Proefschrift Universiteit Maastricht. / Met lit. opg. - Met samenvatting in het Nederlands.

L'inflammation chronique relayée par la glie activée contribue à la dégénérescence des neurones dopaminergiques (ND) au cours de la maladie de Parkinson (MP). L'étendue des dégâts cellulaires provoqués par la réaction inflammatoire dépend de l'efficacité des mécanismes régulateurs de l'inflammation. Les glucocorticoïdes endogènes sont des régulateurs puissants de l'inflammation agissant via le récepteur des glucocorticoïdes (GR). Notre équipe a récemment montré le rôle central du GR microglial dans la régulation de la mort neuronale dont la sévérité est corrélée à l'intensité et la durée de l'inflammation. Mon projet de thèse a été d'étudier le rôle des GR microglial et astrocytaire dans la régulation des réponses inflammatoires au cours de la dégénerescence des ND. Dans la première partie de ma thèse, nous avons effectué une analyse transcriptomique comparative de microglie ex vivo isolée de souris traitées au MPTP (modèle de parkinsonisme) et avons identifié des gènes régulés par le GR microglial, potentiellement impliqués dans l'inflammation chronique. Dans la deuxième partie de ma thèse, nous avons mis en évidence la régulation par le GR microglial de la mort neuronale induite par l'activation de TLR9. L'ADN mitochondrial endogène peut engendrer la mort neuronale en activant le TLR9, en cas de dysfonction du GR microglial. Dans la troisième partie de mon travail, nous avons démontré que le GR astrocytaire régule la survie des ND en modulant l'expression de gènes pro-inflammatoires et l'activité excessive des hémicanaux à connexine 43. Globalement, les GR microglial et astroglial jouent des rôles essentiels dans la régulation de l'inflammation aigue et chronique. / Chronic inflammation, mounted by activated glia, contributes to dopamine neuron (DN) loss, a major hallmark of Parkinson’s disease. It can be postulated that the extent of DN injury inflicted by inflammation is affected by the efficacy of regulatory mechanisms. The activation of hypothalamic–pituitary–adrenal axis results in release of glucocorticoids, which activate glucocorticoid receptor (GR). GR exerts adaptive responses including resolution of inflammation to restore the homeostatic state. We previously demonstrated the role of microglial GR in regulating the intensity and duration of inflammation, which influences DN survival. My thesis was centered on dissecting the roles of microglial and astrocytic GR during DN degeneration in experimental Parkinsonism. In the first part of my thesis, we conducted comparative transcriptome experiments of ex vivo microglia acutely isolated from mice treated with MPTP (model of parkinsonism) and identified genes and pathways in microglia regulated by GR, potentially involved in chronic inflammation in PD. In the second part of my thesis, we found that microglial GR regulates Toll-Like Receptor 9-induced DN loss by regulating the lysosomal compartment and demonstrated that diminished sensitivity of GR in microglia creates a permissive environment for TLR9 activation by endogenous mitochondrial DNA to become lethal for DNs. In the third part of my work, we showed that during DN degeneration, astrocytic GR regulates inflammatory gene expression and prevents connexin-43 hemichannel activity that contributes to DN loss. Overall, both microglial and astrocytic GR play essential roles in regulating chronic and acute inflammation.

Dans les Antilles françaises, une proportion anormalement élevée de parkinsonismes atypiques sporadiques - des tauopathies - est observée. Un lien avec la consommation de plantes de la famille des Annonaceae, en particulier Annona muricata L. (corossol) a été démontré. Les acétogénines d'Annonaceae, des inhibiteurs puissants du complexe I de la chaine respiratoire mitochondriale, sont considérées comme des toxines candidates. L'annonacine, une acétogénine représentative, majoritaire dans A. muricata, est neurotoxique in vitro et in vivo. L'Agence Française de Sécurité Sanitaire des Aliments a exprimé ses doutes quant à ce problème de santé publique. Elle insiste sur l'importance d'évaluer l'exposition des consommateurs d'Annonaceae aux acétogénines, et de déterminer les paramètres pharmacocinétiques de ces molécules. Au cours de cette thèse, nous avons cherché à répondre à ces interrogations, avec l'annonacine pour modèle. Après l'analyse structurale d'acétogénines étalons, une méthode de déréplication puissante et innovante a été mise au point par CLHP-ESI-LTQ-Orbitrap® avec infusion post-colonne de lithium. Les profils complets des acétogénines d'extraits bruts issus d'un nectar d'A. muricata et d'un alcool d'Annona cherimolia Mill. (annone, chérimole) ont été élucidés, mettant en évidence une composition plus complexe et plus variée que celle envisagée dans la littérature. A. cherimolia n'avait pas été identifiée comme une source d'exposition jusqu'à maintenant. Des données quantitatives ont été obtenues par CLHP-DAD-MS, à partir d'une quinzaine d'échantillons de produits commerciaux, confirmant une exposition humaine importante à ces molécules par voie alimentaire, via des produits d'origines géographiques, de statuts et de modes d'obtention variés. Des travaux préliminaires d'étude du passage de l'annonacine à travers des membranes biologiques ont été amorcés (modèles de barrières intestinale - Caco-2 - et hémato-encéphalique - hCMEC/D3). Une étude de métabolisation de phase I de l'annonacine sur microsomes de foie de Rat a permis d'identifier 25 métabolites mono-hydroxylés par CLHP-ESI-LTQ-Orbitrap®. Seuls trois d'entre eux sont observés avec des microsomes humains. Ces métabolites ont été obtenus par hémisynthèse (bioconversion, catalyse par porphyrines) et leur structure a été déterminée. Les résultats montrent que cette étape de métabolisation n'est pas cruciale dans le devenir de l'annonacine, et ne peut expliquer de susceptibilité différentielle aux acétogénines. Après la présentation de rappels concernant les Annonaceae, les parkinsonismes et leurs formes atypiques guadeloupéennes et tropicales, puis d'aspects méthodologiques en spectrométrie de masse, nos travaux de phytochimie analytique, d'analyse métabolique, d'hémisynthèse et de détermination structurale sont présentés, et discutés en regard d'un problème de santé publique potentiellement large et préoccupant.

Une importante proportion de formes atypiques de parkinsonismes a été rapportée en Guadeloupe en 1999. Il ressort des études épidémiologiques menées sur place, que tous les patients atteints étaient de grands consommateurs de produits alimentaires et médicinaux de la famille des Annonaceae, et plus particulièrement du genre Annona. De nombreux genres appartenant à cette famille renferment des molécules fortement cytotoxiques : les acétogénines d’Annonaceae. Leur présence dans les fruits d’Annona muricata a déjà été mise en évidence. Cependant, peu de données existent sur leur teneur en acétogénines dans ces fruits ou dans ceux d’espèces proches. Par ailleurs, bien que l’annonacine, acétogénine principale d’A. muricata soit neurotoxique in vivo, aucune donnée quantitative de son accès au cerveau n’est disponible. Nous nous sommes donc attachés au cours de ce travail, à développer des méthodes de dosage de l’annonacine par LC-MS/MS dans le plasma et le cerveau de Rat pour déterminer sa biodisponibilité et la fraction à tropisme cérébral, dans le but de comprendre pourquoi des molécules si fortement cytotoxiques n’entraînaient pas d’intoxication aigue lors de l’exposition alimentaire. Nous avons par ailleurs développé une méthode de quantification des acétogénines totales par 1H RMN dans des extraits bruts de fruits, appliquée à des lots d’A. muricata d’origines variées. Une méthode par LC-MS/MS a également été développée pour une description plus approfondie des acétogénines présentes dans des extraits bruts de fruits, appliquée à différents lots d’A. squamosa. Les fruits d’A. reticulata et d’A. glabra ont également fait l’objet d’investigations. Ces deux approches combinées ont contribué à améliorer l’estimation de l’exposition aux acétogénines dans le cadre de l’alimentation. / Abstract : A high proportion of atypical parkinsonisms was reported in French West Indies in 1999. Epidemiological studies pointed out an association with the consumption of fruits and medicinal herbs from Annonaceae of the Annona genera. Numerous Annonaceae members contain Annonaceous acetogenins (AAGs), which are highly cytotoxic molecules. They were found in the pulp fruit of Annona muricata. However, scarce only quantitative exist for this fruit and those of related species. Moreover, although annonacin, the major AAG of A. muricata proved neurotoxic in vivo, no quantitative data is available towards its distribution to the brain. We therefore developed a method for annonacin quantitation in Rat plasma and brain homogenate, in order to determine its bioavailability and the fraction reaching the brain, to understand why those highly cytotoxic molecules are not responsible for acute toxicity when fruits are ingested. We then developed a quantitation method for global estimation of AAGs in crude fruit extracts by 1H NMR, which we applied to the fruit pulp of A. muricata batches from diverse locations. An LC-MS/MS method was also developed for the qualitative study of AAGs. It was applied to different batches from A. squamosa fruits. The species A. reticulata and A. glabra were also examined. Those two approaches contributed in a better estimation of AAGs exposure by fruit consumption.

Purpose: Monoamine oxidase (MAO) plays a key role in the treatment of Parkinson‟s disease (PD), since it is the major enzyme responsible for the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO-B may conserve dopamine in the brain and provide symptomatic relief. The MAO-B inhibitors that are currently used for the treatment of PD, are associated with a variety of adverse effects (psychotoxic and cardiovascular effects) along with additional disadvantages such as irreversible inhibition of the enzyme. Irreversible inhibition may be considered a disadvantage, since following treatment with irreversible inhibitors, the rate by which the enzyme activity is recovered may be variable and may require several weeks. In contrast, following the administration of reversible inhibitors, enzyme activity is recovered when the inhibitor is cleared from the tissues. There exists therefore, a need to develop new reversible inhibitors of MAO-B which are considered to be safer than irreversible MAO-B inhibitors. Rationale: Recently discovered reversible MAO-B inhibitors include safinamide and (E)-8-(3-chlorostyryl)caffeine (CSC). Safinamide has a benzyloxy side chain, which is thought to be important for inhibition of MAO-B. CSC, on the other hand, consists of a caffeine moiety with a styryl substituent at C-8, which is also a critical feature for its inhibitory activity. In a previous study, the caffeine ring and the benzyloxy side chain were combined to produce a series of 8-benzyloxycaffeine analogues which proved to be potent new MAO-B inhibitors. In this study, caffeine was substituted with the phenoxymethyl functional group at C-8, instead of the benzyloxy moiety. The aim of this study was therefore to compare the MAO-B inhibition potencies of selected 8-(phenoxymethyl)caffeine analogues with the previously studied 8-benzyloxycaffeine analogues. In the current study, 8-(phenoxymethyl)caffeine (1) and nine 8-(phenoxymethyl)caffeine analogues (2-10) were synthesized and evaluated as inhibitors of recombinant human MAOA and –B. These analogues only differed in substitution on C3 and C4 of the phenoxymethyl phenyl ring. The substituents that were selected were halogens (Cl, F, and Br), the methyl group, the methoxy group and the trifluoromethyl group. These substituents are similar to those selected in a previous study where 8-benzyloxycaffeine analogues were evaluated as MAO inhibitors. This study therefore explores the effect that a variety of substituents on C3 and C4 of the phenoxymethyl phenyl ring will have on the MAO-A and –B inhibition potencies of 8-(phenoxymethyl)caffeine. Based on the results, additional 8-(phenoxymethyl)caffeine analogues with improved MAO-A and –B inhibition potencies will be proposed for investigation in future studies. Methods: The target, 8-(phenoxymethyl)caffeine, analogues were synthesized by reacting 1,3- dimethyl-5,6-diaminouracil with the appropriately substituted phenoxyacetic acid in the presence of a carbodiimide coupling agent. Ring closure was catalyzed in basic conditions and methylation of the resulting theophyline intermediates at C-7 was carried out with iodomethane. The structures and purities of all the target compounds were verified by NMR, MS and HPLC analysis. All of the 8-(phenoxymethyl)caffeine analogues were subsequently evaluated as MAO-A and –B inhibitors using the recombinant human enzymes. The inhibition potencies of the analogues were expressed as the IC50 values (concentration of the inhibitor that produces 50% inhibition). In addition, the time-dependency of inhibition of both MAO-A and –B was evaluated for two inhibitors in order to determine if these inhibitors interact reversibly or irreversibly with the MAO isozymes. A Hansch-type quantitative structure-activity relationship (QSAR) study was carried out in order to quantify the effect that different substituents on the phenyl ring of the 8-(phenoxymethyl)caffeine analogues have on MAO-B inhibition activity. Results: The results showed that among the test compounds, several analogues potently inhibited human MAO-B. The most potent inhibitor was 8-(3-bromophenoxymethyl)caffeine with an IC50 value of 0.148 μM toward human MAO-B. There were also inhibitors which displayed inhibition activities towards human MAO-A with IC50 values ranging from 4.59 μM to 34.0 μM. Compared to the 8-benzyloxycaffeine analogues, that were in general non-selective inhibitors, the 8-(phenoxymethyl)caffeine analogues, evaluated here, were selective for MAO-B. For example, 8-(3-bromophenoxymethyl)caffeine was found to be 141 fold more selective as an inhibitor of MAO-B than of MAO-A. Also, compared to the 8-benzyloxycaffeine analogues, the 8-(phenoxymethyl)caffeine analogues were slightly less potent MAO-B inhibitors. For example, 8-benzyloxycaffeine is reported to have an IC50 value of 1.77 μM for the inhibition of human MAO-B while 8-(phenoxymethyl)caffeine was found to have an IC50 value of 5.78 μM for the inhibition of human MAO-B. This study also shows that two selected analogues bind reversibly to MAO-A and –B, respectively, and that the mode of MAO-B inhibition is competitive for one representative compound. Qualitative inspection of the results revealed interesting structure-activity relationships. For the 8-(phenoxymethyl)caffeine analogues, bearing both the C3 and C4 substituents on the phenyl ring, the MAO-B activity significantly increases with halogen substitution. Furthermore, increased MAO-B inhibition was observed with increased electronegativity of the halogen substituent. To quantify these apparent relationships, a Hansch-type QSAR study was carried out. The results showed that the logarithm of the IC50 values (logIC50) correlated with Hansch lipophilicity (π) and the Swain-Lupton electronic (F) constants of the substituents at C-3 of the phenoxymethyl ring. The correlation exhibited an R2 value of 0.87 and a statistical F value of 13.6. From these results it may be concluded that electron-withdrawing substituents at C3 with a high degree of lipophilicity enhance MAO-B inhibition potency. These results are similar to those previously obtained for the series of 8-benzyloxycaffeine analogues. For this series, the MAO-B inhibition potencies correlated with the Hansch lipophilicity (π) and Hammett electronic (σ) constants of the substituents at C-3 of the benzyloxy ring. Similarly to the 8-(phenoxymethyl)caffeine analogues, electron-withdrawing substituents with a high degree of lipophilicity also enhance the MAO-B inhibition potencies of 8-benzyloxycaffeine analogues. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011

Dans les Antilles françaises, une proportion anormalement élevée de parkinsonismes atypiques sporadiques – des tauopathies – est observée. Un lien avec la consommation de plantes de la famille des Annonaceae, en particulier Annona muricata L. (corossol) a été démontré. Les acétogénines d’Annonaceae, des inhibiteurs puissants du complexe I de la chaine respiratoire mitochondriale, sont considérées comme des toxines candidates. L’annonacine, une acétogénine représentative, majoritaire dans A. muricata, est neurotoxique in vitro et in vivo. L’Agence Française de Sécurité Sanitaire des Aliments a exprimé ses doutes quant à ce problème de santé publique. Elle insiste sur l’importance d’évaluer l’exposition des consommateurs d’Annonaceae aux acétogénines, et de déterminer les paramètres pharmacocinétiques de ces molécules. Au cours de cette thèse, nous avons cherché à répondre à ces interrogations, avec l’annonacine pour modèle. Après l’analyse structurale d’acétogénines étalons, une méthode de déréplication puissante et innovante a été mise au point par CLHP-ESI-LTQ-Orbitrap® avec infusion post-colonne de lithium. Les profils complets des acétogénines d’extraits bruts issus d’un nectar d’A. muricata et d’un alcool d’Annona cherimolia Mill. (annone, chérimole) ont été élucidés, mettant en évidence une composition plus complexe et plus variée que celle envisagée dans la littérature. A. cherimolia n’avait pas été identifiée comme une source d’exposition jusqu’à maintenant. Des données quantitatives ont été obtenues par CLHP-DAD-MS, à partir d’une quinzaine d’échantillons de produits commerciaux, confirmant une exposition humaine importante à ces molécules par voie alimentaire, via des produits d’origines géographiques, de statuts et de modes d’obtention variés. Des travaux préliminaires d’étude du passage de l’annonacine à travers des membranes biologiques ont été amorcés (modèles de barrières intestinale – Caco-2 – et hémato-encéphalique – hCMEC/D3). Une étude de métabolisation de phase I de l’annonacine sur microsomes de foie de Rat a permis d'identifier 25 métabolites mono-hydroxylés par CLHP-ESI-LTQ-Orbitrap®. Seuls trois d’entre eux sont observés avec des microsomes humains. Ces métabolites ont été obtenus par hémisynthèse (bioconversion, catalyse par porphyrines) et leur structure a été déterminée. Les résultats montrent que cette étape de métabolisation n’est pas cruciale dans le devenir de l’annonacine, et ne peut expliquer de susceptibilité différentielle aux acétogénines. Après la présentation de rappels concernant les Annonaceae, les parkinsonismes et leurs formes atypiques guadeloupéennes et tropicales, puis d’aspects méthodologiques en spectrométrie de masse, nos travaux de phytochimie analytique, d’analyse métabolique, d’hémisynthèse et de détermination structurale sont présentés, et discutés en regard d’un problème de santé publique potentiellement large et préoccupant. / In the French West Indies, an unusually high proportion of atypical sporadic parkinsonisms - tauopathies - is observed. A link between these atypical parkinsonisms and the consumption of plants of the Annonacea family, Annona muricata L. (soursop) was demonstrated. The Annonaceous acetogenins are potent inhibitors of complex I of mitochondrial respiratory chain and are considered to be in vitro toxins candidate. The major acetogenin in Annona muricata, annonacin, is neurotoxic in in vitro and in vivo models. Afssa (Agence Française de Sécurité Sanitaire des Aliments) expressed its concern regarding this public health problem. The Agency reports the importance of assessing consumers ‘exposure to Annonaceous acetogenins, and of determining the pharmacokinetic of these molecules. In this thesis, we sintended to answer these questions, with annonacine as a model. After the structural analysis of acetogenins standards, a powerful and innovative method of dereplication was developed by HPLC-ESI-LTQ-Orbitrap ® with post-column infusion of lithium. Complete acetogenins profile in crude extracts from nectar of A. muricata and from an alcohol of Annona cherimolia Mill. (Annona, cherimoya) were elucidated, revealing a more complex and more varied composition than that proposed in the literature. A. cherimolia had not been identified as a source of exposure to date. Quantitative data were obtained by HPLC-DAD-MS, from fifteen samples of commercial products, confirming an important human exposure to these molecules through food products of varied geographical origins, status and methods. Preliminary works to study ability of annonacin to cross biological membranes have been initiated (intestinal barrier models - Caco-2 - and blood-brain barrier - hCMEC/D3). A study of phase I metabolism of annonacin in rat liver microsomes allowed the identification of 25 mono-hydroxylated metabolites by HPLC-ESI-LTQ-Orbitrap®. Only three of them were observed with human microsomes. These metabolites were obtained by semisynthesis (bioconversion, porphyrin mediated catalysis) and their structure was determined. The results show that phase metabolism is not critical in the becoming of annonacin, and cannot explain a differential susceptibility of acetogenins. After a presentation of the Annonaceae family, of parkinsonism – including atypical and guadeloupean forms then of and methodological aspects of mass spectrometry, analytical phytochemistry of our work, metabolic analysis, semisynthesis and structure determination are proposed, and discussed in the context of a public health problem and potentially broad concern.

Parkinson's disease is the most recurrent movement disorder and has a radical effect on the lives of people. This chronic neurological disorder is accompanied by a significant social and financial burden with a negative brunt on sufferers' quality of life. The main cause of Parkinson's disease is still unknown, however, the main goal of existing treatment for Parkinson's disease is to improve the patient's quality of life and ability to go about as normally and easily as possible. The general objective of this study was to investigate the prescribing patterns of medicine items used in Parkinson's disease and other movement disorders associated with Parkinson's disease, as well as the cost associated with the medication in a section of the private health care sector of South Africa. A quantitative, retrospective drug utilisation review (DUR) study was performed according to data obtained from a medicine claims database, of a South African pharmacy benefit management company (PBM) for four consecutive years (i.e. 2005 to 2008). Of all patients on the total database 0.26% (n = 3 993) were Parkinson's disease patients in 2005 (N = 1 509 621), 0.28% (n = 4 423) in 2006 (N = 1 558 090), 0.34% (n = 4 028) in 2007 (N = 1 178 596) and 0.42% (n = 4 072) in 2008 (N = 974 497). Female Parkinson's disease patients were between 56% and 60% of all Parkinson's disease patients from 2005 to 2008. According to age groups, Parkinson's disease patients had the highest representation in age group five (70 80 years) and age group six (> 80 years). In total the number of Parkinson's disease prescriptions claimed through the PMB accounted for 0.3% from 2005 to 2007 and 0.4% in 2008 of all prescriptions claimed on the database. From 2005 (N = R1 819 865 251) to 2008 (N = R1 785 871 013) Parkinson's disease expenditures represented 0.6% (2005, n = R10 459 835; 2006, n = R11 320 616; 2007, n = 11 040 596; 2008, n = 10 697 155) of the total database's prescription expenditure. The female gender and patients of 70 years and older, presented with the highest number of prescriptions claimed and also with the highest costs within the specific age and gender groups. In 2005 the medicine treatment expenditure for a year's Parkinson's disease treatment was approximately R2 619 R4 179, decreasing with 2% to R2 559 R4 237 in 2006, from thereon increasing with 7% to R2 740 R 4 337 in 2007, decreasing again with 4% to R 2 627 R4 424 in 2008. Medicine item analyses indicated that dopaminergic medicine items were the most frequently used antiparkinson medicine items from 2005 to 2008. Carbidopa/levodopa containing medicine items were most frequently claimed throughout the study period. The average cost per tablet increased from 2005 to 2008, with the most expensive tablets during the four–year study period indicated as, Tasmar® 100 mg tab and Permax® 1 mg tab. The PDD of all antiparkinson medicine items appeared intact. There were only two medicine items that indicated a PDD, above the maximum daily dosage, namely Permax® 1 mg tablets and Tasmar® 100 mg tablets. The frequencies of medicine items prescribed in combination decreased rather drastically with an increase of medicine items per prescription throughout the study period. CNS medicine items prescribed together with antiparkinson medicine items per prescription often occurred. The highest frequencies encountered in combination with antiparkinson medicine items were found to include the antidepressants, hypnotics, antipsychotics and anxiolytic medicine items. A majority of antiparkinson medicine items (53.50%, n = 4 691) had low refill–adherence rates below 90% and were therefore unacceptable. These accounted for 41.62% (n = R16 398 512) of the total cost (N = R39 402 898) of all antiparkinson medicine items included in this study. Only 36.78% (n = 3 225) of antiparkinson medicine items had acceptable refill–adherence rates between 90% and 110%. Those with unacceptably high refill–adherence rates accounted for 9.72% (n = 852) of all antiparkinson medicine items and represented 6.5% (n = R2 574 597) of the total cost. Conclusion: It can be concluded that even though antiparkinson medicine items are used by only a small percentage of the total patient population in a section of the private health care sector of South Africa, they are expensive and bear implications for the patient as well as medical schemes. Good prescribing patterns were adhered to, with the exception of the poor refill–adherence of antiparkinsons medication items. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2011.

Parkinson's disease is the most recurrent movement disorder and has a radical effect on the lives of people. This chronic neurological disorder is accompanied by a significant social and financial burden with a negative brunt on sufferers' quality of life. The main cause of Parkinson's disease is still unknown, however, the main goal of existing treatment for Parkinson's disease is to improve the patient's quality of life and ability to go about as normally and easily as possible. The general objective of this study was to investigate the prescribing patterns of medicine items used in Parkinson's disease and other movement disorders associated with Parkinson's disease, as well as the cost associated with the medication in a section of the private health care sector of South Africa. A quantitative, retrospective drug utilisation review (DUR) study was performed according to data obtained from a medicine claims database, of a South African pharmacy benefit management company (PBM) for four consecutive years (i.e. 2005 to 2008). Of all patients on the total database 0.26% (n = 3 993) were Parkinson's disease patients in 2005 (N = 1 509 621), 0.28% (n = 4 423) in 2006 (N = 1 558 090), 0.34% (n = 4 028) in 2007 (N = 1 178 596) and 0.42% (n = 4 072) in 2008 (N = 974 497). Female Parkinson's disease patients were between 56% and 60% of all Parkinson's disease patients from 2005 to 2008. According to age groups, Parkinson's disease patients had the highest representation in age group five (70 80 years) and age group six (> 80 years). In total the number of Parkinson's disease prescriptions claimed through the PMB accounted for 0.3% from 2005 to 2007 and 0.4% in 2008 of all prescriptions claimed on the database. From 2005 (N = R1 819 865 251) to 2008 (N = R1 785 871 013) Parkinson's disease expenditures represented 0.6% (2005, n = R10 459 835; 2006, n = R11 320 616; 2007, n = 11 040 596; 2008, n = 10 697 155) of the total database's prescription expenditure. The female gender and patients of 70 years and older, presented with the highest number of prescriptions claimed and also with the highest costs within the specific age and gender groups. In 2005 the medicine treatment expenditure for a year's Parkinson's disease treatment was approximately R2 619 R4 179, decreasing with 2% to R2 559 R4 237 in 2006, from thereon increasing with 7% to R2 740 R 4 337 in 2007, decreasing again with 4% to R 2 627 R4 424 in 2008. Medicine item analyses indicated that dopaminergic medicine items were the most frequently used antiparkinson medicine items from 2005 to 2008. Carbidopa/levodopa containing medicine items were most frequently claimed throughout the study period. The average cost per tablet increased from 2005 to 2008, with the most expensive tablets during the four–year study period indicated as, Tasmar® 100 mg tab and Permax® 1 mg tab. The PDD of all antiparkinson medicine items appeared intact. There were only two medicine items that indicated a PDD, above the maximum daily dosage, namely Permax® 1 mg tablets and Tasmar® 100 mg tablets. The frequencies of medicine items prescribed in combination decreased rather drastically with an increase of medicine items per prescription throughout the study period. CNS medicine items prescribed together with antiparkinson medicine items per prescription often occurred. The highest frequencies encountered in combination with antiparkinson medicine items were found to include the antidepressants, hypnotics, antipsychotics and anxiolytic medicine items. A majority of antiparkinson medicine items (53.50%, n = 4 691) had low refill–adherence rates below 90% and were therefore unacceptable. These accounted for 41.62% (n = R16 398 512) of the total cost (N = R39 402 898) of all antiparkinson medicine items included in this study. Only 36.78% (n = 3 225) of antiparkinson medicine items had acceptable refill–adherence rates between 90% and 110%. Those with unacceptably high refill–adherence rates accounted for 9.72% (n = 852) of all antiparkinson medicine items and represented 6.5% (n = R2 574 597) of the total cost. Conclusion: It can be concluded that even though antiparkinson medicine items are used by only a small percentage of the total patient population in a section of the private health care sector of South Africa, they are expensive and bear implications for the patient as well as medical schemes. Good prescribing patterns were adhered to, with the exception of the poor refill–adherence of antiparkinsons medication items. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2011.

Purpose: Monoamine oxidase (MAO) plays a key role in the treatment of Parkinson‟s disease (PD), since it is the major enzyme responsible for the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO-B may conserve dopamine in the brain and provide symptomatic relief. The MAO-B inhibitors that are currently used for the treatment of PD, are associated with a variety of adverse effects (psychotoxic and cardiovascular effects) along with additional disadvantages such as irreversible inhibition of the enzyme. Irreversible inhibition may be considered a disadvantage, since following treatment with irreversible inhibitors, the rate by which the enzyme activity is recovered may be variable and may require several weeks. In contrast, following the administration of reversible inhibitors, enzyme activity is recovered when the inhibitor is cleared from the tissues. There exists therefore, a need to develop new reversible inhibitors of MAO-B which are considered to be safer than irreversible MAO-B inhibitors. Rationale: Recently discovered reversible MAO-B inhibitors include safinamide and (E)-8-(3-chlorostyryl)caffeine (CSC). Safinamide has a benzyloxy side chain, which is thought to be important for inhibition of MAO-B. CSC, on the other hand, consists of a caffeine moiety with a styryl substituent at C-8, which is also a critical feature for its inhibitory activity. In a previous study, the caffeine ring and the benzyloxy side chain were combined to produce a series of 8-benzyloxycaffeine analogues which proved to be potent new MAO-B inhibitors. In this study, caffeine was substituted with the phenoxymethyl functional group at C-8, instead of the benzyloxy moiety. The aim of this study was therefore to compare the MAO-B inhibition potencies of selected 8-(phenoxymethyl)caffeine analogues with the previously studied 8-benzyloxycaffeine analogues. In the current study, 8-(phenoxymethyl)caffeine (1) and nine 8-(phenoxymethyl)caffeine analogues (2-10) were synthesized and evaluated as inhibitors of recombinant human MAOA and –B. These analogues only differed in substitution on C3 and C4 of the phenoxymethyl phenyl ring. The substituents that were selected were halogens (Cl, F, and Br), the methyl group, the methoxy group and the trifluoromethyl group. These substituents are similar to those selected in a previous study where 8-benzyloxycaffeine analogues were evaluated as MAO inhibitors. This study therefore explores the effect that a variety of substituents on C3 and C4 of the phenoxymethyl phenyl ring will have on the MAO-A and –B inhibition potencies of 8-(phenoxymethyl)caffeine. Based on the results, additional 8-(phenoxymethyl)caffeine analogues with improved MAO-A and –B inhibition potencies will be proposed for investigation in future studies. Methods: The target, 8-(phenoxymethyl)caffeine, analogues were synthesized by reacting 1,3- dimethyl-5,6-diaminouracil with the appropriately substituted phenoxyacetic acid in the presence of a carbodiimide coupling agent. Ring closure was catalyzed in basic conditions and methylation of the resulting theophyline intermediates at C-7 was carried out with iodomethane. The structures and purities of all the target compounds were verified by NMR, MS and HPLC analysis. All of the 8-(phenoxymethyl)caffeine analogues were subsequently evaluated as MAO-A and –B inhibitors using the recombinant human enzymes. The inhibition potencies of the analogues were expressed as the IC50 values (concentration of the inhibitor that produces 50% inhibition). In addition, the time-dependency of inhibition of both MAO-A and –B was evaluated for two inhibitors in order to determine if these inhibitors interact reversibly or irreversibly with the MAO isozymes. A Hansch-type quantitative structure-activity relationship (QSAR) study was carried out in order to quantify the effect that different substituents on the phenyl ring of the 8-(phenoxymethyl)caffeine analogues have on MAO-B inhibition activity. Results: The results showed that among the test compounds, several analogues potently inhibited human MAO-B. The most potent inhibitor was 8-(3-bromophenoxymethyl)caffeine with an IC50 value of 0.148 μM toward human MAO-B. There were also inhibitors which displayed inhibition activities towards human MAO-A with IC50 values ranging from 4.59 μM to 34.0 μM. Compared to the 8-benzyloxycaffeine analogues, that were in general non-selective inhibitors, the 8-(phenoxymethyl)caffeine analogues, evaluated here, were selective for MAO-B. For example, 8-(3-bromophenoxymethyl)caffeine was found to be 141 fold more selective as an inhibitor of MAO-B than of MAO-A. Also, compared to the 8-benzyloxycaffeine analogues, the 8-(phenoxymethyl)caffeine analogues were slightly less potent MAO-B inhibitors. For example, 8-benzyloxycaffeine is reported to have an IC50 value of 1.77 μM for the inhibition of human MAO-B while 8-(phenoxymethyl)caffeine was found to have an IC50 value of 5.78 μM for the inhibition of human MAO-B. This study also shows that two selected analogues bind reversibly to MAO-A and –B, respectively, and that the mode of MAO-B inhibition is competitive for one representative compound. Qualitative inspection of the results revealed interesting structure-activity relationships. For the 8-(phenoxymethyl)caffeine analogues, bearing both the C3 and C4 substituents on the phenyl ring, the MAO-B activity significantly increases with halogen substitution. Furthermore, increased MAO-B inhibition was observed with increased electronegativity of the halogen substituent. To quantify these apparent relationships, a Hansch-type QSAR study was carried out. The results showed that the logarithm of the IC50 values (logIC50) correlated with Hansch lipophilicity (π) and the Swain-Lupton electronic (F) constants of the substituents at C-3 of the phenoxymethyl ring. The correlation exhibited an R2 value of 0.87 and a statistical F value of 13.6. From these results it may be concluded that electron-withdrawing substituents at C3 with a high degree of lipophilicity enhance MAO-B inhibition potency. These results are similar to those previously obtained for the series of 8-benzyloxycaffeine analogues. For this series, the MAO-B inhibition potencies correlated with the Hansch lipophilicity (π) and Hammett electronic (σ) constants of the substituents at C-3 of the benzyloxy ring. Similarly to the 8-(phenoxymethyl)caffeine analogues, electron-withdrawing substituents with a high degree of lipophilicity also enhance the MAO-B inhibition potencies of 8-benzyloxycaffeine analogues. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011

Les processus compensatoires qui accompagnent les atteintes du système dopaminergique (DA-ergic) nigrostrié illustrent les capacités adaptatives du cerveau adulte. Cette neuroplasticité permet le maintien de la transmission dopaminergique pendant un certain temps de sorte que les symptômes moteurs cardinaux de la Maladie de Parkinson (MP), qui se caractérise par une dégénérescence progressive des neurones DA-ergiques de la substantia nigra (SN), ne se manifestent qu'après une perte neuronale très importante. De ce fait, le diagnostic présymptomatique est une question cruciale pour le développement de traitements neuroprotecteurs. Un autre exemple de neuroplasticité est illustré par la production de nouveaux neurones dans le cerveau adulte (neurogenèse adulte). Cette neurogenèse s’observe principalement dans deux zones: le continuum zone sous-ventriculaire (SVZ)-bulbe olfactif (OB) et le gyrus denté (DG) de l'hippocampe, et se trouve altérée chez les patients parkinsoniens. Ces dernières années, le traitement chirurgical par la stimulation à haute fréquence (SHF) du noyau sous-thalamique (NST) s'est avéré être une option thérapeutique très efficace pour ces patients. Dans ce contexte, mon travail de thèse a été axé sur l’étude de la neuroplasticité dans différents modèles de la maladie de Parkinson et de son traitement avec les objectifs principaux: 1) Développer un modèle de MP présymptomatique; 2) étudier les mécanismes compensatoires impliquant le système nigrostrié; 3) Déterminer les effets de la SHF-NST sur la neurogenèse adulte dans la SVZ-OB et le DG.Dans la première étude, nous avons développé des modèles expérimentaux de la MP à différents stades, basés sur l’administration de MPTP chez la souris. Nous avons montré que le passage du stade avancé présymptomatique au stade symptomatique précoce correspondant au seuil d’atteinte des systèmes DA-ergiques associé à l’apparition des déficits moteurs, se caractérise par : (a) une diminution de DA dans les terminaisons striatales épargnées par la lésion; (b) une augmentation de DA et d’expression de la tyrosine hydroxylase dans les cellules de la SN; (c) une augmentation du renouvellement de la DA dans le striatum et une augmentation moindre dans la SN.La deuxième étude est basée sur un modèle de lésion DA-ergique extensive par injection intranigrale de 6-hydroxydopamine chez le rat, imitant les stades tardifs de la MP. Nous avons étudié séparément les étapes de prolifération et de survie des nouvelles cellules sur des animaux non lésés et des animaux lésés avec ou sans SHF subchronique (8 jours) du NST. Nous avons pu montrer une régulation spécifique des étapes de prolifération et de survie suite à la lésion dopaminergique, et des effets stimulateurs de la SHF du NST sur la survie des cellules néoformées, suggérant un effet neuroprotecteur de ce traitement. / The compensatory processes that accompany a lesion of the nigrostriatal dopaminergic (DA-ergic) system serve to maintain its function and illustrate adult brain neuroplasticity. The typical motor symptoms of Parkinson’s diseases (PD), characterized by progressive degeneration of DA-ergic neurons of substantia nigra (SN), appear only after substantial neuronal loss. Therefore presymptomatic diagnosis is a crucial issue for future neuroprotective therapies. Another good manifestation of neuroplasticity is adult neurogenesis, known to persist in two areas: the subventricular zone (SVZ) – the olfactory bulb (OB) continuum, and the dentate gyrus (DG) of the hippocampus, and to be altered in PD. In recent years, the surgical treatment by high frequency stimulation (HFS) of the subthalamic nucleus (STN) has proven to be an efficient therapeutic option for PD patients. In this context, my PhD work was focused on neuroplasticity under the functional deficiency of the nigrostriatal DA-ergic system (parkinsonism) and its treatment with the following main objectives: 1 - Develop a model of presymptomatic parkinsonism; 2 - study compensatory mechanisms in nigrostriatal system; 3 - Characterize the effects of subchronic STN HFS on adult neurogenesis. In the first part, we have developed models of presymptomatic parkinsonism based on MPTP administration in mice, as defined by sub-threshold DA depletion and degeneration of DA-ergic axons in the striatum followed by a loss of DA-ergic cell bodies in the SN (advanced presymptomatic stage). In the early symptomatic stage, these parameters reach a threshold that is associated with the appearance of motor deficiency. We have shown that the transition from the advanced presymptomatic stage to the early symptomatic stage is characterized by: (a) a decrease of DA content in surviving DA-ergic axons in the striatum; (b) an increase of DA content and TH-expression in surviving neuronal cell bodies in the SN; (c) an increase of DA turnover in the striatum and much less increase in the SN. The last part of my work is based on extensive DA lesion in rats, using intranigral 6-hydroxydopamine injection mimicking late PD stages, to determine a possible effect of STN-HFS on adult neurogenesis. We have completed series of animals with DA lesion either sham implanted or subsequently treated for 8 days by STN-HFS to be compared with unlesioned rats, and studied selective phases of neurogenesis: proliferation and survival. This study demonstrates selective regulation of cell proliferation and survival following DA depletion and provides the first evidence that prolonged STN-HFS might have a neuroprotective action as shown by the selective increase in survival of newly formed cells following this treatment.