This thesis describes the preparation of 5-substituted-amino derivatives of 5-phenylhydantoin. The hydantoin derivatives are to be tested for anticonvulsant activity by the Pharmacology Department of the Eli Lilly Company of Indianapolis, Indiana.

<p>  </p> <p>Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs (ASD) and treatment methods. Therefore, a critical need exists to efficiently screen ASDs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel ASDs. Here, we present an optimized PTZ assay to improve reliability and reproducibility based on work in our laboratory. This optimized assay improves robustness in our screening of anti-seizure drugs (topiramate, lamotrigine, carbamazepine and GS967). These findings show that electroencephalogram (EEG) and calcium sensitive GFP from fusion protein (GCaMP) assays largely correlate with the behavioral findings, helping us connect physiological and behavioral responses to ASDs. Genetic epilepsy syndromes, like voltage gated sodium channel <em>SCN2A</em> and <em>SCN8A</em> pathogenic variants, are often poorly controlled by current medications. Our optimized assay relied on a fast and precise zebrafish seizure model using mRNA overexpression of h<em>SCN2A</em> and h<em>SCN8A</em> variants including: hSCN2A R1882Q and R853Q and hSCN8A R1872Q. All three pathogenic variants increased seizure activity, and the ASDs significantly decreased this seizure activity. This mRNA overexpression assay can be used to quickly evaluate seizure activity induced by pathogenic variants in voltage gated sodium channel genes and test ASDs to determine efficacy. In a separate study, we tested if the addition of the human <em>SCN2A </em>sodium channel could potentially rescue the loss of the zebrafish scn1Lab gene. Our GCaMP assay data indicates that this loss was successfully rescued. Cumulatively, these findings can be used to improve the screening of novel ASDs and treatments for patients with refractory epilepsy.</p>

The use of traditional and natural medicines in primary healthcare or alternative therapy is on the increase. However, the safety and efficacy of these medicines have not yet been confirmed. Pharmacognosy, the study of the properties of drugs, potential drugs or drug substances of natural origin and the search for new drugs from natural resources, is therefore of extreme importance in today’s healthcare environment. Cotyledon orbiculata L. var. orbiculata (Haw.) DC., a succulent shrub that is widely distributed over the whole of southern Africa, is an example of a plant used in traditional medicine for its antiepileptic effects. Oxidative stress can either be the cause of, or be secondary to epilepsy pathogenesis. Lipid peroxidation causes the disruption of cell membranes which leads to cell destruction and, in the case of neurological disorders, neurodegeneration. Reactive species have also been found to influence neurotransmission by affecting neurotransmitter metabolism and functions. Reactive species can therefore be responsible for the development of convulsions. Conventional anti-epileptics have shown to exert neuroprotective effects but information or research regarding their ability to prevent epilepsy from becoming chronic does either not exist or is not promising. Antioxidants have potential in the treatment of epileptic seizures as well as the prevention of chronic epilepsy by preventing the effects that oxidative stress has on neurotransmitter metabolism and functions that cause alterations in neuronal excitability and seizure threshold, ultimately leading to epileptic foci. The aim of this study was to evaluate the potential of the bufadienolide orbicusides of C. orbiculata and analogues as anti-epileptic treatment through antioxidant activity. Initially the isolation of novel antioxidants from C. orbiculata leaf juice was attempted. The antioxidant activity of the concentrated juice and fractions resulting thereof were evaluated with two assays. The thiobarbituric acid (TBA) assay was used to measure the extent of lipid peroxidation and nitroblue tetrazolium (NBT) assay was used to measure superoxide scavenging activity in rat brain homogenate. The low concentrations of orbicusides prompted the determination of the activity of two commercial bufadienolides (bufalin and cinobufotalin) and two bufadienolide analogues, synthesised by the esterification of trans-androsterone and androstanolone, respectively, using coumalic acid, producing Compound 1 and Compound 2. The toxicity of the commercial bufadienolides and synthesised analogues were evaluated by using the MTT assay (a cell viability assay). C. orbiculata juice showed significant pro-oxidant activity in both assays. Bufalin showed significant pro-oxidant activity in the TBA assay. Cinobufotalin showed no significant activity. Compound 1 showed pro-oxidant activity in the TBA assay and Compound 2 showed slight antioxidant activity in the NBT assay. The commercial bufadienolides showed low cell viability, indicating significant toxicity. The synthesised analogues showed a significant reduction in toxicity (despite Compound 2 being moderately toxic) when compared to the toxicity of the commercial bufadienolides. The low concentrations of orbicusides in the plant material and the antioxidant assay results of the two commercial bufadienolides suggested that the orbicusides may not be involved in the antioxidant properties of C. orbiculata. However, the antioxidant activity of Compound 2 showed that altering the pyrone moiety of bufadienolides could possibly improve antioxidant activity. The reduced toxicity and slight antioxidant activity of the synthesised bufadienolide analogues motivates further investigation. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

The use of traditional and natural medicines in primary healthcare or alternative therapy is on the increase. However, the safety and efficacy of these medicines have not yet been confirmed. Pharmacognosy, the study of the properties of drugs, potential drugs or drug substances of natural origin and the search for new drugs from natural resources, is therefore of extreme importance in today’s healthcare environment. Cotyledon orbiculata L. var. orbiculata (Haw.) DC., a succulent shrub that is widely distributed over the whole of southern Africa, is an example of a plant used in traditional medicine for its antiepileptic effects. Oxidative stress can either be the cause of, or be secondary to epilepsy pathogenesis. Lipid peroxidation causes the disruption of cell membranes which leads to cell destruction and, in the case of neurological disorders, neurodegeneration. Reactive species have also been found to influence neurotransmission by affecting neurotransmitter metabolism and functions. Reactive species can therefore be responsible for the development of convulsions. Conventional anti-epileptics have shown to exert neuroprotective effects but information or research regarding their ability to prevent epilepsy from becoming chronic does either not exist or is not promising. Antioxidants have potential in the treatment of epileptic seizures as well as the prevention of chronic epilepsy by preventing the effects that oxidative stress has on neurotransmitter metabolism and functions that cause alterations in neuronal excitability and seizure threshold, ultimately leading to epileptic foci. The aim of this study was to evaluate the potential of the bufadienolide orbicusides of C. orbiculata and analogues as anti-epileptic treatment through antioxidant activity. Initially the isolation of novel antioxidants from C. orbiculata leaf juice was attempted. The antioxidant activity of the concentrated juice and fractions resulting thereof were evaluated with two assays. The thiobarbituric acid (TBA) assay was used to measure the extent of lipid peroxidation and nitroblue tetrazolium (NBT) assay was used to measure superoxide scavenging activity in rat brain homogenate. The low concentrations of orbicusides prompted the determination of the activity of two commercial bufadienolides (bufalin and cinobufotalin) and two bufadienolide analogues, synthesised by the esterification of trans-androsterone and androstanolone, respectively, using coumalic acid, producing Compound 1 and Compound 2. The toxicity of the commercial bufadienolides and synthesised analogues were evaluated by using the MTT assay (a cell viability assay). C. orbiculata juice showed significant pro-oxidant activity in both assays. Bufalin showed significant pro-oxidant activity in the TBA assay. Cinobufotalin showed no significant activity. Compound 1 showed pro-oxidant activity in the TBA assay and Compound 2 showed slight antioxidant activity in the NBT assay. The commercial bufadienolides showed low cell viability, indicating significant toxicity. The synthesised analogues showed a significant reduction in toxicity (despite Compound 2 being moderately toxic) when compared to the toxicity of the commercial bufadienolides. The low concentrations of orbicusides in the plant material and the antioxidant assay results of the two commercial bufadienolides suggested that the orbicusides may not be involved in the antioxidant properties of C. orbiculata. However, the antioxidant activity of Compound 2 showed that altering the pyrone moiety of bufadienolides could possibly improve antioxidant activity. The reduced toxicity and slight antioxidant activity of the synthesised bufadienolide analogues motivates further investigation. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Prior work in the Durst group had generated more than forty analogs of the potent anticonvulsant isoxylitone isolated isolated from a medicinal plant Delphinium denudatum Wall. The nitrile designated as TD532 was the most potent compound generated by A. Saikaley. The starting material for the synthesis of TD532 is isophorone. The observation that TD532 showed considerable potential as an anticonvulsant suggested that other cyclohexenones might have have similar activity. During this project close to fifty derivatives of cyclohex-2-enone, focusing mainly on 3-arylcylohex-2-enones, were prepared. The synthesis of these compounds is described and structure activity relationships are discussed. Based on all the available structure activity data, we have designated the indicated portion of structure A as the pharmacophore for anticonvulsant and anti-epileptic activity. The ester designated as TD561 (compound 40) showed excellent potential in both in vitro and in vivo assays. It has been shown to be a pro-drug of the corresponding acid TD562 (compound 48). These two compounds and the sodium salt of TD562 are currently undergoing final pre-clinical studies at the Center for Drug Research and Development in Vancouver. Five analogs, including TD561 are also under investigation by the Epilepsy and Seizure Division of the US National Institutes of Health.

Die Bedeutung des Immunsystems in der Pathophysiologie von bipolaren Erkrankungen und Epilepsie ist ein aktueller Gegenstand der neuropsychoimmunologischen Forschung. Eine erhöhte Produktion von Zytokinen aufgrund von oxidativem Stress wurde dabei wiederholt als für die Pathophysiologie von Epilepsie und Bipolarer Störung relevant angesehen. In Hinblick auf Veränderungen der Zytokine Interleukin (IL)-1ß, IL-2, IL-4, IL-6 und Tumornekrosefaktor-alpha (TNF-α) wurden z. T. überlappende Ergebnisse bei beiden Erkrankungen beschrieben. Inwiefern diese Zytokine durch Stimmungsstabilisierer und Antiepileptika beeinflusst werden, wurde bisher jedoch nicht systematisch untersucht. In dieser Studie wurden systematisch in-vitro die Konzentrationen von IL-1ß, IL-2, IL-4, IL-6, IL-17, IL-22 und TNF-α im stimulierten Blut 14 gesunder Frauen mittels Vollblutverfahren (whole blood assay) nach Zugabe von Stimmungsstabilisierern bzw. Antiepileptika gemessen. Es wurden dabei die Stimmungsstabilisierer bzw. Antiepileptika Primidon, Carbamazepin, Levetiracetam, Lamotrigin, Valproat, Oxcarbazepin, Topiramat, Phenobarbital und Lithium untersucht. Die Ergebnisse lassen darauf schließen, dass der Mechanismus von erwünschter und unerwünschter anderer Wirkung von Stimmungsstabilisierern und Antiepileptika mit der Regulation von IL-1ß, IL-2, IL-22 und TNF-α in Zusammenhang stehen könnte. Getrennt nach den im Vollblutverfahren verwendeten Stimulanzien – Toxic-Shock-Syndrome-Toxin-1 (TSST-1) vs. monoklonaler Antikörper gegen das CD3-Oberflächenantigen (OKT3) in Kombination mit dem 5C3-Antikörper gegen CD40 (OKT3/CD40) – wurden die Ergebnisse in zwei unterschiedlichen Publikationen berichtet, die dieser Promotion zugrunde liegen.

Die Bedeutung des Immunsystems in der Pathophysiologie von bipolaren Erkrankungen und Epilepsie ist ein aktueller Gegenstand der neuropsychoimmunologischen Forschung. Eine erhöhte Produktion von Zytokinen aufgrund von oxidativem Stress wurde dabei wiederholt als für die Pathophysiologie von Epilepsie und Bipolarer Störung relevant angesehen. In Hinblick auf Veränderungen der Zytokine Interleukin (IL)-1ß, IL-2, IL-4, IL-6 und Tumornekrosefaktor-alpha (TNF-α) wurden z. T. überlappende Ergebnisse bei beiden Erkrankungen beschrieben. Inwiefern diese Zytokine durch Stimmungsstabilisierer und Antiepileptika beeinflusst werden, wurde bisher jedoch nicht systematisch untersucht. In dieser Studie wurden systematisch in-vitro die Konzentrationen von IL-1ß, IL-2, IL-4, IL-6, IL-17, IL-22 und TNF-α im stimulierten Blut 14 gesunder Frauen mittels Vollblutverfahren (whole blood assay) nach Zugabe von Stimmungsstabilisierern bzw. Antiepileptika gemessen. Es wurden dabei die Stimmungsstabilisierer bzw. Antiepileptika Primidon, Carbamazepin, Levetiracetam, Lamotrigin, Valproat, Oxcarbazepin, Topiramat, Phenobarbital und Lithium untersucht. Die Ergebnisse lassen darauf schließen, dass der Mechanismus von erwünschter und unerwünschter anderer Wirkung von Stimmungsstabilisierern und Antiepileptika mit der Regulation von IL-1ß, IL-2, IL-22 und TNF-α in Zusammenhang stehen könnte. Getrennt nach den im Vollblutverfahren verwendeten Stimulanzien – Toxic-Shock-Syndrome-Toxin-1 (TSST-1) vs. monoklonaler Antikörper gegen das CD3-Oberflächenantigen (OKT3) in Kombination mit dem 5C3-Antikörper gegen CD40 (OKT3/CD40) – wurden die Ergebnisse in zwei unterschiedlichen Publikationen berichtet, die dieser Promotion zugrunde liegen.

Disease- and drug-related bone disorders are rapidly increasing in the population. The drugs which are used for the treatment of neurodegenerative diseases and metabolic derangements, may have negative or positive effects on bone tissues. In the first study, the possible side-effects of Carbamazepine and epileptic seizures on bone structure and composition were investigated by FTIR and synchrotron-FTIR microspectroscopy, AFM and micro- and nano-hardness analysis. The effects on the blood parameters, bone turnover and vitamin D metabolism were also investigated by ELISA and western blot analysis. The current study provides the first report on differentiation of the effects of both epileptic seizures and AED therapy on bones. Besides Carbamazepine treatment, seizures also caused a decrease in the strength of bone. The biochemical data showed that both the epileptic and drug-treated groups decreased vitamin D levels by increasing the vitamin D catabolism enzyme / 25-hydroxyvitamin D-24-hydroxylase. In the second study, the possible pleiotropic (positive) effects of cholesterol lowering drug / Simvastatin on bones were investigated by ATR-FTIR spectroscopy. The current study provides the first report on dose-dependent effects of simvastatin on protein structure and lipid conformation of bones. ATR-FTIR studies showed that although both high and low dose simvastatin strengthen bones, low dose simvastatin treatment is much more effective in increasing bone strength. Neural network analysis revealed an increased antiparallel and aggregated beta sheet and random coil in the protein secondary structure of high dose group implying a protein denaturation. Moreover, high dose may induce lipid peroxidation which limit the pleiotropic effects of high dose treatment on bones. This study clearly demonstrated that using low dose simvastatin is safer and more effective for bone health than high dose simvastatin treatment.