Use of hexadeuterated valproic acid and gas chromatography-mass spectrometry to determine the pharmacokinetics of valproic acid

Di-[( 3,3,3-2H3]propyl)acetic acid, a hexadeuterated analogue of valproic acid, was synthesized and its pharmacokinetic properties compared with valproic acid. Concentrations of valproic acid and [2H]valproic acid in serum and saliva were determined by GC-MS using selected-ion monitoring. Saliva drug levels were measured with good precision down to 0.1 microgram/mL. Kinetic equivalence of valproic acid and [2H]valproic acid was demonstrated in a single-dose study in a human volunteer. An isotope effect was observed for omega-oxidation, but the difference in metabolism was not sufficient to make [2H]valproic acid biologically nonequivalent. The application of [2H]valproic acid to determine the kinetics of valproic acid under steady-state concentrations was evaluated in the same volunteer. The kinetic data obtained with [2H]valproic acid was consistent with previously reported values for valproic acid including kinetic differences observed between single-dose and steady-state experiments. Saliva levels of valproic acid were found to give a good correlation (r = 0.953) with total serum valproic acid under multiple-dose conditions. A concentration dependence was found for the ratio of saliva valproic acid to free valproic acid in serum, low ratios being observed at high serum concentrations of valproic acid.     

Pharmacological evaluation of various metabolites and analogues of valproic acid. Anticonvulsant and toxic potencies in mice

Thirty-two metabolites and analogues of the antiepileptic drug valproic acid (2-propylpentanoic acid; VPA) were tested for anticonvulsant and toxic effects in mice, in an attempt to find out if any of these compounds were superior to valproic acid. Valproic acid and ethosuximide, another clinically established antiepileptic drug, were included in these studies for comparison. After intraperitoneal administration, the anticonvulsant potency of the various drugs was determined in three seizure tests: the threshold for maximal electroconvulsions, the maximal electroshock seizure test and seizures induced by subcutaneous injection of pentylenetetrazol. For the most potent compounds, median minimal neurotoxic doses (TD50S) and LD50S (after i.p. and i.v. injection) were determined. Valpramide, the primary amide of valproic acid, proved to be the most potent compound in the three seizure tests, used, being 2-5 times as potent as valproic acid, but valpramide was also considerably more sedative and toxic than valproic acid or ethosuximide. Of the metabolites of valproic acid tested, the unsaturated compounds 4-en-valproic acid (4-en-VPA) and the trans-isomer of 2-en-valproic acid (2-en-VPA) were most potent and, depending on the seizure test used, reached 60-100% of the efficacy of the parent drug. Both metabolites had LD50 values which were similar or greater than those of valproic acid but they were more sedative than the parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simple and accurate quantitative analysis of ten antiepileptic drugs in human plasma by liquid chromatography/tandem mass spectrometry

A simple, accurate, and sensitive liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method has been developed for the simultaneous quantification of 10 antiepileptic drugs (AEDs; gabapentin (GBP), levetiracetam (LEV), valproic acid (VPA), lamotrigine (LTG), carbamazepine-10,11-epoxide (CBZ-epoxide), zonisamide (ZNS), oxcarbazepine (OXC), topiramate (TPM), carbamazepine (CBZ), phenytoin (PHT)) in human plasma as a tool for drug monitoring. d₁₀-Phenytoin (d₁₀-PHT) and d_6-valproic acid (d₆-VPA) were used as internal standards for the positive- and negative-ionization modes, respectively. Plasma samples were precipitated by the addition of acetonitrile, and supernatants were analyzed on a C18 reverse-phase column using an isocratic elution. Detection was carried out in selected reaction monitoring (SRM) mode. The calibration curves were linear over a 50-fold concentration range, with correlation coefficients (r²) greater than 0.997 for all AEDs. The intra- and inter-day precision was less than 12%, and the accuracy was between 85.9 and 114.5%. This method was successfully used in the identification and quantitation of AEDs in patients undergoing mono- or polytherapy for epilepsy.     

抗癫痫中成药中非法添加西药成分的血药浓度监测分析

目的：对长期服用抗癫痫中成药的患者进行血药浓度监测,查明抗癫痫中成药中非法添加的西药成分。方法：通过全自动生化分析仪,采用酶联免疫法测定中成药中添加的丙戊酸、卡马西平、苯巴比妥、苯妥英的种类及血药浓度。结果：所有服用抗癫痫中成药的患者中均检测到上述四种西药成分,其中含丙戊酸17例,血药浓度在有效范围内占23.53%;含卡马西平17例,血药浓度在有效范围内占17.65%;含苯巴比妥17例,血药浓度在有效范围内占41.18%;含苯妥英5例,血药浓度在有效范围内占60.00%。结论：本方法操作简便、准确,可快速筛查抗癫痫中成药中非法添加的西药成分,便于临床指导患者合理用药。     

No pharmacokinetic interaction between lacosamide and valproic Acid in healthy volunteers

Two open-label, randomized, multiple-dose clinical studies evaluated the potential for pharmacokinetic interaction between the antiepileptic drugs lacosamide and valproic acid. The influence of lacosamide on valproic acid pharmacokinetics (trial A) and valproic acid on lacosamide pharmacokinetics (trial B) was investigated in 32 healthy male volunteers, 16 in each trial. Volunteers in trial A received valproic acid (300 mg bid) with randomization to either early or late addition of lacosamide (200 mg bid). Those in trial B received lacosamide (200 mg bid) with randomization to either early or late addition of valproic acid (300 mg bid). Area under the concentration-time curve during a 12-hour dosing interval at steady state (AUC(τ,ss)) and maximum steady-state plasma drug concentration (C(max,ss)) were measured for each drug alone and together and tested for equivalence. The point estimates (90% confidence intervals) for AUC(τ,ss) and C(max,ss) were 104% (99%-109%) and 101% (97%-107%), respectively, for valproic acid and 100% (98%-103%) and 101% (96%-107%), respectively, for lacosamide, which were within the generally accepted equivalence range of 80% to 125%. No changes in the rate or extent of absorption, terminal half-life, or time to maximum concentration were observed. These results suggest that lacosamide and valproic acid have no relevant pharmacokinetic drug-drug interaction.     

Pharmacoepidemiologic investigation of clonazepam relative clearance by mixed-effect modeling using routine clinical pharmacokinetic data in Japanese patients.

The effects of drug-drug interactions on clonazepam clearance were examined through a retrospective analysis of serum concentration data from pediatric and adult epileptic patients. Patients received clonazepam as monotherapy or in combination with other antiepileptic drugs. A total of 259 serum clonazepam concentrations gathered from 137 patients were used in a population analysis of drug-drug interactions on clonazepam clearance. Data were analyzed using a nonlinear mixed-effects modeling (NONMEM) technique. The final model describing clonazepam clearance was CL = 152 x TBW(-0.181) x DIF, where CL is clearance (ml/kg/h), TBWis total body weight (kg), and DIF (drug interaction factor) is a scaling factor for concomitant medication with a value of 1 for patients on clonazepam monotherapy, 1.18 for those patients receiving concomitant administration of clonazepam and one antiepileptic drug (carbamazepine or valproic acid), and 2.12 x TBW(-0.119) for those patients receiving concomitant administration of clonazepam and more than two antiepileptic drugs. Clonazepam clearance decreased in a weight-related fashion in children, with minimal changes observed in adults. Concomitant administration of clonazepam and carbamazepine resulted in a 22% increase in clonazepam clearance. Concomitant administration of clonazepam and valproic acid resulted in a 12% increase in clonazepam clearance. Concomitant administration of clonazepam with two or more antiepileptic drugs resulted in a 23% to 75% increase in clonazepam clearance.     

丙戊酸钠血药浓度监测及意义

目的:为临床癫痫患者丙戊酸钠的合理用药提供参考.方法:采用荧光偏振免疫法对143例癫痫患者服用丙戊酸钠进行血药浓度监测,并对结果及疗效进行分析总结.结果:222例次监测结果在有效浓度范围内,占总例次的68.5%,而有6例次血药浓度在正常范围,未能控制癫痫症状,13例次出现毒性反应.血药浓度高于或低于正常浓度有102例次,占31.5%.结论:丙戊酸钠血药浓度监测对合理用药有重要的指导作用,血药浓度个体差异大,临床用药时需个体化给药.     

Evaluation of assay techniques for the measurement of antiepileptic drugs in serum: a study based on external quality assurance measurements

The accuracy and precision of eight techniques used to measure a range of eight antiepileptic drugs in human serum were compared using data from 80 samples from the Heathcontrol External Quality Assurance scheme. The fluorescence polarization immunoassay was significantly more precise than other techniques for several analytes, producing the lowest number of measurements rejected as outliers and measurements with the lowest coefficient of variation. Other techniques had a significantly lower precision. Nephelometry (Neph) produced most outliers for phenytoin and carbamazepine and had the highest variability for phenytoin. Gas-liquid chromatography (GLC) with derivatization was most variable for phenobarbitone and primidone. Measurements by GLC with or without derivatization contained greater than 10% of outliers and were least precise for carbamazepine. Differences between the majority of techniques were in many cases, however, not significant. The accuracy of techniques was assessed from differences between sample means and spiked drug concentrations. Neph was notable in producing overestimates at lower concentrations. Immunoassay methods had a 16-21% cross-reactivity with carbamazepine 10,11-epoxide when measuring carbamazepine. All techniques reported underestimates for valproic acid that were thought to result from the hygroscopic nature of the salt used for spiking.     

Unbound plasma phenytoin concentrations measured using enzyme immunoassay technique on the cobas MIRA analyser--in vivo effect of valproic acid.

This communication describes a modification of the total plasma phenytoin enzyme immunoassay technique (EMIT) run on the Cobas MIRA analyser that allows quantitation of unbound phenytoin concentrations in human plasma for routine therapeutic drug monitoring (TDM) purposes. An application of this method is also presented to consider the previously described protein binding drug interaction with concomitantly administered valproic acid in patients with epilepsy. The coefficients of variation for the unbound phenytoin assay ranged from 7.5 to 9.6% and the assay had a reproducibility and accuracy similar to the total phenytoin assay, acceptable for routine TDM. Phenytoin protein binding was linear over a range of total plasma concentrations of 3-65 mg/L. Patients also receiving valproic acid (nine patients, 105 specimens) had a significantly (p less than 0.0001) greater mean +/- SD unbound phenytoin fraction (13.3 +/- 3.1%) compared to nine patients (110 specimens) not receiving valproic acid (8.3 +/- 1.6%). There was also a significant correlation (p less than 0.001) between plasma valproic acid concentration and unbound phenytoin fraction, which resulted in greater intrasubject variability in phenytoin protein binding.     

244例抗癫痫药血药浓度监测结果分析

目的：通过对抗癫痫药物血药浓度监测结果分析，为临床提高本类药物的治疗水平作参考。方法：采用荧光偏振免疫法对26例服用苯妥英钠、74例服用卡马西平、144例服用丙戊酸钠的患者血药浓度监测结果及临床疗效分析、评价。结果：本类药物治疗指数小，安全度较低，其作用的个体差异大。结论：癫痫患者应重视血药浓度监测，并结合其他因素调整用药方案。以达到安全、有效、合理应用本类药物。     

Population pharmacokinetics of lamotrigine adjunctive therapy in adults with epilepsy

Plasma concentrations of lamotrigine, an antiepileptic drug obtained in three adult controlled clinical trials conducted in the United States were pooled and analyzed using NONMEM, a population pharmacokinetic computer program, to facilitate development of dosing guidelines. A total of 2,407 lamotrigine plasma concentrations from 527 patients with epilepsy were analyzed. Regression equations for oral clearance were developed as a function of body size, age (18-64 years), gender, race, and use of concomitant antiepileptic drugs. The population mean apparent oral clearance of lamotrigine in adult patients receiving one concomitant enzyme-inducing antiepileptic drug and not valproic acid was estimated to be 1 mL/min/kg. Gender and age did not affect clearance significantly. On average, clearance was reduced by 25% in non-whites and increased by 13% in patients receiving more than one concomitant enzyme-inducing antiepileptic agent. Lamotrigine did not influence the disposition of phenytoin or carbamazepine. Dosing adjustments for lamotrigine in patients receiving concomitant enzyme-inducing antiepileptic drugs and not valproic acid should not be necessary for age, gender, or the number of concomitant enzyme-inducing antiepileptic drugs. Lamotrigine does not influence the dosing requirements for phenytoin or carbamazepine.     

409例次丙戊酸血药浓度监测结果分析

目的：分析癫痫患者丙戊酸血药浓度监测结果,为丙戊酸的合理应用提供参考依据。方法：采用荧光偏振免疫法测定丙戊酸血药浓度,对409例次血药浓度监测结果进行比较分析,并观察其临床疗效和不良反应。结果：409例次中,丙戊酸血药浓度〈50μg·mL-1的占39.85%,50～100μg·mL-1的占52.32%,〉100μg·mL-1的占7.82%。血药浓度〈50μg·mL-1的患者,控制癫痫发作的有效率为44.78%;血药浓度≥50μg·mL-1的患者,控制癫痫发作的有效率为86.99%。结论：丙戊酸血药浓度个体差异大,血药浓度监测对合理用药有重要指导作用,应根据血药浓度个体化用药。     

Evaluation of fluorescence polarization assays for measuring valproic acid,phenytoin, carbamazepine and phenobarbital in serum

The authors evaluated the fluorescence polarization (FP) assay on the COBAS INTEGRA 700 using COBAS INTEGRA reagent system cassettes for estimating the antiepileptic drugs valproic acid, phenytoin, carbamazepine, and phenobarbital in serum. The study comprised the determination of precision, method comparison performed against a conventional HPLC assay and linearity studies, according to the National Committee for Clinical Laboratory Standards (NCCLS) protocols. Precision results were well acceptable for all FP assays. Intra-assay coefficients of variation (CVs) were from 1.3% to 2.4% for valproic acid, from 0.8% to 2.8% for phenytoin, from 1.7% to 3.3% for carbamazepine, and from 1.3% to 2.3% for phenobarbital. Interassay CVs for these drugs ranged from 1.5% to 2.6%, from 2.9% to 6.5%, from 1.8% to 3.7% and from 1.5% to 3.2%, respectively. Results of the FP assays showed excellent correlation with those from HPLC: r = 0.99 for valproic acid, r = 0.98 for phenytoin, r = 0.98 for carbamazepine and r = 0.99 for phenobarbital. Linearity was satisfactory, with all CVs below the acceptable level. With the COBAS INTEGRA 700 analyzer FP assays are fully automated, which is less laborious and saves time compared with HPLC. Moreover, the fast measuring procedure is convenient in short turnaround time (STAT) analysis. It is an analytically reliable and rapid system, which can be used successfully for the therapeutic monitoring of antiepileptic drugs in serum.     

Serum concentrations of Levetiracetam in epileptic patients: the influence of dose and co-medication

Levetiracetam (LEV) is a new antiepileptic drug approved as add-on therapy. Previous studies indicated that LEV has no relevant interactions with other antiepileptic drugs. The aim of this study was to investigate the influence of LEV dose, age, and co-medication on the serum concentration of LEV. In total, 363 samples of 297 inpatients who fulfilled the inclusion criteria (e.g., trough concentration, body weight available) were investigated. A patient was considered twice only if his co-medication had been changed. The LEV serum concentration in relation to LEV dose/body weight [level-to-dose ratio, LDR, (microgram/mL)/(mg/kg)] was calculated and compared for the most frequent drug combinations. Analysis of covariance (using age as covariate) carried out on the log-transformed data showed that co-medication had a highly significant (P < 0.001) effect on LEV serum concentrations. The median LDR of LEV was 0.32 for LEV + phenytoin, 0.32 for LEV + carbamazepine, 0.34 LEV + oxcarbazepine, 0.45 for LEV + lamotrigine, 0.46 for LEV + phenobarital, 0.52 for LEV monotherapy, 0.53 for LEV + valproic acid, and 0.54 LEV + valproic acid + lamotrigine. In co-medication with phenytoin (P < 0.001), carbamazepine (P < 0.001), and oxcarbazepine (P < 0.004), the LDR of LEV was significantly lower than it was with LEV monotherapy, whereas the LDR of LEV of patients on co-medication with valproic acid or lamotrigine did not differ significantly from the LDR of LEV of patients on LEV monotherapy (P > 0.05). Regression analysis including all 363 samples confirmed that other drugs (e.g., phenytoin, carbamazepine) lower LEV concentrations. In addition to co-medication, age had a significant effect on clearance of LEV. Children had lower LEV concentrations than adults on the same LEV dose per body weight. In contrast to other studies, our data point out that other enzyme-inducing antiepileptic drugs (e.g., phenytoin, carbamazepine) can moderately decrease LEV serum concentrations (by 20-30%). However, our observations should be confirmed by prospective pharmacokinetic studies.     

我院432例抗癫痫药血药浓度监测数据分析

目的:分析抗癫痫药血药浓度监测结果,为临床合理用药提供参考。方法:建立抗癫痫药Access数据库,对我院2001年1月～2009年6月4种常用抗癫痫药的血药浓度监测结果进行统计、分析。结果:我院抗癫痫药血药浓度监测例数大体呈逐年上升趋势,共监测432例/次;血药浓度在治疗窗内所占比例为48.6%;卡马西平、苯巴比妥、苯妥英钠、丙戊酸钠血药浓度在治疗窗内的有效率分别为91.3%、77.8%、62.5%、88.2%;儿童(0～10岁)与老人(>60岁)进行血药浓度监测例数占总监测例数的22.2%;有43例采用抗癫痫药联合给药方案,占总例数的10%,其中偏离正常治疗浓度范围的有27例,占62.8%。结论:血药浓度监测结果是指导临床用药的重要依据之一,结合其他临床指标综合分析,可最大限度地促进抗癫痫药合理应用。     

Detection of Carbamazepine-induced Changes in Valproic Acid Relative Clearance in Man by Simple Pharmacokinetic Screening

Selecting the optimum dose of valproic acid is difficult because the pharmacokinetics are complicated by inter-patient variability and by effects arising as a result of co-administration with other antiepileptic drugs. The multiple peak approach has been used to evaluate the effect of age, total body weight, dose, gender and co-medication (carbamazepine-induced change) on population estimates of valproic acid relative clearance. Routine clinical pharmacokinetic data (n = 479) were collected from 207 epilepsy patients on combination therapy. The data were analysed by a simple steady-state pharmacokinetic model with the use of NONMEM, a computer program designed for population pharmacokinetic analysis that enables pooling of data. NONMEM estimates suggested that the rate of valproic acid clearance in patients receiving concomitant administration of valproic acid and carbamazepine decreased non-linearly with increasing total body weight in the maturation process, and increased non-linearly with increasing valproic acid dose. The clearance in females was 5.7% less than in males. NONMEM estimates also suggested that the rate of valproic acid clearance increased non-linearly with increasing carbamazepine dose. Concomitant administration of valproic acid and carbamazepine with other antiepileptic drugs resulted in an increase in valproic acid clearance of 10%. The final regression model of valproic acid relative clearance was CL = 606TBW^0.168 ×DOSE^0.414 × CBZDOSE^0.095 × 0.943^GEN × 1.10^CO, where CL is the clearance (mL kg^? h^?), TBW is the total body weight (kg), DOSE is the dose of valproic acid, CBZDOSE is the dose of carbamazepine, GEN = 0 for males and 1 for females and CO = 0 for concomitant administration of valproic acid and carbamazepine and 1 for concomitant administration of valproic acid and carbamazepine with other antiepileptic drugs. This technique can be used to estimate the pharmacokinetic parameters of a population from sparse data collected during routine clinical care and to determine the extent to which patient characteristics influence drug pharmacokinetics.     

我院4种抗癫痫药物血药浓度监测结果回顾性分析

目的:提高临床对血药浓度监测工作的重视程度。方法:运用回顾性调查方法,对我院自1998年～2005年所监测的应用苯巴比妥、苯妥英钠、卡马西平、丙戊酸钠4种抗癫痫药治疗的1443例/次患者的血药浓度结果进行分析。结果:血药浓度在高、低及正常治疗浓度范围内的患者分别为10.40%、36.59%、53.01%。联合用药的患者中,血药浓度在正常治疗浓度范围内者为28.45%。结论:抗癫痫药的血药浓度监测对临床调整用药剂量有指导意义;及时监测血药浓度,实施个体化给药是确保临床治疗效果和用药安全的重要措施之一。     

丙戊酸钠联用碳青霉烯类药物血药浓度变化特点探讨

目的：探讨丙戊酸钠联用碳青霉烯类药物后引起丙戊酸钠血药浓度变化特点,为临床合理用药提供参考。方法：采用化学发光免疫分析法检测丙戊酸钠联用碳青霉烯类药物前、期间以及停用碳青霉烯类药物后的血药浓度,总结变化特点,并研究给药剂量调整与血药浓度变化情况。结果：丙戊酸钠联用碳青霉烯类药物后,美罗培南使其血药浓度下降（83.2±7.8）%,亚胺培南使其血药浓度下降（71.7±5.3）%,停用碳青霉烯类药物后,血药浓度逐渐恢复,但增加丙戊酸钠给药剂量对血药浓度变化影响不明显。结论：碳青霉烯类药物可致丙戊酸钠血药浓度显著降低,两者不宜联用,以确保临床用药安全、有效。     

Comparison of two extraction procedures for determination of drugs of abuse in human saliva by high-performance liquid chromatography

High performance liquid chromatography in combination with diode array detection (HPLC-DAD) was used to determine morphine, 6-acetylmorphine, cocaine, benzoylecgonine, cocaethylene, methadone and 2-ethylene-1,5-dimethyl-3,3,-diphenylpyrrolidine in human saliva. For comparison, samples were prepared by either liquid-liquid extraction in Toxitubes A or microwave-assisted extraction (MAE), by mixing 1 ml of saliva with 10 ml of chloroform and operating at 100 degrees C for 10 min. Acetonitrile and 0.02 m phosphate buffer at pH 6.5 were used as mobile phase in HPLC in gradient mode. The detector response was linear over the drug concentration range of 0.05-2.0 microg ml(-1) in human saliva. The analytical method was validated by determining its precision and accuracy (n = 5), which were lower than 5% as relative standard deviation and 6% as relative error. Limits of detection ranged from 10 to 35 ng ml(-1); mean recoveries of drugs were from 53 to 95% with Toxitubes A and from 83 to 100% with MAE at two different concentrations (0.1 and 1.0 microg ml(-1)). The proposed method was applied to 24 saliva samples from individuals poisoned with opiates and/or cocaine.     

Relationship between serum and saliva theophylline levels in patients with cystic fibrosis.

Theophylline levels in stimulated and unstimulated mixed saliva were compared with total and free (unbound) serum theophylline levels in 11 outpatients with cystic fibrosis (CF) who were using theophylline regularly. Stimulated saliva from CF patients predicted both total and unbound serum theophylline concentrations to within +/- 1 microgram/ml in 53.3 and 80.0%, respectively, of the samples examined. In addition, the total serum levels from CF patients could be used to predict unbound serum concentrations to within +/- 1 micrograms/ml in 100% of the cases examined. Furthermore, it was observed that prediction equations derived in a previous study with asthmatics employing identical methodology would allow both unbound and total serum theophylline levels to be predicted from saliva levels in CF patients with a degree of accuracy and precision that was approximately equal to or slightly better than the results obtained using prediction equations derived in other CF patients. These results indicate that saliva levels allow predictions of the unbound serum theophylline levels with greater accuracy and precision than they predict total serum theophylline levels. In addition, total serum levels can be used to reliably predict unbound serum levels. The use of mixed stimulated saliva is recommended as a reliable noninvasive method for monitoring unbound serum theophylline levels. The therapeutic range for saliva, which corresponds to the accepted total serum concentration range of 10-20 micrograms/ml, is approximately 5.55-11.3 micrograms/ml.