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Metabolism and pharmacokinetic studies of valproic acid using stable isotope techniques Zheng, Jiaojiao


Valproic acid (VPA) is an anticonvulsant agent widely used in the treatment of several types of epileptic seizures. The drug is unique within its therapeutic class, in terms of its mechanism of action, its chemical structure, as well as its extensive biotransformation into at least 16 different metabolites. The interest in VPA metabolites has been stimulated by the potential of VPA to produce severe hepatotoxicity. Metabolites 4-ene VPA and 2,4-diene VPA are thought to be responsible for the rare but fatal hepatotoxicity associated with VPA. Thus, methodology to study the pharmacokinetics of VPA metabolitesis important to an evaluation of the role that metabolites may play during VPA therapy. Stable isotope techniques and gas chromatography mass spectrometry (GCMS) have been used in several areas of research on VPA. For example the application of a stable isotope labelled analog as a "pulse dose" in antiepileptic drug studies allows the elimination kinetics of the drug to be determined without discontinuing therapy and risking the exacerbation of seizures. In the present study, [21-10PA and [13C4] VPAwere evaluated as to their applicability to pharmacokinetic studies of VPA. Pharmacokinetic parameters of VPA, [21-161VPA were measured in a healthy human volunteer. Potential isotope effects of [2H6JVPA and [13C4] VPA were studied based on the urine recovery ratio or AUC ratio of VPA and its metabolites to their isotope labelled analogs. No apparent isotope effect was found in the metabolism of [13C4]VPA, which makes [13C4]VPA qualified for use in a "pulse dose" manner. Upon [2H6JVPA administration, a large isotope effect was observed in the metabolic formation of [24]5-0H VPA and [2H3]2-PGA, and a small isotope effect was apparent for the formation of [2H6](E)-2,4-diene VPA. Based on the latter observation it was proposed that the formation of 2,4-diene VPA might occur partly from 3-ene VPA. (E)- and (Z)-3-ene VPA were synthesized to test this proposal. The use of stable isotope labelled analogs as internal standards can minimize the variance arising from extraction of VPA metabolites due to a slight pH change or incompleteness of derivatization due to time and temperature. In order to obtain optimal analytical results, eight deuterium labelled VPA metabolites were synthesized as internal standards, which included [2H7]VPA, [2H7]2-ene VPA, [2H7]3-keto VPA,[2H7]3-0H VPA, [2H7]4-ene VPA, [2H7]4-0H VPA, [2H7]4-keto VPA, and[2H7]5-0H VPA. These internal standards were applied to the analysis ofVPA, [13C4]VPA and their metabolites in serum and urine samples collected from two nonpregnant sheep following single dose administration of VPA:[13C4]VPA (50:50). The elimination half-life of VPA in the sheep was estimated to be approximately 2.5-5 hours. GCMS conditions for both electron ionization (EI) and negative chemical ionization (NCI) were optimized to obtain the best resolution and sensitivity for VPA metabolites. A single temperature program with a run time of 47 min was established for NCI analysis of PFB derivatives of VPA, [2H6]VPA and their metabolites. Two temperature programs were investigated for the EI analysis of t-BDMS derivatives. One run time of 35 min was used for VPA unsaturated metabolites, while a run time of 20 min was used for the more polar metabolites of VPA. All the urine and serum samples were analyzed with both El and NCI techniques. PFB derivatives of VPA metabolites analyzed by the NCI technique gave higher sensitivity and better resolution than t-BDMS derivatives of VPA metabolites analyzed by El methods. All urine samples were hydrolyzed with glucuronidase and with NaOH solution. No difference was observed between the results obtained with the different hydrolysis methods, which indicated that there was little or no fl-glucuronidase-resistant conjugate present in the urine samples of this human volunteer after urine samples were kept at -20 °C for about two months. The conjugated fractions were measured, more than 90% of VPAand its unsaturated metabolites were excreted into urine in the form of their glucuronic conjugates. Metabolites 3-0H, 4-0H and 5-0H VPA were excreted partly as glucuronides, while 3-keto, 4-keto VPA, 2-PSA and 2-PGA were excreted mostly as the free metabolites. The present investigation reaffirmed the importance of GCMS assay techniques to studies of VPA pharmacokinetics and disposition. Stableisotope labelled internal standards improved the accuracy and precision of VPA metabolite analysis by GCMS. [13C4JVPA was ideal for "pulsedose" VPA studies of pharmacokinetics, in which techniques it was demonstrated that the pharmacokinetic parameters of VPA metabolites will be obtained for the first time in pediatric patients.

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