Thesis (M. P. H.)--University of Hong Kong, 2008 . / Also available in print.

The severe hepatotoxicity of the antiepileptic drug valproic acid (VPA) is believed to be mediated through chemically reactive metabolites. The monounsaturated metabolite 4-ene VPA is steatogenic in the rat, and in a similar fashion to the hepatotoxin 4-pentenoic acid, is thought to be oxidized by mitochondria to a highly reactive α,β-unsaturated ketone, 3-keto-4-ene VPA. The tripeptide thiol, glutathione (GSH), is known to react with a variety of electrophilic compounds that have the potential to interact with cellular macromolecules. The identification and structural characterization of GSH conjugates provides a means of identifying short-lived unstable electrophiles and thus an insight into the mechanisms of toxicity. This thesis describes the synthesis and characterization of thiol conjugates of reactive metabolites derived from the in vivo metabolism of VPA, 4-ene VPA, (E)-2,4-diene VPA, and 4-pentenoic acid. A negative ion chemical ionization gas chromatographic/mass spectrometric (NICI/GC/MS) method for the determination of VPA and 14 of its metabolites in a single chromatographic run was developed. A combination of pentafluorobenzyl and trimethylsilyl derivatization resulted in the [M-181]̄̄̄ ̄anion as the base peak for all the metabolites measured. When these ions were monitored sensitivities in the low picogram range were achieved. The VPA metabolite profile was determined in pediatric patients on VPA monotherapy and on combined therapy with either carbamazepine or clobazam. 4-Ene- and (E)-2,4-diene-VPA were found to be minor metabolites with serum levels below 1% that of VPA. In patients on combined therapy with carbamazepine, the ω and ω-l pathways of VPA metabolism were induced, while products of β-oxidation were significantly decreased. Polytherapy had no significant effect on the serum levels of 4-ene- or (E)-2,4-diene-VPA. Rats were dosed intraperitoneally with 100 mg/kg of the sodium salts of VPA, 4-ene-, (E)-2,4-diene-VPA, 4-pentenoic or (E)-2,4-pentadienoic acids. Methylated bile extracts were analyzed by high pressure liquid chromatography and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for GSH conjugates while urine samples were analyzed by GC/MS and LC/MS for N-acetylcysteine (NAC) conjugates and other metabolites. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene-VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The structures of these metabolites were confirmed by comparing GC/MS or LC/MS properties of the isolated metabolites to those of synthetic standards. The GSH and NAC conjugates of (E)-2,4-diene VPA were chemically synthesized and their structures established to be (E)-5-(glutathion-S-yl)-3-ene VPA and (E)-5-(N-acetylcystein-S-yl)-3-ene VPA by nuclear magnetic resonance spectroscopy and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo (about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr). In rats treated with 4-pentenoic and/or (E)-2,4-pentadienoic acids the following conjugates were identified and characterized by synthesis: GSH and cysteine conjugates of 3-oxo-4-pentenoic acid, GSH and NAC conjugates of (E)-2,4-pentadienoic acid, and the NAC conjugate of acrylic acid. The results thus provided the first direct biochemical evidence for the in vivo formation of the metabolite of 4-pentenoic acid considered responsible for the irreversible inhibition of fatty acid metabolism. The results also revealed basic differences between the mitochondrial metabolism of 4-ene VPA and 4-pentenoic acid. The 3-keto-4-ene VPA and its GSH and NAC conjugates were synthesized in order to facilitate the in vivo identification of these compounds following the administration of VPA, 4-ene-, or (E)-2,4-diene-VPA to rats. However, neither the 3-keto-4-ene VPA nor its thiol derivatives were evident in any of the treatments. The NAC conjugate of (E)-2,4-diene VPA was also found to be a metabolite of VPA in patients. The level of the conjugate appeared to be higher in two patients who recovered from VPA-induced liver toxicity. The characterization of GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug. / Pharmaceutical Sciences, Faculty of / Graduate

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 164-188).

published_or_final_version / Community Medicine / Master / Master of Public Health

Valproic acid (VPA) is a broad-spectrum anticonvulsant and antiepileptic drug and widely used in many neurological conditions and psychiatric disorders as well as in cancer and HIV treatment. However, despite all its many benefits, VPA also has side effects. It is a strong fetal teratogen that can induce congenital malformation and neurodevelopmental problems. Case reports and population studies have revealed that prenatal exposure to VPA is associated with a higher risk of autism in postnatal life. Animal models also have confirmed that VPA can induce autistic-like features in rodents. Yet, there are some questions remaining unanswered by existing animal studies of prenatal VPA exposure. The embryotoxicity of a drug is not only determined by its own chemical, physiological or pharmacological properties, but also on the dose and the time in development that the exposure happens. The majority of studies investigating the behavioral, neuroanatomical and physiological impact of VPA in animals have examined early gestational exposure to relatively high doses that can cause significant malformation or loss of offspring. Thus, although there are behavioral alterations considered similar to autistic symptoms in humans, these are found in the offspring that have survived a very toxic insult, leading to problems of interpretation. Low dose exposure has not been widely studied, nor has the impact of VPA exposure late in gestation. Moreover, how the age and sex of offspring influences the phenotypic outcome has rarely been considered. Therefore, in the present series of studies, a battery of behavioral tests and in vivo magnetic resonance imaging (MRI) was used to investigate the postnatal consequences of prenatal exposure to lower doses of VPA in mice in early and late gestation. The effects of VPA were examined in female and male mice at juvenile and adult ages. The main findings were, that low doses of VPA in early or late gestation cause no physical malformation and no gross neurological functional impairments, but induce behavioral abnormalities and neuroanatomical differences related to autism. Generally, VPA-treated mice exhibited lower motor activities and higher anxiety levels in the open field test; dislike of novelty in the novel abject exploration test; higher startle response and sensorimotor gating differences; decreased responses to non-social and social odors in the olfactory test; and volumetric changes in brain structures similar to those found in autism. However, the timepoint of exposure, dose of VPA, sex and age of testing influenced the phenotypic outcome. Although largely neglected in previous studies, late gestation exposure to VPA elicited an autistic phenotype. Surprisingly, given the male bias in autism, female mice were often more ‘sensitive’ to VPA. Although the present studies had some limitations, these experiments confirmed that low dose VPA in pregnancy could trigger behavioral abnormalities and brain anatomical differences in mice that resembled a range of features of autism. Importantly, these behaviors were unconfounded by ‘gross’ neurological or physical abnormalities. Further studies to investigate the cellular mechanisms underlying the low dose VPA phenotype will therefore be helpful to shed light on possible causal pathways with specific relevance to autism. / published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

Autism, a neurodevelopmental disorder, is assumed to result from early neural tube damage. Individuals with Autism exhibit macroencephaly during childhood. To examine increased neurogenesis as a factor in macroencephaly, the valproic acid (VPA) model of Autism was used to examine how exposure to enrichment affects neurogenesis in the dentate gyrus. To induce the model, pregnant rats received two 100mg/kg VPA injections on days 11, 12, and 13 of gestation. Half the pups in each group were exposed to enrichment from post-natal days 30-60. Neurogenesis was examined by fluorescence microscopy for the proliferation marker bromodeoxyuridine (BrdU) and neuronal specific nuclear marker (NeuN). Counts of double-labeled cells were done from the dentate gyrus, an area known for adult neurogenesis. Results indicate that neurogenesis is not abnormal in the VPA model and enrichment increases the neurogenesis similarly in both VPA and control animals. This research provides a better understanding of brain plasticity in the VPA model of Autism.

Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70 years). The relative survival of patients with CLL has been shown to decrease with patient age, and this age-related reduction in survival was found to correlate with the levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate positively with patient age, and increased levels were associated with lower overall survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8 secretion. In a search of a treatment regimen that may be effective and readily tolerated by elderly patients, we examined the combination of fludarabine with valproic acid (VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The combination was synergistic against human leukaemic cells, including primary CLL cells. In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine combination induced reduction in the peripheral and lymph node tumour loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes, while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease. The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro), primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine combination also lowered phospho-Akt levels and ATM activation, which also contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels and phospho-Akt levels in vivo. In summary, there lies a biological explanation for the poor survival observed with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.

Autism, a neurodevelopmental disorder, is assumed to result from early neural tube damage. Individuals with Autism exhibit macroencephaly during childhood. To examine increased neurogenesis as a factor in macroencephaly, the valproic acid (VPA) model of Autism was used to examine how exposure to enrichment affects neurogenesis in the dentate gyrus. To induce the model, pregnant rats received two 100mg/kg VPA injections on days 11, 12, and 13 of gestation. Half the pups in each group were exposed to enrichment from post-natal days 30-60. Neurogenesis was examined by fluorescence microscopy for the proliferation marker bromodeoxyuridine (BrdU) and neuronal specific nuclear marker (NeuN). Counts of double-labeled cells were done from the dentate gyrus, an area known for adult neurogenesis. Results indicate that neurogenesis is not abnormal in the VPA model and enrichment increases the neurogenesis similarly in both VPA and control animals. This research provides a better understanding of brain plasticity in the VPA model of Autism.

Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70 years). The relative survival of patients with CLL has been shown to decrease with patient age, and this age-related reduction in survival was found to correlate with the levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate positively with patient age, and increased levels were associated with lower overall survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8 secretion. In a search of a treatment regimen that may be effective and readily tolerated by elderly patients, we examined the combination of fludarabine with valproic acid (VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The combination was synergistic against human leukaemic cells, including primary CLL cells. In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine combination induced reduction in the peripheral and lymph node tumour loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes, while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease. The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro), primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine combination also lowered phospho-Akt levels and ATM activation, which also contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels and phospho-Akt levels in vivo. In summary, there lies a biological explanation for the poor survival observed with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.

Abstract Epilepsy is associated with reproductive disorders and decreased fertility. The role of antiepileptic medication and type of epilepsy in development of these disorders has been widely debated. The effects of oxcarbazepine on reproductive function in women and the effects of antiepileptic medication on male fertility have not been previously studied, and only a few studies have evaluated fertility in subjects with epilepsy in a population based setting. This study aimed to analyze predictors of reproductive disorders and the effects of oxcarbazepine on reproductive function in women. Moreover, the effects of antiepileptic medication on male reproductive health were also evaluated, and finally, the reproductive health of patients with epilepsy and the normal population was compared in a population based setting. The study was conducted in the Departments of Neurology, Gynecology and Obstetrics and Public Health Science and General Practice in the University of Oulu. Studies I–III were cross-sectional studies consisting of 249 subjects with epilepsy and 247 control subjects. Study IV was a retrospective study; the data was based on Northern Finland Birth Cohort 1966(NFBC1966), consisting of 12,058 subjects, of which 222 had epilepsy. In studies I–III all subjects were interviewed, clinical examinations were done, blood samples were analyzed and ovarian ultrasound examination or testicular ultrasound examination and sperm samples were studied. In study IV all subjects with epilepsy were identified from NFBC1966 and patient files were reviewed. Fertility analyses were based on information obtained from the Finnish Population Center and Finnish Birth Register. Reproductive disorders were more common in women with idiopathic generalized epilepsy and in women taking valproate. Also young age increased the risk of these disorders. Oxcarbazepine was associated with reproductive disorders in women with epilepsy. In men all antiepileptic drugs studied were associated with sperm abnormalities, and sperm abnormalities in men taking valproate were associated with decreased testicular volume. In a population based setting active epilepsy and antiepileptic medication during adulthood decreased fertility. The reproductive endocrine effects of AEDs should be taken into consideration when prescribed to fertile aged men and women, especially, if the anticipated duration of treatment is long.