Carbamazepine-10,11-epoxide Research Articles

PharmGKB summary

Carbamazepine (CBZ), a dibenzazepine, is a tricyclic compound used in the treatment of epilepsy, trigeminal neuralgia, and psychiatric mood disorders [1]. Serious adverse events have been reported for CBZ including Stevens–Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and Drug Reaction with Eosinophilia and Systemic Symptoms [2,3]. Other types of hypersensitivity reactions are also associated with CBZ including mild skin rashes, fever, eosinophilia, and cross-reactions to other anticonvulsants. Up to 80% of patients who have an idiopathic drug reaction to CBZ drugs will also have an adverse reaction to other anticonvulsants, further restricting treatment options [4]. In addition to adverse events, lack of efficacy can also be a problem, with as many as 30% of patients with epilepsy experiencing drug-resistance [5,6]. The mechanisms by which these events occur are not entirely clear although several candidate pharmacogenes have been associated with CBZ treatment responses. Current methods to individualize treatment involve therapeutic drug monitoring, the measurement of drug metabolites in patient samples posttreatment, and subsequent dose adjustment. Although this provides an accurate view of the drug-response phenotype, it still risks adverse events and cross-sensitivity. The ability to identify the patients that will benefit from CBZ, not suffer adverse events and define dose before treatment would be a highly valuable clinical tool. Here we present the current knowledge of CBZ pharmacogenomics (PGx) as a gene centered view of the pharmacokinetics of CBZ (Fig. 1) and collate the gene variants associated with CBZ responses. Fig. 1 Stylized liver cell depicting candidate genes involved in the pharmacokinetics of carbamazepine (CBZ). A fully interactive version is available online at http://www.pharmgkb.org/do/serve?objCls=Pathwayo ... Pharmacokinetics CBZ is almost completely metabolized in the liver with only approximately 5% of the drug excreted un-changed [7]. The major route of metabolism is conversion to CBZ 10,11-epoxide (CBZ-E) [1]. This reaction is primarily catalyzed by CYP3A4 although CYP2C8 also plays a role, and involvement of CYP3A5 has also been suggested (Fig. 1) [1,8]. Minor metabolic pathways include ring-hydroxylation to form 2-hydroxy-CBZ (2-OH-CBZ) and 3-hydroxy CBZ (3-OH-CBZ). The formation of each presumably proceeds by an epoxide intermediate (referred to as an arene oxide intermediate), with CYP2B6 and CYP3A4, being the major catalysts of 3-OH-CBZ formation [1] and multiple CYPs involved in 2-OH-CBZ formation [9]. Secondary metabolism of 2-OH-CBZ and 3-OH-CBZ by CYP3A4 represent two distinct potential bioactivation pathways. CYP3A4-dependent secondary oxidation of 2-OH-CBZ leads to the formation of thiol-reactive metabolites by an iminoquinone intermediate [10], whereas CYP3A4-dependent secondary oxidation of 3-OH-CBZ results in the formation of reactive metabolites capable of inactivating CYP3A4 [1] and forming covalent adducts [11]. 3-OH CBZ, and to a lesser extent 2-OH CBZ and CBZ, can be metabolized to form radicals by myeloperoxidase [12]. This releases reactive oxygen species and may lead to the formation of protein adducts. Covalent binding and protein adduct formation has also been observed for another antiepileptic drug, phenytoin, and is generally considered to be a necessary step in the pathogenesis of idiosyncratic reactions to this class of compounds [12]. CBZ stimulates the transcriptional upregulation of genes involved in its own metabolism, with autoinduction of CYP3A4 and CYP2B6, by nuclear receptors NR1I2 (PXR) and NR1I3 (CAR) [13–15]. Drug–drug interactions through CYP3A4 [16] and CYP2B6 [17] are well documented and can complicate the use of CBZ in polytherapy. Some studies have suggested that glucuronidation is likely to play only a minor role in metabolism of CBZ and CBZ-E [7]. But other studies dispute the documenting involvement of UGT2B7 [18,19].

Carbamazepine oxidation catalyzed by manganese porphyrins: Effects of the β-bromination of the macrocycle and the choice of oxidant

Carbamazepine (CBZ), which is one of the most commonly prescribed antiepileptic drugs and also used in the treatment of trigeminal neuralgia and psychiatric disorders, is metabolized primarily by the cytochromes P450. A homologous series of β-brominated porphyrins derived from 5,10,15,20-tetrakis(4-carbomethoxyphenyl)porphyrinatomanganese(III) chloride, i.e., Mn(III)(Br x TCMPP)Cl ( x = 0, 2, 4, 6, and 8), was investigated as cytochrome P450 models for CBZ oxidation in CH 2Cl 2 by iodosylbenzene, iodobenzene diacetate, tert-butyl hydroperoxide, meta-chloroperoxybenzoic acid, and hydrogen peroxide. Unlike previous studies on metalloporphyrin-based CBZ oxidation, which yielded CBZ epoxide (CBZ-EP) as the sole product, the Mn(III)(Br x TCMPP)Cl systems catalyzed both the oxidation of CBZ to CBZ-EP and the formation of the respective vicinal diol, CBZ-DiOH. The influence of β-bromination of the macrocycle and the choice of the oxidant on the catalytic properties of the Mn(III)(Br x TCMPP)Cl ( x = 0, 2, 4, 6, and 8) series were examined. Some partially β-brominated Mn-porphyrins surpass the corresponding octabrominated analogue in efficiency and selectivity, but the extent by which the β-bromination affects the catalytic activity depends on the choice of oxidant. The selectivity for CBZ oxidation to yield the respective epoxide reached 100% or ∼94% by using tert-butyl hydroperoxide or hydrogen peroxide as oxygen donors, respectively.

A chiral HPLC‐UV method for the quantification of dibenz[b,f]azepine‐5‐carboxamide derivatives in mouse plasma and brain tissue: Eslicarbazepine acetate, carbamazepine and main metabolites

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.

Interaction of the anticonvulsants, denzimol and nafimidone, with liver cytochrome P450 in the rat.

The presence of an imidazole moiety in the chemical structure of denzimol and nafimidone suggested that these new anticonvulsants might interfere with cytochrome P450-mediated mixed function monooxygenase activities. We therefore investigated their ability to bind reversibly to rat liver cytochrome P450. Both drugs displayed a type II spectra. The Ks values of binding were 6.66 and 7.00 mM, respectively, for denzimol and nafimidone. In other in-vitro studies the IC50 of the inhibition caused by denzimol and nafimidone was determined on carbamazepine (CBZ) epoxidation and diazepam C3-hydroxylation and N1-dealkylation. The IC50 values for CBZ epoxidation were 4.46 x 10(-7) and 2.95 x 10(-7) M, respectively, in the presence of denzimol and nafimidone. The IC50 values for diazepam C3-hydroxylation were 1.44 x 10(-6) and 1.00 x 10(-6) M, respectively, and those for N1-dealkylation 6.66 x 10(-7) and 5.95 x 10(-7) M. The inhibition of CBZ metabolism was also investigated ex-vivo and in-vivo after single oral doses (15 and/or 60 mg kg-1) of denzimol or nafimidone. Inhibition of CBZ-10,11-epoxidation by the two drugs was time- and dose-dependent. Further studies in-vivo showed that denzimol and nafimidone prolong pentobarbitone sleeping times indicating that both drugs bind to rat liver microsomes and are potent inhibitors in the rat of mixed function monooxygense activities both in-vitro and in-vivo.

Simultaneous Determination of Carbamazepine and Its Major Metabolite in Serum

The simultaneous analysis of carbamazepine and its major metabolite 10, 11-carbamazepine epoxide in serum is described. A high-performance liquid chromatography (HPLC) system based on a μ-bondapack phenyl column (300 × 3·9 mm) and a UV detector (Λ = 214nm) were used. A mixture of methanol-water (50:50 v/v) at a flow rate of 1·5 mL min−1 was used as mobile phase. The proteins were precipitated with an acetonitrile solution containing benzophenone as an internal standard. Serum standard curves were linear over the range 0 to 20 μg mL−1 for carbamazepine and 0 to 5 μg mL−1 for carbamazepine epoxide. The limit of detection for both compounds was less than 10 ng mL−1. Analytical recoveries ranged from 80 to 85% for carbamazepine and 95 to 98% for carbamazepine epoxide. The method is a simple, sensitive and reproducible means of determining carbamazepine and its metabolite in serum.

Input of selected human pharmaceuticalmetabolites into the Norwegian aquatic environment

The occurrence of the metabolites of five human pharmaceuticals was investigated in treated wastewater, surface waters and sediments. Metabolites of carbamazepine (carbamazepine epoxide), diclofenac (4'- and 5-hydroxy diclofenac) and atorvastatin (o- and p-hydroxy atorvastatin) were typically detected in flow proportional 24 h composite samples of wastewater effluent collected from the Norwegian cities of Oslo and Tromsø at higher concentrations than the parent pharmaceutical. The concentrations determined in discharged effluent were as high as 3700 ng L(-1) for 5-hydroxy diclofenac. The overall mean concentration of metabolites being typically higher in the primary treated effluent from the city of Tromsø compared to the tertiary treatment performed on the Oslo effluent. Metabolites of carbamazepine (carbamazepine-10,11-epoxide), metoprolol (α-hydroxy metoprolol) and simvastatin (hydroxy simvastatin) were detected in surface water samples collected from Oslofjord at concentrations of up to 108 ng L(-1), whilst α-hydroxy metoprolol and simvastatin hydroxy carboxylic acid were also detected in sediments at low ng L(-1) concentrations. These screening data show that the metabolites of selected pharmaceuticals are being discharged into the Norwegian coastal environment and that certain metabolites occur in marine surface waters and sediments.

Discordant carbamazepine values between two immunoassays: carbamazepine values determined by ADVIA centaur correlate better with those determined by LC-MS/MS than PETINIA assay

Discordant carbamazepine values as determined by two different immunoassays may be due to different cross-reactivities with the active metabolite carbamazepine 10, 11-epoxide and may cause confusion in interpreting carbamazepine serum levels. In this study, we compared carbamazepine values in samples containing carbamazepine and the epoxide metabolite, as determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and by two commercial carbamazepine immunoassays: the PETINIA and the ADVIA Centaur carbamazepine. Clinical specimens were used for the comparative studies wherein we determined carbamazepine concentrations using the PETINIA, ADVIA Centaur, and LC-MS/MS assays. We observed an excellent correlation between carbamazepine concentrations determined by the ADVIA Centaur and LC-MS/MS methods while carbamazepine values were overestimated using the PETINIA assay. When aliquots of drug-free serum were supplemented with clinically relevant concentrations of the carbamazepine epoxide metabolite, we observed negligible cross-reactivity of epoxide with the ADVIA Centaur assay but over 90% cross-reactivity with the PETINIA assay. We conclude that the ADVIA centaur assay accurately measures carbamazepine concentrations in plasma or serum and that the PETINIA assay significantly overestimates true carbamazepine concentration. Such discordance may cause confusion in interpreting serum carbamazepine levels.

Occurrence and removal of pharmaceuticals and hormones through drinking water treatment

The occurrence of fifty-five pharmaceuticals, hormones and metabolites in raw waters used for drinking water production and their removal through a drinking water treatment were studied. Thirty-five out of fifty-five drugs were detected in the raw water at the facility intake with concentrations up to 1200 ng/L. The behavior of the compounds was studied at each step: prechlorination, coagulation, sand filtration, ozonation, granular activated carbon filtration and post-chlorination; showing that the complete treatment accounted for the complete removal of all the compounds detected in raw waters except for five of them. Phenytoin, atenolol and hydrochlorothiazide were the three pharmaceuticals most frequently found in finished waters at concentrations about 10 ng/L. Sotalol and carbamazepine epoxide were found in less than a half of the samples at lower concentrations, above 2 ng/L. However despite their persistence, the removals of these five pharmaceuticals were higher than 95%.

In vivo solid-phase microextraction for single rodent pharmacokinetics studies of carbamazepine and carbamazepine-10,11-epoxide in mice

The use of solid-phase microextraction (SPME) for in vivo sampling of drugs and metabolites in the bloodstream of freely moving animals eliminates the need for blood withdrawal in order to generate pharmacokinetics (PK) profiles in support of pharmaceutical drug discovery studies. In this study, SPME was applied for in vivo sampling in mice for the first time and enables the use of a single animal to construct the entire PK profile. In vivo SPME sampling procedure used commercial prototype single-use in vivo SPME probes with a biocompatible extractive coating and a polyurethane sampling interface designed to facilitate repeated sampling from the same animal. Pre-equilibrium in vivo SPME sampling, kinetic on-fibre standardization calibration and liquid chromatography–tandem mass spectrometry analysis (LC–MS/MS) were used to determine unbound and total circulating concentrations of carbamazepine (CBZ) and its active metabolite carbamazepine-10,11-epoxide (CBZEP) in mice ( n = 7) after 2 mg/kg intravenous dosing. The method was linear in the range of 1–2000 ng/mL CBZ in whole blood with acceptable accuracy (93–97%) and precision (<17% RSD). The single dose PK results obtained using in vivo SPME sampling compare well to results obtained by serial automated blood sampling as well as by the more conventional method of terminal blood collection from multiple animals/time point. In vivo SPME offers the advantages of serial and repeated sampling from the same animal, speed, improved sample clean-up, decreased animal use and the ability to obtain both free and total drug concentrations from the same experiment.

Carbamazepine

Carbamazepine

Comparison between the immunoassay and high performance liquid chromatography for therapeutic monitoring of carbamazepine and phenytoine

Objective: To investigate the correlation of the immunoassay and chromatography method for quantitative measurement of two antiepileptic drugs (AED), carbamazepine (CBZ) and phenytoin (PHT) and determination of relation between the CBZ and it’s metabolite carbamazepine 10,11epoxide (CBZ-E). Additionally we investigated whether there is a diff erence in the determination of serum concentration of CBZ and PHT when measured in two diff erent labs by high performance liquid chromatography (HPLC). Materials and methods: This study was carried out on 102 blood samples (72 CBZ and 30 PHT) collected from epileptic outpatients. Plasma concentrations of CBZ and PHT were determined by validated HPLC (Shimadzu and Agilent) and the CEDIA-immunoassay method. Results: The correlations of serum concentrations of CBZ between CEDIA and HPLC1 and between CEDIA and HPLC2 were good (R = 0.97 for both techniques). Even better correlation was found between concentrations of CBZ measured by the two HPLC systems (R = 0.99). Similar, for PHT, we found good correlation between CEDIA and the two systems of HPLC (HPLC1 and HPLC2, R = 0.98) and between the two systems of HPLC of R =0.98. The moderate correlation coeffi cient was found between serum concentrations of CBZ and its metabolite CBZ-E, measured in two labs by diff erent HPLC (R = 0.49 and 0.43, respectively; P < 0.001). Conclusion: We observed good correlation for estimation of CBZ and PHT concentration obtained by means the immunoassay and two diff erent HPLC. The possibility of measurement of CBZ-E could be advantage of chromatography in comparison with immunoassay.

Effect of memantine on plasma concentrations of carbamazepine and phenytoin in rats: A controlled experimental study

Background: Elderly patients, especially those with Alzheimer's disease, may be prescribed memantine and an antiepileptic drug concurrently. Objective: The aim of this study was to compare the interaction of memantine with phenobarbital (an enzyme inducer) and chloramphenicol (an enzyme inhibitor) on plasma concentrations of carbamazepine (CBZ), CBZ-10,11-epoxide (CBZE), and phenytoin in an experimental model. Methods: Eight groups of rats (200–230 g) were treated for 14 days each. In groups 1 and 2, phenobarbital 50 mg/kg was administered daily as an enzyme inducer 60 minutes before CBZ 50 mg/kg or phenytoin 30 mg/kg administration, respectively. In groups 3 and 4, chloramphenicol 300 mg/kg was administered daily as an enzyme inhibitor 60 minutes before CBZ or phenytoin administration, respectively. In groups 5 and 6, memantine 20 mg/kg was administered daily 60 minutes before CBZ or phenytoin, respectively. In group 7, CBZ alone was administered daily; in group 8, phenytoin alone was administered daily. Two hours after the last intragastric gavage, animals were anesthetized with ether and 2 mL of blood was drawn from the heart into a syringe containing EDTA. A validated method developed in this study was used for simultaneous determination of CBZ, CBZE, and phenytoin concentrations in rat plasma. Results: The study comprised 8 groups of 9 male adult Wistar rats each. Compared with groups 7 and 8, concurrent use of CBZ or phenytoin with phenobarbital (groups 1 and 2) was associated with significantly lower mean (SEM) plasma concentrations of CBZ (3.45 [0.16] vs 2.20 [0.21] μg/mL; P < 0.001) and phenytoin (3.68 [0.09] vs 1.63 [0.15] μg/mL; P < 0.001) and a significantly higher plasma CBZE concentration (9.85 [0.29] vs 11.18 [0.29] μg/mL; P < 0.05). Concurrent use of CBZ or phenytoin with chloramphenicol (groups 3 and 4) was associated with significantly higher plasma concentrations of CBZ (4.81 [0.17] μg/mL; P < 0.001) and phenytoin (6.24 [0.22] μg/mL; P < 0.001) and a significantly lower plasma CBZE concentration (3.88 [0.25] μg/mL; P < 0.001). Concurrent use of CBZ or phenytoin with memantine (groups 5 and 6) was not associated with a significant change in the plasma concentration of CBZ, CBZE, or phenytoin. Conclusion: Memantine was not associated with a significant change in the plasma concentration of CBZ, CBZE, or phenytoin in this experimental model.

Tetra-crowned porphyrin as P450 biomimetic model for carbamazepine oxidation

A substituted porphyrin bearing four crown ether units, H(2)(TCP), was synthesized from the reaction between (5,10,15,20-tetra(o-aminophenyl) porphyrin) and the acyl derivative of the ether (4-carboxy-18-crown-6). The free-base porphyrin was characterized by C, N, and H elemental analysis; UV-vis and IR spectroscopies; and (1)H NMR. The corresponding ironporphyrin, Fe(TCP)Cl, was obtained via iron insertion into H(2)(TCP). Fe(TCP)Cl was employed as catalyst for carbamazepine (CBZ) oxidation by iodosylbenzene (PhIO), 3-chloroperoxybenzoic acid (m-CPBA) or sodium hypochlorite (NaOCl), in methanol or in a biphasic water/dichloroethane system. The crowned ironporphyrin proved to be a highly efficient and selective catalyst for CBZ epoxidation even in the biphasic dichloroethane /H(2)O system, with no need for an additional phase transfer agent.

Chemometric resolution of fully overlapped CE peaks: Quantitation of carbamazepine in human serum in the presence of several interferences

Drug monitoring in serum samples was performed using second-order data generated by CE-DAD, processed with a suitable chemometric strategy. Carbamazepine could be accurately quantitated in the presence of its main metabolite (carbamazepine epoxide), other therapeutic drugs (lamotrigine, phenobarbital, phenytoin, phenylephrine, ibuprofen, acetaminophen, theophylline, caffeine, acetyl salicylic acid), and additional serum endogenous components. The analytical strategy consisted of the following steps: (i) serum sample clean-up to remove matrix interferences, (ii) data pre-processing, in order to reduce the background and to correct for electrophoretic time shifts, and (iii) resolution of fully overlapped CE peaks (corresponding to carbamazepine, its metabolite, lamotrigine and unexpected serum components) by the well-known multivariate curve resolution-alternating least squares algorithm, which extracts quantitative information that can be uniquely ascribed to the analyte of interest. The analyte concentration in serum samples ranged from 2.00 to 8.00 mg/L. Mean recoveries were 102.6% (s=7.7) for binary samples, and 94.8% (s=13.5) for spiked serum samples, while CV (%)=4.0 was computed for five replicate, indicative of the acceptable accuracy and precision of the proposed method.

High-Speed Simultaneous Determination of Nine Antiepileptic Drugs Using Liquid Chromatography-Mass Spectrometry

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important and widely practiced. However, simultaneous AED assays usually concentrate only on old or new AEDs. A new simultaneous assay was developed to monitor both older and newer AEDs in the same sample. This assay measures zonisamide (ZNS), lamotrigine (LTG), topiramate (TPM), phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), carbamazepine-10,11-diol (CBZ-Diol), 10-hydroxycarbamazepine (MHD), and carbamazepine-10,11-epoxide (CBZ-E). Sample pretreatment consisted of a single solid-phase extraction (SPE) for all AEDs in a 100-microL plasma sample. HPLC separation was achieved on a Shimdazu Shimpack XR-ODS (4.6 id x 50 mm, 2.2-mum) column with a gradient mobile phase of acetate buffer, methanol, acetonitrile, and tetrahydrofuran. Four internal standards were used. Detection was achieved by atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in selected-ion monitoring (SIM) mode with constant polarity switching. High recovery (88%-96%) was obtained for all compounds by SPE. Linearity was observed throughout an 80-fold concentration range, with correlation coefficient (r) values higher than 0.99 for all AEDs. For the standards, the accuracy ranged from 89.3% to 111.8%. The within-run coefficient of variation (CVw) value was < or =9.7%, the between-run coefficient of variation (CVb) was < or =16.2%, and the total variability (CVt) was < or =16.8%. For the quality controls (QCs), accuracy ranged from 89.3% to 108.8%, CVw was < or =9.6%, CVb was < or =14.1%, and CVt was < or =15.1%. The correlation r values for comparison of this assay with existing validated assays in our laboratory (GC-MS or LC-MS) were 0.95, 0.91, 0.87, 0.95, and 0.95 for PHT, LTG, CBZ, CBZ-E, and CBZ-Diol, respectively.

An epoxidation mechanism of carbamazepine by CYP3A4

Human CYP3A4 catalyzes the 10,11-epoxidation of carbamazepine (CBZ). However, the epoxide is less stable in terms of potential energy than hydroxides of the six-membered aromatic ring. To clarify the reason why CYP3A4 produces such an energetically unfavorable compound, the mechanism of epoxidation of CBZ by CYP3A4 was investigated by theoretical calculations. The reaction consisted of two elementary processes in which two C–O bonds were generated stepwise. The rate-determining step was the first one and the activation energy was 21.3 kcal/mol at the DFT (B3LYP/6-31G ∗∗) level. The activation energy level of the first step of the 10,11-epoxidation was lower than that of the hydroxylation of the aromatic ring. For this reason, 10,11-epoxidation is more probable than hydroxylation of the aromatic ring, and only 10,11-epoxide is formed.

Relative Proportions of Serum Carbamazepine and its Pharmacologically Active 10,11-Epoxy Derivative: Effect of Polytherapy and Renal Insufficiency

Background: The proposed action mechanism and pharmacological activity of carbamazepine (CBZ) and its major metabolite, carbamazepine-10,11-epoxide (CBZE), are the same. The aim of our study was the investigation of the effect of concomitant antiepileptic treatment and renal insufficiency on the relative proportions of serum CBZ and CBZE.Methods: Serum trough steady-state CBZ and CBZE concentrations were determined by high-performance liquid chromatography (HPLC) in 140 epileptic patients treated with CBZ in monotherapy (n=100) and polytherapy with phenytoin, phenobarbital and valproate (n=40). The levels of CBZ were also determined using the Dade Behring enzyme multiplied immunoassay technique (EMIT). The glomerular filtration rate (GFR) was estimated from serum cystatin C using the Dade Behring nephelometric immunoassay.Results: The CBZE/CBZ and CBZ+CBZE/CBZEMIT ratios were significantly increased in 7 cases (3 in monotherapy and 4 in polytherapy) with GFR<60 mL/min/1.73m2 in relation to the patients treated in monotherapy or polytherapy having normal or mildly decreased renal function (p<0.001).Conclusions: In patients with moderate to severe renal insufficiency the relative proportion of CBZE with respect to the parent drug is significantly increased. In these cases, the CBZ concentrations obtained using the EMIT, or other immunoassays having low CBZE cross-reactivity, may have an inadequate diagnostic efficiency.

Biotransformation of Sildenafil in the Male Rat: Evaluation of Drug Interactions with Testosterone and Carbamazepine

The biotransformation of sildenafil to its major circulating metabolite, UK-103,320, was studied in male rat liver microsomes. The conversion of sildenafil to UK-103,320 by rat microsomes followed Michaelis–Menten kinetics, for which the parameters were Vmax = 1.96 μM/minand Km = 27.31 μM. Using substrates of CYP3A4 of testosterone and carbamazepine, the active sites on CYP3A4 responsible for metabolizing sildenafil were also evaluated. Sildenafil biotransformation was inhibited in the individual presence of testosterone and carbamazepine. The results showed drug interaction was observed in the sildenafil-testosterone and sildenafil-carbamazepine. Although testosterone and carbamazepine can inhibit sildenafil demethylation in concentration- and incubation time-dependent manners, sildenafil did not inhibit testosterone hydroxylation or carbamazepine epoxidation. These results may be explained by a model in which multiple substrates or ligands can concurrently bind to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 cannot be ruled out in this study.

Identification of primary and sequential bioactivation pathways of carbamazepine in human liver microsomes using liquid chromatography/tandem mass spectrometry

Carbamazepine (CBZ)-induced idiosyncratic toxicities are commonly believed to be related to the formation of reactive metabolites. CBZ is metabolized primarily into carbamazepine-10,11-epoxide (CBZE), 2-hydroxycarbamazepine (2-OHCBZ) and 3-hydroxycarbamazepine (3-OHCBZ), in human liver microsomes (HLM). Over the past two decades, the 2,3-arene oxidation has been commonly assumed to be the major bioactivation pathway of CBZ. Recently, CBZE has been also confirmed to be chemically reactive. In order to identify other possible primary and sequential CBZ bioactivation pathways, individual HLM incubations of CBZ, CBZE, 2-OHCBZ and 3-OHCBZ were conducted in the presence of glutathione (GSH). In the CBZ incubation, a variety of GSH adducts were formed via individual or combined pathways of 10,11-epoxidation, arene oxidation and iminoquinone formation. In the CBZE incubation, the only detected GSH adducts were CBZE-SG1 and CBZE-SG2, which represented the two most abundant conjugates observed in the CBZ incubation. In the incubation of either 2-OHCBZ or 3-OHCBZ, a number of sequential GSH adducts were observed. However, none of the 2-OHCBZ-derived GSH adducts were detected in the CBZ incubation. Meanwhile, several GSH adducts were only observed in the CBZ incubation. In conclusion, CBZ can be bioactivated in HLM via 10,11-epoxidation, 2,3-arene oxidation, and several other pathways. In addition, the sequential bioactivation of 3-OHCBZ appeared to play a more important role than that of either CBZE or 2-OHCBZ in the overall bioactivation of CBZ in HLM. The identification of several new bioactivation pathways of CBZ in HLM demonstrates that possible CBZ bioactivation can be more complex than previously thought.

Determination of Carbamazepine and its Main Metabolite Carbamazepine-10,11-Epoxide in Rat Brain Microdialysate and Blood Using ESI–LC–MS (Ion Trap)

A new analytical method has been developed and validated for determination of the anti-epileptic drug carbamazepine (CBZ) and its main metabolite carbamazepine-10,11-epoxide (CBZ-E) using ESI–LC–MS (ion trap). The compounds were separated on a C18 (150 × 2.1 mm I.D., 3 μm particles) column and were isocratically eluted in the mobile phase consisting of water–acetonitrile–acetic acid (74.5:25:0.5, v/v) using the flow rate of 0.4 mL min−1. The other anti-epileptic drug oxcarbamazepine (OXC) was used as an internal standard. The retention times for CBZ-E, OXC and CBZ were 5.6, 6.8, 12.8 min. Signals of the compounds were monitored under multi-reaction monitoring mode (MRM) of ESI–LC–MS (ion trap) for the quantification. Selected ions of CBZ-E, OXC and CBZ in MRM were m/z 253→210, m/z 253→180 and m/z 237→194. The method was validated over the concentration range of 5.0–500.0 ng mL−1 and was applied to rat brain microdialysate and blood samples for the determination of CBZ and main metabolite. The brain microdialysate and the blood sample were collected simultaneously after intra-peritoneal injection of CBZ (12 mg kg−1) during a period of 10 h. No interference from endogenous substances and matrix effect were found on the separation of microdialysates and blood samples. The consequent signals of the compounds were resolved and integrated clearly. The LC–MS method was presented as an alternative to investigate pharmacokinetic parameters of CBZ and CBZ-E in blood and brain studies.