Carlos Fernandes-Lopes

Pharmacokinetic and safety profile of trans-resveratrol in a rising multiple-dose study in healthy volunteers

This was a double-blind, randomised, placebo-controlled study to investigate the pharmacokinetics and safety of trans-resveratrol. In four groups of ten healthy adult subjects (five males and five females), two subjects were randomized to receive placebo and eight subjects to receive trans-resveratrol 25, 50, 100 or 150 mg, six times/day, for thirteen doses. Peak plasma concentrations of trans-resveratrol were reached at 0.8-1.5 h postdose. Following the 13th dose of trans-resveratrol 25, 50, 100 and 150 mg, mean peak plasma concentration (C(max)) was 3.89, 7.39, 23.1 and 63.8 ng/mL and mean area under the plasma concentration-time curve (AUC(0-tau)) was 3.1, 11.2, 33.0 and 78.9 ng.h/mL. Interindividual variability was high, with coefficients of variation >40%. Trans-resveratrol half-life was 1-3 h following single-doses and 2-5 h following repeated dosing. Trough (C(min)) concentrations were < or = 1 ng/mL following 25 and 50 mg, 3 ng/mL following 100 mg and < 10 ng/mL following 150 mg. Trans-resveratrol pharmacokinetics showed circadian variation. Adverse events were mild in severity and similar between all groups. In conclusion, repeated administration was well-tolerated but produced relatively low plasma concentrations of trans-resveratrol, despite the high doses and short dosing interval used. Bioavailability was higher after morning administration.

Pharmacokinetics of Trans‐resveratrol Following Repeated Administration in Healthy Elderly and Young Subjects

From the Department of Research and Development, S Mamede do Coronado, Portugal (Dr Nunes, Dr Almeida, Mr Rocha, Mr Fernandes-Lopes, Ms Loureiro, Dr Wright, Dr Vaz-da-Silva, Dr Soares-da-Silva); Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal (Dr Almeida, Dr Vaz-da-Silva, Dr Soares-da-Silva); Department of Health Sciences, University of Aveiro, Portugal (Dr Almeida); and 4Health Consulting, BIOCANT, Cantanhede, Portugal (Dr Falcao). Submitted for publication February 27, 2009; revised version accepted May 10, 2009. Address for correspondence: Patricio Soares-da-Silva, MD, PhD, Department of Research & Development, BIAL, A Av da Siderurgia Nacional, 4745-457 S Mamede do Coronado, Portugal; e-mail: psoares.silva@bial.com.DOI: 10.1177/0091270009339191

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.

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.

Hepatic UDP-Glucuronosyltransferase Is Responsible for Eslicarbazepine Glucuronidation

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 β-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 Km 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 (Km = 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 IC50 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.

Pharmacokinetic-Pharmacodynamic Interaction Between Nebicapone and Controlled-Release Levodopa/Benserazide: A Single-Center, Phase I, Double-Blind, Randomized, Placebo- Controlled, Four-Way Crossover Study in Healthy Subjects

BACKGROUND Nebicapone is a reversible catechol-O-methyltransferase (COMT) inhibitor. Coadministration of a COMT inhibitor with levodopa and a dopa-decarboxylase inhibitor (carbidopa or benserazide) increases levodopa exposure and its therapeutic effect. OBJECTIVES The primary objective of this study was to investigate the effect of nebicapone (50, 100, and 200 mg), compared with placebo, on levodopa pharmacokinetics when coadministered with a single dose of controlled-release levodopa 100 mg/benserazide 25 mg. The secondary objectives were to investigate the effect of nebicapone on the erythrocyte-soluble COMT (S-COMT) activity and on the plasma levels of the levodopa 3-O-methylated metabolite (3-O-methyldopa [3-OMD]). Nebicapones tolerability was also assessed. METHODS This was a single-center, Phase I, doubleblind, randomized, placebo-controlled, 4-way crossover study conducted in healthy adult volunteers. Each of the 4 single-dose treatment periods was separated by a washout period of > or = 5 days. During the different treatment periods, subjects received a single dose of controlled-release levodopa 100 mg/benserazide 25 mg concomitantly with nebicapone 50, 100, and 200 mg or placebo. Plasma concentrations of nebicapone, levodopa, and 3-OMD were determined by HPLC. Blood samples (7 mL) for determination of plasma concentrations of levodopa, 3-OMD, and 2258 nebicapone, as well as for the assay of S-COMT activity, were collected in potassium EDTA test tubes at the following times: predose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 hours postdose. S-COMT activity was assessed as the amount of metanephrine formed by the action of S-COMT on an epinephrine substrate. Spontaneously reported clinical adverse events (AEs) were recorded throughout the study. RESULTS Sixteen subjects (8 females, 8 males; mean [SD] age, 26.13 [6.29] years; weight, 69.4 [12.4] kg; body mass index, 24.0 [3.0] kg/m2) completed the 4 treatment periods and had data available for pharmacokinetic and pharmacodynamic analyses. Compared with placebo, levodopa C(max) increased 25%, 30%, and 34%, and AUC increased 14%, 37%, and 42% after administration of nebicapone 50, 100, and 200 mg, respectively. After administration of nebicapone 50, 100, and 200 mg, 3-OMD C(max) decreased 44%, 57%, and 58%, and 3-OMD AUC(0-infinity) decreased 33%, 37%, and 45%, respectively, compared with placebo. Extent of exposure to levodopa, as assessed by using AUC(0-t), increased with all doses of nebicapone in relationship to placebo, but the difference did not reach statistical significance. This may be related to a relatively high inter-subject variability: %CVs ranged from 48.0% with nebicapone 100 mg to 66.8% with placebo. Maximum S-COMT inhibition by nebicapone occurred at approximately 1.5 hours postdose and ranged from 57% with nebicapone 50 mg to 74% with nebicapone 200 mg. There was an inverse correlation between plasma concentrations of nebicapone and S-COMT activity; T(max) of nebicapone plasma concentrations and time to occurrence of the maximum inhibition of S-COMT activity appeared to correlate. Nineteen AEs were reported; 8 were assessed by the investigator as possibly related to treatment. All AEs were mild in severity. There were no serious AEs or discontinuations due to AEs. No abnormalities in liver enzyme levels were found. CONCLUSIONS When administered concomitantly with a single dose of controlled-release levodopa 100 mg/benserazide 25 mg, single doses of nebicapone 50, 100, and 200 mg were well tolerated in these healthy adult volunteers, and dose dependently inhibited S-COMT activity and reduced 3-OMD formation compared with placebo. However, there was no significant difference in levodopa bioavailability.

N-Acetylation of Etamicastat, a Reversible Dopamine-β-Hydroxylase Inhibitor

Etamicastat [(R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1H-imidazole-2(3H)-thione hydrochloride] is a reversible dopamine-β-hydroxylase inhibitor that decreases norepinephrine levels in sympathetically innervated tissues. After in vivo administration, N-acetylation of etamicastat was found to be a main metabolic pathway. The purpose of the current study was to characterize the N-acetylation of etamicastat by N-acetyltransferases (NAT1 and NAT2) and evaluate potential species differences in etamicastat N-acetylation using a sensitive and specific liquid chromatography–mass spectrometry assay. Marked differences in etamicastat N-acetylation were observed among the laboratory species and humans. After oral administration, the rat, hamster, and human subjects presented the highest rates of etamicastat N-acetylation, whereas almost no acetylation was observed in the mouse, rabbit, minipig, and monkey and no acetylation was observed in the dog. In in vitro studies, rats and humans showed similar acetylation rates, whereas no acetylation was detected in the dog. Studies performed with human recombinant NAT1 4 and NAT2 4 enzymes revealed that both were able to conjugate etamicastat, although at different rates. NAT1 had lower affinity compared with NAT2 (Km, 124.8 ± 9.031 µM and 17.14 ± 3.577 µM, respectively). A significant correlation (r2 = 0.65, P < 0.05) was observed in a comparison of etamicastat N-acetylation by human single-donor enzymes and sulfamethazine, a selective substrate to NAT2. No correlation was observed with p-aminosalicylic acid, a NAT1 selective substrate. In conclusion, these results suggest that NAT2 and, to a lesser extent, NAT1 contribute to etamicastat N-acetylation. Furthermore, the high interspecies and intraspecies differences in N-acetylation should be taken into consideration when evaluating the in vivo bioavailability of etamicastat.

Blood pressure-decreasing effect of etamicastat alone and in combination with antihypertensive drugs in the spontaneously hypertensive rat.

Hyperactivation of the sympathetic nervous system has an important role in the development and progression of arterial hypertension. This study evaluated the efficacy of etamicastat, a dopamine-β-hydroxylase (DβH) inhibitor, in controlling high blood pressure in the spontaneously hypertensive rat (SHR), either alone or in combination with other classes of antihypertensives. SHRs were administered with etamicastat by gavage, and its pharmacodynamic and pharmacokinetic properties were evaluated. Etamicastat induced a time-dependent decrease in noradrenaline-to-dopamine ratios in the heart and kidney, and had no effect on catecholamine levels in the frontal cortex of SHRs. Cardiovascular pharmacodynamic effects following administration of etamicastat alone or in combination with other classes of antihypertensive drugs were assessed by telemetry. Etamicastat was evaluated in combination with captopril, losartan, hydrochlorothiazide, metoprolol, prazosin and/or diltiazem. Etamicastat monotherapy induced a dose-dependent reduction in blood pressure without reflex tachycardia. Combination therapy amplified the antihypertensive effects of all tested drugs. In conclusion, inhibition of peripheral DβH with etamicastat, as a monotherapy or combination therapy, may constitute a valid alternative treatment for high blood pressure.

Species differences in pharmacokinetic and pharmacodynamic properties of nebicapone

The present study was designed to characterize pharmacodynamic and pharmacokinetic properties of nebicapone in rats and mice. Upon oral administration of nebicapone the extent of mouse liver catechol-O-methyltransferase (COMT) inhibition is half that in the rat. Nebicapone was rapidly absorbed reaching plasma C(max) within 30min and being completely eliminated by 8h. Nebicapone was metabolized mainly by glucuronidation and methylation in both species, but rat had an extra major metabolite, resulting from sulphation. Administration of nebicapone by the intraperitoneal route significantly increased compound AUC in the rat while in the mouse a significant increase in AUC of metabolites was observed. These results show that nebicapone exhibited marked species differences in bioavailability and metabolic profile. Evaluation of COMT activity in rat and mice liver homogenates revealed that both had similar methylation efficiencies (K(cat) values, respectively 7.3 and 6.4min(-1)), but rat had twice active enzyme units as the mouse (molar equivalency respectively 150 and 83). Furthermore, nebicapone inhibited rat liver COMT with a lower K(i) than mouse liver COMT (respectively 0.2nM vs. 1.2nM). In conclusion, the results from the present study show that mice and rats respond differently to COMT inhibition by nebicapone. The more pronounced inhibitory effects of nebicapone in the rat may be related to an enhanced oral availability and less pronounced metabolism of nebicapone in this specie, but also concerned with the predominant expression of S-COMT over MB-COMT, the latter of which is less sensitive to inhibition by nebicapone than the former.