Proton pump inhibitors (PPIs) are commonly coadministered with clopidogrel, an antiplatelet agent, to patients with acute coronary syndrome (ACS). Mechanistic studies suggest that PPIs have the potential to competitively inhibit the bioactivation of clopidogrel and may attenuate its antiplatelet action in the body. The clinical implications of this drug-drug interaction have been extensively studied; however reported findings are inconsistent. More recently, several studies have questioned whether PPIs are associated with adverse cardiovascular events independent of clopidogrel. Given that PPIs and clopidogrel are widely used, it is critical to better understand the clinical impact of the concomitant treatment with both drugs. This thesis includes four studies that investigate the clinical effects of the drug-drug interaction between clopidogrel and PPIs. Chapter 2, a systematic review and meta-analysis, summarizes findings from 118 studies. Findings do not provide strong evidence for an association between adverse cardiovascular events and the use of PPIs when used alone, in combination with clopidogrel, or in combination with other antiplatelets. Chapters 3, 4, and 5 present analyses of real-world data comprised of electronic medical records. Results of these analyses demonstrate 1) that the concomitant use of clopidogrel and PPIs among inpatients was consistent with clinical guidelines suggested by the FDA (Chapter 3); 2) a lack of association between PPI use vs nonuse and four adverse cardiovascular outcomes among clopidogrel users (Chapter 4); and 3) a lack of association between PPI use vs nonuse and adverse cardiovascular outcomes among prasugrel users or ticagrelor users (Chapter 5). Collectively, our findings do not provide evidence of an elevated risk of adverse cardiovascular outcomes with the combined use of PPIs and clopidogrel. Although pharmacodynamic and pharmacokinetic studies have demonstrated an interaction between these two drugs, our findings support the opinion that the biological interaction does not translate into adverse clinical events among patients with acute coronary syndrome.

Long-term use of proton pump inhibitors (PPIs) is common in patients with muscle wasting-related chronic diseases. We explored the hypothesis that the use of PPIs may contribute to a reduction in muscle mass and function in these patients. Literature indicates that a PPI-induced reduction in acidity of the gastrointestinal tract can decrease the absorption of, amongst others, magnesium. Low levels of magnesium are associated with impaired muscle function. This unwanted side-effect of PPIs on muscle function has been described in different disease backgrounds. Furthermore, magnesium is necessary for activation of vitamin D. Low vitamin D and magnesium levels together can lead to increased inflammation involved in muscle wasting. In addition, PPI use has been described to alter the microbiota’s composition in the gut, which might lead to increased inflammation. However, PPIs are often provided together with nonsteroidal anti-inflammatory drugs (NSAIDs), which are anti-inflammatory. In the presence of obesity, additional mechanisms could further contribute to muscle alterations. In conclusion, use of PPIs has been reported to contribute to muscle function loss. Whether this will add to the risk factor for development of muscle function loss in patients with chronic disease needs further investigation.

Mikrobielle Rhodopsine sind lichtsensitive Membranproteine und agieren als Sensoren, Biokatalysatoren oder Ionentransporter. Die Ionentransporter unterteilen sich in lichtgetriebene Ionenpumpen und in lichtaktivierte Kanalrhodopsine. Besonders die Protonenpumpe Bakteriorhodopsin steht schon lange im Fokus biophysikalischer Untersuchungen. Obwohl die Protonenpumpen seit über 40 Jahren intensiv untersucht werden, ist das Wissen über deren elektrophysiologische Eigenschaften noch immer gering. Aus diesem Grund widmete sich diese Arbeit der elektrophysiologischen Charakterisierung der mikrobiellen Rhodopsine mit dem Fokus auf Protonenpumpen. Hierfür wurden vor allem „Two-Electrode Voltage Clamp“ -Messungen (TEVC) an Oozyten des afrikanischen Krallenfrosches Xenopus leavis durchgeführt. Die Untersuchung verschiedener Protonenpumpen hat gezeigt, dass diese eine unerwartet große Diversität in ihren elektrophysiologischen Eigenschaften aufweisen. Von besonderem Interesse war die Beobachtung, dass einige Protonenpumpen neben Pumpströmen auch passive einwärts gerichtete Photoströme zeigten. Besonders deutlich war der „Pump-Kanal-Dualismus“ bei dem Gloeobacter-Rhodopsin ausgeprägt. Andere Protonenpumpen, wie das Bakteriorhodopsin oder Coccomyxa-Rhodopsin, zeigten keine einwärts gerichteten Photoströme. Das Coccomyxa-Rhodopsin wurde aufgrund seiner hohen Photostrom-Amplituden in Oozyten für eine Mutationsanalyse ausgewählt. Diese Mutationsanalyse verhalf die strukturellen Ursachen für die funktionalen Unterschiede zu identifizieren, welche sowohl zwischen den Protonenpumpen untereinander als auch gegenüber Kanalrhodopsinen beobachtet wurden. Mutationen im Gegenion-Komplex führen zu rein passiven oder inaktiven Transportern. Dagegen übernimmt der extrazelluläre Halbkanal in Protonenpumpe die Aufgabe einen passiven Protonen-Rückfluss während des Pumpzyklus zu verhindern, denn Mutationen in dieser Region verursachen passive Photoströme zusätzlich zum aktiven Pumpstrom. / Microbial rhodopsins are light-sensitive membrane proteins and operate as sensors, enzymes or ion-transporters. The ion transporters are subdivided into light-driven ion pumps and light-gated channels. Biophysical research has put focus on the proton pump bacteriorhodopsin for long time. Despite the fact that light-driven proton pumps are investigated for over 40 years, the knowledge about their electrophysiological properties is surprisingly low. For this reason, this thesis is devoted to the electrophysiological characterization of microbial rhodopsins with special focus on light-driven proton pumps. For this purpose, “Two-Electrode Voltage Clamp”-recordings (TEVC) were primarily performed using oocytes from African clawed frog Xenopus leavis. The investigation of diverse proton pumps has shown that the differences in their electrophysiological behaviors are unexpectedly high. Special interest was laid on proton pumps which show passive inward directed photocurrents when the electrochemical load exceeds a certain level. The dualism of pump and channel activity was particularly pronounced in the proton pump Gloeobacter-rhodopsin. Other proton pumps, for instance bacteriorhodopsin or Coccomyxa-rhodopsin, do not show inward directed photocurrents. Due to high photocurrent amplitudes, the Coccomyxa-rhodopsin was selected for an efficient mutagenesis study. This study allowed the identification of structural key determinants for the differences among proton pumps themselves and for the differences of proton pumps in comparison with light-gated ion channels (channelrhodopsins). Therefore, mutations of the counter-ion-complex cause inactive or purely passive transporters. The extracellular half-channel is the key element in proton pumps which prevents passive proton-backflow during the pump-cycle. Mutations in this region lead to passive leak-currents in overlap with the remaining pump-activity.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid synthase (FASN) is over-expressed in many cancers, and novel inhibitors that target FASN may find use in the treatment of cancers. It has been shown that orlistat, an FDA approved drug for weight loss, inhibits the thioesterase (TE) of FASN, but can be hydrolyzed by TE. To understand the mechanisms of TE action and for designing better FASN inhibitors, I examined the mechanism of orlistat hydrolysis by TE using molecular dynamics simulations. I found that the hexyl tail of orlistat undergoes a conformational transition, destabilizing a hydrogen bond that forms between orlistat and the active site histidine. A water molecule can then hydrogen bond with histidine and become activated to hydrolyze orlistat. These findings suggest that rational design of inhibitors that block hexyl tail transition may lead to a more potent TE inhibitor. To search for novel inhibitors of TE, I performed virtual DOCK screening of FDA approved drugs followed by a fluorogenic assay using recombinant TE protein and found that proton pump inhibitors (PPIs) can competitively inhibit TE. PPIs, which are used for the treatment of gastroesophageal reflux and peptic ulcers, work to decrease gastric acid production by binding irreversibly with gastric hydrogen potassium ATPase in the stomach. Recently, PPIs have been reported to reduce drug resistance in cancer cells when used in combination with chemotherapeutics, although the mechanism of resistance reduction is unknown. Further investigation showed that PPIs are able to decrease FASN activity and cancer cell proliferation in a dose-dependent manner. These findings provide new evidence that FDA approved PPIs may synergistically suppress cancer cells by inhibiting TE of FASN and suggests that the use of PPIs in combinational therapies for the treatment of many types of cancer, including pancreatic cancer, warrants further investigation.

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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A busca por estratÃgias de cultivo que possam contribuir para a aclimataÃÃo de plantas à salinidade à de fundamental importÃncia, pois, alÃm de possibilitar a identificaÃÃo de genes potenciais para guiar ensaios de modificaÃÃo genÃtica, permite selecionar cultivares com maior capacidade de crescer em solos com excesso de sais. A fim de testar a hipÃtese que a nutriÃÃo nitrogenada com NH4+ aumenta a tolerÃncia de plantas de Sorghum bicolor à salinidade, atravÃs da ativaÃÃo de mecanismos voltados ao controle da homeostase iÃnica, estabeleceram-se trÃs etapas experimentais. Na primeira delas, que objetivou definir a relaÃÃo entre as fontes de nitrogÃnio (N), NO3- e NH4+, que proporcionasse melhor crescimento das plantas sob salinidade, observou-se claramente que a nutriÃÃo somente com NH4+ (proporÃÃo NO3-/NH4+ de 0:100) foi mais vantajosa para o crescimento de S. bicolor sob salinidade que a nutriÃÃo apenas com NO3- ou com o regime misto desses dois Ãons, dado os maiores Ãndices de Ãrea foliar e massa seca da parte aÃrea. Verificou-se tambÃm que, sob estresse, as plantas nutridas somente com NH4+ acumularam menos Na+ nas folhas e nas raÃzes, influenciando positivamente a relaÃÃo K+/Na+, e apresentaram maiores teores de aminoÃcidos solÃveis, principalmente aqueles ricos em N (glutamina e asparagina), que contribuÃram para evitar a toxicidade do NH4+ e provavelmente para o ajustamento osmÃtico. AlÃm disso, enquanto plantas nutridas com proporÃÃes NO3-/NH4+ de 100:0, 75:25, 50:50 e 25:75 apresentaram taxas de assimilaÃÃo lÃquida de CO2 inalteradas ou reduzidas pela salinidade, plantas cultivadas somente com NH4+ (proporÃÃo 0:100) apresentaram incrementos nessa variÃvel, em reposta ao estresse. A segunda etapa teve como objetivo principal investigar se a tolerÃncia à salinidade mediada pelo NH4+ era resultante da regulaÃÃo efetiva dos processos relacionados à fotossÃntese. Nessa ocasiÃo, esse argumento foi refutado, pois a melhor eficiÃncia do fotossistema II sob estresse salino foi observada nas plantas cultivadas com a mesma proporÃÃo de NO3- e NH4+ (proporÃÃo 50:50). Nesse grupo de plantas, a reduÃÃo no quenching nÃo fotoquÃmico (NPQ) confirmou a maior eficiÃncia fotoquÃmica, dado o aumento na eficiÃncia quÃntica potencial (Fv/Fm) e efetiva (ΦPSII) do fotossistema II e a elevada taxa de transporte de elÃtrons (ETR). Esse fenÃmeno foi diretamente relacionado com os incrementos nos teores de clorofila b e de antocianinas. Por fim, na terceira etapa, objetivou-se elucidar os mecanismos envolvidos no controle do acÃmulo de Na+, sob salinidade, na cÃlula e na planta inteira, bem como identificar o papel da nutriÃÃo com NH4+ nesses processos. Em estudos com vesÃculas de membrana de raÃzes, verificou-se que plantas estressadas cultivadas somente com NH4+ apresentaram maior ativaÃÃo dos transportadores do tipo antiporte Na+/H+ (SOS1) de membrana plasmÃtica e, em menor proporÃÃo, do antiporte Na+/H+ (NHX) de tonoplasto, ao passo que o oposto foi observado nas plantas nutridas com NO3-. Esses dados sugerem que o cultivo somente com NO3- induziu o mecanismo de compartimentaÃÃo de Na+ no vacÃolo, como evidenciado pela anÃlise dos transcritos da famÃlia NHX, em que a expressÃo do gene SbNHX2 (principal isoforma expressa) nas raÃzes das plantas foi aumentada em quase todos os tempos analisados (24, 48, 120 e 240 horas apÃs exposiÃÃo ao NaCl). Mesmo assim, essa resposta nÃo foi suficiente para o controle do Na+, jà que a entrada contÃnua desse Ãon no xilema radicular afetou o influxo de K+ na seiva e limitou o acÃmulo de K+ nas folhas. Por outro lado, a nutriÃÃo somente com NH4+ ativou potencialmente mecanismos de controle do acÃmulo de Na+, uma vez que houve acionamento efetivo do efluxo de Na+ para o apoplasto via SOS1, que restringiu o carregamento desse Ãon no xilema e, consequentemente, limitou a acumulaÃÃo dele nos tecidos aÃreos. A formaÃÃo do gradiente de potencial eletroquÃmico, essencial para a atividade dos transportadores Na+/H+, foi modulada diferencialmente pela fonte de N. A atividade de bombeamento de prÃtons da H+-ATPase de membrana plasmÃtica (P-ATPase) foi estimulada em maior proporÃÃo pela presenÃa de NH4+, sem haver, contudo, aumento na atividade de hidrÃlise de ATP. Jà o aumento da translocaÃÃo de H+ pela P-ATPase em plantas estressadas cultivadas com NO3- foi diretamente relacionado ao incremento na hidrÃlise de ATP. Esses resultados sugerem que a disponibilidade de NH4+ aumentou a afinidade da P-ATPase por H+, pois houve melhor eficiÃncia de acoplamento H+/ATP, e isso tornou a enzima mais efetiva para transportar H+ com menos gasto de energia. AlÃm disso, esse aumento no bombeamento de prÃtons resultou em um maior potencial eletroquÃmico, e favoreceu diretamente a atividade do antiporte SOS1 de membrana plasmÃtica. Os nÃveis de transcritos dos genes SbPHA3 e SbPHA5 (principais isoformas expressas da famÃlia) foram aumentados nas plantas cultivadas somente com NO3-, nos tempos iniciais de exposiÃÃo ao estresse salino (12 e 24 h), enquanto que, nos cultivos somente com NH4+, essa resposta sà foi detectada apÃs 24 h. No vacÃolo, a principal bomba responsÃvel pela formaÃÃo do gradiente de H+ durante a exposiÃÃo ao estresse salino foi a H+-ATPase (V-ATPase), em comparaÃÃo à H+-PPiase. Nas plantas cultivadas somente com NO3-, observou-se uma melhor regulaÃÃo da V-ATPase, em associaÃÃo à atividade aumentada do antiporte NHX, enquanto que no cultivo com NH4+, a ativaÃÃo do transporte de H+ sob salinidade foi diretamente relacionada a incrementos na atividade de hidrÃlise de ATP da V-ATPase, bem como ao aumento da expressÃo dos transcritos do gene SbVHA2, ao longo de todo o perÃodo experimental. Essas observaÃÃes revelam que o NH4+, como fonte Ãnica de N, ativa mecanismos que envolvem uma regulaÃÃo coordenada, nas raÃzes, da atividade e da expressÃo gÃnica de bombas de H+ e transportadores Na+/H+ de membrana plasmÃtica e de tonoplasto, que culminam no controle do acÃmulo de Na+ na planta inteira e aumentam a tolerÃncia de S. bicolor ao estresse salino. / Over the last decades, several researchers have focused the development of cultivation strategies in order to improve the plantâs ability to withstand salinity. Understanding the plant salt tolerance is one of important trait to enhance productivity of crops in saline soils because it provides molecular basis for plant breeding, as well as allows identify plant species with a greater ability to grown in salinized areas. In order to test the hypothesis that nitrogen nutrition with NH4+ improves the salt tolerance in Sorghum bicolor plants, through the restrict control of ionic homeostasis, three experimental steps were established. In the first one, we investigated what would nitrogen regime, as NO3-:NH4+ ratio, contribute to the better growth of plants under salinity. Our data clearly showed that the nutrition with only NH4+ (NO3-/NH4+ at 0:100) was more advantageous for the growth of S. bicolor under salinity than the supply with solely NO3- or the mixed regimes, as evidenced by the higher leaf area and shoot dry mass. Under salinity, Na+ accumulation was severely limited in presence of NH4+ (0:100), which positively influenced on K+/Na+ homeostasis. In parallel, NH4+-fed plants displayed a substantial accumulation of N-rich amino acids (mainly glutamine and asparagine) in both tissues, which seems to be fundamental in alleviating the NH4+ toxicity. Furthermore, whereas plants treated with NO3-:NH4+ ratio of 100:0, 75:25, 50:50 and 25:75 ratios had their photosynthetic rates (A) unaltered or reduced by salinity, plants supplied with only NH4+ showed an increased CO2 assimilation in response to stress. During the second step, we evaluated if the better salt tolerance in NH4+ cultivated plants was due to an effective regulation of photosynthesis-related processes. This idea was rejected because of the most striking effects of nitrogen regime were observed in plants supplied with equal amounts of NO3-: NH4+ (50:50). Under salt stress, plants from 50:50 NO3-:NH4+ treatments displayed a lower non-photochemical quenching (NPQ) and an improved photosystem II maximum efficiency (Fv/Fm). Their superior performance was also indicated by a higher effective quantum yield of PSII (ΦPSII) and electron transport rate (ETR), as well as increased chlorophyll b and anthocyanins. Finally, at the third step, we supply S. bicolor plants with NO3- or NH4+ to investigate changes in pathways for control of Na+ accumulation, at cell and whole plant level, in response to 75 mM NaCl-stress. By using root membrane-enriched vesicles, it was found that a more pronounced plasma membrane Na+/H+ antiporter (SOS1) activity and low loading of Na+ in the xylem in the NH4+ treated plants, whereas a largest vacuolar Na+/H+ exchanger (NHX) activity was noticed by NO3- grown plants. These data suggest that the NO3- availability induced the compartmentalization of Na+ into the vacuole, as supported by the upregulation of SbNHX2 gene expression over time of NaCl exposure (12, 24, 48, 120 and 240 h). Nonetheless, it composed an inefficient pathway of Na+ control, since the incessant entrance of Na+ in the xylem sap impaired the K+ loading and limited the K+ accumulation in the shoot. On the other hand, the NH4+ supply potentially activated the mechanisms for control of Na+ accumulation, driving an effective efflux of Na+ out of the cell, via SOS1, restricting its loading in the xylem and thus limiting Na+ reach and accumulation in the aerial tissues. Surprisingly, we found that the generation of electrochemical potential gradient for Na+/H+ exchange activity is differentially modulated by the nitrogen source. The H+-pumping activity driven by plasma membrane H+-ATPase (P-ATPase) was greatly stimulated by the presence of NH4+ in growth medium, however, without an increase in ATP hydrolysis activity. Conversely, the improvement of P-ATPase-generated H+-pumping of NO3- fed stressed plants was directly related to the increase of ATP hydrolysis. These data show that the NH4+ availability enhances the H+/ATP coupling efficiency of P-ATPase, i. e. the enzyme displayed a high capacity of transport H+ across plasma membrane with low ATP consumption. Moreover, the bigger H+ translocation resulted in a greater electrochemical potential which in turn favored the SOS1 activity. The expression of SbPHA3 and SbPHA5 genes was upregulated in NO3- grown stressed plants at the beginning of salt exposure (12 and 24 h), whereas it was enhanced in NH4+ supplied stressed plants only after 24 h. At vacuole level, the H+-ATPase (V-ATPase) was the main proton pump responsive to salinity, as compared do H+-Pyrophosphatase (PPase). A better regulation between V-ATPase and NHX antiporter activities was noticed by plants from NO3- treatments. Under NH4+ supply, the increase of H+ pumping was directly associated to the improvement of ATP hydrolysis by V-ATPase, coupled to upregulation of SbVHA2 gene expression over time of salinity exposure. Taken together, our data reveal that the NH4+, as the only nitrogen source, activates an intricate regulation of Na+ control pathways, involving the existence of a robust regulation and systematic mechanism firstly on root cell and subsequently on whole plant in sorghum upon salinity. In conclusion, the NH4+ stimulated salt tolerance is resulted from a more active SOS1 protein and high efficiency of P- and V-ATPase in the roots, which help to efficient Na+ exclusion and counteract net Na+ accumulation in the cytosol, thus preventing the loading of Na+ in the xylem sap and its reach in the photosynthetic tissues.

Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2008.

Acid-related disorders are common, chronic conditions that have considerable impact on a patient's quality of life. In a study conducted by Majumdar et al. (2003:2411) the prevalence of chronic acid-related disorders was 2.3%. Acid-related disorders represent a major financial consideration with respect to the costs of drug prescribing (Whitaker, 1998:6). Health care cost increases each year. This leads to an increased interest in economic evaluation of health care and medical technologies (Anell & Svarvar, 2000:175). Health care providers no longer make treatment decisions independent of the consideration of the resultant cost. The treatment provided must not only provide value but the value must be documented to justify spending money. Economic evaluation research has emerged to offer guidance to policy makers, practitioners, health plans and institutions facing difficult treatment and coverage decisions (Ellis era/., 2002:271). The main objectives of this study were to investigate the prescribing patterns and cost of acid reducing medicine with special reference to proton pump inhibitors and histamine-2 receptor antagonists in a section of the private health care sector of South Africa from 2001 to 2006. A longitudinal retrospective drug utilisation study was done on acid reducing medicine items claimed through a national medicine claims database. The five study years were 2001, 2002, 2004, 2005 and 2006. All the study years stretched from 1 January to 31 December. It was determined that acid reducing medicine items prescribed decreased from 2.74% during 2001 to 2.50% during 2006 of all medicine items claimed. The same decreasing trend was observed regarding the cost of acid reducing medicine items. The cost percentage decreased from 4.89% (2001) to 3.72% (2006). However, the average cost per medicine item for the acid reducers increased by 5.35% from 2001 (R230.04 ± 176.29) to 2002 (R243.72 ± 184.18) and then decreased by 15.23% from 2002 to 2004. It again decreased with 15.05% from 2004 (R206.19 ± 179.42) to 2006 (R175.70 ± 172.55). The changes in the average cost of acid reducers were of no practical significance. Proton pump inhibitors represented about half of the acid reducing medicine items prescribed and more than 70% of the total cost of acid reducing medicine items during the study years. The average cost of PPIs revealed a practical significant decrease (d > 0.8) from 2002 (R372.42 ± 156.62) to 2006 (R241.56 ± 177.21). H2RAs contributed between 15.00% and 18.26% of all acid reducing medicine items while contributing to between 9.68% and 16.85% of the total cost of all acid reducers. The active ingredient most often prescribed was lansoprazole during 2001 and 2002, esomeprazole during 2004 and omeprazole during 2005 and 2006. Lanzor® 30mg was the acid reducer with the highest cost from 2001 to 2005, while Pariet® 20mg took the lead in 2006. Zantac® 150mg effervescent tablets were the H2RA, with the highest cost, during the five study years. The percentage innovator items decreased by 4.50% from 2001 to 2002, increased by 1.01% from 2002 to 2004 and decreased again by 31.06% from 2004 to 2006. The slight increase in the percentage innovator medicine items claimed from 2002 to 2004 may be explained by the introduction of Nexiam® (esomeprazole) into the market in 2002. The total number of generic medicine items claimed contributed between 9.62% (n = R1 788 242.25) in 2001 and 30.75% (n = R3 196 163.34) in 2006 of the total cost of acid reducing medicine items. The average cost per day of innovator medicine items was higher than the average cost per day of generic medicine items. This might be explained by a lower average cost for generic medicine items. It was also determined that the prevalence of the two-drug regimens was the highest during the five study years. The Helicobacter pylori (H.pylori) eradication treatments, which included different antibiotics, increased from 2.72% in 2001 to 5.05% in 2006. The PDD for most of the active ingredients of H2RAs and PPIs remained stable during the study years. However, it appears that the PDDs, of the PPIs, active ingredients were more constant than the PDDs, or the H2RAs, active ingredients. The median of the different PPI active ingredients was reasonably more constant than the median of the different H2RA active ingredients. Thus the changes between the PPIs' and H2RAs' active ingredients might be explained by the variation in the median (the number of days the relevant medicine item was claimed for). It is then also recommended that the aspects of generic substitution as well as the usage of H2RAs as prescribed vs. self medication should be further investigated to increase possible cost savings. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2008.