal do levetiracetam; volume aparente de distribuição e quantidades excretadas na urina como fármaco inalterado e como ucb L057. Comparações entre os grupos foram feitas a partir dos testes t de Student ou Mann-Whitney, conforme apropriado. O grupo em tratamento com DAEs indutoras enzimáticas apresentou clearance oral aparente do levetiracetam significativamente maior e meia-vida de eliminação significativamente menor do que o grupo controle (p < 0,05). As quantidades tanto de levetiracetam quanto de ucb L057 eliminadas na urina não divergiram significativamente entre os dois grupos (p > 0,05). Discussão e Conclusões: estudos têm evidenciado o potencial das DAEs indutoras enzimáticas tanto para estimular a atividade de enzimas hidrolíticas, como as responsáveis pela conversão do levetiracetam a ucb L057, quanto para inibir e/ou competir pelos sítios de ligação dos transportadores presentes nos túbulos renais responsáveis pela secreção ativa do ucb L057. Embora o presente estudo não tenha objetivado identificar e caracterizar as vias de metabolismo e eliminação do levetiracetam, os dados encontrados evidenciam a diferença na disposição plasmática deste fármaco quando associado às DAEs indutoras enzimáticas. Considerando que o levetiracetam é majoritariamente prescrito em associações, as quais geralmente envolvem ao menos uma DAE indutora enzimática, o sucesso da terapêutica dos pacientes em que o levetiracetam for adicionado ao esquema medicamentoso prévio ou em que as DAEs indutoras enzimáticas tenham suas posologias modificadas pode ser prejudicado caso não haja o reconhecimento da possibilidade de ocorrência da alteração de perfil farmacocinético evidenciada. / Introduction: patients with epilepsy treated with two or more antiepileptic drugs (AEDs) associated (politherapy) can show signs of adverse effects and/or therapeutic inefficacy due to possible interactions among the different combined AEDs. As an attempt to handle this problem, the AEDs developed nowadays are showing pharmacokinetic characteristics that decrease their potential to get involved in pharmacological interactions. Levetiracetam is a new AED approved as add-on therapy for the treatment of focal seizures in adults. Its metabolism does not rely significantly on the hepatic microssomal oxidative system, what has been considered a positive aspect in favor of its use in association with other AEDs. However, clinical observations of decreased levetiracetam plasma disposition when it is associated with enzyme inducing AEDs (carbamazepine, phenytoin, phenobarbital and/or primidone) has been confirmed by some studies and considered irrelevant by others. Purpose: to describe and compare the pharmacokinetic profile of levetiracetam among adult patients with epilepsy in treatment with enzyme-inducers AEDs and patients in treatment with AEDs without any impact on enzymes activity or with no pharmacological treatment. Patients and Methods: a single oral dose of levetiracetam 1000 mg was administered for the thirty selected patients (fifteen per group). Over 24 hours, seven blood samples were collected to have their levetiracetam concentrations quantified, and three urine samples were collected to have their levetiracetam and ucb L057 (the main levetiracetam inactive metabolite) amounts quantified. The samples were sent to University of Pavia, Italy, to be analyzed by high performance liquid chromatography (HPLC). Results: the following pharmacokinetics parameters were calculated: the maximum plasma levetiracetam concentration and the time it occurred, elimination half-life, elimination rate constant, area under the curve, levetiracetam apparent oral clearance and renal clearance, volume of distribution and amount excreted in urine as unchanged drug and as ucb L057. Comparisons between the two groups were performed by Student t-test or Mann-Whitney test, as appropriate. The group of patients treated with enzyme-inducers AEDs showed the levetiracetam apparent oral clearance significantly higher and elimination half-life significantly lower than those from control group (p < 0,05). The amount excreted in urine as unchanged drug and as ucb L057 were not significantly different between the two groups (p > 0,05). Discussion: some studies highlight the capacity of enzyme inducing AEDs both to increase the activity of hydrolysis enzymes, such as those responsible for converting levetiracetam to ucb L057, and to inhibit and/or to compete for the binding sites on the transporters responsible for active tubular secretion of ucb L057. Although the present study did not aim to identify and describe the metabolic and elimination pathways of levetiracetam, the data found clearly show the difference in levetiracetam disposition when it is associated with enzyme inducing AEDs. Considering that levetiracetam is mainly prescribed in association with other drugs, and in most of these associations at least one drug is an enzyme inducer, neglecting this evident change in levetiracetam pharmacokinetic in cases such as those which levetiracetam is added to a previous regimen, or those which enzyme-inducers have their prescription changed, can negatively affect the success of the treatment and consequently the patients quality of life.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Levetiracetam (LEV) is a new antiepileptic drug effective as adjunctive therapy for partial seizures and in experimental models of seizures, including pilocarpine-induced seizures in rodents. Objectifying to clarify if anticonvulsant activity of LEV occurs near cholinergic muscarinic alterations, adult male mice received LEV injections before muscarinic agonistsâ administration. Pretreatment with LEV (30, 50, 100 or 200 mg/Kg, i.p.) increased the latency of seizures, latency of status epilepticus and latency of death on the seizure model induced by pilocarpine, 400 mg/Kg, s.c. (P400). LEV (200 mg/Kg, i.p., LEV200) pretreatment abolished seizures in 53% (20/38) of the animals; reduced status epilepticus appearance in 58% (22/38); increased deathâs latency in 116% compared to P400 group; protected 61% (23/38) of the animals from death and also reduced the intensity of tremors induced by oxotremorine (0,5 mg/kg, i.p). [3H]-N-methylscopolamine binding assays in mice hippocampus showed that LEV200 pretreatment reverts the downregulation on muscarinic acetylcholine receptors (mAChR), induced by P400 administration, bringing back these density values to control ones (0,9% NaCl, i.p.). However, subtype-specific binding assays revealed that P400 and LEV200 alone treatments results in M1 and M2 subtypes decrease, respectively. The agonist-like behavior of LEV200 on the inhibitory M2 mAChR subtype, observed in this work, could contribute to explain the reduction on oxotremorine-induced tremors and the delay on pilocarpine-induced seizures, by an increase in the attenuation of neuronal activity mediated by the M1 receptors. [3H]-spiroperidol binding assays also showed a downregulation of dopaminergic D2 receptors, induced by LEV200 alone treatment. Although this effect was reverted on P400 presence, maybe it posses some influence on LEV200 protective effect, since the stimulation of these receptors can reduce the intensity of pilocarpine induced-seizures. Objectifying to investigate if LEVâ mechanism of action also involves anti-oxidant proprieties, neurochemical analysis were carried out in hippocampus and striatum and showed that P400 produced an increased lipid peroxidation in hippocampus, main epileptogenic focus on cholinergic agents induced-seizures, demonstrating and confirming the involvement of free radical oxygen injury in the P400 induced brain injury. However, LEV200 pretreatment abolished this increase, bringing lipid peroxidation levels back to normal values, suggesting an anti-oxidative property of LEV. Our finds also showed a decrease of nitrite levels and stabilization of catalase activity in hippocampus and striatum, beyond an increase on reduced glutathione levels on hippocampus, induced by LEV200 treatment before P400 administration. In this way, the anti-oxidative action of LEV was showed in several stages involved on oxidative injury, suggesting a novel mechanism of its protector effects. / O levetiracetam (LEV) à uma nova droga antiepilÃptica, com eficÃcia na terapia adicional das convulsÃes parciais e em vÃrios modelos experimentais de convulsÃo, inclusive nas convulsÃes induzidas por pilocarpina em roedores. Objetivando investigar se o mecanismo de aÃÃo anticonvulsivante do LEV està relacionado a alteraÃÃes no sistema colinÃrgico muscarÃnico, camundongos machos adultos receberam injeÃÃes de LEV uma hora antes da administraÃÃo de agonistas muscarÃnicos. O prÃ-tratamento com LEV (30, 50, 100 ou 200 mg/Kg, i.p.) aumentou significativamente as latÃncias de convulsÃo, de estatus epilepticus e de morte no modelo de convulsÃes induzidas por pilocarpina, 400 mg/Kg, s.c. (P400). O prÃ-tratamento com LEV (200 mg/Kg, i.p., LEV200) levou à ausÃncia de convulsÃes em 53% (20/38) dos animais; reduziu a ocorrÃncia de estatus epilepticus em 58% (22/38); aumentou a latÃncia de morte em 116% comparada ao grupo P400; protegeu 61% (23/38) dos animais da morte e, ainda, reduziu a intensidade dos tremores induzidos por oxotremorina (0,5 mg/kg, i.p). Ensaios de binding com [3H]-N-metilescopolamina em hipocampo mostraram que o prÃ-tratamento com LEV200 reverte a downregulation dos receptores muscarÃnicos de acetilcolina, induzida por P400, normalizando a densidade desses receptores. Todavia, ensaios para subtipos especÃficos de receptores muscarÃnicos revelaram que a administraÃÃo de P400 ou LEV200, isoladamente, resulta em downregulation dos subtipos M1 e M2, respectivamente. A aÃÃo agonista-sÃmile do LEV nos receptores prÃ-sinÃpticos inibitÃrios M2, observada no presente estudo, poderia contribuir para explicar a reduÃÃo nos tremores induzidos por oxotremorina e o retardo na instalaÃÃo das convulsÃes induzidas por P400, atravÃs da atenuaÃÃo da atividade neuronal mediada pelos receptores M1. Ensaios de binding com [3H]-espiroperidol demonstraram uma downregulation dos receptores dopaminÃrgicos D2, induzida pela administraÃÃo isolada de LEV200. Embora esse efeito tenha sido revertido na presenÃa de P400, talvez possua alguma influÃncia na aÃÃo protetora oferecida por LEV200, considerando que a estimulaÃÃo desses receptores reduz a intensidade das convulsÃes induzidas pela pilocarpina. Objetivando investigar se o efeito protetor demonstrado pelo LEV envolve propriedades antioxidantes, anÃlises neuroquÃmicas foram realizadas em hipocampo e corpo estriado. A administraÃÃo de P400 aumentou a ocorrÃncia de peroxidaÃÃo lipÃdica no hipocampo, considerado principal foco de instalaÃÃo das convulsÃes provocadas por agentes colinÃrgicos, demonstrando e confirmando o envolvimento de espÃcies deletÃrias na injÃria cerebral produzida por esse modelo de convulsÃes. O prÃ-tratamento com LEV200 reverteu esse efeito, fornecendo indÃcios de uma atividade antioxidante dessa droga. Nossos estudos tambÃm mostraram uma diminuiÃÃo da produÃÃo de nitrito e estabilizaÃÃo da atividade da catalase no hipocampo e corpo estriado e o aumento dos nÃveis de glutationa reduzida no hipocampo, decorrentes do tratamento com LEV antes de P400. A aÃÃo antioxidante do LEV foi evidenciada em vÃrias etapas envolvidas no processo de dano oxidativo, sugerindo um novo mecanismo atravÃs do qual essa droga poderia exercer seus efeitos protetores.

Levetiracetam (LEV) is an antiepileptic drug (AED) that has been shown to mainly enhance synaptic depression and modulate certain voltage and ligand-gated channels, after it gains entry into neurons through endocytosis. Since synaptic terminals and distal axons are the first compartments exposed to LEV, we utilized a crayfish motor axon preparation to investigate whether LEV modulates axonal excitability. Two electrode current clamp from the inhibitor axon of the crayfish opener showed that LEV reduced action potential amplitude (APamp) and enhanced synaptic depression, although these events did not occur at the same time, the latter occurred later than the reduction in APamp. Further examinations of these effects and comparison of antidromic and orthodromic conducting action potentials in LEV suggests that this drug preferentially reduces excitability of the proximal axon despite the expectation that it enters the axon at terminals and reaches distal branches first. Results presented here demonstrate that LEV modulates axonal excitability, which may also contribute to its antiepileptic effects.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Levetiracetam (LEV), nova droga antiepilÃptica, apresenta eficÃcia na terapia adicional das convulsÃes e em modelos experimentais. Clonazepam (CNZP) à um benzodiazepÃnico utilizado no tratamento de convulsÃes mioclÃnicas e crises generalizadas. Melatonina (MEL), hormÃnio pineal, demonstra atividade anticonvulsivante em diversos modelos animais. Objetivando investigar novos mecanismos relacionados aos efeitos dessas drogas, foi realizado estudo comparativo, a partir da anÃlise da influÃncia do prÃ-tratamento com LEV, CNZP ou MEL sobre o estresse oxidativo neuronal e sobre a modulaÃÃo de sistemas de neurotransmissÃo (colinÃrgico e dopaminÃrgico) durante as convulsÃes induzidas por pilocarpina (400mg/Kg; P400). Camundongos Swiss, machos, 25-30g receberam LEV, 200 mg/Kg, CNZP, 0,5mg/Kg ou MEL, 25mg/Kg, i.p., (doses escolhidas à partir de curvas dose-resposta) 30min antes de P400. Hipocampo e corpo estriado foram removidos para as anÃlises neuroquÃmicas. Experimentos in vitro, onde homogenatos cerebrais foram incubados com as drogas em estudo (50, 100 ou 200&#61549;g/mL), tambÃm permitiram analisar alteraÃÃes no estresse oxidativo apÃs a induÃÃo de choque tÃrmico e, ainda, a densidade de receptores muscarÃnicos no hipocampo. Os estudos sobre os efeitos sobre o sistema de neurotransmissores colinÃrgicos demonstraram que o prÃ-tratamento com LEV, CNZP ou MEL causou reduÃÃo nos tremores induzidos por oxotremorina e elevou a atividade da acetilcolinesterase no hipocampo. LEV e CNZP alteraram o binding dos receptores muscarÃnicos hipocampais in vivo, revertendo a downregulation induzida por P400, e ensaios in vitro demostraram alteraÃÃo no binding muscarÃnico hipocampal pela prÃvia incubaÃÃo com LEV, CNZP ou MEL. Os ensaios de binding demonstraram, ainda, a downregulation dos receptores D2 hipocampais nos animais tratados LEV, CNZP ou prÃ-tratados com MEL antes de P400. As anÃlises para investigaÃÃo da atividade antioxidante de LEV e CNZP e do papel da aÃÃo antioxidativa da MEL na proteÃÃo contra as convulsÃes permitiram observar que a associaÃÃo com vitamina E potencializou os efeitos anticonvulsivantes de todas as drogas estudas. A administraÃÃo prÃvia de LEV, CNZP ou MEL, antes de P400, reverteu o aumento nos nÃveis de peroxidaÃÃo lipÃdica e nitrito-nitrato e normalizou a atividade da catalase e os nÃveis fisiolÃgicos do antioxidante glutationa em hipocampo e/ou corpo estriado. Nos experimentos de stress oxidativo in vitro, o aumento da peroxidaÃÃo lÃpÃdica, dos nÃveis de nitrito-nitrato e da atividade da catalase nos homogenatos cerebrais submetidos ao choque tÃrmico, foram alterados de forma significativa pela incubaÃÃo prÃvia com LEV, CNZP ou MEL, onde estas drogas foram capazes de reduzir os nÃveis de MDA, de nitrito-nitrato e, ainda, estabilizar a atividade da catalase, potencializando, assim, a atividade enzimÃtica antioxidante endÃgena e a capacidade de inativaÃÃo de radicais livres. Dessa forma, o estudo sugere uma aÃÃo moduladora, exercida por LEV, CNZP e MEL sobre o funcionamento dos sistemas muscarÃnico e dopaminÃrgico, em nÃvel central, como mecanismo alternativo para a proteÃÃo contra as convulsÃes no modelo de P400, bem como a participaÃÃo de propriedades antioxidantes diretas ou indiretas dessas drogas, atravÃs da capacidade de modificar a resposta ao estresse oxidativo neuronal. / Levetiracetam (LEV), nova droga antiepilÃptica, apresenta eficÃcia na terapia adicional das convulsÃes e em modelos experimentais. Clonazepam (CNZP) à um benzodiazepÃnico utilizado no tratamento de convulsÃes mioclÃnicas e crises generalizadas. Melatonina (MEL), hormÃnio pineal, demonstra atividade anticonvulsivante em diversos modelos animais. Objetivando investigar novos mecanismos relacionados aos efeitos dessas drogas, foi realizado estudo comparativo, a partir da anÃlise da influÃncia do prÃ-tratamento com LEV, CNZP ou MEL sobre o estresse oxidativo neuronal e sobre a modulaÃÃo de sistemas de neurotransmissÃo (colinÃrgico e dopaminÃrgico) durante as convulsÃes induzidas por pilocarpina (400mg/Kg; P400). Camundongos Swiss, machos, 25-30g receberam LEV, 200 mg/Kg, CNZP, 0,5mg/Kg ou MEL, 25mg/Kg, i.p., (doses escolhidas à partir de curvas dose-resposta) 30min antes de P400. Hipocampo e corpo estriado foram removidos para as anÃlises neuroquÃmicas. Experimentos in vitro, onde homogenatos cerebrais foram incubados com as drogas em estudo (50, 100 ou 200&#61549;g/mL), tambÃm permitiram analisar alteraÃÃes no estresse oxidativo apÃs a induÃÃo de choque tÃrmico e, ainda, a densidade de receptores muscarÃnicos no hipocampo. Os estudos sobre os efeitos sobre o sistema de neurotransmissores colinÃrgicos demonstraram que o prÃ-tratamento com LEV, CNZP ou MEL causou reduÃÃo nos tremores induzidos por oxotremorina e elevou a atividade da acetilcolinesterase no hipocampo. LEV e CNZP alteraram o binding dos receptores muscarÃnicos hipocampais in vivo, revertendo a downregulation induzida por P400, e ensaios in vitro demostraram alteraÃÃo no binding muscarÃnico hipocampal pela prÃvia incubaÃÃo com LEV, CNZP ou MEL. Os ensaios de binding demonstraram, ainda, a downregulation dos receptores D2 hipocampais nos animais tratados LEV, CNZP ou prÃ-tratados com MEL antes de P400. As anÃlises para investigaÃÃo da atividade antioxidante de LEV e CNZP e do papel da aÃÃo antioxidativa da MEL na proteÃÃo contra as convulsÃes permitiram observar que a associaÃÃo com vitamina E potencializou os efeitos anticonvulsivantes de todas as drogas estudas. A administraÃÃo prÃvia de LEV, CNZP ou MEL, antes de P400, reverteu o aumento nos nÃveis de peroxidaÃÃo lipÃdica e nitrito-nitrato e normalizou a atividade da catalase e os nÃveis fisiolÃgicos do antioxidante glutationa em hipocampo e/ou corpo estriado. Nos experimentos de stress oxidativo in vitro, o aumento da peroxidaÃÃo lÃpÃdica, dos nÃveis de nitrito-nitrato e da atividade da catalase nos homogenatos cerebrais submetidos ao choque tÃrmico, foram alterados de forma significativa pela incubaÃÃo prÃvia com LEV, CNZP ou MEL, onde estas drogas foram capazes de reduzir os nÃveis de MDA, de nitrito-nitrato e, ainda, estabilizar a atividade da catalase, potencializando, assim, a atividade enzimÃtica antioxidante endÃgena e a capacidade de inativaÃÃo de radicais livres. Dessa forma, o estudo sugere uma aÃÃo moduladora, exercida por LEV, CNZP e MEL sobre o funcionamento dos sistemas muscarÃnico e dopaminÃrgico, em nÃvel central, como mecanismo alternativo para a proteÃÃo contra as convulsÃes no modelo de P400, bem como a participaÃÃo de propriedades antioxidantes diretas ou indiretas dessas drogas, atravÃs da capacidade de modificar a resposta ao estresse oxidativo neuronal. / Levetiracetam (LEV), a new antiepileptic drug, shows efficacy in the treatment of additional seizures and in experimental models. Clonazepam (CNZP) is a benzodiazepine used to treat myoclonic seizures and generalized seizures. Melatonin (MEL), the pineal hormone, shows anticonvulsant activity in several animal models. To investigate new mechanisms related to the effects of these drugs, comparative study was conducted, from the analysis of the influence of pretreatment with LEV, CNZP or MEL on the oxidative stress and neuronal modulation of neurotransmitter systems (cholinergic and dopaminergic) during seizures induced by pilocarpine (400mg/Kg; P400). Male Swiss mice, 25-30g received LEV, 200 mg / kg, CNZP, 0.5 mg / kg or MEL, 25mg/kg, ip (doses chosen from the dose-response curves) 30min before P400. Hippocampus and striatum were removed for neurochemical analysis. In vitro experiments, where brain homogenates were incubated with drugs under study (50, 100 ou 200&#61549;g/mL) also allowed us to analyze changes in oxidative stress after induction of heat shock and also the density of muscarinic receptors in the hippocampus. Studies on the muscarinic modulation demonstrated that pretreatment with LEV, CNZP or MEL resulted in lower oxotremorina induced tremors and increased acetylcholinesterase activity in the hippocampus. LEV and CNZP altered the binding of hippocampal muscarinic receptors in vivo, reversing the P400-induced downregulation and in vitro tests showed changes in hippocampal muscarinic binding by previous incubation with LEV, CNZP or MEL. The binding assays also showed a downregulation of hippocampal D2 receptors in treated animals LEV, CNZP or pretreated with MEL before P400. Analyses to investigate the antioxidant activity of LEV and CNZP and role of antioxidative action of MEL in the protection against seizures propose that the association with vitamin E increased the anticonvulsant effects of all studied drugs. The prior administration of LEV, MEL or CNZP before P400, reversed the increased levels of lipid peroxidation and nitrite-nitrate and normalized the activity of catalase and the physiological levels of the antioxidant glutathione in the hippocampus and / or striatum. According to in vitro experiments, increased lipid peroxidation, levels of nitrite-nitrate and catalase activity in brain homogenates subjected to thermal shock were significantly altered by incubation with LEV, CNZP or MEL, where these drugs were able to reduce the levels of MDA, nitrite-nitrate, and also stabilize the activity of catalase, enhancing thus the endogenous antioxidant enzyme activity and the ability to inactivate free radicals.Thus, the study suggests a modulatory action exerted by LEV, CNZP and MEL on the functioning of muscarinic and dopaminergic systems in the central nervous system as an alternative mechanism to protect against seizures in the model P400, and the participation of direct and indirect antioxidant properties of these drugs, through the ability to modify the neuronal response to oxidative stress. / Levetiracetam (LEV), a new antiepileptic drug, shows efficacy in the treatment of additional seizures and in experimental models. Clonazepam (CNZP) is a benzodiazepine used to treat myoclonic seizures and generalized seizures. Melatonin (MEL), the pineal hormone, shows anticonvulsant activity in several animal models. To investigate new mechanisms related to the effects of these drugs, comparative study was conducted, from the analysis of the influence of pretreatment with LEV, CNZP or MEL on the oxidative stress and neuronal modulation of neurotransmitter systems (cholinergic and dopaminergic) during seizures induced by pilocarpine (400mg/Kg; P400). Male Swiss mice, 25-30g received LEV, 200 mg / kg, CNZP, 0.5 mg / kg or MEL, 25mg/kg, ip (doses chosen from the dose-response curves) 30min before P400. Hippocampus and striatum were removed for neurochemical analysis. In vitro experiments, where brain homogenates were incubated with drugs under study (50, 100 ou 200&#61549;g/mL) also allowed us to analyze changes in oxidative stress after induction of heat shock and also the density of muscarinic receptors in the hippocampus. Studies on the muscarinic modulation demonstrated that pretreatment with LEV, CNZP or MEL resulted in lower oxotremorina induced tremors and increased acetylcholinesterase activity in the hippocampus. LEV and CNZP altered the binding of hippocampal muscarinic receptors in vivo, reversing the P400-induced downregulation and in vitro tests showed changes in hippocampal muscarinic binding by previous incubation with LEV, CNZP or MEL. The binding assays also showed a downregulation of hippocampal D2 receptors in treated animals LEV, CNZP or pretreated with MEL before P400. Analyses to investigate the antioxidant activity of LEV and CNZP and role of antioxidative action of MEL in the protection against seizures propose that the association with vitamin E increased the anticonvulsant effects of all studied drugs. The prior administration of LEV, MEL or CNZP before P400, reversed the increased levels of lipid peroxidation and nitrite-nitrate and normalized the activity of catalase and the physiological levels of the antioxidant glutathione in the hippocampus and / or striatum. According to in vitro experiments, increased lipid peroxidation, levels of nitrite-nitrate and catalase activity in brain homogenates subjected to thermal shock were significantly altered by incubation with LEV, CNZP or MEL, where these drugs were able to reduce the levels of MDA, nitrite-nitrate, and also stabilize the activity of catalase, enhancing thus the endogenous antioxidant enzyme activity and the ability to inactivate free radicals.Thus, the study suggests a modulatory action exerted by LEV, CNZP and MEL on the functioning of muscarinic and dopaminergic systems in the central nervous system as an alternative mechanism to protect against seizures in the model P400, and the participation of direct and indirect antioxidant properties of these drugs, through the ability to modify the neuronal response to oxidative stress.

Class of 2013 Abstract / Specific Aims: Guidelines developed by the Brain Trauma Foundation recommend the use of prophylactic anticonvulsants, particularly phenytoin, for the prevention of early posttraumatic seizures for patients with severe traumatic brain injuries. The purpose of this study is to evaluate the effectiveness of levetiracetam, a newer anticonvulsant, for posttraumatic seizure prevention in patients with severe traumatic brain injury. Methods: This study was approved by the University of Arizona Medical Center Institutional Review Board. The project consists of a retrospective cohort analysis from January 1, 2010 to September 30, 2011. We have abstracted data from all patients with traumatic brain injuries over this time period from the University of Arizona Medical Center Trauma Registry, and have matched these patients with their records in the pharmacy database to determine who received levetiracetam versus no prophylaxis. Patients younger than 18 years of age, pregnant women, patients who were deemed to be nonsalvageable, and patients who had a seizure prior to initiation of levetiracetam were excluded from the study. The following data was collected: age, gender, ethnicity, mechanism of injury, injury severity score, ED GCS, ED SBP, ED pulse, ED RR, blood alcohol level, ICU length of stay, number of ventilator days, hospital length of stay, FIM score at discharge (totals, and by component), diagnosis, surgery and complication type, anticonvulsant given, type of beta-blocker given, maximum and minimum dose used, cumulative doses given, and whether there exists a known prior history of anticonvulsant use. All data were recorded without patient identifiers and have been kept confidential. A multivariate logistic regression analysis was used to evaluate a relationship between other data collected from the patients’ medical records and seizure occurrence. Chi Square or Fisher's Exact test will be used in the final analysis to compare the effectiveness of levetiracetam versus no prophylaxis to prevent posttraumatic brain injury seizures. Significance is defined as p<0.05 for all analyses. Main Results: The results are pending the final data analysis. Conclusion: To be determined.

No description available.

Class of 2015 Abstract / Objectives: Guidelines from the Brain Trauma Foundation recommend that after traumatic brain injury (TBI) patients should be given seizure prophylaxis for up to seven days. Currently, phenytoin is the first line therapy for this indication. However, levetiracetam is increasingly being used as an alternative because it does not require serum concentration monitoring and has a desirable safety profile. Studies evaluating levetiracetam have used a loading dose, followed by a maintenance dose of 1000 mg every 12 hours. The primary objective of this study was to evaluate the efficacy of low-dose (500 mg every 12 hours) levetiracetam for seizure prophylaxis after TBI. Methods: This was a retrospective cohort study conducted in a tertiary care, academic institution that is designated as a level 1 trauma center. Institutional review board approval was obtained prior to data collection. Consecutive patients with TBI between 2010 and 2012, who received levetiracetam for seizure prophylaxis, were included. Patients who met at least one of the following criteria were included: cortical contusion on computerized tomography scan, subdural hematoma, epidural hematoma, intracerebral hematoma, depressed skull fracture, penetrating head injury, or Glasgow Coma Scale (GCS) of 10 or less. Patients were excluded if they were less than 16 years of age, had a previous head injury, previous neurosurgery, history of seizure, or anti-seizure medication, or were given a loading dose of levetiracetam, or given a maintenance dose greater than 500 mg every 12 hours. The primary outcome was the occurrence of a seizure within seven days of TBI. A one-sample test of proportions was used to compare the rate of seizures while being treated with levetiracetam to a hypothesized value of 3.6 percent (from previous trials), using an a priori alpha for 0.05. Results: There were a total of 146 patients included in the study, who were treated with levetiracetam 500 mg every 12 hours. The median age was 51 years (interquartile range 31 to 65 years), 110 (75 percent) were male, and the median GCS on admission was 11 (interquartile range 5 to 14). The mechanisms of injury were fall (n equals 49), motor vehicle or motorcycle collisions (n equals 42), pedestrian or bicyclist (n equals19), assault (n equals16), suicide attempt (n equals 2), and other (n equals18). The median time to first dose of levetiracetam was 4 hours after injury (interquartile range 1 to 13 hours). After initiation of levetiracetam, there were 5 (3.4 percent) patients who had a seizure within seven days. This was not significantly different than the hypothesized population value (p equals 0.910). The median length of stay was 13 days (interquartile range 9 to 21) and 7 (4.8 percent) patients died during hospitalization. Conclusions: A low-dose of levetiracetam 500 mg every 12 hours after TBI was effective for early seizure prevention. This regimen may be an appropriate alternative to phenytoin or traditional dose levetiracetam for this indication. Future, prospective studies are needed to confirm these findings.

La réponse à l’agression est à la fois neuroendocrinienne, neurovégétative et comportementale. Elle implique particulièrement l’amygdale. Celle-ci joue en effet un rôle dans l’anxiété et la peur mais également dans la constitution d’un syndrome de stress post-traumatique (i.e. SSPT). La réponse comportementale à une situation de stress a été peu étudiée chez les patients admis dans un service de Réanimation, alors que des études indiquent qu’une réponse inadaptée de l’axe corticotrope ou du système nerveux autonome serait associée à une surmortalité ou la survenue d’une défaillance multiviscérale. Au décours de leur séjour en réanimation, les patients sont à haut risque de développer des troubles psychologiques (i.e. anxiété, dépression et SSPT) et cognitifs (i.e. affectant préférentiellement la mémoire et les fonctions exécutives) qui ont un impact majeur sur leur qualité de vie et qui seraient proportionnels à la sévérité de leur maladie aiguë et de son retentissement neuro-comportemental. L’objet de notre travail a été d’une part, de décrire quantitativement et qualitativement l’anxiété des patients admis de réanimation et d’en déterminer sa valeur pronostique ; d’autre part, d’étudier le lien entre activation amygdalienne et syndrome post-traumatique dans un modèle murin de sepsis par ligature-ponction caecale (i.e. CLP). Notre étude observationnelle a porté sur 354 patients (Age, 63 ans [49-73] ; Sexe F/H, 137/217) admis dans trois services de réanimation d’Île de France entre Janvier 2014 et Septembre 2016. L’anxiété était en médiane d’intensité modérée, selon l’échelle visuelle analogique (4 [1-6) et le questionnaire STAI (41 [31-53]). La moitié des patients rapportaient se sentir vulnérables (54%) ; considérer leur état grave (67 %) et avoir peur de mourir (45%). Une défaillance d’organe a été observée chez 157 (45%) des patients. Une valeur de STAI ≥ 40 (OR 1,69, 95%CI [1,02-2,84]) était associée à la survenue d’une défaillance d’organe après ajustement sur le score de défaillance d’organe SOFA à J1, la mise en route d’une ventilation mécanique non-invasive (OR 4,93 ; 95%CI [2,9-9,4]) et la gravité d’une pathologie préexistante selon le score Knaus (OC2,01 95%CI [1,21-3,33]) et la peur de mourir (OR 0,55 [0,33-0,92]). Celle-ci était significativement associée à un risque moindre de défaillance parmi les patients ayant une maladie aiguë sévère, définie par un score IGS-2 > 30 (58% vs. 37%). Cette étude indique que l’évaluation de l’anxiété est utile pour estimer le risque d’aggravation des patients de réanimation mais indique également que si son intensité est positivement corrélée à la survenue d’une défaillance, un défaut de perception d’un danger (tel que l’exprimerait la peur de mourir) en augmenterait le risque. Notre étude expérimentale a mis en évidence une activation précoce (i.e. à H6 de la CLP) et transitoire des neurones CAMK-II positif du noyau central de l’amygdale (CeA) et des anomalies tardives (i.e. J15 après CLP) de l’open-field test (test en libre champ) et du Fear-conditionning (test de formation de la mémoire aversive), traduisant respectivement un comportement type anxieux et une hypermémorisation de la peur, qui s’apparenterait à un SSPT. Nous avons procédé à une inhibition pharmacogénétique par transfection virale des neurones CAMKII, qui a entraîné une réduction de l’hyper-mémorisation aversive induite par le sepsis. L’enregistrement électrophysiologique intra-amygadalienne a mis en évidence une activité pro-épileptique ou épileptique chez les souris septiques. L’administration d’un antiépileptique, le levetiracetam au cours des 24 premières heures de la CLP a résulté en une diminution de la mortalité, de l’activation des neurones CAMKII du noyau central de l’amygdale et de l’hyper-mémorisation aversive induites par le sepsis. (...) / Systemic insults trigger neuroendocrine, neurovegetative and behavioural responses. Amygdala is particularly involved in anxiety and fear but also in the generation of post-traumatic stress disorder (i.e. PTSD). Amygdala is part of the limbic system and modulates the neuroendocrine and the autonomous nervous system activity. Behavioural changes to critical illness has been poorly studied in ICU-admited patients, despite studies showing that non-adapted corticotropic axis or autonomic nervous system responses correlate with a higher mortality or organ failures. During their ICU stay, patients are at high risk of developing psychological (e.g. anxiety, depression or PTSD), and cognitive alterations (e.g. memory and executive functions) with a major impact on their long-term quality of life. Such alterations intensity is correlated with the severity of critical illness. Our present work aimed, on the one hand at assessing at ICU admission patients’ anxiety and its prognostic value, and on the other hand, at characterizing the link between amygdalar activation and PTSD in a murine model of caecal ligation and puncture (i.e. CLP). 354 patients were included in our observational study (median age 63 [IQR 49-73], sex 137W/217M), from 3 Ile-De-France ICU between January 2014 and September 2016). Median anxiety was moderate according to both visual scale (4 [1-6]) and STAI questionnaire (43[32-53]). Half of participants declared (54%) feeling vulnerable; considered their state to be severe (67%) and feared to die (45%). One organ failure – mostly neurological, but also the need for mechanical ventilation, dialysis or vasopressive catecholamines during the first 7 days – was present in 157 (45%) patients. A STAI index ≥ 40 (OR 4.93 ; 95% CI[1.02 – 2.84]) was associated with the occurrence of an organ failure, even after adjusting for the day-1 SOFA score, the onset of a mechanical ventilation (OR 4.93, 95CI [2.9 – 9.4]), the Knaus score of prior pathologies severity (OC 2.01, 95CI [1.21 – 3.33]) and fear of death. (OR 0.55 [0.33 -0.92]). The latter significantly associating with a decreased risk of organ failure among patients with a severe acute pathology as defined by a IGS-2 >30 (58% vs. 37%). This study shows that evaluating early anxiety can prove useful in predicting patient aggravation risk in ICU, but also indicates that if its intensity positively predicts the onset of organ failures, the lack of perceived severity (lower fear of death) would also associate with an increased risk of failure. Our experimental study higlighted an early (i.e. 6H post CLP) and transitory activation of Central Amygdala (CeA) CAMK-II positive neurons, and delayed (i.e D15 post CLP) alterations in open field and fear-conditioning tests, respectively indicating an anxious behaviour and fear hypermnesia, both critical aspects of PTSD. Pharmacogenetic inhibition of CAMK-II neurons by viral transduction led to a decrease in aversive sepsis-induced hypermnesia. Administration of Levetiracetam, an antipeileptic drug, during the first 24h post-CLP led to a decrease in sepsis-induced mortality, in CAMK-II CeA neurons activation and in aversive memory. Amygdalar neuronal activation was also associated with microglial morphological alterations, partly prevented by levetiracetam, and reminiscent of alterations seen in septic shock autopsic samples. Our experimental work shows an increased activity in CAMK-II amygdalar neurons during early sepsis, potentially implicated in the onset of sepsis-induced anxiety and PTSD. this constitutes a plausible neuro-anatomical and neuro-biological background to our clinical study showing the prognostic interest of early anxiety assessment in ICU patients, as it positively correlates with both stress intensity and the misperception of danger.

M. Tech. (Biomedical technology, Faculty of Applied and Computer Science), Vaal University of Technology / Approximately 1% of the world’s population has epilepsy, the second most common neurological disorder after stroke. In South Africa almost 1 in every 100 people has epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between 12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in patients with poor seizure control or long term treatment. TDM depends on accurate drug concentration measurements. In order to provide an accurate measurement, the High performance liquid chromatography (HPLC) method was developed, compared with a commercially available kit, and the stability of the samples was investigated. Ethical approval was obtained from the Human Research Ethics Committee (Medical), VUT (Ethics reference number: 2015024.4). The study was conducted from January to October 2015. This study involved three groups of volunteers who gave written consent. The first group were fifteen healthy MTech students in the Biomedical Technology Department at the Vaal University of Technology (VUT). Their blood samples were used for the analytical validation of the method and for the stability studies over a 4 weeks period. The second group were six patients from Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used Levetiracetam. Their blood samples were used to investigate the influence of different collection tubes as well as the handling and storage of samples on the LEV concentration. The third group were forty four patients from Pathcare Laboratories, Cape Town. Their blood samples were transported to Clinical Pharmacokinetic Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to compare the newly developed HPLC method and the Commercial kit. The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 = 0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine laboratory analyses in the future. The agreement between the newly developed method and the ClinRep® HPLC complete commercial kit was the same and there was a statistical significant correlation between the two methods (average r=0.999; p-value < 0.0001, F-test with a true value =0). The method was much cheaper than the commercial kit, used less sample (100 μl) and had a longer running time (15 minutes) to ensure no endogenous interference. The costs of the developed method was 71-82% lower than the three commercial kits available in South Africa. Stability experiments were performed to evaluate the stability of LEV in human plasma/serum, simulating the same conditions which occurred during study samples’ analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge, room temperature and auto sampler over the 4 week period. The results showed that both LEV and the I.S (internal standard) were stable in human serum/plasma under all these conditions. The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect. A cost effective and reliable HPLC-method with minimal sample preparation time for the routine determination of LEV in plasma/serum samples was developed. It was also shown that the plasma/serum samples were stable at different temperatures over a time period. The only collection tubes that may interfere with the concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.