ABSTRACT
Carbamazepina, fenitoína, fenobarbital e lamotrigina são bem conhecidos como fármacos anticonvulsivantes usados para o tratamento da epilepsia. A utilização destes fármacos é associada a várias reações adversas, tornando necessário o controle terapêutico. Os ensaios foram realizados por meio de cromatografia líquida de alta eficiência e o método de extração utilizado foi o líquido-líquido. Os fármacos e o padrão interno de zolpidem foram separados por uma coluna de fase reversa (ACE5, C18150x 4,6 mm d.i.). A fase móvel constituída de acetonitrila (30%) e ácido cítrico/tampão fosfato pH 5,0 (70%) foi utilizada sob gradiente de fluxo de 0,7 a 1,2 mL/min e o comprimento de onda utilizado para a detecção dos analitos foi fixado em 210 nm. A técnica apresentou linearidade ao longo do intervalo de 0,5 a 20,0μg/mL para carbamazepina/lamotrigina e de 2,0 a 64,0 μg/mL, para fenitoina/fenobarbital com coeficiente de correlação linear maior que 0,98. As amostras de sangue foram obtidas de pacientes adultos com diagnóstico de epilepsia em acompanhamento no Ambulatório Municipal de Psiquiatria de Goiânia. A média de recuperação obtida foi de 96,8% a 108,5% para carbamazepina/lamotrigina e de 93,8% a 108,8% para a fenitoina/fenobarbital. Os limites de quantificação, precisão (CV< 15,0 %)e exatidão (E > 85,0 %) demonstraram estar em conformidade com as exigências da ANVISA. Nove pacientes foram avaliados para confirmar a validade do método.
Carbamazepine, phenytoin, phenobarbital and lamotrigine are well known anticonvulsant drugs used in the treatment of epilepsy. However, these medications are associated with side effects and therefore require therapeutic monitoring. Blood samples of adult outpatients diagnosed with epilepsy at the Goiânia Municipal Psychiatry Clinic (Brazil) were collected and analyzed using high-performance liquid chromatography. The analytes and internal standard (zolpidem) were extracted by liquid-liquid extraction. A reversed phase column (ACE 5, C18, 150 x 4.6 mm i.d.) was used. The mobile phase was composed of acetonitrile (30%) and citric acid/phosphate buffer, pH 5.0 (70%). The gradient flow rate was from 0.7 to 1.2 mL/min and the detection wavelength was set at 210 nm for all analytes. The analysis revealed a linear range from 0.5 to 20.0μg/mL for carbamazepine/lamotrigine and 2.0 to 64.0 μg/mL for phenytoin/phenobarbital. Mean recovery ranged from 96.8% to 108.5% for carbamazepine/lamotrigine and 93.8% to 108.8% for phenytoin/phenobarbital. The quantification limit, precision (CV < 15%) and accuracy (A >85%) proved to be in accordance with the requirements stipulated by the Brazilian National Health Surveillance Agency (ANVISA). Nine outpatients were evaluated to confirm the validity of the method.
Subject(s)
Carbamazepine/chemistry , Epilepsy/drug therapy , Phenytoin/chemistry , Phenobarbital/chemistry , Chromatography, High Pressure Liquid/methodsABSTRACT
P-glycoprotein (P-gp), an ATP-dependant efflux pump transports a wide range of substrates across cellular membranes. Earlier studies have identified drug efflux due to the over-expression of P-gp as one of the causes for the resistance of phenytoin, an anti-epileptic drug (AED). While no clear evidence exists on the specific characteristics of phenytoin association with the human P-gp, this study employed structure-based computational approaches to identify its binding site and the underlying interactions. The identified site was validated with that of rhodamine, a widely accepted reference and an experimental probe. Further, an in silico proof-of-concept for phenytoin interactions and its decreased binding affinity with the closed-state of human P-gp model was provided in comparison with other AEDs. This is the first report to provide insights into the phenytoin binding site and possibly better explain its efflux by P-gp.
Subject(s)
Binding Sites , Catalysis , Computer Simulation , Humans , Models, Chemical , Models, Molecular , ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/ultrastructure , Phenytoin/chemistry , Protein Binding , Protein ConformationABSTRACT
Phenytoin is commonly used as an antiepileptic drug worldwide. The unique properties of phenytoin such as poor water solubility and zero-order kinetics of its metabolism, together with difference in pharmaceutical formulations can result in dramatic changes in bioavailability of phenytoin capsule. The innovator (Dilantin, Parke Davis), three local brands (brand A, B and C) were investigated for pharmaceutical characteristics including drug content, content uniformity, and dissolution. All these tests were performed as described in the monograph of extended phenytoin sodium capsules in USP XXII with slight modification in HPLC analysis. It was found that all the products, except brand C, had drug content and content uniformity within the standard range. Brand A and brand C failed to meet the USP XXII specification for the dissolution test. Per cent dissolution of brand A was lower whereas per cent dissolution of brand C was much higher than the standard value. The qualities of innovator and brand B were within the pharmacopoeial specification. This result revealed that phenytoin capsules available in Thailand did not have homogeneous pharmaceutical equivalence which may lead to difference in plasma phenytoin levels (see part II: In vivo study). Thus, changing the brand of phenytoin in stable epileptic patients should be performed with caution.
Subject(s)
Anticonvulsants/chemistry , Biological Availability , Capsules , Chromatography, High Pressure Liquid , Phenytoin/chemistry , ThailandABSTRACT
Se describe un método sensible y específico de cromatografía de alta resolución (HPCL) para el análisis en plasma humano de 3-hidroxi, 3-etil, 3-fenil propionami-da (HEPP), un nuevo anticonvulsivante. El estándar interno (2-hidroxi, 2-etil, 2-fenil acetamida HEPA) y la HEPP se extrajeron en acetonitrilo de plasma amortiguado, la extracción fue cercana al 100 por ciento respecto a la cantidad extraída de cada fármaco de solución salina amortiguada. El método consiste en HPCL en fase reversa (Lichro Spher 100 RP-18 en Lichro Cart 125-4), la fase móvil está compuesta de metanol-acetonitrilo-amortiguador de fosfatos (35/15/50 por vol) y detección ultravioleta a 200 nm. Las curvas de calibración fueron lineales y repetibles (coeficiente de correlación > 0.999). Las determinaciones de HEPP en plasma humano o en solución salina fueron lineales en el intervalo 0 - 10 µg/ml y el coeficiente de variación fue menor que 10 por ciento. HEPP es muy estable a temperatura ambiente y a 4 ºC, y puede ser cuantificada en presencia de otros antiepilépticos tales como: difenilhidantoina, clonazepam. carbamezepina y hexobarbital