Simultaneous measurement of epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma by a newly developed ultra-performance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies.

Epinastine is an antiallergic drug with high selectivity for histamine receptors. It has been reported that 9,13b-dehydroepinastine is present as a metabolite in vivo in humans, but there was little information about their pharmacokinetics (PKs) in humans. Although several analytical methods have been reported for epinastine analysis in different matrices, none are available for its metabolite. Therefore, the purpose of this study was to develop an analytical method to simultaneously measure epinastine and its metabolite, 9,13b-dehydroepinastine, in human plasma samples using an ultra-performance liquid chromatography-tandem mass spectrometer. Analytes were separated on a C18 column. Quantification of this analysis was performed on a triple-quadrupole mass spectrometer. Chromatograms showed high sensitivity, selectivity, and resolution with no interference with plasma constituents. Calibration curves for both epinastine and 9,13b-dehydroepinastine in human plasma were 0.1-50 ng/ml and displayed excellent linearity with correlation coefficients (r2 ) >0.99. The developed analytical method satisfied the criteria of international guidance and was validated. The method could be successfully applied to pharmacokinetic studies of epinastine and, for the first time, the metabolite kinetics of epinastine to 9,13b-dehydroepinastine in humans after oral administration of 20 mg epinastine hydrochloride tablets. Our study is expected to be useful in future studies such as dosage settings and clinical pharmacotherapy.

Development of an online solid-phase extraction-liquid chromatography-mass spectrometric analysis of oxcarbazepine and its active metabolite licarbazepine from plasma with a direct injection step.

We developed an online solid phase extraction procedure using a hydrophilic-lipophilic balance sorbent, with reversed-phase liquid chromatography-high-resolution mass spectroscopy for the determination of oxcarbazepine and its active metabolite licarbazepine in plasma samples. The analytes were detected using a high-resolution Q Orbitrap mass spectrometer with targeted-selected ion monitoring (t-SIM) in positive scan mode. Under the optimized conditions, the method was linear with R2 values >0.99. The method was linear from 0.008 to 2.000 µg mL-1 and the lower limit of quantification was 0.008 µg mL-1 for both oxcarbazepine and licarbazepine. Recoveries ranged from 92.34 to 104.27% and from matrix-matched samples from 94.26 to 104.19%. The intraday and interday precision RSD values were <9.13% with an associated accuracy of 92.71 to 104.06%. The total time for the one step online procedure was only 8 min. This method provides a direct and accurate measurement for therapeutic drug monitoring of oxcarbazepine and its active metabolite licarbazepine.

Development and validation of a specific and sensitive LC-MS/MS method for determination of eslicarbazepine in human plasma and its clinical pharmacokinetic study.

In this work, we developed and validated the specific, sensitive and simple LC-MS/MS method for quantification of eslicarbazepine in human plasma. The analyte samples were prepared through a simple one-step protein precipitation method by acetonitrile. The chromatographic separation was operated on an economical Hanbon ODS-2 C18 column (150 mm × 2.1 mm, 10 µm) with isocratic elution using 10 mM ammonium acetate containing 0.01% formic acid and acetonitrile (72:28, v/v) as the mobile phase at the flow rate of 0.5 mL/min. The mass quantification was carried on the multiple reaction monitoring (MRM) of the transitions of m/z 255.1 → 194.1 for eslicarbazepine and m/z 446.1 → 321.1 for glipizide (the internal standard), respectively. The established method was validated with acceptable specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability in accordance with FDA regulations. At last, the validated method was successfully applied to determination of eslicarbazepine in human plasma obtained from clinical study.

UHPLC-MS/MS method for simultaneous determination of carbamazepine and its seven major metabolites in serum of epileptic patients.

Carbamazepine (CBZ) was considered as the drug of choice in the treatment for various forms of epilepsy, yet the non-negligible adverse effects of CBZ suspend as the major concern for rational and efficient clinical medication. This study developed a method for the simultaneous determination of CBZ and its seven metabolites acridine (AI), iminostilbene (IM), acridone (AO), carbamazepine­10,11­epoxide (CBZ­EP), 10,11­dihydro­10,11­trans­dihy­droxy­carbamazepine (CBZ­DiOH), 2­hydroxycarbamazepine (2­OH­CBZ) and 3­hydroxycarbamazepine (3­OH­CBZ) with phenacetin as the internal standard (IS) using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Plasma samples were purified with the one-step 96-well protein precipitation plate. Chromatographic separation was achieved on an Agilent Eclipse XDB-C18 (1.8 µm, 2.1 mm × 50 mm) chromatography column carrying the mobile phrase of acetonitrile and 0.1% formic acid in a gradient elution at a flow rate 0.3 mL/min. Agilent G6460 MS/MS in the positive MRM mode using electrospray ionization (ESI) was applied for quantification of target compounds. CBZ and its seven metabolites showed good linear correlation coefficient (r > 0.990). Intra-day and inter-day precision (CV) were no >15%. This method was successfully accomplished within 5 min which was especially applicable for therapeutic drug monitoring (TDM) of CBZ and its metabolites in epileptic patients, and it provided insights for toxicological studies to achieve a rational and effective individualized administration in clinical practice.

Transfer of epinastine to infants through human breast milk.

The purpose of this study was to develop an analytical method for analyzing epinastine in breast milk and maternal plasma samples to determine the safety of epinastine in breastfed infants. Six nursing mothers took epinastine hydrochloride (20 mg) once a day for 7 days, while a nursing mother took it for 30 days. Breast milk and blood samples were collected 2, 4, and 10 h after administration from the volunteers. A liquid chromatography-mass spectrometry system was used to analyze samples pretreated by liquid-liquid extractions. The concentration of epinastine in human milk was 10.3-33.5 ng/mL after 2 h, 9.1-63.8 ng/mL after 4 h, and 8.3-28.9 ng/mL after 10 h. The increase achieved 4 h after administration indicates that epinastine was transferred into human breast milk. However, the milk-to-plasma ratio had a wide range (0.82-3.39), while the relative infant dose at 4 h was 0.36-2.49%, which is lower than the safety level of transferability (10%). Moreover, the plasma levels of epinastine in two infants were slightly below the quantification limit. Overall, our results suggested that epinastine can safely be used by nursing mothers without affecting their infants.

Population Pharmacokinetic Evaluation and Missed-Dose Simulations for Eslicarbazepine Acetate Monotherapy in Patients With Partial-Onset Seizures.

Given the potential consequences of antiepileptic therapy nonadherence, missed-dose scenarios of 12- to 48-hour dose delays (4-hour intervals) for eslicarbazepine acetate monotherapy were evaluated using simulated plasma concentrations of a population pharmacokinetic model (representing 493 subjects). When 1600-mg doses were delayed 12 to <16 or 36 to <44 hours, simulations showed immediate administration of 1600 mg followed by the same dose at the scheduled time maintained plasma concentrations within the target concentration range. With 16- to 24- or 44- to 48-hour delays, administration of 2400 mg at the scheduled time followed by resumption of 1600 mg/day maintained plasma concentrations within the target concentration range. For exploratory purposes, the population pharmacokinetic model was refined to predict (n = 6 subjects) and also to allow for simulation of cerebrospinal fluid concentrations. Based on the plasma concentration simulations conducted herein, potential dosing recommendations were developed that suggest a missed ESL dose should be taken when remembered, and the usual dose regimen resumed. If it is remembered within 4 hours of the next dose, 1.5 times the usual dose should be taken immediately, the scheduled dose for that day should be skipped, and the usual regimen resumed the next day.

Pharmacokinetic variability, efficacy and tolerability of eslicarbazepine acetate-A national approach to the evaluation of therapeutic drug monitoring data and clinical outcome.

BACKGROUND: Eslicarbazepine acetate (ESL) is a new antiepileptic drug (AED), still insufficiently studied regarding pharmacokinetic variability, efficacy and tolerability. The purpose of this study was to evaluate therapeutic drug monitoring (TDM) data in Norway and relate pharmacokinetic variability to clinical efficacy and tolerability in a long-term clinical setting in patients with refractory epilepsy. METHODS: This retrospective observational study included TDM-data from the main laboratories and population data from the Norwegian Prescription Database in Norway, in addition to clinical data from medical records of adult patients using ESL for up to three years, whenever possible. RESULTS: TDM-data from 168 patients were utilized for assessment of pharmacokinetic variability, consisting of 71% of the total number of patients in Norway using ESL, 2011-14. Median daily dose of ESL was 800mg (range 400-1600mg), and median serum concentration of ESL was 53µmol/L (range 13-132µmol/L). Inter-patient variability of ESL was extensive, with 25-fold variability in concentration/dose ratios. Additional clinical data were available from 104 adult patients out of the 168, all with drug resistant focal epilepsy. After 1, 2 and 3 years follow-up, the retention rate of ESL was 83%, 72% and 64%, respectively. ESL was generally well tolerated as add-on treatment, but sedation, cognitive impairment and hyponatremia were reported. Hyponatremia (sodium <137mmol/L) was present in 36% of the patients, and lead to discontinuation in three. CONCLUSION: Pharmacokinetic variability of ESL was extensive and the demonstration of usefulness of TDM requires further studies. In patients with drug resistant focal Epilepsy, the high retention rate indicated good efficacy and tolerability. Hyponatremia was observed in one third of the patients. The present results point to a need for individualization of treatment and TDM may be useful.

The Impact of Pharmacokinetic Interactions With Eslicarbazepine Acetate Versus Oxcarbazepine and Carbamazepine in Clinical Practice.

BACKGROUND: Eslicarbazepine acetate (ESL) is a new anti-epileptic drug (AED) chemically related to oxcarbazepine (OXC) and carbamazepine (CBZ) and is increasingly used in clinical practice. The purpose of the study was to investigate 2-way pharmacokinetic interactions between ESL and other AEDs as compared to OXC and CBZ. METHODS: Anonymous data regarding age, gender, use of AEDs, daily doses and serum concentration measurements of ESL, OXC, CBZ and lamotrigine (LTG) and other AEDs were retrieved from 2 therapeutic drug monitoring (TDM) databases in Norway. Drugs were categorized according to their known potential for interactions. Concentration/dose (C/D) ratios were calculated. RESULTS: Data from 1100 patients were available. The C/D ratios of ESL and OXC were unchanged in combination with enzyme-inducing AEDs or valproate (VPA). The C/D ratio of CBZ decreased by 40% and 22% in combination with other enzyme-inducing AEDs or VPA, respectively, pointing to an increased clearance. ESL demonstrated no significant enzyme-inducing effect on LTG metabolism although there was a 20% and 34% decrease in the C/D ratio of LTG in combination with OXC and CBZ, respectively. CONCLUSIONS: Possible pharmacokinetic interactions have been studied for ESL as compared to OXC and CBZ. The pharmacokinetics of ESL is not affected by enzyme-inducing AEDs or VPA and does not affect the metabolism of LTG in contrast to OXC and CBZ. The study demonstrates the value of using TDM databases to explore the potential for pharmacokinetic interactions of new AEDs.

Development and validation of an enantioselective SFC-MS/MS method for simultaneous separation and quantification of oxcarbazepine and its chiral metabolites in beagle dog plasma.

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.

Formulation of cyclodextrin inclusion complex-based orally disintegrating tablet of eslicarbazepine acetate for improved oral bioavailability.

The present investigation was aimed towards developing a beta-cyclodextrin (ß-CD) solid dispersion (SD) based orally disintegrating tablet (ODT) of eslicarbazepine acetate (ESL), for improving the dissolution and providing fast onset of anti-epileptic action. Optimum ratio of ESL and ß-CD was determined by Job's plot. Thereafter, solid dispersions were prepared by solvent evaporation method and evaluated for yield, assay, Differential scanning calorimetry (DSC), Fourier transform infra red spectroscopy (FTIR), X-ray diffraction (XRD), and in vitro dissolution. Optimized SD was compressed into ODT by direct compression using super disintegrants and evaluated for wetting time, drug content, in vitro drug release and in vivo studies. The results of DSC, FTIR and XRD analysis supported the formation of inclusion complex. An improved dissolution with 99.95 ± 2.80% drug release in 60 min was observed in comparison to 24.85 ± 2.96% release from a plain drug suspension. Tablets with crosspovidone as a super disintegrant showed the least disintegration time of 24.66 ± 1.52 s and higher in vitro drug release against marketed tablets. In vivo studies indicated that the formulated tablets had 2 times higher bioavailability than marketed tablets. Thus, the developed ß-CD-ESL SD-ODT could provide faster onset of action and higher bioavailability, which would be beneficial in case of epileptic seizures.

Liquid chromatographic assay based on microextraction by packed sorbent for therapeutic drug monitoring of carbamazepine, lamotrigine, oxcarbazepine, phenobarbital, phenytoin and the active metabolites carbamazepine-10,11-epoxide and licarbazepine.

A new, sensitive and fast high-performance liquid chromatography-diode-array detection assay based on microextraction by packed sorbent (MEPS/HPLC-DAD) is herein reported, for the first time, to simultaneously quantify carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), and the active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in human plasma. Chromatographic separation of analytes and ketoprofen, used as internal standard (IS), was achieved in less than 15min on a C18-column, at 35°C, using acetonitrile (6%) and a mixture (94%) of water-methanol-triethylamine (73.2:26.5:0.3, v/v/v; pH 6.5) pumped at 1mL/min. The analytes and IS were detected at 215, 237 or 280nm. The method showed to be selective, accurate [bias ±14.8% (or ±17.8% in the lower limit of quantification)], precise [coefficient variation ≤9.7% (or ≤17.7% in the lower limit of quantification)] and linear (r(2)≥0.9946) over the concentration ranges of 0.1-15µg/mL for CBZ; 0.1-20µg/mL for LTG; 0.1-5µg/mL for OXC and CBZ-E; 0.2-40µg/mL for PB; 0.3-30µg/mL for PHT; and 0.4-40µg/mL for LIC. The absolute extraction recovery of the analytes ranged from 57.8 to 98.1% and their stability was demonstrated in the studied conditions. This MEPS/HPLC-DAD assay was successfully applied to real plasma samples from patients, revealing to be a cost-effective tool for routine therapeutic drug monitoring of CBZ, LTG, OXC, PB and/or PHT.

Pharmacokinetics and tolerability of eslicarbazepine acetate and oxcarbazepine at steady state in healthy volunteers.

PURPOSE: Investigate the pharmacokinetics of once-daily (QD; 900 mg) and twice-daily (BID; 450 mg) regimens of eslicarbazepine acetate (ESL) and BID (450 mg) regimen of oxcarbazepine (OXC) at steady state in healthy volunteers. METHODS: Single-center, open-label, randomized, three-way (n = 12) crossover studies in healthy volunteers. KEY FINDINGS: Mean eslicarbazepine Cmax,ss (in µm) following ESL QD (87.3) was 33.3% higher (p < 0.05) compared to ESL BID (65.5) and 82.1% higher (p < 0.05) compared to OXC BID (48.0). The mean area under the curve (AUC)ss,0-τ (in µmol h/L) following the last dose of an 8-day repeated dosing was 1156.3, 1117.6, and 968.4 for ESL QD, ESL BID, and OXC BID, respectively. The ratio eslicarbazepine plasma exposure (µmol h/L) to ESL daily-dose (µmol) was 0.381 (1156.3:3037.3), 0.368 (1117.6:3037.3), and 0.271 (968.4:3567.6) for ESL-QD, ESL-BID, and OXC-BID, respectively, which translates into a 40.6% increase in the ability of ESL-QD compared to OXC-BID to deliver into the plasma their major active entity eslicarbazepine. The extent of plasma exposure to ESL minor metabolites: (R)-licarbazepine and oxcarbazepine after ESL-QD was 71.5% and 61.1% lower, respectively, than after OXC-BID. Twenty, 24 and 38 treatment emergent adverse events were reported with ESL-QD, ESL-BID, and OXC-BID, respectively. SIGNIFICANCE: ESL-QD resulted in 33.3% higher peak plasma concentration (Cmax,ss ) of eslicarbazepine and similar extent of plasma exposure (AUCss,0-τ ) when compared to ESL-BID, which may contribute to the efficacy profile reported with once-daily ESL. In comparison to OXC-BID, administration of ESL-QD resulted in 40.6% increase in the delivery of eslicarbazepine into the plasma as well as a significantly lower systemic exposure to (R)-licarbazepine and oxcarbazepine.

Effect of eslicarbazepine acetate on the pharmacokinetics of a combined ethinylestradiol/levonorgestrel oral contraceptive in healthy women.

OBJECTIVE: To investigate the effect of once-daily (QD) eslicarbazepine acetate (ESL) 800 mg and 1,200 mg administration on pharmacokinetics of a combined ethinylestradiol/levonorgestrel oral contraceptive (OC) in women of childbearing potential. METHODS: Two two-way, crossover, two-period, randomized, open-label studies were performed in 20 healthy female subjects, each. In one period (ESL+OC period), subjects received ESL 800 mg QD in one study and ESL 1200 mg QD in the other study, for 15 days; concomitantly with the Day 14 ESL dose, an oral single dose of 30 µg ethinylestradiol and 150 µg levonorgestrel was administered. In the other period (OC alone), a single dose of 30 µg ethinylestradiol and 150 µg levonorgestrel was administered. Three weeks or more separated the periods. An analysis of variance (ANOVA) was used to test for differences between pharmacokinetic parameters of 30 µg ethinylestradiol and 150 µg levonorgestrel following ESL+OC and OC alone, and 90% confidence intervals (90%CI) for the ESL+OC/OC alone geometric mean ratio (GMR) were calculated. RESULTS: ESL significantly decreased the systemic exposure to both ethinylestradiol and levonorgestrel. GMR (90%CI) for AUC0-24 of ethinylestradiol were 68% (64%; 71%) following 1,200 mg ESL and 75% (71%; 79%) following 800 mg ESL. GMR (90%CI) for AUC0-24 of levonorgestrel were 76% (68%; 86%) following 1,200 mg ESL and 89% (82%; 97%) following 800 mg ESL. CONCLUSIONS: A clinically relevant dose-dependent effect of ESL administration on the pharmacokinetics of ethinylestradiol and levonorgestrel was observed. Therefore, to avoid inadvertent pregnancy, women of childbearing potential should use other adequate methods of contraception during treatment with ESL, and, in case ESL treatment is discontinued, until CYP3A4 activity returns to normal.

Steady-state plasma and cerebrospinal fluid pharmacokinetics and tolerability of eslicarbazepine acetate and oxcarbazepine in healthy volunteers.

PURPOSE: To evaluate the pharmacokinetics and tolerability of once-daily eslicarbazepine acetate (ESL) and twice-daily oxcarbazepine (OXC) and their metabolites in cerebrospinal fluid (CSF) and plasma following repeated oral administration. METHODS: Single-center, open-label, randomized, parallel-group study in healthy volunteers. Volunteers in ESL group (n = 7) received 600 mg on days 1-3 and 1,200 mg on days 4-9, once daily. Volunteers in the OXC group (n = 7) received 300 mg on days 1-3 and 600 mg on days 4-9, twice daily. Plasma and CSF sampling was performed following the last dose. KEY FINDINGS: Eslicarbazepine was the major drug entity in plasma and CSF, accounting for, respectively, 93.84% and 91.96% of total exposure in the ESL group and 78.06% and 76.42% in the OXC group. The extent of exposure to drug entities R-licarbazepine and oxcarbazepine was approximately four-fold higher with OXC as compared with ESL. There was relatively little fluctuation from peak-to-trough (ratio) in the CSF for both eslicarbazepine (ESL = 1.5; OXC = 1.2) and R-licarbazepine (ESL = 1.2; OXC = 1.2). In contrast, oxcarbazepine showed larger differences between peak and trough (ESL = 3.1; OXC = 6.4). A total of 84 and 24 treatment-emergent adverse events (TEAEs) were reported with OXC and ESL, respectively. SIGNIFICANCE: In comparison to OXC, administration of ESL resulted in more eslicarbazepine, less R-licarbazepine, and less oxcarbazepine in plasma and CSF, which may correlate with the tolerability profile reported with ESL. The smaller peak-to-trough fluctuation of eslicarbazepine in CSF (a measure of sustained delivery to the brain) than in plasma supports once-daily dosing of ESL.

A phase II study of the Ras-MAPK signaling pathway inhibitor TLN-4601 in patients with glioblastoma at first progression.

This phase II trial was undertaken to evaluate the efficacy of TLN-4601 in patients with glioblastoma (GBM) at first progression. TLN-4601 inhibits the Ras-MAPK signaling pathway, and in animal models crosses the blood-brain barrier and accumulates in implanted gliomas, possibly by binding specifically to the peripheral benzodiazepine receptor. A maximum of 40 patients with recurrent GBM were to be enrolled in this study. TLN-4601 was administered at a dose of 480 mg/m(2)/day by continuous intravenous (CIV) administration. Each 21-day cycle consisted of a 14-day CIV administration and a 7-day recovery period. Samples were obtained from all patients for pharmacokinetic evaluations (PK) and for Raf-1 and pERK biomarker assessment using immunohistochemistry and flow cytometry. Following enrollment of 20 patients, this study was terminated due to a lack of efficacy. Of 17 evaluable patients, 14 had MR scans performed after two cycles of TLN-4601. Of these 14 patients, three had stable disease and 11 had disease progression. Only three patients had MR scans performed after four cycles and all had evidence of radiographic progression. Serum PKs confirmed that patients were exposed to TLN-4601 at targeted drug levels. TLN-4601 was generally well tolerated although two patients discontinued treatment due to adverse events. Biomarker analysis did not show consistent changes. TLN-4601 infused via CIV at 480 mg/m(2)/day for 14 of 21 days is well tolerated by patients with progressive GBM. However, this agent is ineffective in progressive GBM when administered as monotherapy in this schedule.

Three-phase hollow fiber microextraction based on two immiscible organic solvents for determination of tricyclic antidepressant drugs: comparison with conventional three-phase hollow fiber microextraction.

The aim of this research was to compare the extraction efficiencies of two modes of three-phase hollow fiber microextraction (HF-LLLME) based on aqueous and organic acceptor phases for analysis of tricyclic antidepressant (TCA) drugs. High-performance liquid chromatography with photodiode array detection (HPLC-DAD) was applied for determination of the drugs. In order to examine the ability of the new concept of HF-LLLME based on organic acceptor solvent in comparison with aqueous acceptor phase to extract the analytes, four TCAs were selected. The effect of different extraction conditions (i.e., type of acceptor phase, hollow fiber length, ionic strength, stirring rate, and extraction time) on the extraction efficiency of the TCAs was investigated and optimized using central composite design (CCD) as a powerful tool. Both methods were characterized by good linearity and high repeatability, but HF-LLLME with organic acceptor provided higher extraction efficiency and thus lower limits of detection (LODs). Calibration curves were linear (r(2)>0.996) in the range of 0.2-200 µgL(-1). LODs for all the TCAs ranged from 0.08 to 0.2 µgL(-1) using HPLC-DAD. Also an improvement in sensitivity of several orders of magnitude was achieved using single-ion monitoring GC-MS analyses (0.04 µgL(-1)) due to compatibility of this technique with GC instrument. The applicability of the proposed HF-LLLME/GC-MS and HPLC-DAD methods was demonstrated by analyzing the drugs in spiked urine and plasma samples. The obtained recoveries of the drugs in the range of 87.9-109.2% indicated the excellent capability of the developed method for extraction of TCAs from complex matrices.

A chiral liquid chromatography method for the simultaneous determination of oxcarbazepine, eslicarbazepine, R-licarbazepine and other new chemical derivatives BIA 2-024, BIA 2-059 and BIA 2-265, in mouse plasma and brain.

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.

Evaluation of Eslicarbazepine acetate on cardiac repolarization in a thorough QT/QTc study.

This study investigated the effect of eslicarbazepine acetate (ESL) on cardiac repolarization in healthy adult volunteers. A randomized, placebo/active-controlled, 4-period crossover study was conducted in 67 participants. In 3 periods, participants received once-daily doses of ESL 1200 mg, ESL 2400 mg, and placebo for 5 days; in 1 period, participants received placebo on days 1 to 4 and a 400-mg moxifloxacin single dose on day 5. In each period, 24-hour 12-lead Holter monitoring was performed on days -;1 (baseline) and 5. There was no clinically relevant effect of ESL 1200 mg and 2400 mg versus placebo on cardiac depolarization or repolarization as measured by the QRS or QTc intervals, respectively. Mean PR interval increased following ESL 1200 mg and 2400 mg, but there was no participant with a PR interval above the upper limit of the normal range (200 ms). The upper bound of the 95% confidence interval for the placebo-corrected change from baseline of the individually corrected QT interval (QTcI) following administration of ESL 1200 mg and ESL 2400 mg was <10 ms at every time point. Moxifloxacin caused an increase in QTcI above the 10-ms threshold for clinical significance at several time points, demonstrating assay sensitivity. It is concluded that administration of ESL 1200 mg and ESL 2400 mg did not induce a clinically significant prolongation of the QTcI interval.

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.

Pharmacokinetics of eslicarbazepine acetate at steady-state in adults with partial-onset seizures.

OBJECTIVE: To evaluate the pharmacokinetics of eslicarbazepine acetate (ESL) at steady-state in adults with partial-onset seizures who have taken ESL for at least 1 year with one or two concomitant antiepileptic drugs (AEDs). METHODS: Blood samples for the pharmacokinetic assessment were taken at pre-dose, and 1, 2, 3, 4, 6, 8, 12 and 24h post-dose at steady-state in 51 patients stabilised on chronic (beyond 1 year) treatment with ESL 400mg (n=7), 800mg (n=26) or 1200mg (n=18) once-daily. Most patients (n=29, 56.9%) were receiving 2 concomitant AEDs, and most frequent co-medications were carbamazepine (n=34, 66.7%) and valproic acid (n=19, 37.3%). Plasma concentrations of ESL and its metabolites eslicarbazepine, R-licarbazepine and oxcarbazepine (OXC) were determined by a validated chiral method using liquid chromatography coupled to mass spectrometry. RESULTS: Similarly to earlier findings in healthy subjects, plasma ESL concentrations were consistently below the lower limit of quantification (50ng/mL). The major compound in plasma was the active metabolite eslicarbazepine, which reached maximum concentrations (C(max)) 2h post-dose; thereafter, its plasma concentrations declined with a mean apparent half-life of 13, 14, and 20h in patients receiving ESL doses of 400, 800, and 1200mg once daily, respectively. Eslicarbazepine C(max) were 9.7, 15.5 and 23.0µg/mL, and areas under the plasma concentration-time curve over the dosing interval (AUC(0-24)) were 132.5, 205.4 and 336.1µgh/mL in patients receiving ESL doses of 400, 800 and 1200mg once-daily, respectively. Eslicarbazepine main pharmacokinetic parameters (C(max) and AUC(0-24)) were dose-proportional. R-licarbazepine and OXC were minor metabolites. CONCLUSIONS: Following once-daily oral administration of ESL 400mg, 800mg and 1200mg to epilepsy patients treated concomitantly with one or two other AEDs, ESL was rapidly converted to eslicarbazepine, which was the primary active compound found in plasma. Systemic exposure to eslicarbazepine was dose-proportional.