RESUMEN
Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other four substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. SIGNIFICANCE STATEMENT: This is the first study to clarify that arylacetamide deacetylase (AADAC) is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Dibenzazepinas/metabolismo , Microsomas Hepáticos/metabolismo , Polimorfismo Genético/fisiología , Adulto , Anciano , Células Cultivadas , Dibenzazepinas/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Femenino , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Hidrólisis/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Persona de Mediana Edad , Polimorfismo Genético/efectos de los fármacosRESUMEN
Although the pathophysiology of carbamazepine-induced idiosyncratic or hypersensitivity reactions is unclear, they are presumed to be immune mediated, involving a complex interaction between drug metabolism and activation of the immune system. Cell stress can be caused by reactive metabolites, and this has the potential to release damage-associated molecular patterns (DAMPs), which are responsible for activation of the immune system. Idiosyncratic drug reactions occur mainly in the liver because of its role in drug metabolism and reactive metabolite formation. DAMPs can activate inflammasomes, which may be a common mechanism by which DAMPs lead to an immune response. In the present study, we investigated whether carbamazepine induces the release of DAMPs by using human hepatocarcinoma functional liver cell-4 (FLC-4) cells for bioactivation of carbamazepine. THP-1 cells, a human macrophage cell line, were used for detecting inflammasome activation. We found that increased caspase-1 activity and production of interleukin-1ß by THP-1 cells were caused by the supernatant from the incubation of carbamazepine with FLC-4 cells. In the supernatant, heat shock protein 60 was significantly increased. In addition, 2-hydroxyiminostilbene, which is a metabolite of carbamazepine, activated inflammasomes. These results suggest that the reactive iminoquinone metabolite can directly activate inflammasomes or that stressed hepatocytes cause the release of DAMPs, which are responsible for inflammasome activation. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients. SIGNIFICANCE STATEMENT: A metabolite of carbamazepine, 2-hydroxyiminostilbene itself, and the damage-associated molecular patterns released from hepatocytes incubated with carbamazepine activated inflammasomes. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients.
Asunto(s)
Anticonvulsivantes/efectos adversos , Carbamazepina/efectos adversos , Dibenzazepinas/metabolismo , Hipersensibilidad a las Drogas/inmunología , Inflamasomas/efectos de los fármacos , Alarminas/inmunología , Alarminas/metabolismo , Anticonvulsivantes/farmacocinética , Carbamazepina/farmacocinética , Línea Celular Tumoral , Epilepsia/tratamiento farmacológico , Hepatocitos/metabolismo , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismoRESUMEN
In this study, interface-assembled carbonyl reductase (IACR) was prepared and used in the synthesis of S-licarbazepine in a toluene/Tris-HCl biphasic system. The carbonyl reductase (CR) was conjugated with polystyrene to form a surfactant-like structure at the interface of the toluene/Tris-HCl biphasic system. The interface-assembled efficiency of IACR reached 83% when the CR (180 U/mg) and polystyrene concentration were 8 × 10² g/ml and 3.75 × 10³ g/ml, respectively. The conversion reached 95.6% and the enantiometric excess of S-licarbazepine was 98.6% when 3.97 × 106 nmol/l oxcarbazepine was converted by IACR using 6% ethanol as a co-substrate in toluene/Tris-HCl (12.5:10) at 30°C and 43 ×g for 6 h. IACR could be reused efficiently five times.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Dibenzazepinas/metabolismo , Tolueno/química , Trometamina/química , Oxidorreductasas de Alcohol/química , Carbamazepina/análogos & derivados , Carbamazepina/metabolismo , Dibenzazepinas/química , Etanol , Concentración de Iones de Hidrógeno , Hidroxiapatitas , NAD/química , Oxcarbazepina , Poliestirenos/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
A LC method using a chiral stationary phase (CSP) with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector in polar organic mode (POM) was developed for the separation of the biopharmaceutic classification system (BCS) class II chiral prodrug eslicarbazepine acetate (ESL) and its main metabolites, namely eslicarbazepine, its optical antipode, (R)-licarbazepine, and the achiral oxcarbazepine (OXC). The percentage of methanol (MeOH) in the mobile phase containing acetonitrile (ACN) as the main solvent was found to significantly influence analyte retention and resolution. A reversal of elution order of OXC and (R)-licarbazepine was observed, depending on the MeOH percentage in the mobile phase. The optimized mobile phase consisted of ACN/MeOH/acetic acid/diethylamine (95/5/0.2/0.07; v/v/v/v). The potential of this chemo- and enantioselective LC method combined with solid-phase extraction (SPE) was then evaluated for in vitro metabolism studies using ESL as a model case. Only eslicarbazepine could be detected after incubation of ESL in human liver microsome systems.
Asunto(s)
Dibenzazepinas/aislamiento & purificación , Profármacos/aislamiento & purificación , Ácido Acético , Acetonitrilos , Carbamazepina/análogos & derivados , Carbamazepina/química , Carbamazepina/aislamiento & purificación , Celulosa/análogos & derivados , Celulosa/química , Celulosa/aislamiento & purificación , Cromatografía Liquida/métodos , Dibenzazepinas/química , Dibenzazepinas/metabolismo , Dietilaminas , Humanos , Microsomas Hepáticos/química , Oxcarbazepina , Fenilcarbamatos/química , Fenilcarbamatos/aislamiento & purificación , Profármacos/metabolismo , Extracción en Fase Sólida , Solventes , EstereoisomerismoRESUMEN
A rapid and sensitive assay based on supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) has been developed and validated for the determination of oxcarbazepine (OXC) and its chiral metabolite licarbazine (Lic) in beagle dog plasma using carbamazepine as internal standard. Chiral analysis in a run time of only 3 min was performed on an ACQUITY UPC(2) ™ Trefoil™ CEL2 column (3.0 × 150 mm, 2.5 µm) at 50 °C by isocratic elution with a mobile phase of supercritical carbon dioxide (purity ≥ 99.99%) and methanol (60:40, v/v) at a flow rate of 2.3 mL/min. The assay was linear over the concentration ranges 5-1000 ng/mL for OXC and 0.5-100 ng/mL for the enantiomers of Lic with corresponding lower limits of quantitation of 5 ng/mL and 0.5 ng/mL. Intra- and inter-day precisions were in the range 0.78-14.14% with accuracies in the range -10.80% to 0.42%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 16 mg/kg OXC as Trileptal(@) tablets to beagle dogs.
Asunto(s)
Carbamazepina/análogos & derivados , Cromatografía con Fluido Supercrítico/métodos , Dibenzazepinas/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Carbamazepina/sangre , Carbamazepina/química , Carbamazepina/farmacocinética , Dibenzazepinas/química , Dibenzazepinas/metabolismo , Perros , Límite de Detección , Modelos Lineales , Oxcarbazepina , Reproducibilidad de los ResultadosRESUMEN
The fluorescence, ultraviolet (UV) absorption, time resolved techniques, circular dichroism (CD), and infrared spectral methods were explored as tools to investigate the interaction between histamine H1 drug, epinastine hydrochloride (EPN), and bovine serum albumin (BSA) under simulated physiological conditions. The experimental results showed that the quenching of the BSA by EPN was static quenching mechanism and also confirmed by lifetime measurements. The value of n close to unity indicated that one molecule of EPN was bound to protein molecule. The binding constants (K) at three different temperatures were calculated (7.1 × 10(4), 5.5 × 10(4), and 3.9 × 10(4) M(-1)). Based on the thermodynamic parameters (ΔH(0), ΔG(0), and ΔS(0)), the nature of binding forces operating between drug and protein was proposed. The site of binding of EPN in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz, warfarin, ibuprofen, and digitoxin. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (EPN) was evaluated and found to be 4.48 nm. The UV-visible, synchronous fluorescence, CD, and three-dimensional fluorescence spectral results revealed the changes in secondary structure of the protein upon its interaction with EPN.
Asunto(s)
Dibenzazepinas/química , Dibenzazepinas/metabolismo , Imidazoles/química , Imidazoles/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/metabolismo , Animales , Bovinos , Dicroismo Circular , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Latrunculin A microperfusion of the hippocampus induces acute epileptic seizures and long-term biochemical changes leading to spontaneous seizures. This study tested the effect of eslicarbazepine acetate (ESL), a novel antiepileptic drug, on latrunculin A-induced acute and chronic seizures, and changes in brain amino acid extracellular levels. Hippocampi of Swiss mice were continuously perfused with a latrunculin A solution (4 µM, 1 µl/min, 7 h/day) with continuous EEG and videotape recording for 3 consecutive days. Microdialysate samples were analyzed by HPLC and fluorescence detection of taurine, glycine, aspartate, glutamate and GABA. Thereafter, mice were continuously video monitored for two months to identify chronic spontaneous seizures or behavioral changes. Control EEG recordings (8 h) were performed in all animals at least once a week for a minimum of one month. RESULTS: Oral administration of ESL (100 mg/kg), previous to latrunculin A microperfusion, completely prevented acute latrunculin A-induced seizures as well as chronic seizures and all EEG chronic signs of paroxysmal activity. Hippocampal extracellular levels of taurine, glycine and aspartate were significantly increased during latrunculin A microperfusion, while GABA and glutamate levels remained unchanged. ESL reversed the increases in extracellular taurine, glycine and aspartate concentrations to basal levels and significantly reduced glutamate levels. Plasma and brain bioanalysis showed that ESL was completely metabolized within 1 h after administration to mainly eslicarbazepine, its major active metabolite. CONCLUSION: ESL treatment prevented acute latrunculin A-induced seizures as well as chronic seizures and all EEG chronic signs of paroxysmal activity, supporting a possible anti-epileptogenic effect of ESL in mice.
Asunto(s)
Aminoácidos/metabolismo , Anticonvulsivantes/farmacología , Dibenzazepinas/farmacología , Espacio Extracelular/metabolismo , Hipocampo/efectos de los fármacos , Convulsiones/tratamiento farmacológico , Enfermedad Aguda , Animales , Ácido Aspártico/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes , Enfermedad Crónica , Dibenzazepinas/metabolismo , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Glicina/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Convulsiones/metabolismo , Taurina/metabolismo , Tiazolidinas , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The present work describes, for the first time, the simultaneous separation of oxcarbazepine (OXC) and its active metabolite 10-hydroxy-10,11-dihydrocarbamazepine (licarbazepine, Lic) by chiral CE. The developed method was employed to monitor the enantioselective biotransformation of OXC into its active metabolite by fungi. The electrophoretic separations were performed using 10 mmol/L of a Tris-phosphate buffer solution (pH 2.5) containing 1% w/v of ß-CD phosphate sodium salt (P-ß-CD) as running electrolyte, -20 kV of applied voltage and a 15°C capillary temperature. The method was linear over the concentration range of 1000-30 000 ng/mL for OXC and 75-900 ng/mL for each Lic enantiomer (r ≥ 0.9952). Within-day precision and accuracy evaluated by RSD and relative errors, respectively, were lower than 15% for all analytes. The validated method was used to evaluate the enantioselective biotransformation of OXC, mediated by fungi, into its active metabolite Lic. This study showed that the fungi Glomerella cingulata (VA1) and Beuveria bassiana were able to enantioselectively metabolize the OXC into Lic after 360 h of incubation. Biotransformation by the fungus Beuveria bassiana showed 79% enantiomeric excess for (S)-(+)-Lic, while VA1 gave an enantiomeric excess of 100% for (S)-(+)-Lic. This study opens a new route to the drug (S)-(+)-licarbazepine.
Asunto(s)
Carbamazepina/análogos & derivados , Dibenzazepinas , Electroforesis Capilar/métodos , Phyllachorales/metabolismo , Biotransformación , Carbamazepina/análisis , Carbamazepina/química , Carbamazepina/metabolismo , Dibenzazepinas/análisis , Dibenzazepinas/química , Dibenzazepinas/metabolismo , Modelos Lineales , Oxcarbazepina , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
The bacterium Micromonospora sp. RV115, isolated from a marine sponge, produces the unusual metabolite diazepinomicin, a prenylated benzodiazepine derivative. We have cloned the prenyltransferase gene dzmP from this organism, expressed it in Escherichia coli, and the resulting His8-tagged protein was purified and investigated biochemically. It was found to catalyze the farnesylation of the amide nitrogen of dibenzodiazepinone. DzmP belongs to the ABBA prenyltransferases and is the first member of this superfamily which utilizes farnesyl diphosphate as genuine substrate. All previously discovered members utilize either dimethylallyl diphosphate (C5) or geranyl diphosphate (C10). Another putative diazepinomicin biosynthetic gene cluster was identified in the genome of Streptomyces griseoflavus Tü4000, suggesting that the formation of diazepinomicin is not restricted to the genus Micromonospora. The gene cluster contains a gene ssrg_00986 with 61.4% identity (amino acid level) to dzmP. The gene was expressed in E. coli, and the purified protein showed similar catalytic properties as DzmP. Both enzymes also accepted other phenolic or phenazine substrates. ABBA prenyltransferases are useful tools for chemoenzymatic synthesis, due to their nature as soluble, stable biocatalysts. The discovery of DzmP and Ssrg_00986 extends the isoprenoid substrate range of this superfamily. The observed prenylation of an amide nitrogen is an unusual biochemical reaction.
Asunto(s)
Benzodiazepinas/metabolismo , Vías Biosintéticas/fisiología , Dibenzazepinas/metabolismo , Dimetilaliltranstransferasa/metabolismo , Micromonospora/genética , Familia de Multigenes/genética , Streptomyces/genética , Secuencia de Aminoácidos , Animales , Benzodiazepinas/química , Vías Biosintéticas/genética , Catálisis , Análisis por Conglomerados , Biología Computacional , Escherichia coli , Micromonospora/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Filogenia , Prenilación/fisiología , Alineación de SecuenciaRESUMEN
Radioing in on NPY: Attachment of a [2,3-(3)H]propionyl group through an appropriate linker to the guanidine group of an (S)-argininamide-type neuropeptide Y (NPY) Y(2) receptor antagonist resulted in a subtype-selective radioligand.
Asunto(s)
Arginina/análogos & derivados , Dibenzazepinas/química , Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/antagonistas & inhibidores , Arginina/síntesis química , Arginina/química , Arginina/metabolismo , Señalización del Calcio , Dibenzazepinas/síntesis química , Dibenzazepinas/metabolismo , Cinética , Ligandos , Neuropéptido Y/metabolismo , Unión Proteica , Receptores de Neuropéptido Y/metabolismo , Relación Estructura-Actividad , Tritio/químicaRESUMEN
Recently, in silico models have been developed to predict drug pharmacokinetics. However, before application, they must be validated and, for that, information about structurally similar reference compounds is required. A chiral liquid chromatography method with ultraviolet detection (LC-UV) was developed and validated for the simultaneous quantification of BIA 2-024, BIA 2-059, BIA 2-265, oxcarbazepine, eslicarbazepine (S-licarbazepine) and R-licarbazepine in mouse plasma and brain. Compounds were extracted by a selective solid-phase extraction procedure and their chromatographic separation was achieved on a LiChroCART 250-4 ChiraDex column using a mobile phase of water-methanol (92:8, v/v) pumped at 0.7 mL/min. The UV detector was set at 235 nm. Calibration curves were linear (r(2) ≥ 0.996) over the concentration ranges of 0.2-30 µg/mL for oxcarbazepine, eslicarbazepine and R-licarbazepine; 0.2-60 µg/mL for the remaining compounds in plasma; and 0.06-15 µg/mL for all the analytes in brain homogenate. Taking into account all analytes at these concentration ranges in both matrices, the overall precision did not exceed 9.09%, and the accuracy was within ±14.3%. This LC-UV method is suitable for carrying out pharmacokinetic studies with these compounds in mouse in order to obtain a better picture of their metabolic pathways and biodistribution.
Asunto(s)
Anticonvulsivantes/metabolismo , Encéfalo/metabolismo , Carbamazepina/análogos & derivados , Cromatografía Liquida/métodos , Dibenzazepinas/metabolismo , Animales , Anticonvulsivantes/sangre , Calibración , Carbamazepina/sangre , Carbamazepina/metabolismo , Dibenzazepinas/sangre , Ratones , Oxcarbazepina , Control de Calidad , Estándares de Referencia , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-ß activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-ß activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.
Asunto(s)
Señalización del Calcio/fisiología , Receptores de Amina Biogénica/clasificación , Receptores de Amina Biogénica/metabolismo , Receptores Dopaminérgicos/clasificación , Receptores Dopaminérgicos/metabolismo , Secuencia de Aminoácidos , Animales , Abejas , Sitios de Unión , Calcio/metabolismo , Señalización del Calcio/genética , Línea Celular , AMP Cíclico/metabolismo , Dibenzazepinas/metabolismo , Flupentixol/farmacología , Humanos , Imidazoles/metabolismo , Datos de Secuencia Molecular , Filogenia , Receptores de Amina Biogénica/química , Receptores de Amina Biogénica/genética , Receptores Dopaminérgicos/química , Receptores Dopaminérgicos/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Espiperona/farmacologíaRESUMEN
OBJECTIVE: To determine effects of various concentrations of retinoic acid (RA) or a synthetic RA receptor antagonist (LE135) on equine chondrocytes or bone marrow-derived equine mesenchymal stem cells (BMDMSCs) in monolayer cultures. SAMPLE: Articular cartilage and BMDMSCs from 5 clinically normal horses. PROCEDURES: Monolayers of chondrocytes cultured in standard media and of BMDMSCs cultured in chondrogenic media were treated with RA at concentrations of 0, 0.1, 1, or 10 µM or LE135 at concentrations of 0, 0.1, 1, or 10 µM on day 0. On days 7 and 14, samples were analyzed for DNA concentration, chondrocyte morphology or features consistent with chondrogenesis (ie, chondral morphology [scored from 0 to 4]), and gene expression of collagen type Ia (CI), collagen type II (CII), and aggrecan. RESULTS: Chondrocytes treated with RA had more mature chondral morphology (range of median scores, 3.0 to 4.0) than did untreated controls (range of median scores, 0.5 to 0.5). Chondrocytes treated with LE135 did not sustain chondrocyte morphology. All BMDMSCs had evidence of chondral morphology or high CII:CI ratio. Retinoic acid (1 or 10 µM) or LE135 (10 µM) treatment decreased DNA content of BMDMSC cultures. At 0.1 and 1 µM concentrations, LE135 weakly but significantly increased chondral morphology scores, compared with untreated controls, but lack of aggrecan expression and lack of increased CII:CI ratio, compared with that of controls, did not affect chondrogenesis. CONCLUSIONS AND CLINICAL RELEVANCE: RA promoted maturation and hypertrophy in chondrocytes but not BMDMSCs in monolayer cultures. Deficiency or blockade of RA may prevent hypertrophy and maturation of differentiated chondrocytes.
Asunto(s)
Agrecanos/metabolismo , Condrogénesis , Dibenzazepinas/metabolismo , Caballos/fisiología , Células Madre Mesenquimatosas/efectos de los fármacos , Receptores de Ácido Retinoico/antagonistas & inhibidores , Tretinoina/farmacología , Agrecanos/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Técnicas de Cultivo de Célula , Diferenciación Celular , Condrocitos/citología , Condrocitos/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulación de la Expresión Génica , Caballos/anatomía & histología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Receptores de Ácido Retinoico/genética , Tretinoina/metabolismoRESUMEN
PURPOSE: Antiepileptic drugs (AEDs) are widely used not only in the treatment of epilepsy but also as treatments for psychiatric disorders. Pharmacoresistance of AEDs in the treatment of epilepsy and psychiatric disorders is a serious problem. Transport of antiepileptic drugs by P-glycoprotein (Pgp, ABCB1, or MDR1), which is overexpressed in the blood-brain barrier, may be a mechanism for resistance of AEDs. For most AEDs, conflicting evidence precludes consensus on whether they are substrates of Pgp. The objective of this study was to evaluate whether analogs and metabolites of the AED carbamazepine are substrates of human Pgp. METHODS: Polarized cell lines MDCKII and LLC transfected with the human MDR1 gene were used in the bidirectional transport assay and concentration equilibrium transport assay. The expression of Pgp was detected by real-time polymerase chain reaction (PCR) and immunofluorescent staining. Rhodamine-123 uptake was also determined. KEY FINDINGS: Pgp did not transport carbamazepine, but it did transport its active metabolite carbamazepine-10,11-epoxide. Pgp also pumped eslicarbazepine acetate and oxcarbazepine, as well as their active metabolite (S)-licarbazepine. Transport of the drugs was in the order of ESL>OXC>S-LC>CBZ-E in concentration equilibrium conditions. The transport of these drugs was blocked by Pgp inhibitors tariquidar and verapamil. SIGNIFICANCE: All carbamazepine analogs or metabolites tested are Pgp substrates, except for carbamazepine. These data suggest that resistance to carbamazepine, oxcarbazepine, or eslicarbazepine acetate may be attributed to increased efflux function of Pgp because they or their active metabolites are Pgp substrates.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Anticonvulsivantes/metabolismo , Carbamazepina/análogos & derivados , Carbamazepina/metabolismo , Dibenzazepinas/metabolismo , Animales , Transporte Biológico , Línea Celular , Resistencia a Medicamentos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Células LLC-PK1/metabolismo , Oxcarbazepina , Reacción en Cadena de la Polimerasa , PorcinosRESUMEN
Eslicarbazepine acetate (ESL) is a once-daily novel antiepileptic drug approved in Europe for use as adjunctive therapy for refractory partial-onset seizures with or without secondary generalization. Metabolism of ESL consists primarily of hydrolysis to eslicarbazepine, which is then subject to glucuronidation followed by renal excretion. In this study, we have identified that human liver microsomes (HLM) enriched with uridine 5'-diphosphoglucuronic acid give origin to a single Escherichia coli ß-glucuronidase-sensitive eslicarbazepine glucuronide (most likely the O-glucuronide). The kinetics of eslicarbazepine glucuronidation in HLM was investigated in the presence and absence of bovine serum albumin (BSA). The apparent K(m) were 412.2 ± 63.8 and 349.7 ± 74.3 µM in the presence and absence of BSA, respectively. Incubations with recombinant human UDP glucuronosyltransferases (UGTs) indicated that UGT1A4, UGT1A9, UGT2B4, UGT2B7, and UGT2B17 appear to be involved in eslicarbazepine conjugation. The UGT with the highest affinity for conjugation was UGT2B4 (K(m) = 157.0 ± 31.2 and 28.7 ± 10.1 µM, in the absence and presence of BSA, respectively). There was a significant correlation between eslicarbazepine glucuronidation and trifluoperazine glucuronidation, a typical UGT1A4 substrate; however, no correlation was found with typical substrates for UGT1A1 and UGT1A9. Diclofenac inhibited eslicarbazepine glucuronidation in HLM with an IC(50) value of 17 µM. In conclusion, glucuronidation of eslicarbazepine results from the contribution of UGT1A4, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, but the high-affinity component of the UGT2B4 isozyme may play a major role at therapeutic plasma concentrations of unbound eslicarbazepine.
Asunto(s)
Dibenzazepinas/metabolismo , Glucuronosiltransferasa/metabolismo , Hígado/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Anticonvulsivantes/metabolismo , Escherichia coli/metabolismo , Glucuronidasa/metabolismo , Glucurónidos/metabolismo , Humanos , Cinética , Hígado/enzimología , Ratones , Microsomas Hepáticos/enzimología , Albúmina Sérica Bovina/metabolismo , Trifluoperazina/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
For the first time, a selective and sensitive chiral HPLC-UV method was developed and fully validated for the simultaneous quantification of eslicarbazepine acetate (ESL), carbamazepine (CBZ), S-licarbazepine (S-Lic), R-licarbazepine (R-Lic), oxcarbazepine (OXC) and carbamazepine-10,11-epoxide (CBZ-E), in mouse plasma and brain homogenate supernatant. After the addition of chloramphenicol as the internal standard, samples were processed using an SPE procedure. The chiral chromatographic analysis was carried out on a LiChroCART 250-4 ChiraDex column, employing a mobile phase of water and methanol (88:12, v/v) pumped at 0.9 mL/min and the UV detector set at 235 nm. The assay was linear (r(2) ≥0.995) for ESL, CBZ, OXC, S-Lic, R-Lic and CBZ-E in the range of, respectively, 0.2-4, 0.4-30, 0.1-60, 0.2-60, 0.2-60 and 0.2-30 µg/mL, in plasma, and of 0.06-1.5 µg/mL for ESL, 0.12-15 µg/mL for CBZ and CBZ-E and 0.06-15 µg/mL for OXC and both licarbazepine (Lic) enantiomers in brain homogenate supernatant. The overall precision was within 8.71% and accuracy ranged from -7.55 to 8.97%. The recoveries of all the compounds were over 92.1%. Afterwards, the application of the method was demonstrated using real plasma and brain samples obtained from mice administered simultaneously with ESL and CBZ.
Asunto(s)
Anticonvulsivantes/análisis , Química Encefálica , Carbamazepina/análisis , Cromatografía Líquida de Alta Presión/métodos , Dibenzazepinas/análisis , Animales , Anticonvulsivantes/sangre , Anticonvulsivantes/metabolismo , Encéfalo/metabolismo , Carbamazepina/sangre , Carbamazepina/metabolismo , Cromatografía Líquida de Alta Presión/instrumentación , Dibenzazepinas/sangre , Dibenzazepinas/metabolismo , Epilepsia/tratamiento farmacológico , Humanos , Masculino , Ratones , Modelos AnimalesRESUMEN
Dibenzazecines are a novel class of dopamine receptor antagonists, characterized by their high affinities as well as their tendency for D(1) selectivity. Hitherto, the most active dibenzazecines were phenolic in nature; a 3-OH substituent was found to result in the highest affinities. However, the phenolic nature of these compounds mostly renders them unsuitable for in vivo application, due to the poor pharmacokinetic profile, imparted by the phenolic group. A novel dibenzazecine derivative was prepared, with methylenedioxy moiety, connecting C(2) amd C(3), instead of the 3-OH group. The newly synthesized derivative 3 showed high affinities similar to the lead LE404, displaying nanomolar affinities for all dopamine receptor subtypes. Its dibrominated derivative 4, though exhibiting almost a fivefold decrease in affinities, still displayed nanomolar ones for all dopamine receptors, except for D(4) . In a functional Ca(2+) assay, both compounds 3 and 4 were found to possess antagonistic properties towards the dopamine receptors.
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Dibenzazepinas/química , Dibenzazepinas/farmacología , Antagonistas de Dopamina/química , Antagonistas de Dopamina/farmacología , Receptores Dopaminérgicos/química , Dibenzazepinas/síntesis química , Dibenzazepinas/metabolismo , Antagonistas de Dopamina/síntesis química , Antagonistas de Dopamina/metabolismo , Humanos , Estructura Molecular , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The effectiveness of precursor-directed biosynthesis to generate diazepinomicin (1) analogues with varied ring-A substitutents was investigated by feeding commercially available, potential ring-A precursors such as fluorinated tryptophans, halogenated anthranilates, and various substituted indoles into growing actinomycete culture DPJ15 (genus Micromonospora). Two new monofluorinated diazepinomicin analogues (2 and 3) were identified and characterized by spectroscopic methods. Both derivatives showed modest antibacterial activity against the Gram-positive coccus Staphylococcus aureus with MIC values in the range 8-32 microg/mL.
Asunto(s)
Dibenzazepinas/aislamiento & purificación , Hidrocarburos Fluorados/aislamiento & purificación , Indoles/aislamiento & purificación , Micromonospora/química , Dibenzazepinas/química , Dibenzazepinas/metabolismo , Dibenzazepinas/farmacología , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/metabolismo , Hidrocarburos Fluorados/farmacología , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Staphylococcus aureus/efectos de los fármacosRESUMEN
Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of (15)N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous. In the case of the mono-(15)N-labeled diazepinomicin (1) derived from labeled tryptophan, tandem mass spectrometry located this (15)N label at the non-amide nitrogen. Through the use of exceptionally high mass resolving power of over 125,000, the isotopic fine structure of the molecular ion cluster of 1 was revealed. Separation of the (15)N(2) peak from the isobaric (13)C(15)N peak, both having similar abundances, demonstrated the presence of a minor amount of doubly (15)N-labeled diazepinomicin (1). Tandem mass spectrometry amplified this isotopic fine structure (Deltam=6.32 mDa) from mDa to 1 Da scale thereby allowing more detailed scrutiny of labeling content and location. Tandem mass spectrometry was also used to assign the location of deuterium labeling in two deuterium-labeled diazepinomicin (1) samples. In one case three deuterium atoms were incorporated into the dibenzodiazepine core; while in the other a mono-D label was mainly incorporated into the farnesyl side chain. The specificity of (15)N-labeling in dioxapyrrolomycin (2) and the proportion of the (15)N-label contained in the nitro group were determined from the measurement of the relative abundance of the (14)NO(2)(1-) and (15)NO(2)(1-) fragment ions.
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Productos Biológicos/biosíntesis , Ciclotrones , Dibenzazepinas/metabolismo , Espectrometría de Masas en Tándem/métodos , Deuterio , Fermentación , Análisis de Fourier , Pirroles/metabolismoRESUMEN
Eslicarbazepine acetate is a promising antiepileptic drug structurally related to carbamazepine and oxcarbazepine, which is in the final phase of clinical development. The metabolism of eslicarbazepine acetate is clearly species dependent and, in this case, among small laboratory animals, the mouse seems to be the most relevant species to humans. Hence, the aim of this study was to investigate the plasma, brain and liver disposition of eslicarbazepine acetate in mice to better understand its disposition in humans. Adult male CD-1 mice were treated orally with a single dose of eslicarbazepine acetate 350 mg/kg. Blood samples, brain and liver tissues were taken at 0.25, 0.5, 0.75, 1, 2, 4, 6, 10, 16 and 24 h post-dose. Plasma and tissue levels of eslicarbazepine acetate and its metabolites (S-licarbazepine, R-licarbazepine and oxcarbazepine) were assessed by using high-performance liquid chromatography-ultraviolet detection. Both eslicarbazepine acetate and R-licarbazepine concentrations were below the limit of quantification of the assay in all matrices. Eslicarbazepine acetate was rapidly and extensively metabolized to S-licarbazepine (major metabolite), which was oxidized to oxcarbazepine to a small extent. The brain/plasma ratios suggest that the brain exposure to S-licarbazepine and oxcarbazepine was approximately 30% of their total systemic exposure. However, S-licarbazepine crossed the blood-brain barrier (BBB) less efficiently than oxcarbazepine. On the other hand, the liver/plasma ratios support the notion that S-licarbazepine undergoes hepatic accumulation, whereas oxcarbazepine appears to leave this compartment twice as fast as S-licarbazepine. Thus, the diffusion through the BBB is favourable to oxcarbazepine and the liver acts like a deposit of the pharmacologically active metabolite of eslicarbazepine acetate (S-licarbazepine).