Distribution and pharmacokinetics of etamicastat and its N-acetylated metabolite (BIA 5-961) in dog and monkey.

1. The disposition etamicastat was evaluated in the Cynomolgus monkey after intravenous and oral administration of [(14)C]-etamicastat. The pharmacokinetics of etamicastat and its N-acetylated metabolite BIA 5-961 were also evaluated in monkeys and dogs. 2. In the monkey, 7 days after intravenous and oral administration of [(14)C]-etamicastat, 76.6-91.1% of the etamicastat-related radioactivity had been excreted mainly in urine. The radioactivity peaked in plasma between 4- and 8-h post-dosing followed by a quick decline and a slow terminal phase (half-life of 68.7 h). The calculated oral bioavailability for etamicastat was 46.1%. Etamicastat was quickly absorbed in monkeys and dogs with a half-life ranging from 5.2 to 9.9 h in monkeys and 6.9 to 11.4 h in dogs over. 3. The N-acetylated metabolite of etamicastat, represented 4-7% of the extent of exposure of etamicastat in the monkey, but was not found detectable in dogs. Gender did not influence etamicastat exposure and the concentration versus time curves fitted a dose-dependent pharmacokinetics in the dog, but not in the monkey. 4. In conclusion, etamicastat is rapidly absorbed and primarily excreted via urine in monkeys. Similarly, to humans, monkeys, unlike dogs, N-acetylate etamicastat and evidence that etamicastat pharmacokinetics is less than dose proportional.

Pharmacological profile of opicapone, a third-generation nitrocatechol catechol-O-methyl transferase inhibitor, in the rat.

BACKGROUND AND PURPOSE: Catechol-O-methyltransferase (COMT) is an important target in the levodopa treatment of Parkinson's disease; however, the inhibitors available have problems, and not all patients benefit from their efficacy. Opicapone was developed to overcome those limitations. In this study, opicapone's pharmacological properties were evaluated as well as its potential cytotoxic effects. EXPERIMENTAL APPROACH: The pharmacodynamic effects of opicapone were explored by evaluating rat COMT activity and levodopa pharmacokinetics, in the periphery through microdialysis and in whole brain. The potential cytotoxicity risk of opicapone was explored in human hepatocytes by assessing cellular ATP content and mitochondrial membrane potential. KEY RESULTS: Opicapone inhibited rat peripheral COMT with ED50 values below 1.4 mg⋅kg(-1) up to 6 h post-administration. The effect was sustained over the first 8 h and by 24 h COMT had not returned to control values. A single administration of opicapone resulted in increased and sustained plasma levodopa levels with a concomitant reduction in 3-O-methyldopa from 2 h up to 24 h post-administration, while tolcapone produced significant effects only at 2 h post-administration. The effects of opicapone on brain catecholamines after levodopa administration were sustained up to 24 h post-administration. Opicapone was also the least potent compound in decreasing both the mitochondrial membrane potential and the ATP content in human primary hepatocytes after a 24 h incubation period. CONCLUSIONS AND IMPLICATIONS: Opicapone has a prolonged inhibitory effect on peripheral COMT, which extends the bioavailability of levodopa, without inducing toxicity. Thus, it exhibits some improved properties compared to the currently available COMT inhibitors.

Development and validation of an enantioselective liquid-chromatography/tandem mass spectrometry method for the separation and quantification of eslicarbazepine acetate, eslicarbazepine, R-licarbazepine and oxcarbazepine in human plasma.

The purpose of this study was develop and validate a sensitive and specific enantioselective liquid-chromatography/tandem mass spectrometry (LC-MS/MS) method, for the simultaneous quantification of eslicarbazepine acetate (ESL), eslicarbazepine (S-Lic), oxcarbazepine (OXC) and R-licarbazepine (R-Lic) in human plasma. Analytes were extracted from human plasma using solid phase extraction and the chromatographic separation was achieved using a mobile phase of 80% n-hexane and 20% ethanol/isopropyl alcohol (66.7/33.3, v/v). A Daicel CHIRALCEL OD-H column (5 µm, 50 mm × 4.6 mm) was used with a flow rate of 0.8 mL/min, and a run time of 8 min. ESL, S-Lic, R-Lic, OXC and the internal standard, 10,11-dihydrocarbamazepine, were quantified by positive ion electrospray ionization mass spectrometry. The method was fully validated, demonstrating acceptable accuracy, precision, linearity, and specificity in accordance with FDA regulations for the validation of bioanalytical methods. Linearity was proven over the range of 50.0-1000.0 ng/mL for ESL and OXC and over the range of 50.0-25,000.0 ng/mL for S-Lic and R-Lic. The intra- and inter-day coefficient of variation in plasma was less than 9.7% for ESL, 6.0% for OXC, 7.7% for S-Lic and less than 12.6% for R-Lic. The accuracy was between 98.7% and 107.2% for all the compounds quantified. The lower limit of quantification (LLOQ) was 50.0ng/mL for ESL, S-Lic, OXC and R-Lic in human plasma. The short-term stability in plasma, freeze-thaw stability in plasma, frozen long-term stability in plasma, autosampler stability and stock solution stability all met acceptance criteria. The human plasma samples, collected from 8 volunteers, showed that this method can be used for therapeutic monitoring of ESL and its metabolites in humans treated with ESL.

Effect of food on the pharmacokinetic profile of trans-resveratrol.

OBJECTIVE: It has been postulated that trans-resveratrol may act as an antioxidant, cardioprotective, neuroprotective and cancer chemopreventive agent. The objective of this study was to investigate the effect of food on the bioavailability of trans-resveratrol following oral administration. MATERIAL AND METHODS: Single-centre, open-label, randomized, 2-way crossover study on 24 healthy subjects. The study consisted of two consecutive treatment periods separated by a washout of 7 days or more. On each of the study periods subjects were administered a single-dose of 400 mg of trans-resveratrol following either a standard high fat content meal or 8 hs of fasting. RESULTS: There was a large interindividual variability in the trans-resveratrol pharmacokinetic parameters. Mean +/- SD maximum plasma concentration (Cmax) was 42.2 +/- 36.6 ng/ml in fed and 47.3 +/- 30.0 ng/ml in fasting conditions. Median time to Cmax (tmax) was 2.0 h in fed and 0.5 h in fasting (p < 0.0001). The fed/fasting geometric mean ratio (GMR) and 90% confidence interval (90% CI) were 79.4 and 53.8, 117.0% for Cmax, and 106.0 and 86.8, 128.0% for the area under the plasma concentration-time curve (AUC0- yen). The 90% CI for the GMR of AUC0- yen and Cmax fall outside the usual bioequivalence acceptance range of 80, 125%, but that of AUC0- yen was close to the bioequivalence standard. CONCLUSION: The rate of absorption of trans-resveratrol following an oral 400 mg single-dose was significantly delayed by the presence of food, as reflected by Cmax and tmax. However, the extent of absorption, as reflected by AUC- yen, was not affected in a relevant way.

Human metabolism of nebicapone (BIA 3-202), a novel catechol-o-methyltransferase inhibitor: characterization of in vitro glucuronidation.

Nebicapone (BIA 3-202; 1-[3,4-dihydroxy-5-nitrophenyl]-2-phenylethanone), a novel catechol-O-methyltransferase inhibitor, is mainly metabolized by glucuronidation. The purpose of this study was to characterize the major plasma metabolites of nebicapone following p.o. administration of nebicapone to healthy volunteers, and to determine the human UDP-glucuronosyltransferase (UGT) enzymes involved in nebicapone glucuronidation. Plasma samples were collected as part of a clinical trial at different time points postdose and were analyzed for nebicapone and its metabolites using a validated method consisting of a solid-phase extraction, followed by high-performance liquid chromatography/mass spectrometry detection. The primary metabolic pathways of nebicapone in humans involve mainly 3-O-glucuronidation, the major early metabolite, and 3-O-methylation, the predominant late metabolite. Of the nine commercially available recombinant UGT enzymes studied (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A9 exhibited high nebicapone glucuronosyltransferase specific activity (24.3 +/- 1.3 nmol/mg protein/min). UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B7, and UGT2B15 exhibited low activity (0.1-1.1 nmol/mg protein/min), and UGT1A1 and UGT1A3 showed extremely low activities (less than 0.03 nmol/mg protein/min). The results show that nebicapone is mainly glucuronidated in humans and that multiple UGT enzymes are involved in this reaction.

Comparative study of ortho- and meta-nitrated inhibitors of catechol-O-methyltransferase: interactions with the active site and regioselectivity of O-methylation.

In this work, we present a comparative case study of "ortho-" and "meta-nitrated" catecholic inhibitors of catechol-O-methyltransferase (COMT), with regard to their interaction with the catalytic site of the enzyme and the in vitro regioselective formation of their mono-O-methyl ether metabolites. In particular, the effects of altering the attachment position of the inhibitors' side-chain substituent, within the classic nitrocatechol pharmacophore, were investigated. For this purpose, we compared two simple regioisomeric nitrocatechol-type inhibitors of COMT, BIA 3-228 and BIA 8-176, which contain the benzoyl substituent attached at the meta and ortho positions, respectively, relative to the nitro group. The two compounds were slowly O-methylated by COMT in vitro, but the particular substitution pattern of each compound was shown to have a profound impact on the regioselectivity of their O-methylation. To provide a plausible interpretation of these results, a comprehensive analysis of the protein-inhibitor interactions and of the relative chemical susceptibility to O-methylation of the catechol hydroxyl groups was performed by means of docking simulations and ab initio molecular orbital calculations. The major structural and chemical factors that determine the enzyme regioselectivity of O-methylation were identified, and the X-ray structure of the complex of COMT with S-adenosyl-l-methionine and BIA 8-176 is herein disclosed. This is the first reported structure of the soluble form of COMT complexed with a nitrocatecholic inhibitor having a bulky substituent group in adjacent position (ortho) to the nitro group. Structural and dynamic aspects of this complex are analyzed and discussed, in the context of the present study.

Molecular modeling and metabolic studies of the interaction of catechol-O-methyltransferase and a new nitrocatechol inhibitor.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a central role in the metabolic inactivation of neurotransmitters and neuroactive xenobiotics possessing a catechol motif. 1-(3,4-Dihydroxy-5-nitrophenyl)-2-phenyl-ethanone (BIA 3-202) is a novel nitrocatechol-type inhibitor of COMT, the potential clinical benefit of which is currently being evaluated in the treatment of Parkinson's disease. In the present work we characterize the molecular interactions of BIA 3-202 within the active site of COMT and discuss their implication on the regioselectivity of metabolic O-methylation. Unrestrained flexible-docking simulations suggest that the solution structure of this complex is better described as an ensemble of alternative binding modes, in contrast to the well defined bound configuration revealed by the X-ray structures of related nitrocatechol inhibitors, co-crystallized with COMT. The docking results wherein presented are well supported by experimental evidence, where the pattern of in vitro enzymatic O-methylation and O-demethylation reactions are analyzed. We propose a plausible explanation for the paradoxical in vivo regioselectivity of O-methylation of BIA 3-202, as well as of its related COMT inhibitor tolcapone. Both compounds undergo in vivo O-methylation by COMT at either meta or para catechol hydroxyl groups. However, results herein presented suggest that, in a subsequent step, the p-O-methyl derivatives are selectively demethylated by a microsomal enzyme system. The overall balance is the accumulation of the m-O-methylated metabolites over the para-regioisomers. The implications for the general recognition of nitrocatechol-type inhibitors by COMT and the regioselectivity of their metabolic O-methylation are discussed.

Metabolism of 10,11-dihydro-10-hydroxyimino-5H-dibenz/b, f/azepine-5-carboxamide, a potent anti-epileptic drug.

1. 10,11-Dihydro-10-hydroxyimino-5H-dibenzo/b, f/azepine-5-carboxamide (BIA 2-024) is a new anti-epileptic drug similar to oxcarbazepine (OXC) in structure and efficacy, but with a preferred pharmacodynamic profile. It possesses high in vitro activity, but since oximes are usually metabolized to their corresponding ketones, it is important to know whether its is vivo potency is a result of acting as a prodrug of OXC or if it is acting on its own. 2. The drug was given orally to rats, mice and rabbits, the metabolites identified and pharmacokinetic profiles compared between those species. Furthermore, the pharmacokinetic profile of the main metabolite was established in the rat. The results were compared to in vitro metabolism studies with liver microsomes from different mammalian species and humans. 3. In an atypical reaction for oximes, BIA 2-024 in rats was rapidly (t(max) = 2h) metabolized to the non-active 10-nitro-derivative (BIA 2-254), whereas rabbits and particularly mice oxidized the oxime moiety to a much lower extent. BIA 2-254 was then transformed to OXC and subsequently to the 10-hydroxy derivative and other minor metabolites. 4. In vitro data showed a very similar cross-species behaviour as the in vivo results; human liver microsomes catalysed the oxidation of BIA 2-024 to the nitro metabolite only at a low rate, and the same was observed for the subsequent metabolism to OXC. 5. The results allow prediction of the in vivo metabolism of BIA 2-024 in humans, where this drug is most likely absorbed efficiently and excreted mainly as the parent compound with a relatively low hepatic clearance. With the exception of rat, BIA 2-024 does not act as a prodrug of OXC.

BIA 3-202, a novel catechol-O-methyltransferase inhibitor, enhances the availability of L-DOPA to the brain and reduces its O-methylation.

1-[3,4-Dihydroxy-5-nitrophenyl]-2-phenyl-ethanone (BIA 3-202) is a new long-acting catechol-O-methyltransferase (COMT) inhibitor with limited access to the brain. The present study evaluated the interference of BIA 3-202 upon levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and metabolites in plasma (3-O-methyl-L-DOPA) and brain [3-O-methyl-L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] in rats orally treated with L-DOPA (20 mg/kg) plus benserazide (30 mg/kg). At different time points (1, 3 and 6 h) after the administration of BIA 3-202 (0, 3, 10 and 30 mg/kg) or L-DOPA plus benserazide, rats were sacrificed and the right striatum was quickly dissected out and stored for the assay of L-DOPA, 3-O-methyl-L-DOPA, dopamine and amine metabolites. Levels of L-DOPA, 3-O-methyl-L-DOPA, dopamine, DOPAC and HVA in the striatum in L-DOPA plus benserazide-treated rats were higher than in vehicle-treated rats. However, this increase in striatal L-DOPA, dopamine, DOPAC and HVA was, in a dose- and time-dependent manner, even higher (P<0.05) in rats given BIA 3-202 (3, 10 and 30 mg/kg). This effect was accompanied by a marked decrease in 3-O-methyl-L-DOPA levels in the striatum of L-DOPA plus benserazide-treated rats. Increases in levels of L-DOPA and decreases in 3-O-methyl-L-DOPA levels in plasma also accompanied the administration of BIA 3-202. BIA 3-202 did not significantly affect levels of DOPAC and HVA in the striatum in vehicle-treated rats. It is concluded that administration of BIA 3-202 enhances the availability of L-DOPA to the brain by reducing its O-methylation in the periphery, which may prove beneficial in parkinsonian patients treated with L-DOPA plus an aromatic amino acid decarboxylase inhibitor.