Di-(3-²H₃-propyl)acetic acid was synthesized and used in pharmacokinetic and metabolism studies of dipropyl acetic acid (valproic acid). Kinetic equivalence of valproic acid and valproic acid-²Hg₆ was demonstrated in a single dose study in a human volunteer. An isotope effect was observed for w-oxidation but the difference in metabolism of the two isotopic forms was not sufficient to make valproic acid-²Hg₆ biologically nonequivalent. In a multiple dose study, the kinetics of valproic acid-²Hg₆ were determined in the presence of steady state concentrations of valproic acid in the same volunteer. Concentrations of valproic acid and valproic acid-²Hg₆ in serum and saliva were determined by gas chromatography mass spectrometry using selected ion monitoring. Saliva drug levels were measured with good precision down to 0.1 μg/ml. Compared to single dose kinetic data, the total body clearance of valproic acid-²Hg₆ increased by 33% at steady state. This could be explained by an increase in the serum free drug fraction. At steady state, intrinsic clearance was found to decrease. Good correlation was found between concentrations of valproic acid in saliva and serum. Serum and urinary metabolites were characterized as their methyl, trimethylsilyl or tert-butyldimethylsilyl derivatives. The metabolism study was facilitated by using the stable isotope tracer technique. A diunsaturated metabolite was identified in serum and urine. The presence of a molecular ion doublet in the mass spectrum reduces the possible structures for this metabolite. A new metabolite, 2-propyl-4-keto-pentanoic acid, was detected in serum and urine and 2-propylsuccinic acid and 2-propylmalonic acid were characterized as metabolites. The identification of metabolites was also verified using synthesized reference compounds. / Pharmaceutical Sciences, Faculty of / Graduate

Thesis (MAppSc in Pharm) -- University of South Australia, 1993

Valproic acid (VPA) is a broad spectrum antiepileptic agent used widely in the treatment of absence and tonic-clonic seizures. VPA is extensively metabolized and forms 17 metabolites in man. A monounsaturated metabolite, (E)-2-ene VPA, is at least as potent as the parent drug VPA in several animal models of epilepsy. Moreover, (E)-2-ene VPA appears to be free of two serious side effects of VPA, namely hepatotoxicity and teratogenicity. Another monounsaturated metabolite of VPA, 4-ene VPA, has been incriminated in the pathogenesis of fatal hepatic failure in children on VPA therapy. This thesis describes the synthesis of (E)-2-ene VPA and 4-ene VPA and the development of a simple and sensitive capillary gas chromatographic-mass spectrometric (GCMS) assay method for the estimation of (E)-2-ene VPA and 4-ene VPA in the biological fluids of the rat. This thesis also describes the pharmacokinetics of (E)-2-ene VPA and 4-ene VPA at two dose levels of 20 and 100 mg/kg in normal and bile exteriorized rats. A simple capillary GCMS assay method was developed that involves a single extraction of 80 µL of plasma, urine or bile with ethyl acetate followed by derivatization with MTBSTFA (N-tertiarybutyldimethylsilyl-N-methyl-trifluoroacetamide). For an 80 µL biological sample employed for extraction, the lowest detection limit for (E)-2-ene VPA was 60 ng/mL and for 4-ene VPA, 100 ng/mL. The calibration curves for (E)-2-ene VPA were linear over a fairly wide concentration range of 0.4-35 /µg/mL in plasma and 2-200 µg/ml in urine of the rat. Standard curves for 4-ene VPA were prepared in concentration ranges of 0.5-45 µg/mL in plasma and 2-80 µg/ml in urine. The assay method is reliable, reproducible, and is able to separate the diene metabolites of (E)-2-ene VPA. For pharmacokinetic studies, a single intravenous (IV) bolus dose of either (E)-2-ene VPA or 4-ene VPA was administered to normal or bile-exteriorized rats. On increasing the dose from 20 to 100 mg/kg in normal rats, the apparent plasma clearance of (E)-2-ene VPA changed from 4.9 ± 1.7 (SD) to 3.0 ± 0.3 mL/min.kg, and of 4-ene VPA decreased from 8.7 ± 0.6 to 5.9 + 0.5 mL/min.kg. A total (conjugates and unconjugates) of 32 + 6% of the low dose and 50 ± 11% of the high dose of (E)-2-ene VPA was recovered in the urine of the rat. The second metabolite, 4-ene VPA, was eliminated in the urine to a relatively smaller extent (22 ± 3% of the low dose and 28 ± 6% of the high dose). In bile-duct cannulated rats, the apparent plasma clearance of (E)-2-ene VPA was 7.7 ± 1.8 mL/min.kg at the low dose and 6.0 ± 1.1 mL/min.kg at the high dose. The corresponding values for 4-ene VPA were 11 ± 1.8 mL/min.kg and 7.4 ± 1.1 mL/min.kg, respectively. The apparent elimination half-life of (E)-2-ene VPA remained unchanged at 20-21 min at the two dose levels, compared to a 1.5 fold increase in the t½ °f 4-ene VPA from 13 ± 2 to 19 ± 3 min. The fraction of the low dose (29 ± 5%) eliminated in bile was significantly larger than at the high dose (21 ± 4%), when calculated as the sum of conjugated and unconjugated 4-ene VPA. The biliary elimination of (E)-2-ene VPA showed a non-significant change from 38 ± 10 to 31 ± 9% on increasing the dose. Like the parent drug VPA, (E)-2-ene VPA and 4-ene VPA showed enterohepatic recirculation in the rat which produced secondary plasma peaks in normal animals. Moreover, both (E)-2-ene VPA and 4-ene VPA showed a rapid but transient choleretic effect in the rat. The plasma protein binding of 4-ene VPA was apparently low (14-25%), in the concentration range of 20-350 µg/mL. The results indicate that 4-ene VPA is cleared much faster from the plasma than (E)-2-ene VPA in the rat. The plasma levels of 4-ene VPA required to show a non-linear decline (>200 µg/mL) in the rat are two orders of magnitude higher than 4-ene VPA levels (<1 µg/ml) seen in patients on VPA therapy. It is, therefore, unlikely that 4-ene VPA is eliminated more slowly than VPA in man. On the other hand, the plasma elimination t½ of (E)-2-ene VPA in bile-exteriorized rats is longer than that reported for VPA, indicating that (E)-2-ene VPA may have a longer lasting pharmacologic effect than VPA. / Pharmaceutical Sciences, Faculty of / Graduate

Modifications to the GCMS assay for valproic acid and 12 metabolites were attempted with respect to internal standards and derivatizing reagents. Four new internal standards, octanoic acid and 2-methylglutaric acid for analysis of VPA and metabolites and hexanoic acid and di-ռ-butylacetic acid for the analysis of hexadeuterated VPA and metabolites were used. Two new derivatizing reagents, MSTFA and MTBSTFA, were tested as alternatives to the reagent previously used. TMS (MSTFA) and tBDMS derivatives were compared with respect to sensitivity, stability, and chromatographic time. The derivatives formed from MTBSTFA were extremely stable a major drawback was the formation of a diderivative of 3-keto VPA upon increased heating time and storage. Preliminary data on the metabolism of D₆-VPA was obtained in one volunteer. The substitution of six deuterium atoms for six hydrogen atoms resulted in an isotope effect with decreased serum trough concentrations of 4-ene VPA and 2,4-diene VPA. Valproic acid and carbamazepine are frequently coadministered in efforts to optimize seizure control. VPA is extensively metabolized while CBZ is known to induce the hepatic microsomal enzyme system, and thus, this is a potentially toxic interaction. Pharmacokinetic parameters for VPA were obtained before and after CBZ administration in five, healthy male volunteers. Increased plasma clearance of VPA accompanied by decreased plasma concentrations, serum half-life, and AUC values were observed after CBZ comedication. This was consistent with the ability of CBZ to induce the hepatic microsomal enzyme systems in a manner similar to phenobarbital. Serum trough and steady state concentrations and AUC values for 12 metabolites were determined before and after CBZ administration. The AUC values for the monounsaturated metabolites decreased after CBZ administration while the AUC values of the polar metabolites increased. The amount of 4-ene VPA, a potential hepatotoxin, was not increased in the serum after administration of CBZ. The amounts of the two diunsaturated metabolites, 2,3'-diene VPA and 2,4-diene VPA, were increased in the serum of the volunteers after CBZ administration. The amount of 2-ene trans VPA in the serum was significantly decreased after CBZ administration, while the amount of 3-keto VPA did not increase. Urinary metabolic profiles were determined individually and grouped in pathways for the five volunteers before and after CBZ administration. Increased recoveries of 4-ene VPA, 4-keto VPA, and 2-PSA after CBZ administration were consistent with enhanced ω-1 oxidation. Formation clearance, metabolic clearance, and fraction metabolized were determined for the metabolic pathways and for the individual metabolites. CBZ adminstration resulted in increased formation clearances for all pathways. The results obtained from this study indicate that CBZ caused a general induction of VPA metabolism and did not specifically affect a particular pathway. The effect of CBZ on the beta-oxidation pathway is not clearly understood. CBZ may cause a metabolic shift away from beta-oxidation, or actually inhibit beta-oxidation to some extent. As well, peroxisomal beta-oxidation may be involved. / Pharmaceutical Sciences, Faculty of / Graduate

Thesis (Ph. D.) -- Virginia Commonwealth University, 2005. / Title from title-page of electronic thesis. Prepared for: Dept. of Pharmaceutics Includes bibliographical references.

Valproic acid (VPA), an antiepileptic drug, possesses a delay in maximum pharmacological response upon initial drug administration, and a prolonged duration of activity following discontinuation of the drug. Metabolites of VPA are thought to be involved as evidence from previous studies in mice demonstrated that (E)-2-ene VPA and (E,E)-2,3'-diene VPA, major products of VPA metabolism in serum, exerted some degree of anticonvulsant activity against pentylenetetrazole (PTZ)-induced seizures. Also associated with VPA therapy is a fatal idiosyncratic hepatotoxicity possibly involving two metabolites, 4-ene VPA and (E)-2,4-diene VPA. Preliminary tissue distribution studies had suggested that (E)-2-ene VPA may not be as hepatotoxic as VPA based solely on (E)-2-ene VPA concentrations in liver. The main objectives of this study were to investigate the kinetic and metabolic profiles, disposition, and anticonvulsant activity of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA in rats. Results of these experiments were intended to provide insight into the possible contributions of these metabolites towards VPA activity or toxicity. Synthesis of (E)-2-ene VPA and (E,E)-2,3'-diene VPA was accomplished by the regiospecific addition of propionaldehyde to an ester enolate, followed by nucleophilic elimination of the mesylate ester with l,8-diazabicyclo[5.4.0]undec-7-ene or potassium hydride. The synthesis provided good yields and was stereoselective. The isomeric purity of the synthesized compounds was found to be 95 - 97% based on nuclear magnetic resonance and gas chromatographyc-mass spectrometric data. The assay of VPA and its metabolites in rat plasma and tissue homogenate extracts was achieved by negative ion chemical ionization gas chromatography-mass spectrometry. This method proved to be selective, sensitive, reproducible, and amenable to automation. In order to compare the disposition and pharmacokinetics of VPA and its analogues, VPA was administered intraperitoneally to rats and the kinetic profiles in plasma, liver, heart, lungs, and nine brain regions were determined. Selective binding of VPA to liver was observed with the liver/plasma ratio at 10 hours after dosing being 4.6. VPA did not persist in brain and the distribution in brain tissue appeared uniform. Metabolites of VPA also were not retained in brain. A most interesting observation was the absence of (E,E)-2,3'-diene VPA in brain while a minor plasma metabolite, (E,Z)-2,3'-diene VPA, was the only detectable diene. A stereoselective active transport mechanism could account for this unusual result. Present in plasma but not detected in liver was (E)-2,4-diene VPA, the hepatotoxic metabolite of VPA. It was proposed that the diene may be covalently bound to liver tissue. Following single dose administration to rats, (E)-2-ene VPA appeared to persist in all tissues assayed following an initial decline phase. The prolonged terminal elimination phase may be attributed to the extensive plasma protein binding of (E)-2-ene VPA (>99%). No selective binding of (E)-2-ene VPA in brain was observed. Brain/plasma ratios at 10 hours after dosing did not exceed 0.03. Metabolites of (E)-2-ene VPA were mainly products of β-oxidation and reduction. Both hepatotoxic metabolites were observed in plasma with concentrations of 4-ene VPA in liver higher than normally seen following VPA administration. Questions arise regarding the potential hepatotoxicity of (E)-2-ene VPA. After single dose administration of (E,E)-2,3'-diene VPA to rats, clearance of the diene was rapid compared to that of VPA or (E)-2-ene VPA. Selective binding of the diene was observed in the superior and inferior colliculus and substantia nigra but the concentrations were too low to be considered clinically significant. Reduction of (E,E)-2,3'-diene VPA appeared to be the main route of metabolism. 4-Ene VPA and (E)-2,4-diene VPA were not detected in plasma or tissues suggesting (E,E)-2,3'-diene VPA may have a lower potential for liver toxicity. The anticonvulsant activities of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA were compared in rats by the PTZ-induced seizure test. Based on ED50 values, the anticonvulsant potencies of VPA and (E)-2-ene VPA were comparable and significantly greater than (E,E)-2,3'-diene VPA. The detection of (E,Z)-2,3'-diene VPA in brain following VPA administration led to the testing of this diene isomer. The potency of the (E,Z)-isomer was found to be equivalent to VPA and (E)-2-ene VPA. Sedation was a severe side effect of (E)-2-ene VPA and the (E,E)-2,3'-diene VPA was stereoselectively unique in causing skeletal muscle rigidity. Sedation was minimal and muscle rigidity was not a property of the (E,Z)-isomer over the dose range studied. Based on the results of these studies, it can be concluded that neither (E)-2-ene VPA nor (E,E)-2,3'-diene VPA is responsible for the pharmacodynamic effects of VPA. From the metabolism of (E,E)-2,3'-diene VPA and the results of anticonvulsant testing, it was proposed that (E,Z)-2,3'-diene VPA may have potential as a relatively safe and useful anticonvulsant drug. / Pharmaceutical Sciences, Faculty of / Graduate

An assay has been developed for the simultaneous quantitative analysis of VPA and twelve of its metabolites namely 2-ene VPA, 3-ene VPA, 4-ene VPA, (E)-2,4-diene VPA, (E,E)-2,3'-diene VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3-keto VPA, 4-keto VPA, 2-propylsuccinic acid and 2-propylglutaric acid. The assay is accomplished in a single run using the Hewlett-Packard 5987A GC-MS. VPA and its metabolites were measured using selected ion monitoring of the characteristic (M-57)⁺ ions of the tertbutyldimethylsilyl (tBDMS) derivatives. The 4-OH VPA was measured as the underivatized ϒ-lactone. [²H₆]-VPA, [²H₃]-2-ene VPA and 3-octanone served as internal standards. In a typical assay 1 mL of urine or serum is adjusted to pH 13 to hydrolyze the conjugates, then acidified to pH 2 and extracted with ethyl acetate. The solvent is then dried, concentrated to 200 μL and derivatized with tBDMS reagent. The chromatographic run time with a 25 m x 0.32 mm I.D. bonded phase OV 1701 capillary column was 22 minutes. All metabolites were well resolved and extracts of control serum or urine showed no interferences. The tBDMS and TMS derivatives of VPA metabolites were compared. Mixed tBDMS-TMS derivatives were also investigated. This assay has been applied to the analysis of urine and serum samples from pediatric patients on VPA therapy. The effects of acetyl salicylic acid (ASA) on the metabolism of VPA were investigated. Urine samples of six pediatric patients under valproic acid therapy and a normal adult subject taking valproic acid were obtained before and after administration of ASA. The analyses showed that the excretion of conjugated and unchanged VPA was enhanced in all subjects following administration of ASA. The 3-keto VPA, the second major urinary metabolite after VPA-glucuronide, was markedly reduced. The average excretion of 2-ene VPA and 3-OH VPA was also lowered. These results suggest that the β-oxidation pathway which includes 2-ene VPA, 3-OH VPA and 3-keto VPA was inhibited by salicylate at some stage prior to the formation of 2-ene VPA. Since serum samples of the normal subject were available the kinetics of VPA metabolites before and after administration of ASA were determined. It was found that the formation clearance, the fraction metabolized and the elimination clearance of the β-oxidation metabolites were decreased following ASA administration. These results confirmed the urinary data obtained with the seven subjects. Salicylate was found to inhibit the β-oxidation of VPA. Other metabolic pathways of VPA were not significantly affected by ASA administration. The mechanism by which salicylate affects the β-oxidation of valproic acid is discussed. / Pharmaceutical Sciences, Faculty of / Graduate

Valproic acid (2-propylpentanoic acid) is an antiepileptic drug widely used for treatment of absence seizures. Valproic acid has a unique chemical structure which does not contain the imide structure found in most conventional antiepileptic drugs. An in vivo study of the antagonism of pentylenetetrazol-induced clonic seizures by alkyl-substituted carboxylic acids and tetrazoles was of interest owing to the known bioisosterism between the carboxylic and the tetrazolyl moiety. The main objective of this study was to investigate the role played by the lipophilicity, the electronic properties and the steric influence of compounds on their anticonvulsant potency. Quantitative structure-activity relationships of the aliphatic and alicyclic substituted carboxylic acids and tetrazoles have been performed using the Hansch linear free-energy relationships model. The study proceeded by synthesis of compounds using known procedures. The di-unsaturated derivatives of valproic acid, 2-[(E)-l'-propenyl]-(E)-2-pentenoic acid and 2-[(Z)-l'-propenyl]-(E)-2-pentenoic acid were prepared via a stereoselective synthetic route. The synthesized di-unsaturated acids were used in identification of the major diunsaturated metabolite of valproic acid as 2-[(E)-l'-propenyl]-(E)-2-pentenoic acid. The anticonvulsant potency of test compounds was determined in mice (CD1 strain, 20-32g) by the standard subcutaneous pentylenetetrazole seizure threshold test. The pentylenetetrazole clonic seizure test was found to be more sensitive to structural effects than the pentylenetetrazole mortality assay. The lipophilicity (octanol-water partition coefficient) of compounds was determined indirectly by reversed phase liquid chromatography employing an octadecylsilane column (Hypersil ODS) and mobile phase as 70% methanol : 30% phosphate buffer (pH 3.5). The electronic character of the compounds was monitored by the apparent acid ionization constant obtained from potentiometric titration in 10% raethanol-water system. The ED₅₀ of 0.70 mmol/kg found for valproic acid was similar to literature values. 5-Heptyltetrazole was found to be the most potent compound in the series of analogues studied. The carboxylic plus tetrazole group gave a low correlation (r = 0.63) between the anticonvulsant potency and a linear combination of lipophilicity and apparent ionization constant. However, in the series of active carboxylic acids, the anticonvulsant activity was noted to be significantly correlated with lipophilicity and apparent ionization constant (r = 0.91). The usefulness of the electronic parameters, acid ionization constant and dipole moment, were explored in an extensive set of alkyl-substituted anticonvulsant compounds with different polar moieties. Addition of the dipole moment term to the lipophilicity term led to significantly better correlations (r = 0.81) as compared to that with an added pKa term. The negative dependency of anticonvulsant activity on dipole moment supported previous findings in studies of 1,4-benzo-diazepines and phenyl-substituted anticonvulsant compounds. There were some exceptions to the dependence of anticonvulsant activity on lipophilicity and dipole moment or pKa. N,N-dibutyl-succinamic acid showed convulsant properties at sublethal doses. The lack of activity of cyclohexylacetic acid and 5-cyclohexylmethyltetra-zole, in comparison to the active l-methylcyclohexanecarboxylic acid, has some pharmacological significance. It shows a certain degree of molecular specificity in the anticonvulsant action of valproic acid analogues. The cyclohexylmethyl conformation was suggested, from aproposed model, to be less effective in hydrophobic binding due to a steric effect at a stereoselective position on the hydrophobic site of the GABA receptor complex. Thus it can be concluded that while lipophilicity governed access to sites of action, the dependence of activity on the polar character may explain the diverse structures of anticonvulsants provided that the steric requirements of the hydrophobic binding site are met. Steric effects may lead to inactivity or even convulsant properties of alkyl-substituted compounds. / Pharmaceutical Sciences, Faculty of / Graduate

Valproic acid (VPA) is a major anticonvulsant drug widely used in the treatment of absence seizures. VPA is extensively metabolized in humans. Several VPA metabolites possess anticonvulsant activity and other metabolites are implicated in rare but fatal cases of hepatotoxicity. A highly sensitive and more specific analytical method was required to analyze the large number of VPA metabolites, some of which are present at trace levels. The objective of this study was to develop such a method and to make a preliminary application of the method to the determination of trace VPA levels and to search for new VPA metabolites. The suitability of analyzing halogenated derivatives of VPA and its metabolites by negative ion chemical ionization (NICI) GCMS was evaluated for the desired sensitivity and specificity. An assay was thus developed for VPA in serum and saliva based on NICI-GCMS of the pentafluorobenzyl (PFB) derivative. The NICI spectrum of the PFB ester of VPA was dominated by a single fragment ion, the m/z 143 ([M-181]⁻) ion. When the m/z 143 ion was monitored the lower limit of detection was 2 ng/mL of VPA in serum or saliva. Using [²H₆]-VPA as the internal standard, the intra- and inter-assay variations were less than 10 % at serum VPA concentrations of 10 to 800 ng/mL. Linearity was observed over the concentration range of 10 ng/mL to 25 µg/mL. The NICI assay was employed to quantitate VPA in serum (total and free) and saliva in five healthy volunteers who took part in a drug interaction study between VPA and carbamazepine (CBZ). A total of 63 paired saliva and serum samples were analyzed by NICI-GCMS; 33 before the administration of CBZ and 30 after CBZ. The % decrease in the average VPA concentration after CBZ was 27.91 ± 3.48, 36.85 ± 13.64, and 48.13 ± 7.70, for serum total, serum free and saliva VPA, respectively. There was a significant reduction (p<0.025) in the average VPA concentration in all three biological fluids. The average saliva to serum free VPA ratio was 18.92% ± 6.25 before CBZ and 16.37% ± 2.82 following CBZ. The average saliva to serum total VPA ratio was 2.43% ± 0.86 before CBZ and 1.67% ± 0.50 following CBZ, indicating that the saliva to serum total VPA ratio was concentration dependent. A strong correlation was found between saliva and both serum free (r = 0.9035 ± 0.0784) and serum total VPA (0.9058 ± 0.0450) (after CBZ). The free fraction of VPA did not increase after CBZ administration suggesting that the decrease in VPA concentration after CBZ was not related to changes in the free fraction of VPA. PFB derivative formation of VPA metabolites was facile and resulted in uniform derivatization of all metabolites studied. In the NICI mass spectra most of the ion current was carried by the [M-181]⁻ fragment ion, the only exception being that of 3-keto VPA. The base peak in the NICI spectrum of PFB derivatized 3-keto VPA was [M-181-C0₂]⁻. Isolated metabolites were identified with the help of twin ions (deuterated and undeuterated) in the mass spectra and by comparison of mass spectra and retention times with synthetic reference compounds. Urine or serum metabolites were analyzed in one chromatographic run and SIM chromatograms obtained. Serum and urine controls showed no interfering peaks and the analytical method appears suitable for a sensitive assay of VPA metabolites. The NICI method employing PFB derivatives was sensitive enough to detect VPA metabolites in saliva. Seven metabolites were detected. The ratio of Z to E isomers of 2-ene VPA was much greater in saliva than in serum (3.82 vs. 0.458), suggesting differences in the transport or plasma protein binding properties of these two isomers. A new VPA metabolite, assigned the structure 4¹-keto-2-ene VPA was detected in urine. The mass spectrum and retention time of this new metabolite matched that of one compound which was present in a synthetic mixture containing 4¹-keto-2-ene VPA. Another new metabolite which appears to be 2-(2¹-propenyl)-glutaric acid was also detected in urine. The synthesis of 4¹-keto-2-ene VPA was attempted using two different synthetic methods. The first method which involved the dehydrogenation of the 0-TMS dialkyl ketene acetal of ethyl 2-propyl-4-oxopentanoate apparently resulted in the formation of the positional isomer, 4-keto-2-ene VPA. The second synthetic route was based on the dehydration of 4-carboethoxy-2-ethylenethioketal-5-hydroxyheptane and produced 4¹-keto-2-ene VPA. However, it was not possible to isolate sufficient product for NMR characterization. / Pharmaceutical Sciences, Faculty of / Graduate

丙戊酸 (VPA) 是一種在臨床上被用來抗驚厥和穩定情緒的藥物。除了治療神經功能紊亂的功效以外, 最近的研究發現VPA可以導致癌細胞的凋亡及分化，增加胚胎幹細胞的分化，並在提升由成體細胞誘導成多功能幹細胞的效率。至今，VPA在幹細胞發育的作用並不十分清楚。在這項研究中，我們使用體外培養體系，來研究VPA在小鼠神經幹細胞，神經前體細胞和胚胎幹細胞的生物學效應。 / 實驗發現VPA可以抑制神經前體細胞株C17.2和神經幹細胞的增生。另一方面，VPA可以增加這兩類細胞分化形成神經元，但同時並沒有增加神經膠質細胞和少突膠質細胞的分化。另一方面，VPA抑制胚胎幹細胞向神經類細胞分化。實驗顯示這種抑制作用源自於VPA對胚胎幹細胞早期譜系分化的抑制。 / 胚胎幹細胞分化實驗顯示，VPA可以抑制胚胎幹細胞向神經外胚層譜系分化，並促進對中胚層和內胚層譜系的分化。經VPA處理後的胚體不能夠產生β-微管蛋白III陽性樹突狀突起。我們還發現，在含血清的培養液中分化的胚體會出現跳動的細胞群。此類細胞群在VPA組裡面的比例較高。因此可以推斷VPA有區增強心肌分化的功能。免疫熒光染色也顯示，VPA處理組中較多細胞為內胚層蛋白標記 FOXA2和甲胎蛋白(AFP)陽性。經VPA處理六天後的胚體胚層標誌基因表達量顯示，神經外胚層標記 Pax6和Noggin在VPA處理組中表達量降低。VPA處理組的中胚層標記brachyury和Mef2c以及內胚層標記GATA4和FOXA2胚層標記的表達量均有上調。 / 在神經幹細胞, 神經前體細胞以及胚胎幹細胞的分化中，可以從VPA處理組觀察到經典 wnt信號通路上調。定量逆轉錄聚合酶鏈反應檢測顯示在VPA處理組裡面, 神經幹細胞, 神經前體細胞以胚體的 wnt信號分子的表達水平的上調。免疫墨點法也檢測到VPA處理組中活化β-catenin較對照組為高。 / Wnt信號通路抑製劑 IWP-2可以破壞棕櫚酰化wnt信號，從而抑制信號分子的成熟和分泌。IWP-2抵銷了VPA 在pNSC中促進神經元的分化的作用。另一方面，抑制wnt信號通路增加了VPA處理組胚體中β-微管蛋白III陽性樹突狀突起的生成, 並且減少跳動細胞群以及內胚層標記蛋白FOXA2和甲胎蛋白陽性的細胞比例。定量逆轉錄聚合酶鏈反應檢測顯示，IWP-2處理組的胚體和對照組相比，神經外胚層標記Pax6和Noggin被上調，中胚層標記 brachyury和Mef2c和內胚層標記 GATA4和FOXA2均被下調。IWP-2 VPA雙重處理組和VPA處理組相比也呈現相同趨勢。這些數據表明，VPA對於神經幹細胞的神經元分化和胚胎幹細胞的胚層譜系分化的影響是通過wnt信號通路介導的。 / 體內研究可以觀察到VPA處理會導致小鼠胚胎中出現的一系列畸形，其中出現有關神經管閉合，異常出血和小腸生長畸形的症狀。胚層標誌分析指出，從VPA注射組孕鼠體內取出的早期階段胚胎具有和在胚胎幹細胞實驗中類似的胚層標記基因表達模式。中胚層和內胚層標記上調而外胚層標記下調。另一方面，VPA在全腦缺血小鼠的治療方面有幫助改善認知功能的作用。 / 這項研究為說明 VPA的致畸作用提供了一個潛在的機制。同時也能夠顯示VPA對全腦缺血患者的潛在治療效果。 / Valproic acid (VPA) is commonly used as a mood stabilizer and anticonvulsant. Despite the clinical relevance to neural disorders, recent studies of VPA revealed the apoptotic and differentiating effects on cancer cells, enhancement of lineage commitments of embryonic stem cells and augmentation of the efficiency in the induction of somatic cells into pluripotent stem cells. Up to date the roles of this small molecule in stem cell development are not well understood. In this study, in vitro culture system was used to elucidate the biological effects of VPA on mouse primary neural stem cells (pNSC), neural precursor cells C17.2 and embryonic stem cells, D3 and E14Tg2a. / VPA was found to inhibit the proliferation of NPC C17.2 and pNSC. On the other hand, it enhanced the differentiation into neurons but not astrocytes and oligodendrocytes. The differentiation of ESC revealed that VPA treatment inhibited the differentiation of ESC into the neuro-ectodermal lineage but promoted the commitment towards the mesodermal and endodermal lineages. / Embryoid bodies (EB) derived from VPA-treated ESC displayed less cell foci with β-tubulin III+ protrusions, but an increase of beating cell clusters, suggesting that VPA enhanced cardio differentiation. Immunofluorescence staining demonstrated that a higher portion of the cells in the VPA-treated group were positive for endodermal markers, FOXA2 and α-fetoprotein. Quantitative RT-PCR (q-PCR) for dermal markers in EB differentiated and treated with VPA for six days showed that neural-ectodermal markers, Pax6 and Noggin, were down-regulated, whereas mesodermal markers, brachyury and Mef2c, and endodermal markers, GATA4 and FOXA2, were all up-regulated. / In studies of the molecular signalling mediated by VPA, quantitative RT-PCR revealed an up-regulation in the gene expression level of wnt molecules in VPA-treated pNSC, NPC and EB. Western blotting also envisaged a higher level of activated β-catenin proteins in VPA treated cells. / The wnt pathway inhibitor IWP-2 was employed to disrupt the palmitoylation of wnt and block the maturation and secretion of the signalling molecule. It was noted that the application of IWP-2 negated the VPA-enhanced neuronal differentiation of pNSC. On the other hand, wnt inhibition increased the incidence of EB having β-tubulin III⁺ protrusions and reduced the numbers of beating EB, FOXA2⁺ and α-fetoprotein⁺ cells in EB-derived cultures of both controls and VPA-treated group. Besides, IWP-2 enhanced the gene expression of neural-ectodermal markers, Pax6 and Noggin, but repressed the gene expression of mesodermal markers, brachyury and Mef2c, and endodermal markers, GATA4 and FOXA2, in EB cultures treated with and without VPA. Data suggest that VPA modulated the neuronal differentiation of pNSC and the dermal commitment of ESC via the wnt pathway. / In vivo study demonstrated that VPA mediated malformations in mouse embryos including deficits in neural tube closure, abnormal bleeding and intestine outgrowth. Dermal marker analysis of VPA-treated embryos at the early stage of development displayed a similar pattern of gene expression noted in in vitro ESC study. Mesodermal and endodermal genes were up-regulated while the ectodermal genes were down-regulated. In global brain ischemic mice, VPA helped restore the cognitive impairment, suggesting the potential therapeutic effect of VPA in global brain ischemia. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lau, Shong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 182-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / List of publications --- p.ii / Abstract --- p.iii / 綜述 --- p.vi / Table of content --- p.viii / Lists of figures --- p.xii / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.2 --- Embryonic stem cells --- p.2 / Chapter 1.3 --- Application of stem cells for therapeutic purpose --- p.2 / Chapter 1.3.1 --- Application of stem cells in neurological disorders --- p.3 / Chapter 1.3.2 --- Application of stem cells in cardiac repair --- p.6 / Chapter 1.3.3 --- Application of stem cells in diabetes --- p.8 / Chapter 1.3.4 --- Application of haematopoietic stem cells --- p.9 / Chapter 1.3.5 --- Application of messenchymal stem cells --- p.10 / Chapter 1.4 --- Reprogramming --- p.11 / Chapter 1.4.1 --- Somatic cell nucleus transfer (SCNT) --- p.11 / Chapter 1.4.2 --- Altered nucleus transfer --- p.11 / Chapter 1.4.3 --- Induced pluripotent stem cells --- p.12 / Chapter 1.5 --- Propagation of ESC --- p.15 / Chapter 1.6 --- Molecular mechanism of ESC pluripotency and self-renewal --- p.16 / Chapter 1.6.1 --- Oct3/4 and Sox2 --- p.16 / Chapter 1.6.2 --- Nanog --- p.17 / Chapter 1.6.3 --- LIF/JAK/STAT3 --- p.17 / Chapter 1.6.4 --- Wnt --- p.18 / Chapter 1.6.5 --- Basic fibroblast growth factor (bFGF) --- p.18 / Chapter 1.6.6 --- TGF-β/Activin/Nodal --- p.18 / Chapter 1.7 --- Wnt signalling pathway --- p.19 / Chapter 1.7.1 --- Canonical wnt pathway --- p.20 / Chapter 1.7.2 --- Planar cell polarity pathway --- p.21 / Chapter 1.7.3 --- Wnt/Ca²⁺ Pathway --- p.22 / Chapter 1.7.4 --- Signal specificity --- p.23 / Chapter 1.7.4.1 --- Wnt 1 --- p.24 / Chapter 1.7.4.2 --- Wnt 2 --- p.25 / Chapter 1.7.4.3 --- Wnt3/wnt3A --- p.25 / Chapter 1.7.4.4 --- Wnt4 --- p.27 / Chapter 1.7.4.5 --- Wnt5 --- p.28 / Chapter 1.7.4.6 --- Wnt7 --- p.29 / Chapter 1.7.4.7 --- Other wnts --- p.31 / Chapter 1.8 --- Valproic acid --- p.33 / Chapter 1.8.1 --- Effect of VPA on histone modification --- p.34 / Chapter 1.8.2 --- Interaction of VPA with extracellular signal-regulated kinase pathway --- p.35 / Chapter 1.8.3 --- Effect of VPA on PI3K pathway --- p.37 / Chapter 1.8.4 --- Effect of VPA on wnt pathway --- p.37 / Chapter 1.8.5 --- VPA on stem cells --- p.38 / Chapter 1.8.6 --- VPA on brain ischemia animal model --- p.39 / Chapter 1.9 --- Overall aim and design of the Study --- p.41 / Chapter 2.1 --- Cells /cell lines --- p.53 / Chapter 2.1.1 --- Neural precursor cell line C17.2 --- p.53 / Chapter 2.1.2 --- Primary neural stem cells --- p.53 / Chapter 2.1.3 --- Primary mouse embryonic fibroblast --- p.53 / Chapter 2.1.4 --- Embryonic stem cell line D3 --- p.54 / Chapter 2.1.5 --- Embryonic stem cell line E14TG2a --- p.54 / Chapter 2.2 --- Cell cultures and assays --- p.54 / Chapter 2.2.1 --- Medium and solutions --- p.54 / Chapter 2.2.2 --- Isolation of primary neural stem cells --- p.58 / Chapter 2.2.3 --- Maintenance and passaging of primary neural stem cells --- p.60 / Chapter 2.2.4 --- Cryopreservation and thawing of primary neural stem cells --- p.60 / Chapter 2.2.5 --- Coating of coverslips using ornithine and laminin (O&L) --- p.61 / Chapter 2.2.6 --- Differentiation of primary neural stem cells --- p.61 / Chapter 2.2.7 --- Isolation of mouse embryonic fibroblasts --- p.62 / Chapter 2.2.8 --- Maintenance and passaging of mouse embryonic fibroblasts --- p.63 / Chapter 2.2.9 --- Cryopreservation and thawing of mouse embryonic fibroblasts --- p.63 / Chapter 2.2.10 --- Preparation of gelatin-coated culture wares and cover slips --- p.64 / Chapter 2.2.11 --- Preparation of irradiated mouse embryonic fibroblast feeder layer --- p.64 / Chapter 2.2.12 --- Maintenance and passaging of neural precursor cell C17.2 --- p.64 / Chapter 2.2.13 --- Cryopreservation and thawing of neural precursor cell C17.2 --- p.64 / Chapter 2.2.14 --- Differentiation of neural precursor cell C17.2 --- p.65 / Chapter 2.2.15 --- Maintenance and passaging of mouse embryonic stem cell line --- p.65 / Chapter 2.2.16 --- Cryopreservation and thawing of mouse embryonic stem cell line --- p.65 / Chapter 2.2.17 --- Spontaneous differentiation of embryonic stem cells --- p.66 / Chapter 2.2.18 --- Neural differentiation of embryonic stem cells --- p.66 / Chapter 2.2.19 --- WST-1 proliferation assay --- p.67 / Chapter 2.2.20 --- Colony formation assay --- p.67 / Chapter 2.2.21 --- Two-stage neural differentiation assay --- p.68 / Chapter 2.3 --- Molecular analysis --- p.68 / Chapter 2.3.1 --- In-house prepared solutions and reaction mixes --- p.68 / Chapter 2.3.2 --- RNA extraction --- p.72 / Chapter 2.3.3 --- Synthesis of complementary DNA (cDNA) by reverse transcription --- p.73 / Chapter 2.3.4 --- Polymerase chain reaction (PCR) --- p.74 / Chapter 2.3.5 --- Quantitative polymerase chain reaction (qPCR) --- p.74 / Chapter 2.3.6 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting --- p.75 / Chapter 2.4 --- Microscopy and Immunofluorescence staining --- p.77 / Chapter 2.5 --- In vivo Study --- p.77 / Chapter 2.5.1 --- Embryo malformation study --- p.77 / Chapter 2.5.2 --- Establishment of a mouse global brain ischemia model --- p.78 / Chapter 2.5.3 --- Behaviour study --- p.79 / Chapter 2.5.4 --- Histological analysis --- p.79 / Chapter 2.5.4.1 --- Animal sacrifice for brain harvest --- p.79 / Chapter 2.5.4.2 --- Paraffin sectioning --- p.80 / Chapter 2.5.4.3 --- Haematoxylin and eosin staining --- p.80 / Chapter 2.5.4.4 --- Cresyl violet staining --- p.81 / Chapter 2.5.4.5 --- TUNEL assay --- p.81 / Chapter 2.6 --- Statistical analysis --- p.82 / Chapter 2.7 --- Equipments used in this study --- p.82 / Chapter Chapter 3 --- Effects of VPA on proliferation and differentiation of mouse NPC, pNSC and ESC --- p.88 / Chapter 3.1 --- Effects of VPA on mouse ESC proliferation --- p.88 / Chapter 3.1 --- Effects of VPA on mouse ESC proliferation --- p.88 / Chapter 3.2 --- Effects of VPA on differentiation and proliferation of neural lineage cells --- p.89 / Chapter 3.2.1 --- Effects of VPA on differentiation and proliferation of NPC C17.2 --- p.89 / Chapter 3.2.2 --- Effects of VPA on differentiation of pNSC --- p.89 / Chapter 3.2.3 --- Effects of VPA on proliferation and stemness of pNSC --- p.90 / Chapter 3.2.4 --- Effects of VPA on neural differentiation of mouse ESC --- p.91 / Chapter 3.2.5 --- Two-stage neural differentiation assay of mouse ESC --- p.91 / Chapter 3.3 --- VPA-mediated lineage commitment of mouse ESC --- p.92 / Chapter 3.3.1 --- Cardio differentiation of ESC --- p.92 / Chapter 3.3.2 --- Endodermal commitment of mouse ESC --- p.93 / Chapter 3.3.3 --- Germ layer marker expression in mouse ESC --- p.93 / Chapter 3.3.4 --- Up-regulation of histone acetylation in VPA-treated mouse ESC --- p.94 / Chapter 3.6 --- Summary --- p.94 / Chapter Chapter 4 --- Wnt signalling played as a mediator of VPA in the differentiation of ESC, NPC and pNSC differentiation. --- p.118 / Chapter 4.1 --- VPA treatment up-regulated wnt pathway in mouse ESC, NPC and pNSC --- p.118 / Chapter 4.2 --- Effect of wnt inhibitor IWP-2 on stem cells in differentiation cultures supplemented with VPA. --- p.119 / Chapter 4.2.1 --- Effects of wnt inhibitor IWP-2 on the neural differentiation of NPC C17.2 --- p.119 / Chapter 4.2.2 --- Effect of wnt inhibitor on differentiation of pNSC --- p.120 / Chapter 4.2.3 --- Effect of IWP-2 on the differentiation culture of EB --- p.121 / Chapter 4.2.4 --- Effect of wnt inhibitor on the lineage commitment of mouse ESC --- p.121 / Chapter 4.2.5 --- Effects of IWP-2 on VPA-induced lineage markers expressed by mouse ESC --- p.123 / Chapter 4.3 --- Effects of wnt inhibitor IWP-2 on ESC proliferation. --- p.124 / Chapter 4.4 --- Summary --- p.125 / Chapter Chapter 5 --- In vivo effects of VPA --- p.154 / Chapter 5.1 --- Effects of VPA on mouse embryo development --- p.154 / Chapter 5.1.1 --- Effects of VPA at the daily dose of 300mg/kg body weight --- p.154 / Chapter 5.1.2 --- Effects of VPA at the daily dose of 600 mg/kg body weight --- p.155 / Chapter 5.1.3 --- Effects of VPA at twice daily dose of 600 mg/kg body weight --- p.155 / Chapter 5.2 --- Effects of VPA on ischemic mice --- p.156 / Chapter 5.2.1 --- Behaviour test --- p.156 / Chapter 5.2.2 --- Histological and molecular assessment --- p.157 / Chapter 5.3 --- Summary --- p.157 / Chapter Chapter 6 --- Discussion --- p.171 / Chapter 6.1 --- Modulation of ESC fate at early developmental stage --- p.171 / Chapter 6.2 --- Neural differentiation of pNSC and NPC upon VPA treatment --- p.172 / Chapter 6.3 --- VPA-mediated wnt signaling on the differentiation of mouse ESC --- p.173 / Chapter 6.4 --- VPA-mediated wnt signalling in the neuronal differentiation of mouse neural stem/progenitor cells --- p.175 / Chapter 6.5 --- Effects of VPA on the proliferation of mouse ESC, pNSC and NPC --- p.176 / Chapter 6.6 --- Effects of VPA on mouse embryo --- p.177 / Chapter 6.7 --- Therapeutics of VPA in ischemic stroke --- p.178 / Chapter Chapter 7 --- Conclusion --- p.180 / References --- p.182