Identification of fungal metabolites of anticonvulsant drug carbamazepine

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1-C3 and M1-M3). All six metabolites showed [M + H](+) at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography-mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.     

Effect of perinatal asphyxia and carbamazepine treatment on cortical dopamine and DOPAC levels

Background

One of the most important manifestations of perinatal asphyxia is the occurrence of seizures, which are treated with antiepileptic drugs, such as carbamazepine. These early seizures, combined with pharmacological treatments, may influence the development of dopaminergic neurotransmission in the frontal cortex. This study aimed to determine the extracellular levels of dopamine and its main metabolite DOPAC in 30-day-old rats that had been asphyxiated for 45 min in a low (8%) oxygen chamber at a perinatal age and treated with daily doses of carbamazepine. Quantifications were performed using microdialysis coupled to a high-performance liquid chromatography (HPLC) system in basal conditions and following the use of the chemical stimulus.

Results

Significant decreases in basal and stimulated extracellular dopamine and DOPAC content were observed in the frontal cortex of the asphyxiated group, and these decreases were partially recovered in the animals administered daily doses of carbamazepine. Greater basal dopamine concentrations were also observed as an independent effect of carbamazepine.

Conclusions

Perinatal asphyxia plus carbamazepine affects extracellular levels of dopamine and DOPAC in the frontal cortex and stimulated the release of dopamine, which provides evidence for the altered availability of dopamine in cortical brain areas during brain development.     

Effect of carbamazepine and gabapentin on excitability in the trigeminal subnucleus caudalis of neonatal rats using a voltage-sensitive dye imaging technique

Background

The antiepileptic drugs carbamazepine and gabapentin are effective in treating neuropathic pain and trigeminal neuralgia. In the present study, to analyze the effects of carbamazepine and gabapentin on neuronal excitation in the spinal trigeminal subnucleus caudalis (Sp5c) in the medulla oblongata, we recorded temporal changes in nociceptive afferent activity in the Sp5c of trigeminal nerve-attached brainstem slices of neonatal rats using a voltage-sensitive dye imaging technique.

Results

Electrical stimulation of the trigeminal nerve rootlet evoked changes in the fluorescence intensity of dye in the Sp5c. The optical signals were composed of two phases, a fast component with a sharp peak followed by a long-lasting component with a period of more than 500 ms. This evoked excitation was not influenced by administration of carbamazepine (10, 100 and 1,000 μM) or gabapentin (1 and 10 μM), but was increased by administration of 100 μM gabapentin. This evoked excitation was increased further in low Mg²⁺ (0.8 mM) conditions, and this effect of low Mg²⁺ concentration was antagonized by 30 μM DL-2-amino-5-phosphonopentanoic acid (AP5), a N-methyl-d-aspartate (NMDA) receptor blocker. The increased excitation in low Mg²⁺ conditions was also antagonized by carbamazepine (1,000 μM) and gabapentin (100 μM).

Conclusion

Carbamazepine and gabapentin did not decrease electrically evoked excitation in the Sp5c in control conditions. Further excitation in low Mg²⁺ conditions was antagonized by the NMDA receptor blocker AP5. Carbamazepine and gabapentin had similar effects to AP5 on evoked excitation in the Sp5c in low Mg²⁺ conditions. Thus, we concluded that carbamazepine and gabapentin may act by blocking NMDA receptors in the Sp5c, which contributes to its anti-hypersensitivity in neuropathic pain.     

Chemometrics-assisted simple UV-spectroscopic determination of carbamazepine in human serum and comparison with reference methods

In the present report, carbamazepine is determined on serum samples of real patients by a procedure completely assisted by chemometric tools. First, a response surface methodology based on a mixture design was applied in order to select the best conditions for the extraction step. Finally, partial least squares multivariate calibration (PLS-1) was applied to second-derivative UV spectra, eliminating a shift baseline effect that originated in the extraction procedure. The performance assessment included: (a) a three-level precision study, (b) a recovery study analyzing spiked samples, and (c) a method comparison with high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) applied on real patient samples. The obtained results show the potentiality of the presently studied methodology for the monitoring of patients treated with this anticonvulsant.     

High-performance liquid chromatographic determination of carbamazepine and metabolites in human hair

To study the use of hair analysis in monitoring drug compliance and historical changes in pharmacokinetics we developed a method for the quantitative determination of the anti-epileptic drug carbamazepine (CBZ) and trans-10,11-dihydro-10,11-dihydroxy-carbamazepine (CBZ-diol) in hair from carbamazepine users. Digestion by 1 M NaOH was found to be the best method for isolating CBZ and CBZ-diol from hair, followed by solid-phase extraction and reversed-phase HPLC with UV detection. Recoveries from spiked hair samples were 76–86%. Within-day precision (C.V.; n=10) for CBZ and CBZ-diol in hair of a CBZ user containing 10.9 μg/g CBZ and 3.2 μg/g CBZ-diol were 1.7 and 5.0%, respectively. Sectional hair analysis of a patient on a constant dosage of CBZ demonstrates an exponential decrease in hair concentrations of CBZ and CBZ-diol with increasing distance from the root, probably caused by shampooing. No CBZ-10,11-epoxide (CBZ-epox) could be detected. However, one component in the chromatogram is probably CBZ-β-hydroxythioether, an adduct of CBZ-epox with cysteine, or acridinethioacetal, its rearrangement product. The concentration of this component does not decrease with increasing distance from the root.     

Simultaneous high-performance liquid chromatography determination of carbamazepine and five of its metabolites in plasma of epileptic patients

A high-performance liquid chromatographic method with UV detection for the simultaneous analysis of the antiepileptic drug carbamazepine and five of its metabolites in human plasma has been developed. The analysis was carried out on a reversed-phase column (C₈, 150×4.6 mm I.D., 5 μm) using acetonitrile, methanol and a pH 1.9 phosphate buffer as the mobile phase. Under these chromatographic conditions, carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 2-hydroxycarbamazepine, 3-hydroxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine are baseline separated in less than 18 min. The extraction of the analytes from plasma samples was performed by means of an original solid-phase extraction procedure using Oasis HLB cartridges. The method requires only 250 μl of plasma for one complete analysis. The repeatability (RSD%<2.4), intermediate precision (RSD%<3.5) and extraction yield (84.8–103.0%) were very good for all analytes. The method is suitable for reliable therapeutic drug monitoring of patients undergoing chronic treatment with carbamazepine and for kinetic–metabolic studies of this drug.     

Antiepileptic carbamazepine drug treatment induces alteration of membrane in red blood cells: Possible positive effects on metabolism and oxidative stress

Carbamazepine (CBZ) is an iminostilbene derivative commonly used for treatment of neuralgic pain and bipolar affective disorders. CBZ blood levels of treated patients are within the range of micromolar concentrations and therefore, significant interactions of this drug with erythrocytes are very likely. Moreover, the lipid domains of the cell membrane are believed to be one of the sites where iminostilbene derivatives exert their effects. The present study aimed to deeply characterize CBZ effects on erythrocytes, in order to identify extra and/or cytosolic cell targets. Our results indicate that erythrocyte morphological changes promoted by the drug, may be triggered by an alteration in band 3 functionality i.e. at the level of anionic flux. In addition, from a metabolic point of view this perturbation could be considered, at least in part, as a beneficial event because it could favour the CO₂ elimination.     

托吡酯、卡马西平与丙戊酸钠治疗脑炎继发癫痫的疗效比较

目的:观察和比较托吡酯、卡马西平与丙戊酸钠对治疗脑炎继发癫痫的临床疗效及安全性。方法:选择2013年1月~2015年9月在我院进行诊治的脑炎继发癫痫患者80例,随机分为托吡酯组、卡马西平组和丙戊酸钠组,分别采用托吡酯、卡马西平与丙戊酸钠治疗,比较三组的治疗有效率、执行能力与视空间、命名、抽象、注意、定向、语言以及延迟回忆等认知功能评分及不良反应的发生情况。结果:托吡酯组的有效率最高,为80.65%(25/31),卡马西平组的有效率最低,为70.00%(21/30),但三组间有效率相比差异无统计学意义(P0.05)。治疗后,托吡酯组患者的执行能力与视空间、命名、抽象、注意、定向、语言以及延迟回忆等认知功能评分均明显高于卡马西平组和丙戊酸钠组(P0.05);托吡酯组的不良反应发生率(12.90%)明显低于卡马西平组(36.67%)和丙戊酸钠组的(29.62%)(P0.05)。结论:托吡酯、卡马西平以及丙戊酸钠治疗脑炎继发癫痫疗效相当,但托吡酯对患者认知功能损害最小,安全性最高。     

High-speed liquid chromatographic method for the monitoring of carbamazepine and its active metabolite, carbamazepine-10,11-epoxide, in human plasma

The assays of antiepileptic drugs, which are performed by central laboratories in Phase II and III clinical trials, require both a very fast turn-around time and a suitable specificity. In order to decrease the run time and to keep the powerful specificity of the liquid chromatography (HPLC), the use of a reversed-phase 1.5 μm monosized non-porous silicon dioxide microspheres column instead of regular columns containing spherical porous C₁₈ material was studied. The determination of carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZ-E), in human plasma or serum was chosen to demonstrate the utility of these columns. As a prerequisite of this work, no modification of a regular HPLC system was allowed. The samples were prepared in autosampler vials by protein precipitation with acetonitrile, followed by a quick centrifugation. Without any change to a conventional HPLC system, CBZ and CBZ-E are well separated in less than 2.5 min using a Kovasil MS C₁₄ column. No interference was observed with endogenous compounds and with nine antiepileptic drugs commonly prescribed as co-medication, and their metabolites. Due to the very low specific surface area of the packing, the required organic modifier volume per chromatographic run was decreased by a factor of 25. The method was validated. The developed method is well suited for the determination of CBZ and CBZ-E in clinical trials. It can be easily adapted to the monitoring of other antiepileptic drugs. No modification of a regular HPLC system was required.